Saxon Biotechnology Symposium

2 May, 2011

ABSTRACTS

Editors:Michael BrandMichael SchroederRalf SeidelAndrea A. RobitzkiAndreas ReichenbachNorbert Sträter

Prof. Michael BrandDirectorBIOTEChnology Center, Technische Universität Dresden

Prof. Michael SchroederProfessorship for BioinformaticsBIOTEChnology Center, Technische Universität Dresden

Dr. Ralf SeidelJunior Group LeaderBIOTEChnology Center, Technische Universität Dresden

Prof. Andrea A. RobitzkiDirectorCenter for Biotechnology and Biomedicine, Universität Leipzig

Prof. Andreas ReichenbachProfessorship for NeurophysiologyPaul Flechsig Institute of Brain Research, Universität Leipzig

Prof. Norbert SträterProfessorship for structural analysis of biopolymersCenter for Biotechnology and Biomedicine, Universität Leipzig

Dr. Sabine MatthiäHead of AdministrationBIOTEChnology Center, Technische Universität Dresden

Dr. Svenne EichlerChief EXECUTIVE OfficerCenter for Biotechnology and Biomedicine, Universität Leipzig

Robert MännelBIOTEChnology Center, Technische Universität Dresden

Robert MännelBIOTEChnology Center, Technische Universität Dresden

Addprint AG

April 25, 2011

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CONTENT

Foreword

Program

Presentations

1. Biophysics

1 Feeling for cell function with light Jochen Guck 18

2 Torwards multifunctional lab-in-a-tube systems and artificial micromachinery Oliver Schmidt 19

3 Intelligent Glue - using Lipophilic Oligonucleotides to build biocompatible Nanostructures Daniel Huster 20

4 Nanoscale observation with Far-Field (STED) Fluorescence Microscopy: Unraveling membrane heterogenity Christian Eggeling 21

2. Cell Biology

5 Tissue Engineering for the Nervous System Burkhard Schlosshauer 24

6 MCPH1 regulates neuroprogenitor division mode Zhao-Qi Wang 25

7 Molecular and Cellular Pathways of Foxp3+ Regulatory T Cell Generation Karsten Kretschmer 26

8 Human tissue slice cultures: A novel test system Ingo Bechmann 27

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3. Bioinformatics

9 Regulatory Genomics: Computational approaches to gene regulation Martin Vingron 30

10 The complex world of long non-coding RNAs Peter Stadler 31

11 Cellular stress response and regulation of metabolism Edda Klipp 32

12 Disentangling genetic networks controlling complex traits Andreas Beyer 33

Posters

4. Molecular Medicine

13 Impairment of NFκB activity by unsaturated fatty acids Julia Schumann 36

14 Human fibroblasts support the expansion of IL-17 producing T-cells via up-regulation of IL-23 production by dendritic cells Christine Schirmer 37

15 Understanding the molecular basis of brain regeneration in adult zebrafish Caghan Kizil 38

16 T helper cells and macrophages play an important role in IL-4R- dependent pathology in pulmonary cryptococcosis Uwe Müller 39

17 Th1 and Th17 cells are protective and precede fatal eosinophil-modulated Th2 responses in a model of bronchopulmonary disease Daniel Piehler 40

18 Using Next Generation Sequencing Technology to analyze Zebrafish Central Nervous System Regeneration after Traumatic Brain Injury Nikos Kyritsis 41

19 Development of a 7-(2-[18F]Fluoroethoxy)-6-Methoxy-Quinazoline derivated as a pet radiotracer for PDE10A: Synthesis, Potency and Radiolabelling Gregor Schwan 42

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20 A new conditional photoreceptor degeneration and regeneration model in the adult zebrafish retina Sarah Hochmann 43

21 Establishing and characterizing a light lesion paradigm to study retina regeneration in adult zebrafish Anke Weber 44

22 Alteration of gene expression in murine macrophages by fatty acid supplementation Axel Schöniger 45

23 Mechanism of Stem Cell Activation in Burn and Scald Injuries using Erythropoietin Katja Scheffler 46

24 Chemosensitivity testing of novel kinase-inhibitors for the individua- lized therapy of the malignant melanoma Sarah Pönick 47

25 Defining the role of the two distinct fragments of the histone methyl- transferase Mll2 and their activities in regulating and maintaining mouse ES cells. Helmut Hofemeister 48

26 Development of Ghrelin Inverse Agonists against Obesity Constance Chollet 49

27 Regeneration of the adult zebrafish brain after a traumatic brain injury Volker Kroehne 50

28 PDE10A inhibitors demonstrate efficacy in an animal model of negative symptoms in schizophrenia. Barbara Langen 51

5. Bioanalytics

29 Assay of a MCF-7 rho zero cell line Sandra Heller 54

30 Identification of Glycosaminoglycan binding sites on Interleukin-8 using solution NMR spectroscopy Annelie Pichert 55

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31 Micro-fluidic contact printing of Saccharomyces cerevisiae on trans- parent matrices for whole-cell biosensors Mahipal Ganji & Martin Mkandawire 56

32 A novel two-dimensional liquid chromatography- mass spectrome- try based method for site specific mapping of protein carbonylation sites Ravi Chand Bollineni 57

33 Uv/vis spectral imaging of cytochrome c in retinal cells Julia Schweizer 58

34 Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases Marco Kloos 59

35 Novel strategies for identification of dermatophytes, yeasts and mould in context of primer design Janine Brettschneider 60

36 Benzene and toluene cause anti-apoptosis in human lung epithelial cells at non acute toxic concentrations Stefanie Lauckner 61

37 NTPDases of microbial pathogens Ulrike Krug 62

38 Structural studies of DnaK in complex with proline rich antimicrobial peptides Michael Zahn 63

39 Mass spectrometric characterization of peptide-bound lipid peroxidation products Ivana Milic 64

40 The A-YES Assay – an innovative biological measurement system for the detection of estrogenic activity in mineral water Karina Wolfram 65

41 White Biotechnology with Plant Cells – Development and transfer of innovative methods for the application of plant cell secondary metabolites Felix Lenk 66

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6. Bioinformatics

42 Machine learning approach for RNA secondary structure prediction - A case study Kornelia Lindemann 70

43 Correlation between sequence motifs in membrane proteins and PROSITE Anne Nöldner 71

44 Correlation of specific surface parameters and cell adhesion force parameters Maria Beier & Tommy Hofmann 72

45 The novel approach eHMM for analyzing membrane proteins in case of HP_0565. Anne-Marie Pflugbeil 73

46 Novel prediction algorithm eGOR – from sequence to stable regions of membrane proteins Riccardo Brumm, Eric Frenzel & Florian Heinke 74

47 Energy based Prediction and Comparison of Protein Structures Florian Heinke 75

48 Disentangling complex regulatory traits in QTL analysis Mathieu Clément-Ziza 76

49 Analysis of membrane protein stability in Diabetes insipidus Florian Heinke 77

50 Computational analysis of Interleukin-8 interactions with hyaluronan and chondroitin-sulfate derivatives Sergey Samsonov 78

51 Elucidating the regulatory mechanisms of transcription factor activity in hematopoietic stem cell differentiation Marit Ackermann & Weronika Sikora-Wohlfeld 79

52 Joining experts: Improved Biomarker discovery with Power Graphs and NetRank Matthias Reimann & Janine Roy 80

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53 Identification of protein complexes maintaining Oct4 expression in mouse ES cells Weronika Sikora-Wohlfeld 81

54 The Power of Three: Binding Site Comparison, Text Mining and Net work Analysis for Drug Repositioning Joachim Haupt & Simone Daminelli 82

55 Lineage-specific changes in primate transcription factor genes and the evolution of species-specific traits Katja Nowick 83

56 MAGE - A Model of the Ageing Epigenome Thimo Rohlf 84

57 A novel informatics concept for high-throughput shotgun lipidomics based on the molecular fragmentation query language Ronny Herzog 85

58 Automatic Classification of Embryonic Fruit Fly Gene Expression Patterns Andreas Heffel, Peter F. Stadler & Sonja J. Prohaska 86

7. Tissue and Cell Engineering

59 The impact of PUFA supplementation on the oxidative metabolism of macrophages Stephanie Adolph 88

60 Visualisation of megamitochondria with Laser-Scanning-Microscopy. Susanna Schubert 89

61 A neuronal organotypic, tau transgenic in vitro cell culture model for drug screening in Alzheimer’s disease and related disorders Diana Seidel 90

62 Lineage commitment of conditionally immortalized murine bone marrow mesenchymal stromal cells Maria Rostovskaya 91

63 Initiation of X Chromosome Inactivation Sarah Tsurkan 92

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64 Effect of erythropoietin on expansion and neural-like differentiation of ovine bone-marrow-derived mesenchymal stem cells Oliver Petters & Amir Mofidi 93

65 New micro particle based cell separation method sufficient for direct and simultaneously isolation of multiple targets from whole blood. Viability of separated lymphocytes. Jan-Michael Heinrich 94

8. Genome and Protein Engineering

66 Generation of conditional knockout alleles in zebrafish Peggy Jungke 96

67 Direct Cloning of Silent Gene Clusters and Heterologous Expression Jun Fu 97

68 Pseudomonas putida – development of a heterologous expression system for complex natural products Frank Groß 98

69 The Role of Histone Methyltransferases Mll1 and Mll2 in Neural Differentiation and Reprogramming of Mouse Neural Stem Cells Katrin Neumann 99

70 Setd1 histone methyltransferases are required for embryonic stem cells and mammalian development Anita S. Bledau 100

71 Surface supercharged human enteropeptidase light chain shows im proved solubility and refolding yield Peter Simeonov 101

72 Junior Research Group „White Biotechnology“ – New approaches to functional screening Antje Eichler, Karen Stumm & Thorsten Oeser 102

73 Role of efnb2 during Zebrafish development Annekathrin Kraenkel 103

74 Expression of the vast majority of genes in embryonic stem cells does not depend on MLL complex-mediated H3K4 methylation Andrea Kranz 104

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75 Notum expression and its effect on fgf8 gradient and signalling in Danio reiro. Shradha Das 105

76 Protein Tagging and Gene KO strategies for Systems Biology Ashish Gupta 106

9. Biophysical Technologies

77 When cell extracts diffract: Probing the mechanical behavior and structure of mammalian membranes Suzanna Balko 108

78 Getting closer to the nature of specific bonds: Dynamic force spectroscopy on the binding of antibodies and tau peptides Carolin Wagner 109

79 Real-time 3-dimensional particle tracking with subnanometer accuracy at 5,000 frames per second Alexander Huhle 110

80 DNA unwinding mechanism of a RecQ helicase from Arabidopsis thaliana investigated with magnetic tweezers Daniel Klaue 111

81 Scanning evanescent fields in TIRF microscopy using a single point- like light source and a DNA worm gear Hergen Brutzer 112

82 Monitoring of bioelectrical properties of murine embryonic stem cell-derived 3D cell cultures for in vitro drug testing Silvia Vinz 113

83 High resolution optical coherence tomography for in vivo monitoring of retinal degeneration in rodents Peter Cimalla 114

84 Fast and Precise Optical Tracking of Motor Proteins and Motor Systems using Gold Nanoparticles René Schneider 115

85 Under-filling trapping objectives optimizes the use of the available laser power in optical tweezers Mohammed Mahamdeh 116

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86 3D imaging of lung tissue structure and dynamics using non-invasive optical techniques Maria Gärtner, Sven Meissner & Christian Schnabel 117

87 Mechanical measurements reveal high bending but low twisting rigidity of 3D DNA-origami Dominik Kauert, Tim Liedl & Ralf Seidel 118

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FOREWORD

After two successfull meetings in Dresden 2007 and Leipzig 2009, the Saxon Biotechnology Series continues in Dresden again in 2011.

The Center for Biotechnology and Biomedicine (BBZ) at the University Leipzig and the BIOTEChnology Center of the Technische Universität Dresden are continuing cooperation to hold the joint Biotechnology Symposium in Saxony. The main topics of the conference, hosted in Dresden this year, will be “Biophysics”, “Cell Biology” and “Bioinformatics”.

It is the purpose of the 2011 Biotechnology Symposium to further bridge the gap between basic, applied and integrative research in the fields of nanobiotechnology / nanomedicine and molecular bioengineering. This will ingreasingly strengthen regional networks and make Saxony well-known on a supraregional and also on an international level as a place where biotechnology ranks high. Saxony is an excellent location for research and a growing biotechnological industry, providing a platform for the transfer of knowledge and technology in the fields of molecular design, the development and testing of active substances, genomics, proteomics, diagnostics, cell techniques, nano-bioelectronics, biophysics, cellular machines, tissue engineering and bioinformatics.

Biotechnology is emerging as a key technology for the future, inventions and innovations being the motors for biotechnological progress. We hope this symposium will give the Saxon biotechnological sector a boost and encourage the universities, research centers and companies.

Prof. Dr. Michael Brand Prof. Dr. Andrea A. RobitzkiDirector DirectorBIOTEChnology Center Center for Biotechnology and Biomedicine (BBZ)

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PROGRAM 9.30 – 10.00 Opening Prof. Michael Brand, Director BIOTEC Dresden Stanislav Tillich, Prime Minister Saxony Prof. Kai Simons, board member biosaxony e.V. Prof. Gerhard Rödel, Vice-Rector for Research TU Dresden Prof. Dr. Matthias Schwarz, Vice-Rector for Research and Young Academics University Leipzig 10:00-12.00 Biophysics (chair: Ralf Seidel)

10.00-10.30 Jochen Guck, University of Cambridge Feeling for cell function with light10.30-11.00 Oliver Schmidt – IFW Dresden Towards multifunctional lab-in-a-tube systems and artificial micromachinery11.00-11.30 Daniel Huster, Universität Leipzig Intelligent Glue - Using Lipophilic Oligonucleotides to Build Biocompatible Nanostructures11.30-12.00 Christian Eggeling, MPI for Biophysical Chemistry Göttingen Nanoscale Observation with Far-Field (STED) Fluorescence Microscopy: Unraveling membrane heterogeneity

12.00-13.30 Lunch Break und Poster Session

13.30-15.30 Cell Biology (chair: Andreas Reichenbach)

13.30-14.00 Burkhard Schlosshauer, Natural and Medical Sciences Institute Tübingen Tissue Engineering for the Nervous System14.00-14.30 Zhao-Qi Wang, Leibniz Institute for Age Research Jena MCPH1 regulates neuroprogenitor division mode14.30-15.00 Karsten Kretschmer, CRTD Dresden Molecular and Cellular Pathways of Foxp3+ Regulatory T Cell Generation15.00-15.30 Ingo Bechmann, University Leipzig Human tissue slice cultures: A novel test system

15.30-16.00 Coffee Break

16.00-18.00 Bioinformatics (chair: Michael Schroeder)

16.00-16.30 Martin Vingron, MPI for Molecular Genetics Berlin Regulatory Genomics: Computational approaches to gene regulation 16.30-17.00 Peter Stadler, University Leipzig The Complex World of Long Non-coding RNAs17.00-17.30 Edda Klipp, Humboldt University Berlin Cellular stress response and regulation of metabolism17.30-18.00 Andreas Beyer, BIOTEC Dresden Disentangling genetic networks controlling complex traits

Poster Price Awarding and Social Event

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Presentations

1. BIOPHYSICS

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1 Feeling for cell function with light

Jochen Guck

Light has been central in biological sciences to visually investigate cells by microscopy. In recent years it has increasingly been used to also manipulate biological samples by optically induced forces. Besides trapping, moving, and rotating cells, optical traps can even be used to deform cells in a controlled and nondestructive way. The deformability of cells with such an optical stretcher turns out to be a very sensitive inherent cell marker for any physiological or pathological change in cells that is mirrored in the cytoskeleton or in nuclear structure. For example, cancer cells are much more deformable than normal cells because they need to be able to squeeze through small gaps in the tissue to form metastases. An optical stretcher, integrated into an appropriate micro-fluidic system for cell delivery, can thus be used to diagnose cancer, to detect malaria-infections, or to identify and sort stem cells from heterogeneous populations with relatively high-throughput. Recently, we have even shown that we can monitor the epigenetic state of the nucleus by mechanical phenotyping, with relevance for the pluripotency of embryonic stem cells. In this way, we now cannot only look for changes in cell function, but feel for such changes.

Dr. Jochen Guck

University of CambridgeDepartment of PhysicsCavendish Laboratoryjg473@cam.ac.ukhttp://www.bss.phy.cam.ac.uk/

Biophysics

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2 Towards multifunctional lab-in-a-tube systems and artificial micromachinery

Oliver Schmidt

Lab-on-a-chip as a highly integrative technology platform enables laboratory operations on a small scale and has attracted tremendous interest in basic biomedical and pharmaceutical research as well as in micro-/nanofluidic studies over the past years. By using rolled-up nanotechnology, we propose and promote a disruptive technology called Lab-in-a-tube, which could serve for new applications in chemistry, biology, medicine and engineering. The concept is size scalable from micro-/ to nanometers, fully integrative and comprises a wealth of functionalities, ranging from advanced photonic/electronic to chemical/biological components. Catalytic activity inside rolled-up tubes is exploited to manufacture fast and versatile self-propelled micro-/nanoengines allowing full kinematic control and cargo transport. Even on-board energy storage seems feasible by a unique 3D fabrication process.

Prof. Dr. Oliver Schmidt

IFW Dresden Leibniz Institut for Solid State and MaterialsResearch DresdenInstitute for Integrative Nanoscienceso.schmidt@ifw-dresden.dehttp://www.ifw-dresden.de/institutes/iin/

Biophysics

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3 Intelligent Glue - Using Lipophilic Oligonucleotides to Build Biocompatible Nanostructures

Daniel Huster

Single stranded DNA carries multiple potential for numerous applications in nanobio-technology. Here, we used lipid modified oligonucleotides to functionalize lipid membranes and membrane coated particles. Membrane anchoring of the oligonucleotides is carried out by insertion of covalently attached lipophilic moieties. Membrane coated particles are stable and carry a biocompatible surface for numerous applications in engineering, diagnostics, or therapy. In plasma membranes, lipid domains serve as platforms for specific recruitment or separation of proteins involved in various functions. We have applied this natural strategy of lateral separation to functionalize lipid membranes at micrometer scale in a switchable and reversible manner. Membrane-anchored peptide nucleic acid (PNA) and DNA, differing in their lipophilic moieties, partition specifically into different lipid domains in model and biological membranes. Separation was visualized by hybridization with the respective complementary fluorescently labeled DNA strands. Upon heating, domains vanished, and both lipophilic nucleic acid structures intermixed with each other. Re-formation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. By linking appropriate structures/functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces.

Prof. Dr. Daniel Huster

Universität LeipzigFaculty of MedicineInstitute for Medical Physics and Biophysicsdaniel.huster@medizin.uni-leipzig.de http://www.uni-leipzig.de/~biophys

Biophysics

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4 Nanoscale Observation with Far-Field (STED) Fluorescence Microscopy: Unraveling membrane heterogeneity

Christian Eggeling

Far-field fluorescence microscopy is a non-invasive and very sensitive analysis technique, allowing for the disclosure of complex (biological) systems. Its only drawback is the limited spatial resolution: the diffraction of light prevents that objects closer than about 200 nm can be discerned. A remedy to this physical limit is the exploitation of the fluorescent label’s properties. Specifically, utilizing at least two distinguishable molecular states, such as a ‘bright’ and a ‘dark’ state, it is possible to ensure that the measured signal stems from a region of the sample that is much smaller than these 200 nm. Examples are based on Stimulated Emission Depletion (STED), on the use of photoswitchable fluorescent markers, or on optical shelving into the marker’s dark triplet state. The presented results range from imaging with molecular resolution to single-molecule spectroscopy in dynamically reduced sub-diffraction foci. Special attention is drawn to heterogeneous molecular diffusion of lipids and proteins in the plasma membrane of living cells.

Dr. Christian Eggeling

Max Planck Institute for Biophysical Chemistry Department of NanoBiophotonicsceggeli@gwdg.dehttp://www.mpibpc.mpg.de/groups/hell/

Biophysics

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2. CELL BIOLOGY

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5 Tissue Engineering for the Nervous System

Burkhard Schlosshauer

In the peripheral nervous system lesioned axons can regenerate if the continuity of the nerve tissue is preserved. Currently, nerve gaps are bridged by autologous nerve transplants with considerable side effects. To improve the therapeutic performance of resorbable nerve tube implants as alternative to autologous nerve transplants, we employ three tissue engineering strategies based on the combination of microstructured biomaterials, signalling molecules and regulatory glia cells. The three strategies are: 1) The instantaneous induction of Schwann cell bands as guiding cues inside the nerve tube. 2) The desensitization of regrowing axons with regard to inhibitory proteins of fibrotic and glial scars. 3) The induction of neovascularization in the implant biomaterial to foster rapid nutritional support. Implant tubes (1 mm diameter) were made from resorbable synthetic or extracellular matrix derived polymers. Schwann cell bands were formed on hundreds of longitudinally microstructured polymer filaments (0.02 mm diameter) which were inserted into the tube lumen. Desensitization of axons will be achieved by siRNA nanoparticles attached to the implant biomaterial. The siRNA shall silence a central messenger pathway onto which various repulsive signals converged. Otherwise the activation of this pathway leads to the collapse of actin filaments and consequently, of axonal growth cones. Neovascularization was achieved in a novel biomaterial sponge which might surround the internal implant tube. The indicated approaches were evaluated by gel retardation assays, RT-PCR, western blotting, single cell- and explant culture systems, time lapse video recording in vitro, a chorioallantois membrane transplantation system in chick eggs to monitor angiogenesis, rat sciatic nerve implantation, histology, immuno fluorescence- and confocal laser scanning microscopy and other techniques. Our results suggest that the novel tissue engineering approaches have the potential to substantially improve the therapeutic performance of peripheral nerve implants.

Partly supported by the german ministry BMBF (0313728) and the European programme EuroNanoMedicine.

Prof. Dr. Burkhard Schlosshauer

University of TübingenNatural and Medical Science InstituteRegenerative Medicine Ischlosshauer@nmi.dehttp://www.nmi.de

Cell Biology

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6 MCPH1 regulates neuroprogenitor division mode

Zhao-Qi Wang

Mutations in the microcephalin gene cause human type 1 Primary Microcephaly (MCPH1), an autosomal recessive disorder, characterized by a reduction of the brain size and milod mental retardation. A hallmark of MCPH1 patient cells is premature chromosome condensation (PCC), likely due to a premature metaphase entry. MCPH1, encoded by microcephalin, has been shown to be an important regulator of the DNA damage response, cell cycle progression, genomic stability and cancer. MCPH1 forms nuclear foci after ionizing and UV irradiation, which colocalize with molecules involved in the ATM- and ATR-mediated DNA damage response pathways. Further, MCPH1 was shown to function in homologous recombination. How mutations of MCPH1 cause microcephaly phenotype in humans is not known. To study the function of MCPH1 in the development of the central nervous system, we disrupted the Mcph1 gene in mice. Mutant mice exhibit microcephaly, due to defects in the development of the cerebral cortex and increased apoptosis in the ventricular and subventricular zones of the embryonic cortex. While neural progenitors proliferate normally in Mcph1-deficient mice, they show a severely constraint self-renewal capacity due to a premature cell cycle exit. Furthermore, deletion of Mcph1 promotes asymmetric neurogenic cell division, which can be corrected by interfering Chk1 downstream effector Cdc25 in vivo. In neural progenitor cells, Mcph1-deficiency compromises the Chk1-Cdk1-mediated G2-M transition resulting in spindle alignment defects and mitotic catastrophe. However, to our surprise, MCPH1 seems to have almost no impact on canonical ATM- or ATR-mediated DNA damage response in neurons or neuroprogenitor cells, which does not seem to be the major cause of the microcephaly phenotype. In summary, our study shows that the physiological function of MCPH1 is to modulate the neuroprogenitor division mode via Chk1-Cdc25-Cdk1 in the centrosome, thereby controlling the neuron production during mammalian brain development.

Dr. Zhao-Qi Wang

Leibniz Institute for Age Research Jena Fritz Lippman Institute (FLI)zqwang@fli-leibniz.dehttp://www.imb-jena.de/groups/wang_en.php

Cell Biology

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7 Molecular and Cellular Pathways of Foxp3+ Regulatory T Cell Generation

Karsten Kretschmer

Single stranded DNA carries multiple potential for numerous applications in nanobio-technology. Here, we used lipid modified oligonucleotides to functionalize lipid membranes and membrane coated particles. Membrane anchoring of the oligonucleotides is carried out by insertion of covalently attached lipophilic moieties. Membrane coated particles are stable and carry a biocompatible surface for numerous applications in engineering, diagnostics, or therapy. In plasma membranes, lipid domains serve as platforms for specific recruitment or separation of proteins involved in various functions. We have applied this natural strategy of lateral separation to functionalize lipid membranes at micrometer scale in a switchable and reversible manner. Membrane-anchored peptide nucleic acid (PNA) and DNA, differing in their lipophilic moieties, partition specifically into different lipid domains in model and biological membranes. Separation was visualized by hybridization with the respective complementary fluorescently labeled DNA strands. Upon heating, domains vanished, and both lipophilic nucleic acid structures intermixed with each other. Re-formation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. By linking appropriate structures/functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces.

Dr. Karsten Kretschmer

Technische Universität DresdenCenter for Regenerative Therapies Dresdenc/o Medical Theoretical Centerkarsten.kretschmer@crt-dresden.de http://www.crt-dresden.de/research/crtd-core-groups/kretschmer/

Cell Biology

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8 Human tissue slice cultures: A novel test system

Ingo Bechmann

The London Tragedy in which six volunteers experienced sever toxic shock syndromes in response to a novel drug (TGN1412) has emphasized the urgent need for human test systems: the drug was well tolerized in rodents and monkeys! We has recently detected a similarly crucials species difference: The death ligand TRAIL which was well tolerized in mouse exhibited severe toxic effects in human brain slices obtained from epilepsy surgery (Nitsch et al., Lancet 2000). Since then we have set up various culture models of human tissue designed to test drugs and heavy ions (with GSI, Darmstadt) in tonsils (funded by BMBF) and brain (funded by ESA) and glioblastomas. The talk will cover the basic technology, application, and some novel insights into tissue organization.

Prof. Dr. Ingo Bechmann

University LeipzigInstitute for AnatomyIngo.Bechmann@medizin.uni-leipzig.dehttp://www.uni-leipzig.de/~anatomie/

Cell Biology

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3. BIOINFORMATICS

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9 Regulatory Genomics: Computational approaches to gene regulation

Martin Vingron

Genome sequence encodes not only genes but also the regulatory relationships among genes. Thus, the time and spatial patterns of gene expression are also encrypted in the DNA sequence. In order to unravel this other genetic code, regulatory genomics attempts to integrate functional genomics data with sequence data. This talk will summarize several approaches developed in our group, starting with a biophysically motivated method for prediction of transcription factor binding sites. Main applications are the identification of tissue specific transcription factors and the prediction of regulatory changes due to SNPs. Further, the talk will describe some indications that the division of promoters into two classes with high and low CpG contents, respectively, is of functional importance and helps in understanding mammalian promoters. In fact, the two classes of promoters display different features when it comes to binding site usage and tissue specific regulation. The dichotomy is further supported by an analysis of histone modifications in the promoters. Taken together, we interpret this as indication that different regulatory mechanisms govern transcription in these two classes of promoters.

Prof. Dr. Martin Vingron

Max Planck Institute for Molecular GeneticsComputational Molecular Biology Departmentmartin.vingron@molgen.mpg.dehttp://www.molgen.mpg.de/~vingron/

Bioinformatics

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10 The Complex World of Long Non-coding RNAs

Peter Stadler

ENCODE and FANTOM showed that nearly the mammalian genomes genomes are almost completely discribed, putting an end to idea of Junk DNA. Since then, we have learned that transcription is more extensive and more complex also in other eukaryotes and even in prokaryotes. In contrast to the common organizational principles govering the protein-coding minority, the collection of transcripts forms a surprisingly heterogenous zoo of RNAs differing in processing, transport, and function. Complex hierarchical processing pathways, furthermore, generate multiple RNA species from the same genomic information that can act in ways that are unrelated in both biochemical mechanism and biological function. For bioinformaticians, new challenges keep popping up, ranging from the technicalities of analysing huge amounts of high throughput sequencing data to ncRNA annotation and the quest for a sensible taxonomy of ncRNA classes. I will focus on novel approaches to analysing long ncRNAs and on the relation of short RNAs to their long precursors.

Prof. Dr. Peter Stadler

University Leipzig Institute of Computer ScienceBioinformaticspeter.stadler@bioinf.uni-leipzig.dehttp://www.bioinf.uni-leipzig.de/

Bioinformatics

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11 Cellular stress response and regulation of metabolism

Edda Klipp

Cellular responses to various types of stress such osmotic stress, nutrient changes, or changes of oxygen levels primarily exploit signalling pathways, but also include adaptation on the metabolic level. The information transfer from the signalling pathway perceiving the extracellular signal to the metabolic pathway responding to the stimulation in order to counterbalance the cellular perturbation may occur on different paths. A common pattern is the activation of transcription factors that regulate the expression of genes coding for metabolic enzymes. It has turned out that signalling pathways have also direct crosslinks to metabolic pathways, modulating the catalytic activity of individual enzymes and thereby providing a shortcut metabolic regulation after stress. Using mathematical models in form of ordinary differential equations in comparison to experimental data, we study the quantitative contribution of the individual information transfer paths to the metabolic adaptation. We can show that the contribution of metabolic and genetic regulation vary over time, allowing to distinguish between short-term responses and more costly long-term adaptation.

Prof. Dr. Edda Klipp

Humboldt-University BerlinInstitute of BiologyTheoretical Biophysicsedda.klipp@rz.hu-berlin.de http://www2.hu-berlin.de/biologie/theorybp/

Bioinformatics

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12 Disentangling genetic networks controlling complex traits

Andreas Beyer

Although the technique of quantitative trait locus (QTL) analysis has helped understanding the regulation of complex traits, it has proven difficult to comprehensively determine their complex genetic architecture, partially because phenotype variation is often the result of multiple variable processes. However, many existing QTL mapping methods do not account for the complexity of regulatory networks where multiple genes (and in consequence multiple loci) may regulate a single trait. Yet, in order to understand mechanisms of complex traits such as common diseases, we need innovative computational methods for elucidating the interactions between multiple loci, environment and the phenotype. Recently, we have developed new methods for the detection of QTL assuming complex, non-additive interactions between loci. We have developed a battery of tests based on measured data for scoring different QTL mapping schemes. Based on this analysis we could show that multi-locus mapping methods perform significantly better than old-style single-locus mapping methods, which are still widely used. Subsequently, we have applied this method to the understanding of post-transcriptional expression regulation in yeast. Natural genetic variation has a strong impact on transcription and on post-transcriptional processes such as splicing, translation and protein turnover. Our analysis separates protein concentration changes that are due to variations of RNA concentrations from changes that are triggered post-transcriptionally. Mapping this inferred post-transcriptional contribution revealed 36 loci that post-transcriptionally affect 64 proteins. We identified regulatory hotspots that control many genes, and a candidate master regulator of amino-acid metabolism genes. Our work presents an example of how to disentangle related (yet different) complex traits in order to reveal their genetic basis.

Dr. Andreas Beyer

Technische Universität Dresden BIOTEChnology Center Cellular Networks & Systems Biologyandreas.beyer@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/beyer/

Bioinformatics

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Posters

4. Molecular Medicine

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13 Impairment of NFKB activity by unsaturated fatty acids Julia Schumann, Herbert Fuhrmann

Polyunsaturated fatty acids (PUFA) are known to modulate lymphocyte proliferation, antigen presentation, cytokine synthesis, oxidative burst as well as expression of adhesion molecules. In this regard, PUFA are speculated in part to exert their effects on inflammatory gene expression through direct actions on intracellular signaling pathways. However, concerning the action of PUFA on NFKB conflicting data do exist. In fact, it is not known if the interrelation between fatty acids and NFKB is restricted to special fatty acid families. In addition, the impact of the degree of saturation of a fatty acid is unidentified so far.Here we present the first systematic study investigating acute as well as long-term effects of PUFA from the n-3, the n-6 as well as the n-9 family on NFKB activity by mean of a luciferase reporter cell line.We identified PUFA to impair NFKB signaling. Furthermore, we could demonstrate the PUFA ability to derogate NFKB activity to be independent from the family the fatty acid belongs to. Instead, we found a correlation between the number of bis-allyl-methylene positions of the PUFA added and the NFKB activity of stimulated, long term supplemented cells.The data provide new insights into the biological mechanisms PUFA exert their anti-inflammatory effects. Since suppression of NFKB activity could be of benefit in a number of inflammatory diseases as well as cancer, our findings are of clinical implication. According to our data dietary supplementation with PUFA-containing oils is likely to provide an at least palliative therapy for disorders linked to inappropriate NFKB signaling.

Dr. Julia Schumann

University LeipzigFaculty of Veterinary Medicine Institute of Physiological Chemistryjulia.schumann@vmf.uni-leipzig.dehttp://www.vetmed.uni-leipzig.de

Molecular Medicine

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14 Human fibroblasts support the expansion of IL-17 producing T-cells via up-regulation of IL-23 production by dendritic cells

Christine Schirmer, Claudia Klein, Martin von Bergen, Jan C. Simon, Anja Saalbach

The initiation of immune responses is associated with the maturation of dendritic cells (DC) and their migration to draining lymph nodes. En route activated DC encounter cells of the tissue microenvironment such as fibroblasts. Since we have shown that DC interact with fibroblasts during immune responses we studied the impact of skin fibroblasts on human monocyte-derived DC function and subsequent human T cell (TC) differentiation.We show that fibroblasts support IL-23 secretion from DC preactivated by lipopolysaccharide (DCact) compared to LPS-activated DC alone. The underlying complex feedback-loop mechanism involves IL-1b/TNFa (from DCact) which stimulate fibroblasts Prostaglandin E2 production. Prostaglandin E2, in turn, acts on DCact and increases their IL-23 release. Furthermore, fibroblast-stimulated DCact are far superior to DCact alone, in promoting the expansion of Th17 cells in a cox-2-, IL-23-dependent manner. Using CD4+CD45RO+ memory TC and CD4+CD45RA+ naïve TC we showed that fibroblasts induce a phenotype of DCact that promote the expansion of Th17 cells. In conclusion, skin fibroblasts are involved in regulation of IL-23 production in DC and - as a result- of Th17 expansion.

Christine Schirmer

University LeipzigMedical FacultyClinic for Dermatology, Venerology and Allergologychristine.schirmer@medizin.uni-leipzig.dehttp://hautklinik.uniklinikum-leipzig.de/

Molecular Medicine

37

15 Understanding the molecular basis of brain regeneration in adult zebrafish Caghan Kizil, Nikos Kyritsis, Stefanie Dudczig, Dorian Freuden- reich, Volker Kroehne, Jan Kaslin, Stefan Hans, Michael Brand

Upon injury, zebrafish initiate a pronounced regeneration response in central nervous system utilizing the proliferative progenitors that include the radial glia. We are interested in analyzing the molecular events associated with initiation and progression of such a response in the zebrafish telencephalon. By using microarray approach for transcriptome profiling, we identified a set of candidate genes that are expressed in the ventricular progenitor zones after the stab-wound injury. We are analyzing the spatiotemporal expression dynamics of candidate genes and generating cre/lox-based genetic tools to analyze the function of those genes during the course of regeneration of the adult zebrafish telencephalon. As a complementary approach, we are utilizing the deep sequencing technology to characterize the expression profiles of proliferating radial glia cells.

Dr. Caghan Kizil

Technische Universität DresdenCenter for Regenerative Therapies Dresdencaghan.kizil@crt-dresden.de http://www.crt-dresden.de/research/crtd-core-groups/brand/

Molecular Medicine

38

16 T helper cells and macrophages play an important role in IL-4R-dependent pathology in pulmonary cryptococcosis

Uwe Müller, Daniel Piehler, Werner Stenzel, Gabriele Köhler, Oliver Frey, Thomas Kamradt, Andreas Grahnert, Maria Eschke, Tina Richter, Frank Brombacher, Gottfried Alber

Cryptococcus neoformans is widely spread in the environment. This opportunistic fungal pathogen can lead to lethal meningoencephalitis in immunocompromised patients. Even in healthy people cryptococci can persist latently in the lung. The focus of our work is the role of pathogenicity factors of the immune system that support fungal persistence. We found that interleukin 4 receptor (IL-4R) is an important factor for susceptibility. In the lung IL-4R supports the induction of alternative activation of macrophages, mucus production by goblet cells, and dissemination of cryptococcal cells from the lung into brain. Latent pulmonary cryptococcal infection might also be a risk factor for immunocompetent patients by favouring the development of local allergic inflammation or asthma. We recently found a gene-dosage effect of IL-4R for cryptococcosis in a murine infection model, i.e. the IL-4R expression level correlates with severity of disease. In the present study we were interested in the role of IL-4R on T helper cells and macrophages in pathogenesis during pulmonary cryptococcal infection. Therefore, we used cell-specific knockout mice that are deficient in the IL-4R on CD4+ T cells or macrophages, which both are more resistant than littermate controls. Mice lacking the IL-4R on T helper cells or macrophages, show diminished eosinophil recruitment, reduced goblet cell mucus production, absence of alternative activation of macrophages, prolonged survival time and enhanced survival rate. This study highlights the importance of IL-4R expression on T helper cells and macrophages in the pathogenesis of pulmonary cryptococcosis and reveals new targets for anticryptococcal and antiasthmatic therapy.

Dr. Uwe Müller

University LeipzigInstitute for Immunology/Molecular Pathogenesisu.mueller@vetmed.uni-leipzig.dehttp://www.uni-leipzig.de/~blessing/

Molecular Medicine

39

17 Th1 and Th17 cells are protective and precede fatal eosinophil-modulated Th2 responses in a model of bronchopulmonary disease Daniel Piehler, Werner Stenzel, Andreas Grahnert, Josephin Held, Lydia Richter, Gabriele Köhler, Tina Richter, Maria Eschke, Gottfried Alber, Uwe müller

Resistance to the opportunistic fungus Cryptococcus neoformans is mediated by Th1 responses and to a lesser degree also by Th17-mediated immunity. Surprisingly, even susceptible mice mount a robust and combined Th1/Th17 response following intranasal infection, although they ultimately succumb to infection. This unusual outcome relies on a late but fulminant onset of IL-4 producing Th2 cells. In this study we investigated the time course of IL-4 production and its innate cellular source in mice infected intranasally with C. neoformans. We show that pulmonary IL 4 production starts surprisingly late after 6 weeks of infection. Interestingly, in lungs of infected mice not only pulmonary Th cells but also eosinophils produce significant amounts of IL-4. In eosinophil-deficient ΔdblGATA mice, IL 33 receptor-expressing Th2 cells are significantly reduced albeit not absent, while protective Th1 and Th17 responses are enhanced. In addition, recruitment of pulmonary inflammatory cells during infection with C. neoformans is reduced in the absence of eosinophils. These data expand previous findings emphasizing an exclusively destructive effector function by eosinophilic granulocytes. Moreover, in ΔdblGATA mice fungal control is enhanced in the lung but dissemination of Cryptococcus is not prevented. Therefore, eosinophils play an immunoregulatory role which contributes to Th2-dependent susceptibility in allergic inflammation during bronchopulmonary mycosis. Together, the late eosinophil-modulated Th2 response appears to phenotypically dominate the earlier developing and even continuing Th1/Th17 response. We further aim to analyze whether the observed Th subpopulations show plasticity.

Daniel Piehler

University LeipzigInstitute for Immunology/Molecular Pathogenesispiehler@vetmed.uni-leipzig.dehttp://www.vmf.uni-leipzig.de/ik/wimmunologie/

Molecular Medicine

40

18 Using Next Generation Sequencing Technology to an alyze Zebrafish Central Nervous System Regeneration after Traumatic Brain Injury

Nikos Kyritsis, Caghan Kizil, Dorian Freudenreich, Jan Kaslin, Volker Kroehne, Michael Brand

In the past years, zebrafish has become a very useful model to study central nervous system regeneration. In contrast to mammals, in which proliferation and neurogenesis are restricted to 2 zones in the telencephalon, 16 germinal niches have been found in zebrafish all along the neuraxis. Work in our lab indicates that after a traumatic brain injury (TBI) in the zebrafish telencephalon lost neurons can be replaced by a sub-population of radial glial progenitors. In these cells a lesion-induced proliferation response that peaks at 3 days post lesion (dpl) is detected.Our aim is to identify the molecular mechanisms which trigger the proliferation response after TBI. For this reason, we used transgenic lines (Tg(PCNA:egfp) and Tg(her4.1:mcherry)) and FACS to isolate the proliferating and non-proliferating progenitor cells at 1 and 3 dpl as well as in the unlesioned situation. Subsequently, we performed a transcriptome analysis using next generation sequencing technology (RNA-seq) to identify genes that are differentially expressed in progenitor cell populations. We will present the deep sequencing strategy with GO term and cluster analysis of the sequence reads, which will be instrumental in examining the signaling pathways that are involved in the regeneration response of the adult zebrafish brain.

Nikos Kyritsis

Technische Universität DresdenBIOTEChnology Center Dresdennikos.kyritsis@biotec.tu-dresden.de http://www.biotec.tu-dresden.de/research/brand/

Molecular Medicine

41

19 Development of a 7-(2-[18F]Fluoroethoxy)-6-Methoxy- Quinazoline derivated as a pet radiotracer for PDE10a: Synthesis, Potency and Radiolabelling.

Gregor Schwan, Winnie Deuther-Conrad, U. Egerland, Karen Nieber, Norbert Sträter, Peter Brust, Detlef Briel

The phosphodiesterase (PDE) 10A has an important role in neurotransmission by regulation of intracellular levels of the cyclic nucleotides cAMP and cGMP in dopaminergic neurons. Thus, PDE10A is associated with dopamine related central nervous diseases such as Huntington´s disease and schizophrenia. PDE10A is a promising candidate for drug development. Therefore a variety of selective PDE10 inhibitors was published in the last years[1,2]. The aim of the herewith presented work is the development of a positron emission tomography (PET) radiotracer for imaging of PDE10A in vivo. Based on a previously published lead structure[3] (IC50 = 16 nM) three nonradioactive fluoroalkoxy derivatives (1, 2, 3) were enantioselectively synthesized over 11-14 steps and characterized regarding their potency and selectivity to inhibit PDE10A in a cAMP competition assay. A prolonging of the alkyl chain from 1 to 3 by one methylen group each resulted in decreased inhibitory potency from IC50 = 24 nM over 106 nM to 144 nM. With regard to radiolabelling derivative 2 seems to be the most promising candidate because of its radiochemical accessibility under standard conditions. Initially a two step reaction consisting of 18F-labelling of 1,3-bistosyloxyethane and following coupling with the phenol 4 was carried out over 20-25 min with yields of 30-45%. For a one-step procedure the tosylethoxy precursor 5 was used for 18F-labelling. The radiochemical yields were improved to 42-72% by shortening the reaction time to 15 min. First animal experiments in female CD-1 mice revealed high initial brain uptake. However it was not significantly inhibited by homologous competition with 2 as well as pre-treatment with MP-10, a high PDE10A specific inhibitor indicating lack of specificity in vivo. In conclusion, these results motivate further structural variation of the lead compound to make it suitable for neuroimaging of PDE10A with PET.

References: [1] Chappie et al., Current Opinion in Drug Discovery & Development 2009, 12, 458–467. [2] Kehler and J. P. Kilburn, Expert Opinion on Therapeutic Patents 2009, 19, 1715–1725. [3] Chappie et al., Journal of Medicinal Chemistry 2006, 50, 182–185.

Gregor Schwan

University LeipzigFaculty of Biosciences, Pharmacy and Psychology Institute of Pharmacygschwan@uni-leipzig.dehttp://www.uni-leipzig.de/~pharm/

Molecular Medicine

42

20 A new conditional photoreceptor degeneration and regeneration model in the adult zebrafish retina

Sarah Hochmann, Stefan Hans, Jan Kaslin, Anja Machate, Anke Weber, Michael Brand

Purpose: Since photoreceptor cell loss during adulthood is one of the major causes for blindness in humans, it is of huge interest to investigate retinopathy studies in regenerating model organisms. Unlike mammals, zebrafish gain a tremendous ability to regenerate parts of their central nervous system (CNS) and retina. Furthermore, the wealth of tools available and the cone-dominance of the zebrafish retina make it a promising model to investigate human retinopathies.

Method: We developed in our lab a new conditional genetic lesion model in the Fgf pathway with the aim to analyze photoreceptor degeneration and disorganization in the adult retinal tissue. We characterized our model using BrdU birthdating experiments, apoptosis assays and immunohistochemistry. Furthermore our model allows us to do lineage tracing experiments of different cell populations in the adult retina.

Results: Using conditional transgene expression we specifically ablated photoreceptor cells, while other cells are largely unaffected. Our experiments revealed that photoreceptor cells were gradually dying and almost completely lost within one week after onset of transgene expression. More importantly, we could also show that these processes are completely reversible in the adult zebrafish and regeneration can be traced from dividing progenitors into newly differentiated photoreceptors. Thus, a very fast and profound regeneration response is able to restore the layered structure by integrating new photoreceptor cells into the existing adult retina.

Conclusion: Here we introduce a new transgenic and inducible genetic lesion model to study adult photoreceptor degeneration and regeneration. This model will be used to investigate photoreceptor maintenance and the mechanisms that allow regeneration to occur in the adult vertebrate retina.

Sarah Hochmann

Technische Universität DresdenBIOTEChnology Center Dresdensarah.hochmann@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/brand/

Molecular Medicine

43

21 Establishing and characterizing a light lesion paradigm to study retina regeneration in adult zebrafish Anke Weber, Sarah Hochmann, Michael Brand

Purpose: Loss of photoreceptor cells is a common symptom in hereditary and age related human eye diseases. In contrast, Zebrafish is capable of regenerating the whole retina after injury and therefore serves as an important model organism to understand the principles of regeneration. Light lesions cause extensive cell death in the photoreceptor cell layer and trigger a special photoreceptor regeneration response that we want to study in further detail. We characterized and compared two different lesion paradigms, the previously described method by Bernardos and colleagues and our own approach. Methods: The light lesion method previously described by Bernardos et. al. was slightly modified and will be referred to as direct illumination. Our own light lesion paradigm employs a microscope to direct the light on one eye of an anaesthetized fish leaving the other eye as a control and will be called indirect illumination. All fish have been dark adapted for 5 days and were exposed to intense white light for 30 minutes. Results: The exposure of zebrafish retinas to intense focused light using indirect illumination causes a small lesion in the central part of the retina, anterior to the optic nerve head. Transgenic fish expressing GFP in rods or UV cones as well as immunohistochemistry show that photoreceptors die in a distinct area. Furthermore, we show that cones are more sensitive to intense light treatment than rods. Cell death initiates within 12 hours post lesion (hpl) and proceeds until 24 hpl. Proliferation of cells in all layers begins within the first 24 hpl and peaks at 3 days post lesion. Regeneration after indirect illumination completes within 28 days. These observations are confirmed in the direct illumination approach.Conclusions:The light lesion paradigms induce cell death selectively in the photoreceptors of the zebrafish retina. The new approach using indirect illumination is as reliable as the direct illumination approach and has additional advantages. We will use this model to understand the role of Fgf signalling in the regenerating retina by interfering with the Fgf pathway after inducing a lesion. Furthermore we hope to elucidate the molecular mechanisms of regeneration in more detail by the help of transgenic lines.

Anke Weber

Technische Universität DresdenBIOTEChnology Center Dresdenanke.weber@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/brand/

Molecular Medicine

44

22 Alteration of gene expression in murine macrophages by fatty acid supplementation

Axel Schöniger, Herbert Fuhrmann, Julia Schumann

Polyunsaturated fatty acids (PUFAs) have been shown to exert several effects in the body. They affect various health-related processes e.g. inflammation and immune response. The potential of these effects contingent upon the respective fatty acid. Recent studies indicate, that fatty acids can be used to modulate immune response, mainly by influencing respiratory burst or cytokine release (Walloschke et al., 2009). These modulating effects are speculated to arise from the incorporation of fatty acids into the phospholipid bilayer of certain immune cells possibly influencing membrane fluidity and the structure of membrane receptors (Grimm et al., 2002). Moreover, fatty acids have been proposed to alterate the expression of several genes thereby acting as universal cellular regulators (Sessler et al., 1998).Methods: Subject of the study are the effects of PUFA on gene expression of immunoregulation-related genes in the murine macrophage cell line RAW 264.7. RAW, supplemented with alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) and arachidonic acid (AA), respectively are going to be subsequently stimulated with one of the following: phorbol-myristate-acetate (PMA), lipopolysaccharide (LPS), Rhodococcus equi or Pseudomonas aeruginosa. Unsupplemented and unstimulated macrophages serve as reference. Total RNA, isolated from the cells is transcribed into complementary DNA (cDNA) by conventional PCR. The obtained cDNA is than amplified by RT qPCR. For quantification of gene expression mathematical delta-delta Ct method is to be deployed.The study will show whether and to what extend PUFA supplementation of cells will modify the expression of certain immunoregulatory genes. In particular, new knowledge is expected in the field of macrophage activation by pathogens regarding adapter proteins (MyD88, RICK), surface molecules (MHC II, B7, Fc-receptor), enzymes of respiratory burst (superoxid dismutase, myeloperoxidase) and antimicrobial peptides (lysozyme).Based on the new findings an assessment of the underlying processes of immunomodulation by PUFA will become possible. At this, the inclusion of pathogens allows new insights into host-pathogen interaction. This is the precondition of a targeted use of PUFA as supportive therapy of chronic diseases caused by various bacteria.

Axel Schöniger

University LeipzigFaculty of Veterinary MedicineInstitute of Physiological Chemistryaxel.schoeniger@vetmed.uni-leipzig.dehttp://www.uni-leipzig.de/~vpci/

Molecular Medicine

45

23 Mechanism of Stem Cell Activation in Burn and Scald Injuries using Erythropoietin Katja Scheffler, Petra Radehaus, Hans-Günther Machens, Augustinus Bader

The human body has control over a toolbox that allows it to appropriately react to injury with an amazing site specifity to achieve a regenerative response. Today it is not clear how local stem cells are awakened in such instances of need. It is well-known that local trauma leads to the release of inflammatory cytokines including IL-6, IL-1beta and TNF-alpha. During recent years the understanding of erythropoietin (EPO) and its function in wound healing has significantly changed. In addition to regulating the erythropoiesis, EPO shows various effects on the reaction to acute and chronic tissue damages. Recent experimental data indicate that recombinant human EPO supports wound healing by angiogenesis and has a possible vasculogenetic effect by recruitment of endothelial progenitor cells into injured tissue. In previous experiments we studied whether EPO activates stem cells and exposed human dermally derived stem cells (FmSCs - fibroblastic mesenchymal stem-cell-like cells) to IL-6, IL-1beta and TNF-alpha in the presence of EPO. EPO alone had a strong inhibitory effect on FmSC growth. With IL-6 we could observe a switch from inhibitory to stimulatory effect of EPO on stem cell proliferation. The next step will be the analysis of tissue samples using histological and molecular biological methods as part of the clinical study “A Multicenter Study on Regenerative Effects of Low-dose Erythropoietin (LDE) in Burn and Scald Injuries”. This study investigates the EPO-supported wound regeneration after serious burns. To better understand the cellular mechanisms of EPO in thermal wounds, the histological analyses concentrate on the investigation of markers for endothelial and epithelial cells, mesenchymal stem cells and components of extracellular matrix.

Katja Scheffler

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Cell Techniques and Applied Stem Cell Biologykatja.scheffler@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~bader/index.htm

Molecular Medicine

46

24 Chemosensitivity testing of novel kinase-inhibitors for the individualized therapy of the malignant melanoma

Sarah Pönick, Heinz-Georg Jahnke, Jan Maschke, Michael Kendler, Jan C. Simon, Andrea A. Robitzki

Today cancer is the second most common cause of death. Due to the fact that each tumour has its own characteristics concerning genetic properties and sensitivity to active pharmaceutical ingredients (API), there is a strong demand for personalized therapies. In this context we developed a screening system that allows direct testing of oncogene directed therapeutics on cell lines derived from melanoma biopsy material.Therefore, we established protocols for the detection of b-raf and c-kit mutations, for analysis of tumour heterogeneity and patient dependent target validation of novel APIs like the kinase inhibitors PLX4032 and Imatinib. Using impedance spectroscopy as a non-invasive, label-free detection technique in combination with our self-developed microelectrode array we are able to monitor the drug efficacy for more than four days. Therefore, we could correlate oncogene directed mutations like b raf V600E within melanoma metastasis derived cell lines with specific sensitivity to the mutation specific kinase inhibitor PLX4032. Furthermore, these activating mutations like b-raf V600E could be correlated with the activity of downstream kinases like MAPK.Regarding the chip-based sensitivity screening using melanoma metastasis derived cell lines with or without specific mutations we could quantitatively determine the efficacy of new APIs reflecting the individual patient dependent response to the tested API.

Sarah Pönick

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Molecular biological-biochemical Processing Technologysarah.poenick@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~dmpt/

Molecular Medicine

47

25 Defining the role of the two distinct fragments of the histone methyltransferase Mll2 and their activities in regulating and maintaining mouse ES cells. Helmut Hofemeister, Jun Fu, Konstantinos Anastassiadis, Francis Stewart

The histone 3 lysine 4 methyltransferase Mll1 (mixed lineage leukemia) and Mll2/ Wbp7 are essential epigenetic regulatory enzyme in embryogenesis and development. Both proteins become cleaved by protease Taspase1, resulting in a large N-terminal and small C-terminal fragment. For the functional analysis of the single cleavage fragments as well as the function of the cleavage, different Mll2 constructs were tagged with EGFP were generated. We found that both fragments localize independently in the nucleus. Interestingly, the N-terminal fragment of Mll2 is more tightly bound and remains associated with mitotic chromatin. Mass spectrometric analysis confirmed that both tagged fragments assemble into a multiprotein complex, described for Mll/ Mll2. But neither of the single fragments alone could rescue impaired cell viability, gene expression or the impaired cell differentiation potential in Mll2 knockout ES cells (embryonic stem). Only constructs containing both fragments together were able to rescue for the loss of endogenous Mll2. A non-cleavable Mll2 construct could also rescue for the loss of endogenous Mll2 in mouse ES cells, indicating that cleavage of Mll2 is not essential to maintain cell viability and expression of specific target genes. However, ES cells expressing non-cleavable Mll2 cells showed an increased level of Nanog expression and a delay in exiting the pluripotency cycle. Thus cleavage of Mll2 is important for the maintainance and modification of Mll2 activity.

Dr. Helmut Hofemeister

Technische Universität DresdenBIOTEChnology Center Dresdenhelmut.hofemeister@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Molecular Medicine

48

26 Development of Ghrelin Inverse Agonists against Obesity

Constance Chollet, Sylvia Els, Ralf Bergmann, Annette Beck-Sickinger

Ghrelin, a gastrointestinal peptide hormone is the only known endogenous orexigenic signal. It plays a central role in the short and long term regulation of hunger and energy homeostasis.[1] Ghrelin receptor (GHSR-1a) possesses a high constitutive activity representing 50% of its maximal activity.[2] Therefore, ghrelin antagonists and inverse agonists have emerged as potential anti-obesity drugs.[3]

The European project GIPIO (gastro-intestinal peptides in obesity) aims to understand hormonal dysfunctions involved in obesity and to design therapeutic peptides/peptidomimetics against this disease. In this context, we are developing short peptides possessing a high inverse agonist activity at ghrelin receptor. Modification as PEGylation and lipidation are also performed to increase peptides bioavailability. In addition, the combination of a ghrelin inverse agonist with a PYY agonist in a double-drug motif is currently studied. PYY3-36 is an anorexigenic gastrointestinal peptide, acting on the same neuronal cell population than ghrelin, with an opposite effect. Combination of the two peptides is indeed considered to be a new strategy against obesity.

In parallel, ghrelin agonist and inverse agonist radiotracers are developed for PET imaging to give an insight in the peptide behaviour and mode of action in vivo. Moreover, knowing the pharmacokinetic of ghrelin inverse agonist tracers will help to develop druggable peptides.

References:1. Cummings, D. E., Physiol Behav, 2006, 89, 71-84.2. Holst, B.; Cygankiewicz, A.; Jensen, T. H., et al., Mol Endocrinol, 2003, 17, 2201-10.3. Chollet, C.; Meyer, K.; Beck-Sickinger, A. G., J Pept Sci, 2009, 15, 711-30.

Dr. Constance Chollet

University LeipzigFaculty of Biosciences, Pharmacy and PsychologyInstitute of Biochemistrychollet@rz.uni-leipzig.dehttp://www.biochemie.uni-leipzig.de/agbs/

Molecular Medicine

49

27 Regeneration of the adult zebrafish brain after a traumatic brain injury Volker Kroehne, Jan Kaslin, Dorian Freudenreich, Stefan Hans, Michael Brand

Severe traumatic injury to the adult mammalian CNS leads to life-long loss of function. In contrast, it is long known that adult teleosts and amphibians can functionally regenerate complex body structures, including significant portions of their central nervous system (CNS). However, the cellular and molecular mechanisms that enable or prevent CNS regeneration are largely unknown. A common feature of adult zebrafish is that new neurons are continuously added to many zones along the whole anterior-posterior axis of the brain. It is unknown how constitutive neurogenesis relates to neuronal regeneration. To investigate regenerative processes in the CNS of adult zebrafish we have established a traumatic stab-wound lesion paradigm in the telencephalon. After lesion a marked increase in proliferation is detected in the constitutive neurogenic zones at the ventricle, as well as ectopic proliferation proximal to the lesion site. The identity of cells that are induced to proliferate was determined by combining BrdU, PCNA and cell type specific marker protein analysis. We find that ventricular radial glia, which are a constitutive neuronal progenitor cell population, increase proliferation and neurogenesis after lesion. To investigate whether newborn neurons derive from a radial glia population we established a genetic fate mapping strategy based on the conditional Cre/lox technology. Indeed, we find many newly generated, radial glia-derived neurons 3 weeks after injury at the lesion site. The newly generated neurons survive for at least 3 months, are decorated with synaptic contacts and express mature neuronal markers. Thus, regeneration after traumatic lesion of the adult zebrafish brain occurs efficiently from radial glial stem/progenitor cells.In summary our results show that neuronal regeneration in the adult zebrafish CNS is a neural progenitor-based process, suggesting a direct link of constitutive neurogenesis and regeneration.

Volker Kroehne

Technische Universität DresdenBIOTEChnology Center Dresdenvolker.kroehne@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/brand/

Molecular Medicine

50

28 PDE10A inhibitors demonstrate efficacy in an animal model of negative symptoms in schizophrenia

Barbara Langen, Doris Zschaber, Hans Stange, Norbert Hoefgen, Ute Egerland, Tom Kronbach, Thorsten Hage

Methods: Chronic treatment with PCP leads to an increased immobility in mice when tested in the classic forced swim test paradigm (Noda et al. 1995). This parameter can actually be used as a surrogate for the depression-like negative symptoms in schizophrenia. In the present study, we exchanged PCP for the selective NMDA antagonist MK-801 (0.2 mg/kg ip OD over 15 days). To evaluate depression-like behavior mice were forced to swim for 3 min during which immobility was recorded. The effect of haloperidol, clozapine and risperidone were evaluated in this model. Additionally, we examined the effect of PDE10A inhibition in this model. In previous studies PDE10A inhibitors have shown a efficacy in animal model of positive symptoms in schizophrenia.To further analyze the influence of repeated MK-801 treatment on the dopaminergic signaling pathways mice were also treated later on with the D1 agonist A-68930, the D2 agonist quinpirole, the D1 antagonist SCH-23390 and the D2 antagonist haloperidol. The effect of these agents on the locomotor activity on mice receiving a chronic MK-801 treatment were recorded and compared to control groups.Results: Repeated MK-801 treatment significantly increased immobility in the forced swim test without affecting open field activity. This effect is long lasting (>24days after end of treatment). In addition, chronic MK-801 treatment also induces a hypersensitivity towards dopamine D1 agonist A-68930 indicating that repeated treatment regime induces a hypofunction of the D1 pathway in mice. This effect can possibly be linked to the hypofrontality in schizophrenic patients. The increase of immobility in the forced swim test is reversed by the atypical antipsychotic clozapine but not by the typical antipsychotic haloperidol or atypical risperidone. PDE10A inhibitors tested significantly reversed the immobility induced by repeated MK-801 treatment.

Dr. Barbara Langen

biocrea GmbHRadebeulbarbara.langen@biocrea.comhttp://www.biocrea.com

Molecular Medicine

51

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5. Bioanalytics

53

29 Assay of a MCF-7 rho zero cell line Sandra Heller, Susanna Schubert, Mario Krehan, Ingo Schäfer, Peter Seibel

The mitochondrion, one important organelle of eukaryotic cells harbours important biochemical processes and acts as key-player in the ageing process and the programmed cell death. The human mitochondrial genome displays a size of 16569bp and contains 37 genes that are important for normal mitochondrial function. Apart from genes for rRNA and tRNA the mitochondrial DNA (mtDNA) encodes 13 polypeptides that are essential enzymatic subunits of the respiratory chain.Cells without mitochondrial DNA are termed rho zero cells (ρ0-cells) according to the genetics of yeast. Therefore, cells with ρ0-genotype lack a functional respiratory chain and require metabolic supplementation for cell viability. Human ρ0-cells show an auxotrophy for pyrimidines because the enzyme activity of dihydroorotate dehydrogenase (pyrimidine biosynthetic pathway) is coupled to a functional respiratory chain. Additionally ρ0-cells show a requirement for pyruvate. It is proposed that the amount of pyruvate that is available for the tricarboxylic cycle is reduced by the oxidation of NADH through the activity of lactate dehydrogenase.One possible method to generate cells without mitochondrial DNA (mtDNA) is to cultivate cells on growth medium that includes chemicals like ethidiumbromide or ditercalinium that interfere with DNA replication.

Sandra Heller

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Molecular Cell Therapysandra.heller@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~mct/mct/

Bioanalytics

54

30 Identification of Glycosaminoglycan binding sites on Interleukin-8 using solution NMR spectroscopy

Annelie Pichert, Stephan Theisgen, Sergey Samsonov, Jürgen Schiller, M. Teresa Pisabarro, Daniel Huster

Interleukin-8 is a well known cytokine, that triggers acute inflammatory reactions by recruiting and activating neutrophils. Previous studies have shown an interaction of IL-8 with highly sulphated glycosaminoglycans (GAGs) such as heparin or heparan sulphate. Hence, for the development of artificial matrices, favouring the healing process in regenerative medicine, it is important to find GAGs with weaker binding affinities. Therefore the interactions of IL-8 with hyaluronic acid (HA), chondroitin-4-sulphate (C4S) and chondroitin-6-sulphate (C6S) as hexasaccharides were investigated by doing solution NMR experiments. 15N-labelled IL-8 was used to record 1H-15N HSQC (Heteronuclear Single-Quantum Coherence) spectra in the presence of varying GAG concentrations. Due to the full resonance assignment of IL-8, changes in the chemical shifts induced by ligand binding could be used to identify the interacting amino acids. While hyaluronic acid showed no interaction with IL-8, clear shifts of certain peaks could be observed in the presence of C4S and C6S hexasaccharides. In both cases the largest chemical shift changes were found for residues K59, V66, V67, K69, A74, E75. Despite the occurrence of some binding sites in the loop area and in the beta-sheets, most interactions took place at the c-terminal alpha-helix.

Binding measurements of IL-8 with low or un-sulphated hexasaccharides of glycosaminoglycans were done by solution NMR experiments. While no interaction between IL-8 and hyaluronic acid was observed, the c-terminal alpha helix could be identified as the main binding site of chondroitin-6-sulphate and chondroitin-4-sulphate.

Annelie Pichert

University LeipzigMedical FacultyInstitute for Medical Physics and Biophysicsannelie.pichert@medizin.uni-leipzig.dehttp://www.uni-leipzig.de/~biophys

Bioanalytics

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31 Micro-fluidic contact printing of Saccharomyces cere- visiae on transparent matrices for whole-cell biosensors Mahipal Ganji, Martin Mkandawire, Steffen Howitz, Bettina Soltmann, Klaus P. Kuehn, Hans-Georg Braun, Wolfgang Pompe

Patterning of cells on transparent matrices for whole-cell biosensors has been mainly done using a layer by layer approach with the Micro-Contact Printing (μ-CP) technology using the Poly(dimethylsiloxane) (PDMS) stamping procedure. For instance, Saccharomyces cerevisiae have been successfully patterned on different functionalized glass and silicon surfaces using μ-CP technology. However, the force applied during stamping in μ-CP reduces the cells viability. In view of this, we are investigating the potential of immobilizing the cells through Micro-Fluidic Contact Printing (μ-FCP) technology to reduce the negative impact of the force from μ-CP. In the μ-FCP procedure, the PDMS stamp is designed to have micro-channels through which the media with cells is injected, and allows the cells to settle on the glass or the wafer by gravity. Then, the cells adhere onto the matrix surface through electrostatic and van der Waals interactions. Furthermore, an immobilization procedure was tested, where, instead of functionalizing the glass, the cells surface were functionalized with Al(NO3)3 to neutralize the surface charge. Thus, a solution of S. cerevisiae (OD600 = 2) was incubated in a solution of 0.07 mM Al(NO3)3 at pH 4 for one hour at 30°C in a shaker rotating at 100 rpm. Afterwards, the functionalized cells were patterned on clean glass slide using μFCP techniques. This procedure results in higher survival rate with higher strength of immobilization than the previous procedure of stamping yeast cells on functionalized glass or wafers with μCP technique. Unlike the μ-CP technique, there is no application of pressure and no drying step. The cells with neutralized surface charges can adsorb on the negatively charged glass surface without relevant electrostatic repulsion. Based on the current preliminary results, the μ-FCP technology seems to have application potential for immobilization of yeast cells for whole-cell fluorescence based biosensors.

Mahipal Ganji & Martin Mkandawire

Technische Universität DresdenFaculty of Mechanical EngineeringInstitute for Materials Sciencemahipal.ganji@nano.tu-dresden.de martin.mkandawire@nano.tu-dresden.dehttp://nano.tu-dresden.de/

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32 A novel two-dimensional liquid chromatography- mass spectrometry based method for site specific mapping of protein carbonylation sites

Ravi Chand Bollineni, Maria Federova, Ralf Hoffmann

Carbonylation refers to the post-translational introduction of reactive aldehyde and ketone groups in to proteins. Protein carbonylation is a major class of irreversible oxidative protein modification, that is universally accepted as biomarker of oxidative stress and several other age-related neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases. Despite of the technological advances, site specific mapping of protein carbonylation by mass spectrometry still remains as a challenging task. Complexity in their analysis arises due their low abundance, poor ionization efficiency and a various routes by which proteins can be carbonylated.Here we present a new method based on 2, 4-dinitrophenyl hydrazine (DNPH), a widely used derivatization reagent for carbonyl groups. Absorption maximum of DNPH (365 nm), in principle could be used for UV laser-desorption/ionization (LDI-) mass spectrometry (MS), which will then selectively ionizes DNPH derivatized carbonylated peptides while suppressing the signals from abundant unmodified and thus underivatized peptides. Briefly, tryptic digests from in vitro oxidized bovine serum albumin (BSA) and β-lactoglobulin (β-LG) were derivatized with DNPH, separated by hydrophilic interaction chromatography (HILIC) and all fractions were analyzed by LDI-MS. The mass list generated by LDI-MS, was used in the following nano reversed phase chromatography (RPC)-ESI-Orbitrap-MS analysis of each fraction from HILIC to retrieve the sequences and thereby for site specific mapping of carbonylation sites.Employing this novel 2D-LC-MS set up, in total three carbonylation sites were identified in native β-LG and nine in native BSA, whereas oxidized β-LG, BSA contained eleven and 32 carbonylation sites, respectively. Solvent accessible area calculations also confirmed higher solvent accessibility for these residues.Combining the sensitivity and specificity of LDI-MS for DNPH-derivatized peptides with the advantages of inclusion list-based data dependent acquisition in HILICxRPC-ESI-Orbitrap-MS/MS setup allowed identification of protein carbonylation sites with high sentivity, reproducibility and confidence levels.

Ravi Chand Bollineni

University LeipzigFaculty of Chemistry and MineralogyInstitute of Bioanalytical Chemistryravi.bollineni@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~bioanaly/

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33 Uv/vis spectral imaging of cytochrome c in retinal cells Julia Schweizer, Julia Hollmach, Gerald Steiner, Lilla Knels, Richard Funk, Edmund Koch

Age-related macular degeneration (AMD) is the leading cause for blindness in people over 65 years in the western society. The most risk factors for AMD include uv-light exposure, overweight, diabetes and smoking. Furthermore, the prevalence of AMD arises in the older age, due to a generally increased oxidative cell stress. In case of AMD, the results are irreversible degenerative cell processes, like apoptosis, and morphological changes in the eye-background. Currently there is no commonly used method to detect AMD in early states and there are no effective treatments to regenerate the visual function of patients with this disease. Therefore, it is essential to develop methods for an early detection of AMD in order to stop degeneration processes on a very low level.The cell protein cytochrome-c (cyt-c) has been identified as a key signaling molecule of degeneration processes and apoptosis. Cyt-c is an electron transfer protein within the respiratory chain of cells. Upon its oxidative state, the protein exhibits several strong absorbance bands in the spectral range between 400 and 700 nm. All these bands arise from π → π* transitions in the porphyrinic ring. Treating retinal rat ganglion cells with glyoxal-solution, the induced oxidative stress, can be monitored in real time with a high performance uv/vis spectral imaging system in situ. Using this spectral imaging setup, we can detect changes of the concentration and the oxidative state of cyt-c in cells. With these spectral signals, we will be able to draw conclusions of the biochemical status and degeneration processes in cells. In this study, we demonstrate that uv/vis spectral imaging has a high potential for sensitive and non-invasive measurements of the macular to detect cell degeneration processes in the early state, thus the aim of this study is to use uv/vis spectroscopy as an early detection method of AMD.

Julia Schweizer

Technische Universität DresdenMedical FacultyDepartment of Clinical Sensoring and Monitoringjulia.schweizer@tu-dresden.dehttp://www.tu-dresden.de/medksm/

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34 Molecular architecture and structural basis of allosteric regulation of eukaryotic phosphofructokinases

Marko Kloos,

Eukaryotic ATP-dependent 6-phosphofructokinases (Pfks) differ from their bacterial counterparts in a much more complex structural organization and allosteric regulation. Pichia pastoris Pfk (PpPfk) is, with ~1 MDa, the most complex and probably largest eukaryotic Pfk. We have determined the crystal structure of full-length PpPfk to 3.05 Å resolution in the T state. PpPfk forms a (abg)4 dodecamer of D2 symmetry with dimensions of 161 x 157 x 233 Å mainly via interactions of the g-chains. The N-terminal domains of the a- and b-chains have folds that are distantly related to glyoxalase I, but the active sites are no longer functional. Interestingly, these domains located at the 2 distal ends of this protein along the long 2-fold axis form a (ab)2 dimer as does the core Pfk domains; however, the domains are swapped across the tetramerization interface. In PpPfk, the unique g-subunit participates in oligomerization of the ab-chains. This modulator protein was acquired from an ancient S-adenosylmethionine-dependent methyltransferase. The identification of novel ATP binding sites, which do not correspond to the bacterial catalytic or effector binding sites, point to marked structural and functional differences between bacterial and eukaryotic Pfk.

Marko Kloos

University LeipzigFaculty of Chemistry and MineralogyInstitute of Bioanalytical Chemistrymarko.kloos@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~bioanaly/

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35 Novel strategies for identification of dermatophytes, yeasts and mould in context of primer design

Janine Brettschneider, Bianca Liebscher, Doreen Kropp, Werner Brabetz, Dirk Labudde

Developing a fast, powerful and specific detection strategy is a key concern in fungal diagnostics. Our aim is to design a microarray based on species and group specific DNA oligonucleotide probes for the identification of the most important human pathogen fungi. For that purpose a multiplex PCR (polymerase chain reaction) for amplifying classes of organism is required. To realize group specific amplification as well as species specific differentiation target sequences like the well known phylogenetic markers ribosomal DNA (rDNA), beta-tubulin and chitin synthase which include both, highly conserved and variable regions, were chosen. The established microbiological and DNA sequence based fugal identification methods are hampered by serveral problems. They are often extremely time-consuming, sometimes no cultivation is possible, quantification is subjective, database do not contain all fungi sequences (difficult comparison with sequenced samples) or there are no results in case of contaminated samples in sequencing and mixture samples. Our approach was based on new findings of whole genome sequencing. These data sets allowed us to assemble and align larger genomic DNA segments. Thus, especially in the case of rDNA additional DNA regions which lie in between well characterized segments could be exploited for the design of new PCR primers and hybridization probes. Based on the conserved and non conserved segments on the target sequences, we generate primer to identify groups and specific organism of fungi. Our primers identify a huge number of fungi in one sample without interacting with sequences from humans, animals and plants.

We develop a fast and efficient computer tool to automate the whole process of aligning, assembling, primer/probe design and microarray analysis.

Janine Brettschneider

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Sciencejbrettsc@htwm.dehttps://www.mni.hs-mittweida.de/

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36 Benzene and toluene cause anti-apoptosis in human lung epithelial cells at non acute toxic concentrations

Stefanie Lauckner, Kalaimathi Murugesan, Iljana Mögel, Irina Lehmann, Martin von Bergen, Janina Tomm

Due to changes in life-style, people in industrialized countries spend around 90% of the time indoors. Therefore humans are exposed to volatile organic compounds (VOCs) which are emitted from sources like furniture and paints. Because of this exposure many people show symptoms like headaches or mucosa-irritation, which are referred to the sick building syndrome. As VOCs are mainly uptaken by inhalation human lung epithelial cells were used as cellular model. In this study we analyzed the cellular response of those cells to the exposition of the VOCs benzene and toluene on the protein level. To simulate the inhalation a transwell-model was chosen, where the epithelial cells were exposed via gas phase. Changes in the proteome were analyzed using two-dimensional-difference-gel-electrophoresis (2D-DIGE). To ensure that the used (0,1 mg/m3 and 0,1 mg/m3) benzene and toluene concentrations were not acute toxic to human lung epithelial cells, a lactate dehydrogenase assay was performed. The cells were exposed to concentrations stated above for 24 h. As negative control cells were exposed to MetOH. After cell lysis and protein determination 2D-DIGE was utilized to quantify the changes in the protein expression following the VOC exposure. Differentially regulated protein spots were quantified with MALDI-MS/MS and identified via Mascot search. Applying the DIGE-technology we detected 334 protein spots for benzene of which 34 significantly regulated proteins could be identified. For toluene we found 688 protein spots of which 102 differentially regulated proteins were identified. According to the chemical and structural similarity of the analyzed compounds we expected nearly the same effect on the proteome level of the epithelial cells. Previous studies showed that aromatic compounds e.g. 1,2-dichlorobenzene or chlorobenzene [1] lead to oxidative stress in lung epithelial cells, caused by side production of reactive oxygen species (ROS). Thus we expected that our cells also might respond via oxidative stress to benzene and toluene. However, most of the proteins found to be regulated for both VOCs could be related to the anti-apoptotic pathway, e.g. VDAC2 and PGD (both up regulated). Besides anti-apoptosis benzene altered the expression of proteins involved in protein quality control, oxidative stress and metabolism. Toluene induced proteins which could be related to inflammation and RNA regulation.

Stefanie Lauckner

Helmholtz Centre for Environmental ResearchDepartment of Proteomicsstefanie.lauckner@ufz.dehttp://www.ufz.de/index.php?de=6693

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37 NTPDases of microbial pathogens Ulrike Krug, Matthias Zebisch, Norbert Sträter

Nucleoside triphosphate diphosphohydrolases (NTPDase) catalyze the hydrolysis of extracellular nucleotides. These enzymes are found in mammals as well as in microbial pathogens where they are thought to modulate the host-response to an infection by hydrolysis of host ATP. We aim to determine crystal structures of microbial NTPDases to use them for structure-guided drug design.For the production of NTPDases from Legionella pneumophila, Schistosoma mansoni, Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi we have established E. coli expression systems. NTPDases of Toxoplasma gondii and Trichomonas vaginalis were expressed as insoluble inclusion bodies. Using rapid dilution systems it was possible to refold the denatured protein to an active form. NTPDase from Legionella pneumophila was expressed in soluble form by secretion to the periplasm. Subsequently the proteins were purified by chromatographic methods and applied to crystallization screens. We were able to determine the crystal structures by different phasing methods, namely SAD and MIRAS. Furthermore, the proteins could be crystallized in complex with substrate analogs. These crystal structures provide insight into the domain motion of the enzyme and broaden our knowledge about the catalytic mechanism and substrate specificity (as well as promiscuity) already derived from the crystal structure of rat NTPDase2.

Ulrike Krug

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Structural Analysis of Biopolymersulrike.krug@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~straeter/

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38 Structural studies of DnaK in complex with proline rich antimicrobial peptides

Michael Zahn, Daniel Knappe, Nicole Ereth, Ralf Hoffmann, Norbert Sträter

Bacterial infections are a major cause of death worldwide. Due to increasing resistance against the commercially available antibiotics over the past few decades, novel antimicrobial drug classes with new mode of actions are required for future treatments. Small proline rich antimicrobial peptides (PR-AMPs) from mammals and insects were identified to target the E.coli Hsp70 chaperone DnaK after cell penetration. Binding of the peptides to DnaK compromises the activity of the chaperone and thus the viability of the bacterial cells, in particular under conditions of stress. The non-lytic cell penetration of PR-AMPs to Gram-negative bacteria makes them a promising drug candidate against human infections. Therefore, structural informations about the interactions between peptide inhibitors and DnaK are necessary for a better understanding of the mode of action. After recombinant expression of the substrate binding domain in E.coli and subsequent purification by IMAC and gelfiltration, we aim to crystallize the domain with several PR-AMPs. Elucidation of the binding mode of the peptides and characterization of the substrate specificity of DnaK will allow a structure-guided development of peptide inhibitors as antimicrobial agents targeting DnaK.

Michael Zahn

University LeipzigFaculty of Chemistry and MineralogyInstitute of Bioanalytical Chemistrymichael.zahn@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~straeter/

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39 Mass spectrometric characterization of peptide-bound lipid peroxidation products

Ivana Milic, Kristin Teuber, Jürgen Schiller, Ralf Hoffmann, Maria Federova

Background: High levels of ROS during oxidative stress are a non-enzymatic mediator of lipid peroxidation. Especially lipids containing unsaturated fatty acyl residues are very susceptible targets for free radicals. Lipid peroxidation products (LPPs), especially those containing a double bond, are very strong electrophiles that readily react with nucleophilic groups in proteins to form Michael adducts or Schiff bases. These modifications can influence the structure and the functional activity of proteins and cause or promote diseases, such as neurodegenerative and cardiovascular diseases. Regardless of their relevance and occurrence in many proteins, LPP and especially reactive carbonyl compounds are poorly characterized and poorly classified. Objective: The aim of this study was the detection of LPP and specific nucleophilic amino acid residues (e.g. Cys, His and Lys) by oxidizing 1-palmitoyl-2-linoleoyl-sn-glycero¬phosphatidyl¬choline (PLPC) and free linoleic acid under different conditions in vitro. Methods: PLPC was oxidized in water by air, H2O2, Cu(I) or a combination of both H2O2 and Cu(I) (Fenton reagent) and the reaction mixture analyzed by ESI-LTQ-Orbitrap-MS/MS in positive ion mode. Products containing reactive carbonyl groups (e.g. aldehydes and ketones) were identified by DNPH derivatization. The reaction products of LPP and nucleophilic residues in three model peptides were analyzed by nanoRP-UPLC-ESI-LTQ-Orbitrap-MS/MS.Results: The oxidation of PLPC with Cu(I)-ions resulted in LPPs without causing significant oxidation of the peptides (e.g. at Pro, Thr, Cys, His and Lys residues). In total 19 different LPP, belonging to six structural groups, reacted with Lys, His and Cys residues in significant quantities. Lys- and His-residues were mainly modified by early stage lipid peroxidation products, such as hydroxyl/oxo-alkenals and the corresponding hydroxyl/oxo-carboxylic acids. Additionally, the primary amino groups of lysine formed Michael and Schiff base adducts with LPP that retained the glycerophosphatidylcholine moiety. Furthermore, low molecular weight LPP modified mostly the thiol group of the Cys-containing peptide including three previously unidentified LPP with mass shifts of 86, 88 and 102 u.

Ivana Milic

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Structural Analysis of Biopolymersivana.milic@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~straeter/

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40 The A-YES Assay – an innovative biological measure- ment system for the detection of estrogenic activity in mineral water

Karina Wolfram, Kirsten Simon, Steffen Uhlig

quo data - Quality Management and Statistics GmbH - is an innovative software developer and statistical consultant for process optimization, quality assurance, experimental and sampling design as well as development and validation of measurement methods especially for biochemical and biological measurement systems. Due to our comprehensive mathematical and statistical experiences we take part at several R&D projects. In this context we are developing biochemical measurement systems for the detection of hormonal and pharmaceutical substances such as estrogens, androgens and antibiotics. The detection of estrogens and androgens in drinking and surface water is an important task due to their potential to alter the endocrine system of humans and animals causing e. g. feminization of fishes. In cooperation with the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK Gatersleben) we developed an innovative yeast based assay (A-YES) for the detection of estrogenic disruptors in mineral, curative and ultrapure water. The assay is available in two versions, which differ in the sensitivity of the method, whereby the version 2.0 is more sensitive than the version 1.0. For both assays, quo data performed the validation study to determine important quality characteristics like accuracy, precision, limit of detection and quantification as well as robustness. With the hypersensitive A-YES 2.0 it is possible to detect estrogens in the range of 0.8 till 21 ng/L 17-ß-Estradiol. The assay is distributed through new diagnostics GmbH. The A-YES is currently adapted for complex matrices like waste water, surface water, urine and plasma. Quo data accomplishes the validation after successful method development. Furthermore a second test system based on A. adeninivorans for detection and quantification of androgenic substances (A-YAS) will be validated within a short time and is afterwards available through new diagnostics GmbH. Beside this project, quo data is intended to collaborate with companies, which are looking forward for a competent service partner in the field of validation of new biological and analytical measurement methods. We are also interested in cooperations with diverse companies and institutions which aim to generate high quality data analysis due the application of statistical methods. In the case of R&D-projects we support our partners in planning, realization and analysis of experiments as well as the statistical assessment of results. We also provide our competences for medical and diagnostic applications, e. g. planning and statistical analysis of clinical trials.

Karina Wolfram

Quo Data Gesellschaft für Qaulitätsmanagement und Statistik mbHwolfram@quodata.dehttp://www.quodata.de/

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41 White Biotechnology with Plant Cells – Development and transfer of innovative methods for the application of plant cell secondary metabolites Felix Lenk, Thomas Bley, Juliane Steingroewer, Chistiane Haas, Katja Geipel, Sibylle Schulz

Plants bare a wide range of nutritional-physiological and pharmaceutical relevant secondary metabolites. However conventional industrial production is limited by environmental and geographical influences. The production of secondary metabolites in vegetable cell- and tissue-cultures can be considered alternatively to classical technologies. In this case a year-round cultivation in the bioreactor under optimal conditions is possible. This approach also does not use harmful substances such as pesticides and therefore allows a sustainable and gentle-on-resource production.Scientists at the Chair of Bioprocess Engineering at TU Dresden in cooperation with the University of Kaiserslautern and the Bulgarian Academy of Sciences deal with the cultivation as well as the theoretical and practical analysis of different cell-cultures. Focusing on the vitamin E producing sunflower and the substances oleanolic and ursolic acid contained in sage investigations on callus- and hairy root-cultures for optimization of growth and secondary metabolite production is carried out. Goal is a macroscopic, theoretic model of the cell’s metabolism as well as a comparison of numeric simulations and experimental data. Chromatographic methods (HPLC, GC-MS) get used to determine metabolic profiles. Interested industrial companies have access to the gained know-how via a platform for knowledge transfer.

Felix Lenk

Technische Universität DresdenFaculty of Mechanical EngineeringInstitute of Food Technology and Bioprocess Engineeringfelix.lenk@tu-dresden.dehttp://tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_maschinenwesen/ilb

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42 Machine learning approach for RNA secondary structure prediction - A case study Kornelia Lindemann, Nadin D. Exner, Susanne Klemm, Dirk Labudde

The RNA is a very important macromolecule that carries many functions. It is not only responsible for transport and transformation of the genetic information and thus for protein biosynthesis, but also regulates numbers of cellular processes. Like a protein the RNA has also enzymatic functions and is involved in mechanisms for gene regulation. Another important point is the defense of foreign-RNA that was injected for example by viruses. Because of the simple configuration it has a high diversity respective it’s folding and functions. In many cases the base pair interactions lead to secondary and tertiary structures that are fundamental for the correct biological function. [1; 2]

In this work we address the secondary structure prediction of RNAs. We compared the results of different programs by using the MC-Annotate as reference. We also used a machine learning approach. For the prediction of the secondary structure of the investigated base we constructed different decision trees by determining the influence of the antecessor, the successor, their secondary structures and chemical shifts measured by NMR experiments. We used the operator Cross-Validation of the program RapidMiner5 to evaluate our results.

Our results show that the single base or base-duplets doesn’t suffice, so that analyzing by triplets is superior. The accuracy of the estimated decision tree is about 84%. However, the prediction of paired bases is more assured than the prediction of non-paired bases (e.g. loops and bulges).

References:[1] Seifert, Michael. Gene und Proteine erklären nicht alles. [Online] 28. 03. 2011. http://idw-online.de/pages/de/news343127

[2] Steger, Gerhard. Bioinformatik, Methoden zur Vorhersage von RNA- und Proteinstruktur. Basel : Birkhäuser, 2003

Kornelia Lindemann

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Scienceklindem1@hs-mittweida.dehttps://www.mni.hs-mittweida.de/

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43 Correlation between sequence motifs in membrane proteins and PROSITE

Anne Nöldner, Christin Türke, Dirk Labudde

The analysis of motifs in membrane proteins can strongly facilitate the characterisation of novel membrane proteins. Especially the knowledge of packing geometry of transmembrane helices and the preferred helices-forming sequence motifs will provide a suggestion about function and structure of proteins.In this work we analysed pattern of three different Pfam-families, based on the result of Gerstein et al. They analysed membrane proteins, which can be related to each other on the basis of the number of the transmembrane helices and sequence similarities. Furthermore, their analysis of motifs of transmembrane helix segments showed that 50 regular expressions were found. Therefrom the pattern G-x-x-x-G (GG4) and G-x-x-x-x-x-x-G (GG7) are the most prevalent, whereas G presents the amino acid Glycine and x the variable position [1]. In contrast to Gerstein et al. we distinguished in transmembrane and non-transmembrane motifs, based on variable positions in their 50 pattern. This list was organized by a well-defined score and threshold function and leads to a new ranking of the investigated pattern [2]. From the new arrangement list regular expressions were realised for transmembrane and non-transmembrane regions. These were converted in PROSITE pattern and equalized with this database.In summary, we show that the pattern LF10, VF8, LF8, LF9, VL4 and IL4 can be established as characteristic pattern of transmembrane regions and PG10, PG6, SA6, AS4, GG7 and GG5 as significant for non-transmembrane parts, for our three families. The correlation of derived motifs with the PROSITE leads to usable results and needs a stronger discussion. [3]

References:[1] Yang Liu, Donald M Engelman and Mark Gerstein. Genomic analysis of membrane protein families: abundance and conserved motifs. In: Genome Biology – 19 September 2002, 3(10): re-search0054 [2] Konstantinos P. Exarchos, Themis P. Exarchos, Costas Papaloukas, Anastassios N. Troganis und Dimitrios I. Fotiadis. Detection of discriminative sequence patterns in the neighborhood of proline cis peptide bonds and their functional annotation. In: BMC Bioinformatics – 20 April 2009, 10:113[3] Bachelor Thesis: Anne Nöldner. Korrelation von Sequenzmotiven mit PROSITE-Motiven von Membranproteinen. Fakultät Mathematik/Naturwissenschaften/Informatik, Hochschule Mittwei-da, University of Applied Sciences, 2010

Anne Nöldner

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Scienceanoeldne@hs-mittweida.dehttps://www.mni.hs-mittweida.de/

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44 Correlation of specific surface parameters and cell adhesion force parameters Maria Beier, Tommy Hofmann, Dirk Labudde

Cellular adhesion is responsible for united cell structure and the coherence of organs and therefore for the possibility of differentiation, which is decisive for complex life. This is the reason the cellular adhesion is one of the most treated mechanisms of cell biology and omnipresent in all other disciplines of biology. The aim of this work was to provide a reliable mathematical relation that describes the forces that appear during cellular adhesion on glass and to define surface determined parameters. Glass is one of the most common materials used for medical instruments and cell culture equipment. In previous works we discovered structural changes, which are the result of commonplace use glass is exposed to.In this work we analyzed 150 force-distance-curves. These curves had been measured by Single Cell Force Spectroscopy (SCFS) and display the adhesion forces which occur between cells and a glass body. The effect of different sterilization methods (steam, plasma and gamma) on the adhesion forces on the glass was in the point of interest. It was shown that the sterilization with each method caused structural changes which led to various surface conditions. These structural changes varied in their intensity depending on the three sterilization methods. The surfaces can be described by different parameters like roughness, height and average area. Plasma sterilized glass surfaces have shown the largest roughness, followed by gamma sterilized glass. The steam sterilization didn’t change the surface recognizable and left a homogeneous surface. The force-distance-curves confirmed the previous assumption that these changes led to different adhesion forces and adhesion work.We associated the characteristically trajectory of the force-distance-curves into a mathematical model including the cell adhesion force parameters. For each sterilization method we found a mathematical relation to describe the trajectory of the force-distance-curves and we had been able to show the correlation between the adhesion force and sterilization methods.

Maria Beier

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Sciencembeier1@hs-mittweida.dehttps://www.mni.hs-mittweida.de/

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45 The novel approach eHMM for analyzing membrane proteins in case of HP_0565

Anne-Marie Pflugbeil, Florian Heinke, Nora Heinig, Dirk Labudde

Membrane proteins are essential elements in metabolic pathways and play a main role in current protein research. But just a few mechanisms and structures are clarified to date. Single Molecule Force Spectroscopy (SMFS) allows detecting molecular protein stability [1]. In this work we present analyzed SMFS experiments and a new energy profile based description of a putative transmembrane protein of Helicobacter pylori, called HP_0565. This protein is assumed to be a main virulence factor of Helicobacter pylori. To this date, there are no structural information of HP_0565 in data bases [2].We applied SMFS data from HP_0565 to gather information about possible existing unfolding events that are corresponding to the secondary structure elements. The prediction was performed by existing prediction methods, such as TMHMM2.0, HMMTop, MEMSAT3, ConPred II and SOSUI [2]. In this work we predicted a so called energy profile by the sequence of HP_0565. An energy profile is a schematic plot of the interaction energy of each residue as a function of the residue position in the sequence. Actually these residue-residue-interaction energies are calculated by coarse grained models derived by known protein structures. Because of missing three-dimensional structural information, we used a novel GOR-algorithm based prediction method (eGOR) which calculates an energy profile by the amino acid sequence [3]. Furthermore, we developed a hidden markov model (eHMM) which predicts outer- and inner membrane regions by energy profiles. Tested on an evaluation set of 110 known membrane protein structures, our membrane region prediction method showed an accuracy of 77%. The eHMM was applied to predict membrane regions by the energy profile of HP_0565.These predicted regions showed strong correlations to predictions made by known methods mentioned above. By a statistical analysis of unfolding events observed in force curves, we were able to classify unfolding pathways. Using information achieved by the secondary structure of HP_0565, the mapping of unfolding barriers to structure elements, derived from the experimental data, was realized by applying these observations to results gathered from our new eHMM approach. We used this mapping to cluster the observed unfolding pathways to main- and side-pathways of HP_0565.

The eGOR-algorithm and eHMM are available at http://bioservices.hs-mittweida.de/Epros/.

Anne-Maria Pflugbeil

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Scienceapflugbe@hs-mittweida.dehttps://www.mni.hs-mittweida.de/

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46 Novel prediction algorithm eGOR – from sequence to stable regions of membrane proteins Riccardo Brumm, Eric Frenzel, Florian Heinke, Dirk Labudde

Membrane proteins are important elements in signal transduction and transfer of agents between cells. Often the occurrence of point mutations is sufficient to influence protein stability or functionality [1]. Thus, detecting stable regions in protein structures play an important role in understanding protein functionality. Membrane proteins pose an experimental challenge and lead to limited data and knowledge. Therefore deriving data by other non-experimental methods is indispensable. The claim of this work is to detect stable regions in membrane proteins by a new prediction algorithm.We calculated amino acid interaction potentials based on residue-residue contacts in known membrane protein structures. Using these potentials an energy profile of a membrane protein based on its structural information can be derived. An energy profile is a schematic plot of the interaction energy of each residue as a function of the residue position in the sequence. In conclusion, stable regions of membrane proteins can be predicted. These predictions were evaluated against MD simulation and single molecular force spectroscopy (SMFS). We showed that stable regions detected by SMFS correlate with stable regions predicted by energy profiles [2]. Furthermore, we developed a GOR-based prediction algorithm, which performs an energy profile prediction based on an amino acid sequence – eGOR. The predicted energy profiles can be applied to our approach. In conclusion unfolding barriers and structural information can be derived by eGOR [3]. The eGOR prediction algorithm and some more energy profile based tools are available at http://bioservices.hs-mittweida.de/Epros/.

References:[1] Bachelor Thesis: Riccardo Brumm. Energieprofilbasierende Stabilitätsanalyse von Membran-proteinen. Fakultät Mathematik/Naturwissenschaften/Informatik, Hochschule Mittweida, Univer-sity of Applied Sciences : 2010.[2] F. Dressel, A. Marsico, A. Tuukkanen, M. Schroeder, D. Labudde. Understanding of SMFS bar-riers by means of energy profiles. Proc GCB 2007.[3] Bachelor Thesis: Florian Heinke. Energieprofilbasierende Methoden zur Analyse von Proteinfa-milien. Fakultät Mathematik/Naturwissenschaften/Informatik, Hochschule Mittweida, University of Applied Sciences : 2010.

Riccardo Brumm, Eric Frenzel & Florian Heinke

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Sciencerbrumm@hs-mittweida.dehttps://www.mni.hs-mittweida.de/

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47 Energy based Prediction and Comparison of Protein Structures

Florian Heinke, Stefan Schildbach, Dirk Labudde

A lot of tools and methods in the field of bioinformatics and structure biology are based on structure and/ or sequence comparison. In our work we demonstrate a new method based on so called energy profiles for comparison and prediction of globular protein structures. Those profiles are calculated by coarse grained models [1,2]. Based on the residue contacts in known protein structures, we calculated the potential for pair wise residue-residue-interactions [2]. An energy profile is a schematic plot of the interaction energy of each residue as a function of the residue position in the sequence. As an abstraction of protein sequence and structure information, each energy profile represents a protein as a fingerprint.In our new approach, we perform an energy profile alignment based on the Needleman-Wunsch-Algorithm by using self constructed cost-schemes [3]. These cost schemes are derived by the statistical energy distribution of more than 4000 known structures. We showed that the z-scores of these pair wise energy profile alignments correlate with structural alignment scores performed by known methods.

Additionally, we developed a modified GOR Algorithm for the prediction of energy profiles. By using the energy profile alignment algorithm, this prediction leads to a hit list of similar structures [3].These described tools can be accessed at http://bioservices.hs-mittweida.de/Epros/.

References:[1] Wertz, D. H. and Scheraga, H. A. (1978). Influence of water on protein structure. An analysis of the preferences of amino acid residues for the inside or outside and for specific conformations in a protein molecule. Macromolecules, 11(1), 9–15.[2] F.Dressel, A. Marsico, A. Tuukkanen, M. Schroeder and Dirk Labudde; Understanding of SMFS barriers by means of energy profiles; Proc. GCB (2007)[3] Bachelor Thesis: Florian Heinke, Hochschule Mittweida, 2010, Energieprofilbasierende Meth-oden zur Analyse von Proteinfamilien.

Florian Heinke

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Sciencefheinke@htwm.dehttps://www.mni.hs-mittweida.de/

Bioinformatics

75

48 Disentangling complex regulatory traits in QTL analysis

Mathieu Clément-Ziza, Andreas Beyer

Most phenotypes show complex inheritance and natural continuous variation which play a central role in susceptibility to diseases. Although the technique of quantitative trait locus (QTL) mapping has helped understanding the regulation of complex traits, it has proven difficult to comprehensively determine their complex genetic architecture, partially because phenotype variation is often the result of multiple variable processes. Gene expression is a good example of such complex process: it involves several steps each of which is regulated by the cell. Expression QTL studies have been carried out using either transcript or protein abundance to monitor gene expression. However, different biological processes underlie those traits: at steady state, the transcript abundance is mainly dependent on transcription and RNA degradation whereas protein levels are also affected by post-transcriptional processes such as translation or protein degradation. In this work, we dissected gene expression traits from which we isolated the post-transcriptional component in order to better understand its specific regulation. Our work is based on integrating published data obtained from a yeast population genotyped at 1106 markers and phenotyped at the transcriptomic and proteomic levels of 137 genes. We modeled post-transcriptional variation as the residuals after regressing on RNA levels. Mapping this composite trait revealed 36 loci that post-transcriptionally affect 64 proteins. We identified two regulatory hotspots that control many genes. The first locus harbors Leu2, which was deleted in one of the two parental strains. The second locus contains a new candidate master regulator of amino-acid metabolism genes. This is the first QTL study systematically separating transcriptional and post-transcriptional regulation as distinct traits and our work presents a general framework for disentangling related (yet different) complex traits in order to reveal their genetic basis.

Dr. Mathieu Clément-Ziza

Technische Universität DresdenBIOTEChnology Center Dresdenmathieu.clement-ziza@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/beyer/

Bioinformatics

76

49 Analysis of membrane protein stability in Diabetes insipidus

Florian Heinke, Anne Tuukanen, Dirk Labudde

Diabetes insipidus (DI) is a rare endocrine disorder with an incidence in a general population assessed on one case per 25.000-30.000 people. It is a disease characterized by polyuria and compensatory polydipsia. The underlying causes of DI are diverse and can be a central defect in which no functional arginine-vasopressin is released from the pituitary. It may be caused by defects in the kidney (nephrogenic DI, NDI) as well. Four different types of NDI are known. First, acquired NDI can originate as a side-effect of drugs with the most prominent being the antibipolar drug lithium. Second and third, autosomal recessive and dominant inheritable NDI is caused by gene mutations in the AQP2 gene. Finally, mutations in the AVPR2 gene, which encodes V2R, is the cause of the X-linked inheritable form of NDI. V2R is the key player in triggering the transecullar water transport by the availability of binding to the hormone arginine-vasopressin and releasing cAMP to the water resorption cascade. Known from literature, there are about 20 known mutations in this receptor, which are involved in NDI. Furthermore mutations in the aquaporin proteins, which realize the transcellular water transport in the V2R triggered cascade, lead to the loss of transport activity.In our work we demonstrate new methods based on so called energy profiles. Our approach and developed tools (http://bioservices.hs-mittweida.de) deal with the influence of mutations on protein stability. These tools and algorithms perform the calculation and pair wise comparison of protein energy profiles of known structures or models. Those energy profiles lead to the opportunity to compare and detect energetic divergences induced by mutations. Energetic divergences correspond with the stability of the V2R receptor and mutants. So we get information and understanding of influence of mutations and destabilized regions. By that, it is possible to discuss the linkage between function or loss of function and mutations in Diabetes insipidus. We analyzed known mutations by energy profiles in the V2R receptor and the aquaporins with respect to protein mechanisms.

Florian Heinke

University of Applied Science MittweidaFaculty of Mathematics, Natural Science, Computational Sciencefheinke@htwm.dehttps://www.mni.hs-mittweida.de/

Bioinformatics

77

50 Computational analysis of Interleukin-8 interactions with hyaluronan and chondroitin-sulfate derivatives Sergey Samsonov, Annelie Pichert, Stephan Theisgen, Jügen Schiller, Daniel Huster, M. Teresa Pisabarro

Glycosaminoglycans (GAGs) represent a class of linear negatively charged polysaccharides, which play a key role in cell adhesion and proliferation by interactions with their protein targets. Interleukin-8 (IL-8) is a chemotactic cytokine involved in inflammation processes by recruiting and activating neutrophil granulocytes via binding a G protein-coupled receptor. This activation is strongly influenced by the interaction of IL-8 with GAGs, which makes these molecular interactions of a great potential for the engineering of new scaffolds in biomaterials to promote bio-specific cell behaviour. Although the 3D-structure of IL-8 is available in the PDB, there are no experimental structures of IL-8 complexes with GAGs. However, mutagenesis studies have indicated several IL-8 residues to be responsible for heparin binding. We use molecular modelling and computer simulation approaches in combination with NMR experimental techniques to study molecular recognition of several modified GAGs on monomeric and dimeric IL-8. For the analyzed GAGs, we find a common representative and high scoring docking binding pose similar to the previously proposed for heparin. We analyze the energetics of this binding pose using MM-PBSA free energy calculations and per residue energy decomposition. Our results are in agreement with binding data obtained by NMR and show: i) an electrostatics-driven general improvement of binding with the increase of GAGs sulfation; ii) binding patterns, which are GAGs sulfation position specific but not purely electrostatics dependent. In addition, we investigate how the conformational space available to GAGs glycosidic linkages is affected by GAGs binding, the effect of GAGs elongation on binding to IL-8, the role of solvent in GAGs binding and the impact of GAGs binding on the thermodynamics of IL-8 dimerization. Our results highlight the importance of combining theoretical with experimental approaches to obtain a better understanding of the molecular recognition properties of GAG-protein systems.

Dr. Sergey Samsonov

Technische Universität DresdenBIOTEChnology Center Dresdensergey.samsonov@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/pisabarro/

Bioinformatics

78

51 Elucidating the regulatory mechanisms of transcrip- tion factor activity in hematopoietic stem cell differentiation

Marit Ackermann, Weronika Sikora-Wohlfeld, Andreas Beyer

The differentiation of hematopoietic cells is regulated by a number of cross-acting transcription factors. Discovering the regulatory programs that control the activity of these factors is crucial for understanding hematopoiesis. This project aims at unravelling the influence of genetic variability on the different stages of hematopoietic cell differentiation and the transitions between them. Our work is based on eQTL data obtained by the group of Gerald de Haan. The de Haan study profiled 4 hematopoietic cell types (stem cells, progenitor cells, myeloid and erythroid cells) in about 20 recombinant inbred mouse lines. Using such data it is possible to correlate the expression of each gene with each locus in the mouse genome, thereby identifying genetic regions specifically controlling the expression of a given gene. We have integrated these expression quantitative trait locus (eQTL) data with transcription factor binding data (ChIP-Seq) in order to unravel the regulatory programs that drive the development of hematopoietic cells.First, we have used a new method for mapping eQTL based on the Random Forest machine learning, which identifies more high-scoring eQTL than traditional methods. In order to further improve the eQTL mapping we developed a new method for inferring missing data. Next, we developed an approach that quantifies the activity of transcription factors (TF) based on the expression levels of target genes. Target genes are identified using a large compendium of ChIP-Seq studies measuring TFs with known functions in the hematopoietic system. Using our newly developed method we can iteratively learn the activity of each TF specifically for each cell type and each mouse line. Using the cell type specific TF activity as a trait we can find genetic loci controlling TF activity. We are able to show that the resulting ‘TF activity QTL’ coincide with loci regulating cell type specific physiological traits.

Marit Ackermann & Weronika Sikora-Wohlfeld

Technische Universität DresdenBIOTEChnology Center Dresdenmarit.ackermann@biotec.tu-dresden.deweronika.sikora@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/beyer/

Bioinformatics

79

52 Joining experts: Improved Biomarker discovery with Power Graphs and NetRank Matthias Reimann, Janine Roy, Simone Daminelli

Background: Interpretation of microarray experiments for identifying genes, which can serve as biomarkers, is an important task in biomedical problems. Biomarkers are able to distinguish between different phenotypes or traits. The state of the art in microarray analysis includes calculation of fold change, t-test, or other mechanisms that use both gene expression variability and effect size of an outcome variable.

Methods: A new algorithm for microarray analysis is NetRank. NetRank is a modification of Google’s PageRank algorithm. It combines gene expression from microarray experiments with network structure. Thus, NetRank calculates the importance of a gene for outcome prediction by accessing the gene’s expression and the expression of the interacting genes.Power Graphs are a lossless transformation for undirected graphs that compresses redundant information based on cliques and bi-cliques. By explicitly representing these re-occurring motifs, Power graphs unravel the network’s structure and give new insights into the underlying biology.

Conclusion: Identifying significant and reliable biomarkers with the power to ease clinical routine may improve cancer treatment and lead to personalized medicine. NetRank is a powerful tool to identify reliable biomarkers -- power graphs enhance automatic and visual analysis of complex networks -- together, NetRank and power graphs improve biomarker identification and outcome prediction.

Identifying significant and reliable biomarkers with the power to ease clinical routine may improve cancer treatment and lead to personalized medicine. NetRank is a powerful tool to identify reliable biomarkers -- power graphs enhance automatic and visual analysis of complex networks -- together, NetRank and power graphs improve biomarker identification and outcome prediction.

Matthias Reimann & Janine Roy

Technische Universität DresdenBIOTEChnology Center Dresdenmatthias.reimann@biotec.tu-dresden.dejanine.roy@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/schroeder/

Bioinformatics

80

53 Identification of protein complexes maintaining Oct4 expression in mouse ES cells

Weronika Sikora-Wohlfeld, Andreas Beyer

Octamer binding transcription factor-4 (Oct4) is known to be one of the key factors maintaining the pluripotency of embryonic stem (ES) cells. The mechanisms regulating its expression, however, have not been fully elucidated yet.Our work aims at finding the genes and protein complexes involved in the regulation of Oct4 expression. The study is based on two independent genome-wide siRNA screens reporting the change of Oct4 expression upon the knock-down of query genes in mouse ES cells.Direct comparison of the results from both screens at the gene level did not show a statistically significant consistency. One of the possible causes of this disagreement are the false discoveries in siRNA screens. We reasoned that incorporation of orthogonal information in the analysis might remove noise and improve the consistency between screens. We therefore mapped the genes tested in siRNA screens to known protein complexes, assuming that genes participating in the same complex should cause similar phenotypes. To identify complexes enriched with high-scoring genes, we tested several set enrichment methods (hypergeometric test, weighted Kolmogorov-Smirnov statistic, Bayesian network and regularized linear regression). The resulting scoring of protein complexes showed considerably greater consistency between screens than the original gene scores. Subsequently we combined the results from both screens in order to obtain a single set of high-confidence complexes enriched for genes causing Oct4-related phenotypes. Thereby we obtained several complexes with known functions related to cell-cycle or stem cell maintenance. Importantly, these complexes contain many genes that were not identified as significant “hit genes” in the original screens.

The performed analysis reveals that combining results of siRNA screens and adding external data helps to extract more comprehensive information from the experiments. Our analysis identifies a catalogue of protein complexes potentially important for ES cells maintenance.

Weronika Sikora-Wohlfeld

Technische Universität DresdenBIOTEChnology Center Dresdenweronika.sikora@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/beyer/

Bioinformatics

81

54 The Power of Three: Binding Site Comparison, Text Mining and Network Analysis for Drug Repositioning Joachim Haupt, Simone Daminelli, Sainitin Donakonda, Xiaofeng Yu, Bingding Huang, Michael Schroeder

Developing a new drug is a laborious and costly endeavor. Therefore, a promising and valuable solution is drug repositioning, i. e. finding new uses for already known drugs (e.g. Viagra). The benefits of drug repositioning are manifold: Pharmaceutical companies can save around 60% of drug development costs, orphan diseases become treatable, and patients profit from a comprehensive knowledge of drug side effects.The prerequisite for drug repositioning is drug promiscuity – the binding of a drug to more than one target. We show that this is a widespread phenomenon among the drugs in the Protein Data Bank (PDB) with over 50% of them binding to two or more distinct proteins.Here, we combine three in silico strategies to find new uses for already known drugs: Text mining (side effect similarity), protein local structural alignment (binding site similarity) and network analysis (Power Graphs).Text mining is a high-throughput technique to extract information from millions of biomedical documents. This permits profiling of protein and drugs. Matching similar profiles using ontologies allows to identify indirect links between entities, thus suggesting novel drug-target interactions.One reason for drug promiscuity is the similarity of binding sites among unrelated proteins. Binding site comparison of a known target protein to other proteins yields candidates for drug repositioning if the similarity is high.Network analysis with Power Graphs is applied to integrate information from the previous methods. The aim is to complement the data to recover missing links. Exploiting the clustering of similar drugs or proteins, Power Graph Analysis permits to find candidates for drug repositioning.We show examples for each of the approaches to demonstrate their applicability and outline limitations. In particular, we present the case study of an antiviral drug successfully repositioned as an anticancer drug.

Joachim Haupt & Simone Daminelli

Technische Universität DresdenBIOTEChnology Center Dresdenjoachim.haupt@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/schroeder/

Bioinformatics

82

55 Lineage-specific changes in primate transcription factor genes and the evolution of species-specific traits

Katja Nowick

Transcription factors (TF) form gene regulatory networks to regulate the expression of many target genes. Therefore, changes in a single TF protein can potentially alter pathways affected by them and play an important role in defining species differences. My work focuses on the molecular evolution of TFs in primates, including the identification of lineage-specific TF genes, analysis of sequence and expression differences, and the investigation of differences in the networking interactions of TFs between humans and other primates. TFs with interesting molecular differences are followed up by experimental investigation in human and chimpanzee cell lines. My goal is to understand how changes in TF genes cause phenotypic differences between primates and to gain insight into the evolution of human-specific traits.

Dr. Katja Nowick

University LeipzigBioinformaticsnowick@bioinf.uni-leipzig.dehttp://www.nowick-lab.info/

Bioinformatics

83

56 MAGE - A Model of the Ageing Epigenome Thimo Rohlf, Sonja Prohaska, Jörg Galle

Ageing is characterized by a gradual functional decline of virtually every tissue system and increased vulnerability and susceptibility to numerous diseases. Various experiments demonstrated that restrictions in regenerative mechanisms are fundamental in mammalian ageing, affecting whether tissue homeostasis can be maintained by the organism. These regenerative mechanisms predominately rely on an appropriately regulated balance between self-renewal and differentiation of functional adult stem cells. During ageing, gene expression profiles of stem cells undergo changes that are accompanied by decreased chromatin stability. This deregulation impairs stem cell function and can eventually result in loss of stem cell pools, which potentiates the ageing process dramatically. The objective of the project MAGE is to develop and validate a mathematical model of stem cell ageing. Our approach bases on the observation of an unstable stem cell epigenome. In order to understand its dynamics, we combine methods of stem cell biology, molecular biology of evolution, quantitative analysis of transcriptional data and multi-scale agent-based modelling. This gives us the opportunity to follow a systems biology approach to stem cell ageing that explains age-related phenomena at the cellular and tissue level by complex molecular scenarios of transcriptional regulation.

Dr. Thimo Rohlf

University LeipzigInterdisciplinary Centre for Bioinformaticsrohlf@izbi.uni-leipzig.dehttp://www.izbi.uni-leipzig.de/index.php

Bioinformatics

84

57 A novel informatics concept for high-throughput shot- gun lipidomics based on the molecular fragmentation query language

Ronny Herzog, Dominik Schwudke, Kai Schuhmann, Andrej Shevchenko

Lipids play a crucial role in cell physiology and are involved in numerous cellular process-es. Lipids have an enormous structural diversity, originating mostly from various combi-nations of fatty acid chain lengths and possible head groups that are linked to a glycerol backbone. Shotgun lipidome profiling relies on direct mass spectrometric analysis of total lipid extracts from cells, tissues or organisms and is a powerful tool to elucidate the molecular composition of lipidomes. We present a novel informatics concept of the mo-lecular fragmentation query language implemented within the LipidXplorer open source software kit that supports accurate quantification of individual species of any ionizable lipid class in shotgun spectra acquired on any mass spectrometry platform.

Ronny Herzog

Max-Planck Institute for Molecular Cell Biology and Genetics Dresdenherzog@mpi-cbg.dehttp://www.mpi-cbg.de/research/research-groups/andrej-shevchenko.html

Bioinformatics

85

58 Automatic Classification of Embryonic Fruit Fly Gene Expression Patterns Andreas Heffel, Peter Stadler, Sonja Prohaska

Carefully studied in-situ hybridization Gene expression patterns (GEP) can provide a first glance at possible relationships among genes. Automatic comparative analysis tools are an indispensable requirement to manage the constantly growing amount of such GEP images. We present here an automated processing pipeline for Segmenting, Classification, and Clustering large-scale sets of Drosophila melanogaster GEP images that facilitates automatic GEP analysis.

Andreas Heffel, Peter Stadler & Sonja Prohaska

University LeipzigInterdisciplinary Centre for Bioinformaticsheffel@bioinf.uni-leipzig.dehttp://www.bioinf.uni-leipzig.de/

Bioinformatics

86

6. Tissue and Cell Engineering

87

59 The impact of PUFA supplementation on the oxidative metabolism of macrophages Stephanie Adolph, Julia Schumann, Herbert Fuhrmann

Polyunsaturated fatty acids (PUFAs) exhibit various immunmodulating properties. In phagocytes PUFAs may modulate the intracellular production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during oxidative burst. Favoured positions for radical attacking are found at the bis-allyl-methylene positions between adjacent double bounds of the fatty acids, defined as the Methylene Bridge Index (MBI). However, the impact of the carbon chain length and number of double bounds of PUFAs is unknown so far. Furthermore, it is not clear if PUFAs from the n-3 and the n-6 family modulate ROS and RNS formation differentially. The aim of the present study is to compare the effects of the n-3 PUFAs alpha-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and the n-6 PUFAs linoleic acid (LA), arachidonic acid (AA) on the oxidative metabolism of macrophage cell line RAW 264.7.RAW 264.7 were cultured in RPMI medium containing 5% FKS and supplemented for 72h with 15μM LNA, EPA, DHA, LA and AA respectively. Supplemented cells were stimulated with PMA (1μM) or LPS (1μg/ml) for 15/30/45min and 6/24h. ROS production was detected by the fluorogenic probe Dihydrorhodamine 123; RNS formation was determined using Griess reagent (N=6, n=3). ROS production of supplemented and stimulated cells was also determined by 24h of fluorescence microscopic life cell imaging. Two-way analysis of variance followed by student’s t-Test was used to identify significant differences between means, p<0.05 was assumed to indicate significant differences.Enrichment in PUFA content was connected with an increase in MBI of the cells as well as a significant increase of ROS production. At this, a relation between ROS production and MBI could be seen. Stimulation of unsupplemented cells with PMA or LPS resulted in a significant increase in ROS production. Of note, for PUFA enriched cells a significant repressive effect on ROS production was to ascertain. The n-3 PUFA DHA was identified to be most effective, at this totally inhibiting ROS boost in context of stimulation. The modulating action of PUFAs on ROS production could also be confirmed by means of fluorescence microscopic life cell imaging. However, no effects on RNS production could be observed for all PUFAs tested.Our results underline the modulating influence of PUFA supplementation on the oxidative metabolism of macrophages which depends on the MBI of the fatty acids as well as the activation status of the cells. ROS production of unstimulated cells correlates with the number of bis-allyl-methylene positions of the fatty acid supplemented. After stimulation this dependency is lost. The supplementation of DHA completely prevents the increase of ROS production.

Stephanie Adolph

University LeipzigFaculty of Veterinary MedicineInstitute of Physiological Chemistryadolph@vetmed.uni-leipzig.dehttp://www.uni-leipzig.de/~vpci/

Tissue and Cell Engineering

88

60 Visualisation of megamitochondria with Laser-Scanning-Microscopy

Susanna Schubert, Sandra Heller, Mario Krehan, Ingo Schäfer, Peter Seibel

Mitochondria are the primary energy-generating system in most eukaryotic cells. Additionally they participate in intermediary metabolism, calcium signalling, apoptosis and aging.Less than 5 % of all mitochondrial proteins are encoded by the genome of these organelles. More than 95 % are encoded by the nuclear genome and have to be transported into the mitochondria. Mitochondrial DNA (16569 bp in Homo sapiens) contains 37 genes that are important for normal mitochondrial function. Thirteen of these genes are coding for essential enzymatic subunits involved in oxidative phosphorylation. Depending on the tissue, one mitochondrion can contain up to ten copies of its genome.

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 and valinomycin.In our studies we induce large mitochondria by chemical acidification. The effects were visualised by laser-scanning-microscopy.

Susanna Schubert

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Molecular Cell Therapysusanna.schubert@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~mct/mct/

Tissue and Cell Engineering

89

61 A neuronal organotypic, tau transgenic in vitro cell culture model for drug screening in Alzheimer’s disease and related disorders Diana Seidel, Dana Krinke, Heinz-Georg Herzog, Andrea A. Robitzki

The abnormal hyperphosphorylation of the microtubule-associated protein tau is a basic hallmark of Alzheimer’s disease (AD). In recent years, some tau-focused therapeutic strategies have been developed with the aim to stop disease progression. However, the lack of suitable screening platforms and human in vitro organotypic cell culture models are limiting factors for today’s high throughput and high content drug screening. Therefore, we developed an in vivo-like three-dimensional (3D) cell culture model consistent of SH SY5Y neuroblastoma cell lines stably transduced with wild type (WT), single (P301L) and quadruple (P301L, dK280, R406W, V337M) mutated tau. Neurodegenerative as well as regenerative effects could be analyzed with high sensitivity using microcavity array (MCA)-based electrochemical impedance spectroscopy (EIS). With our impedance platform, we were able to quantify a time- and concentration-dependent relative impedance decrease when an AD-like tau pathology was induced in our neuronal 3D cell culture model applying the selective protein phosphatase 2A inhibitor okadaic acid (OA). In contrast, regenerative effects caused by tau-focused therapeutics could be characterized by a time- and mutation-dependent increase of relative impedance. These results indicate a promising implementation of our human organotypic SH SY5Y 3D in vitro cell culture model in combination with the non-invasive, label-free and real-time impedance screening platform for tau-focused drug development in neurodegenerative pathologies including Alzheimer’s disease.

Diana Seidel

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Molecular biological-biochemical Processing Technologydiana.seidel@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~dmpt/

Tissue and Cell Engineering

90

62 Lineage commitment of conditionally immortalized murine bone marrow mesenchymal stromal cells

Maria Rostovskaya, Francis Stewart, Konstantinos Anastassiadis

Adult bone marrow stroma contains a population of mesenchymal stem cells capable to self-renew and to differentiate into haematopoietic-supportive stroma, osteo, adipo- and chondrocytes. However, the identity of mesenchymal stem cells still remains uncertain due to the inability to define stem and progenitor cell hierarchy. The experiments using bone marrow mesenchymal stromal cells (BM MSCs) are often hampered by their limited expansion capacity ex vivo, hence immortalization provides an invaluable tool to study these cells. Transgenic mice with a double ligand (Dox/Dex) inducible SV40 Large T-antigen were generated recently in our group and implemented for isolation of rare and low-proliferative cell types. We were able to isolate and expand BM MSCs from the transgenic mice, and confirmed their differentiation potential into osteo- (“O”), adipo- (“A”) and chondrocytes (“C”). Using cellular cloning we established homogeneous subpopulations of BM MSCs and demonstrated stable distinct properties in differentiation potential. We identified 7 types of lineage-restricted progenitors based on their lineage commitment (tripotential “OAC”, bipotential “OA”, “OC”, “AC”, and monopotential “O”, “A”, “C”), which represent all possible combinations of differentiation properties. Gene expression profiling of each cell type was performed using RNA-Seq (Illumina platform). Gene Ontology term analysis showed an enrichment of genes involved in development and differentiation. Our results revealed transcription signatures of osteogenic lineage (“OAC”, “OA”, “OA” and “O” clones) and adipogenic lineage (“OAC”, “OA”, “AC” and “A” clones). The cells that possess both potentials (“OAC”, “OA”) expressed a combination of both signatures, but did not exhibit a unique identity.We hypothesize that lineage commitment of BM MSC progenitors is determined by the expression of signatures specific for each differentiation pathway (osteo-, adipo-, chondro-), which can be combined in the cells in non-exclusive manner. The diversity of the differentiation properties within a progenitor pool implies a complex model of BM MSC hierarchy.

Dr. Maria Rostovskaya

Technische Universität DresdenBIOTEChnology Center Dresdenmaria.rostovskaya@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/anastassiadis/

Tissue and Cell Engineering

91

63 Initiation of X Chromosome Inactivation Sarah Tsurkan, Kaj Bernhardt, Francis Stewart

Female embryonic stem (ES) cells of the mouse contain two active X chromosomes. During differentiation, one X is inactivated to achieve equal doses of X-chromosomal genes. The pluripotency network of transcription factors, more specifically Oct4, Sox2, Nanog as well as Rex1, Klf4, Myc, is intricately linked with the X inactivation machinery to control expression of the non-coding RNAs Xist and Tsix, respectively(1). Xist and Tsix are antisense counterparts that repress each other and thus facilitate both X chromosome inactivation (XCI) and re-activation of the inactive X during somatic cell reprogramming to induce pluripotent stem cells(2). At the onset of inactivation, before Xist upregulation, the X inactivation centers (XIC) of the X chromosomes transiently co-localize in a process known as X paring. A region of interest, including the gene xpct (slc16a2) was identified as involved in XIC pairing(3). We designed a series of targeting constructs that knock out the gene and its 5’ region when site-specifically recombined. Functional studies of these cells will elucidate the specific genetic elements involved in pairing. To further investigate the initiation of XCI, we generated a g6pdx-venus knock-in ES cell line by gene targeting. The X-linked g6pdx gene is silenced early during XCI and Venus fluorescence thus reports the state of the chromosome that harbors the g6pdx-venus allele. During differentiation of homogeneously fluorescent g6pdx-venus ES cells, a non-fluorescent population of cells will appear, representing silencing of the targeted X chromosome at 50 % chance due to random XCI. Functional studies with these reporter cells are under way to identify new actuators and repressors of XCI.

References:1 P. Navarro et al., Nature 468, 457ff. (2011).2 M. Stadtfeld et al., Cell Stem Cell 2 (3), 230ff. (2008)3 S. Augui, et al., Science 318 (5856), 1632ff. (2007).

Sarah Tsurkan

Technische Universität DresdenBIOTEChnology Centersarah.tsurkan@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Tissue and Cell Engineering

92

64 Effect of erythropoietin on expansion and neural-like differentiation of ovine bone-marrow-derived mesen- chymal stem cells

Amir Mofidi, Oliver Petters, Sanja Pavlica, Augustinus Bader, Henrike Horn, Heidi Mergentaler

Stem cells are capable of repairing injured nervous tissue by replacing damaged cells, neuroprotection or the creation of an environment conducive to regeneration by endogenous cells. Erythropoietin (EPO) is known to have a significant role in tissue outside the hematopoietic system and it is possible therapeutic strategy for neuronal protection and regeneration. Ovine MSCs represent a potentially useful cell population for CNS regeneration studies. To date, data on utilization of oMSC for CNS therapy are still lacking. Also, there is no data available on neuron-like differentiation of ovine MSC. So, in this work, we investigated the effect of EPO on proliferation and neuronal-like differentiation of ovine bone marrow derived mesenchymal stem cells.First, we showed the multipotency of oMSC and their neural-like differentiation. We observed that co-incubation with EPO has no negative effect on oMSC proliferation. During the incubation of oMSC in neuronal induction medium, the cells have turned their shape from fibroblastic-like cells to dendritic-like cells. Using light-microscopy, we detected an increasing number of cell-cell contacts. Furthermore, in addition to EPO, we tested the effect of CoCl2 on neuronal differentiation, too. Interestingly, both compounds exhibited no visible effect on morphology change of oMSC. It is likely that EPO has no effect on cell proliferation and neural differentiation of oMSC in vitro. Further studies are needed to reveal the neuroprotective potential of this exciting cytokine utilising an ovine model of PD and SCI resembling these diseases in the clinical setting.

Amir Mofidi & Oliver Petters

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Cell Techniques and Applied Stem Cell Biologyamir.mofidi@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~bader/

Tissue and Cell Engineering

93

65 New micro particle based cell separation method sufficient for direct and simultaneously isolation of multiple targets from whole blood. Viability of sepa- rated lymphocytes. Jan-Michael Heinrich, Christoph Mohr, Anne Rasser, Maria Kotova, Nadezhda Frolova

Currently there are several cell separation methods available. They are great tools for cultivation and functional analysis of defined cell types, for removal of unwanted cell types from samples or for the diagnosis of certain diseases. Mostly they are based on physical attributes like size or density of the targets or on biological attributes like surface receptors. But their shortcoming is the need for pre-treatment of samples to achieve sufficient purity, especially for whole blood. The removal of cell debris and other distracting components is often time consuming and error prone. We are developing a new easy and time saving bead based cell separation method which combines physically and affinity attributes. It does not acquire any sample pre-treatment. The system is suitable for simultaneously fractionation of multiple targets, for example cells and/or proteins, from one sample. The sample could be whole blood or aqueous media. The targets are captured by specifically affinity functionalized micro particles of distinct sizes. During 30 minutes of incubation each size of particle binds to its belonging kind of target. Thus after incubation the captured targets could be separated from each other via cascade wet sieving by size. The cells can be removed from the micro particles and subsequent cultivation or analysis is possible. Here we describe the isolation of CD4+ and CD8+cells directly from whole blood. We evaluate throughput, purity and viability of separated cells.

Dr. Jan-Michael Heinrich

pluriSelect GmbHjm.heinrich@pluriselect.dehttp://www.pluriselect.de/

Tissue and Cell Engineering

94

7. Genome and Protein Engineering

95

66 Generation of conditional knockout alleles in zebrafish Peggy Jungke, Stefan Hans, Michael Brand

Gene trapping with the Tol2 transposon system has been used to introduce insertional mutations across the zebrafish genome. The gene trap vector consists of a splice acceptor site upstream of a promoterless reporter gene with a transcriptional termination sequence and polyA signal. When inserted into an expressed gene, the gene trap cassette is transcribed from the endogenous promoter in the form of a fusion transcript encoding a truncated and presumably nonfunctional protein and the fluorescent reporter. In this way gene traps simultaneously inactivate and report the expression of the trapped genes and can be rapidly identified. In mouse, gene trap vectors have been generated that can induce conditional mutations employing two directional site-specific recombination systems, Cre/lox and Flp/FRT . After a successful gene trap event, the gene trap is inverted from its mutagenic orientation to a nonmutagenic orientation by Flp. Reconstitution of the mutagenic orientation is later mediated by Cre at a particular time and place in somatic cells. The aim of the proposed project combines these two successful approaches, called flip-excision (FlEx), to generate conditional knock-outs in zebrafish. The zebrafish gene trap vector has been equipped with the target sites of Cre and Flp required for conditionality, and a pilot screen has been performed resulting in 14 different traps. One of these trapping events was chosen to provide the proof-of-principle and the obtained results will be presented.

Peggy Jungke

Technische Universität DresdenCenter for Regenerative Therapies Dresdenpeggy.jungke@crt-dresden.dehttp://www.uni-leipzig.de/~vpci/

Genome and Protein Engineering

96

67 Direct Cloning of Silent Gene Clusters and Heterologous Expression

Jun Fu, Jia Yin, Youming Zhang, Xiaoying Bian, Rolf Müller, Francis Stewart

The next-generation sequencing technologies allow the acquisition of genome sequencing data without genomic library construction. More than 1100 bacterial genomes have been sequenced, of which many contain secondary metabolite pathway gene clusters, though majority of them are unknown or silent. These biosynthetic gene clusters, which encode nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), are often very large and impossible to be PCR-amplified. Here we present systematical study of the RecE function. Based on the understanding of full-size RecE in linear plus linear homologous recombination, a direct cloning platform has been established. The high efficiency of the system was demonstrated by successfully cloning of all the 10 PKS/NRPS gene clusters from Photorhabdus luminescens. Gene cluster as large as 32 kb was cloned into a multiple-copy vector in one step. A 49 kb gene cluster could be cloned in two steps. In heterologous hosts two of the gene clusters produced compounds which have anticancer activity. This direct cloning platform can also be applied to clone DNA sequence from a mammalian genomic DNA pool.

Jun Fu

Technische Universität DresdenBIOTEChnology Centerjun.fu@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Genome and Protein Engineering

97

68 Pseudomonas putida – development of a heterologous expression system for complex natural products Frank Groß, Dominik Pistorius, Youming Zhang, Francis Stewart, Rolf Müller

Microorganisms produce an immense number of secondary metabolites, which are of interest to humans because of their biological activities, e.g. as therapeutics in medicine or agrochemicals. These secondary metabolites span a wide range of chemical classes in which polyketides (PK) and nonribosmal peptides (NRP) are two prominent representatives. Both types of natural products are synthesized by megasynthases, polyketide synthases (PKS) for PKs and nonribosomal peptide synthetases (NRPS) for NRPs, which show an analogous enzyme build up and reaction scheme. Both megasynthases use simple building blocks to produce an astonishing combinatorial array of products [1,2].In recent years the growing number of sequenced microorganisms revealed a large number of so called “silent” gene clusters of secondary metabolites or at least they are not expressed under conditions so far administered in the laboratory. In addition to these, one has also to consider that the number of microorganisms we can cultivate in the lab is believed less than 1% of the existing species. It is a fair assumption that this large untouched pool also has a great potential for natural products of special value for human kind. This potential can be harvested by the isolation of environmental DNA libraries, the so-called metagenomics approach [3].In both cases there is a need for suitable heterologous expression systems to uncouple the biosynthesis genes from their natural repressing regulation circuit in case of the “silent” gene clusters or to clone the gene clusters from the “unculturable” microorganisms for their expression. From the past experiences it can be concluded that there is not one system that suits all purposes, so it is necessary to develop a variety of systems, which can be used as a surrogate for the wild type strain. We chose amongst others Pseudomonas putida as a heterologous host because the genus itself is a producer of secondary metabolites; it harbors the necessary phosphopantetheinyl transferase with broad substrate specificity for posttranslational activation of PKS and NRPS [3] and has a similar GC content to the two prominent bacterial families producing secondary metabolites streptomycetes and myxobacteria [4].We already engineered a strain of P. putida harboring the methylmalonyl-CoA biosynthesis genes of Sorangium cellulosum So ce56 and showed it’s value by expressing the myxothiazol biosynthetic gene cluster in this strain [5]. Presently we improve this strain and can show here first results for the new strains regarding their biosynthesis capacity.

Dr. Frank Groß

Technische Universität DresdenBIOTEChnology Center Dresdenfrank.gross@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Genome and Protein Engineering

98

69 The Role of Histone Methyltransferases Mll1 and Mll2 in Neural Differentiation and Reprogramming of Mouse Neural Stem Cells

Katrin Neumann, Andrea Kranz, Henk Stunnenberg, Francis Stewart, Konstantinos Anastassiadis

In embryonic stem cells (ESC) promoters of lineage-specific genes are characterized by both transcriptional activating histone 3 lysine 4 methylation (H3K4me) and repressing histone 3 lysine 27 methylation (H3K27me). These so-called bivalent domains are thought to prime promoters for fast activation or final silencing upon differentiation of ESC. Upon neural differentiation, neural lineage-specific genes loose H3K27me and maintain H3K4me allowing their expression. During reprogramming of neural stem cells (NSC) to induced pluripotent stem cells (iPSC) H3K4 methyltransferase activity might be required for maintaining H3K4me in order to re-establish bivalent domain character. This implicates a possible role of histone methyltransferases in both differentiation and reprogramming.To test this hypothesis we are using conditional knock-out strategies for H3K4 methyltransferases Mixed Lineage Leukemia 1 (Mll1) and Mll2 (Wbp7). The Cre-ERT2 fusion protein is ubiquitously expressed from Rosa26 locus and activated by 4-OH-tamoxifen removing exon 2 from the genome which is flanked by LoxP sites. Cre recombination thus creates a frame shift and premature stop codon in Mll1 or Mll2 transcripts.Both knock-out ESC lines are able to self-renew implicating a certain redundancy of the six H3K4 methyltransferases found in mammals. Chromatin immunoprecipitation revealed that loss of Mll1 causes only minor chromatin changes in ESC. However, ESC lacking Mll2 specifically loose H3K4me on neural lineage-specific genes. Accordingly, differentiation towards the neural lineage is delayed and less efficient in Mll2 knock-out ESC and they fail to generate NSC in vitro. Due to the conditional knock-out strategy NSC lacking Mll2 can nevertheless be established and propagated in vitro without any evidence for self-renewal defects. In contrast, differentiation of Mll1 knock-out ESC to NSC was not impaired.NSC were reprogrammed by introducing the four cDNAs of Oct4, Klf4. Sox2 and c-Myc by PiggyBac transposition. NSC lacking Mll2 can be reprogrammed to an embryonic stem cell-like state albeit with lower efficiency the earlier the knock-out is induced. In contrast knock-out of Mll1 improves reprogramming efficiency hinting towards a role of Mll1 in stabilizing the NSC chromatin state thus counteracting reprogramming.

Katrin Neumann

Technische Universität DresdenBIOTEChnology Center Dresdenkatrin.neumann@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/anastassiadis/

Genome and Protein Engineering

99

70 Setd1 histone methyltransferases are required for embryonic stem cells and mammalian development Anita S. Bledau, Giovanni Ciotta, Andrea Kranz, Kerstin Schmidt, Francis Stewart, Konstantinos Anastassiadis

Epigenetic modifications of chromatin are heritable signatures that provide information beyond the underlying DNA sequence. Activation or silencing of specific gene loci correlates with posttranscriptional modifications at histone tails of the eukaryotic chromatin. Among those modifications, histone tail methylation originating from trithorax group (TrxG) proteins function is crucial to the developing embryo. These TrxG proteins specifically methylate nucleosomes at their histone tail 3 at lysine residue 4 (H3K4) that is associated with active gene expression. However, how functional TrxG methylation complexes accomplish precise gene activation is still unclear. There are six functional H3K4 histone methyltransferases (HMTs), namely Mll1, Mll2, Mll3, Mll4, Setd1a and Setd1b. So far, only Mll1 and Mll2 were studied. They are epigenetic regulators, essential for the developing embryo and fulfill important functions in adult mice, although they serve completely different gene targets. Here we report results from conditional mutagenesis of Setd1a and Setd1b, the mammalian orthologs of yeast Set1, in embryonic stem (ES) cells and mice to study their requirements during mammalian development. Both Setd1a and Setd1b are expressed during the earliest stages of murine embryonic development and their deficiency in mice results in lethality. While Setd1a deficient embryos are lethal at E7.5 and absent at E8.5, Setd1b knock out embryos appear normal at E7.5 but show severe retardations at E8.5 and E9.5. Further, conditional knockout of Setd1a in ES cells results in apoptosis due to proliferation defects. Conditional mutagenesis of Setd1b in ES cells has not been tested yet. Further, we were able to rescue the fatal phenotype of Setd1a by BAC transgenesis. Individual ES clones that express one or two copies of YFP-tagged Setd1a are sufficient to prevent the cells to undergo apoptosis. Moreover, we used a GFP tagging method of the unique subunit of the Setd1a/b complexes, Cxxc1, combined with Mass Spectrometry (MS) and ChIP-seq to decipher Set1 complex interactions. We showed that Set1 immunoprecipitates with Mll2 (Wbp7) indicating a cross-talk between Setd1 and Mll2 complexes. Amongst the three common H3K4 HMT subunits, Ash2, Wdr5, and RbBP5, Setd1 protein complexes seem to accommodate several other novel proteins that are under further investigation. Our results prove that Setd1a is an indispensable factor for the maintenance of ES cells and provide novel insights into the composition and function of H3K4 methyltransferase complexes. The tagging approach will assist to dissect the complexity of H3K4 HMTs in regulating the pluripotency network in mouse ES cells. This study consequently highlights the importance of tightly regulated epigenetic mechanisms for gene activation during mammalian development.

Dr. Anita S. Bledau

Technische Universität DresdenCenter for Regenerative Therapies Dresdenanita.bledau@crt-dresden.dehttp://www.crt-dresden.de/research/crtd-core-groups/anastassiadis/

Genome and Protein Engineering

100

71 Surface supercharged human enteropeptidase light chain shows improved solubility and refolding yield

Peter Simeonov, Renate Berger-Hoffmann, Ralf Hoffmann, Norbert Sträter, Thole Züchner

Enteropeptidase is a membrane-bound highly specific serine protease of the small intestine. It appears as a disulfide-linked heterodimer consisting of a non catalytic heavy chain (86,7 kDa) and a catalytic light chain (26,2 kDa). For many different biotechnological applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Unfortunately recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe several protein modifications that lead to improved solubility and refolding yield of human hEPL whilst retaining the enzyme activity. Protein surface supercharging (N6D, G21D, G22D, N141D, and K209E) of the protein for example increased the solubility more than 100-fold while the replacement of a free cysteine residue with serine (C112S) improved the refolding yield by 50%. The heat stability of this C112S variant was also significantly improved by supercharging. This study shows that even mild protein surface supercharging can have pronounced effects on protein solubility and stability.

Peter Simeonov

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Institute of Bioanalytical Chemistryp.simeonov@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~straeter/

Genome and Protein Engineering

101

72 Junior Research Group „White Biotechnology“ – New approaches to functional screening Antje Eichler, Karen Stumm, Thorsten Oeser

The Junior Research Group “White Biotechnology” of the University of Leipzig was founded in 2006 within a Biotechnological Cluster in Leipzig as part of the InnoProfile initiative of the Federal Ministry of Education and Research. The aim of the Junior Research Group was to develop innovative products and technologies within the field of biocatalysis, especially the discovery, identification, production and characterization of novel enzymes. Since May 2010 the group is financed by projects in cooperation with industrial and governmental agencies. We want to present a profile of our competences, technical skills and give an overview of our actual research topics.

Dr. Antje Eichler

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Institute of Biochemistryeichler@uni-leipzig.dehttp://www.biochemie.uni-leipzig.de/nwg_wb/home.asp

Genome and Protein Engineering

102

73 Role of efnb2 during Zebrafish development

Annekathrin Kraenkel, Michael Brand

The Eph proteins, Eph receptor and efrin ligands, play an important role in the early boundary formation, especially the midbrain-hindbrain boundary (MHB) and in the regulation of cytoskeleton and embryonic vasculature (Holder et al, 1999).The Eph receptors are the largest family of receptor tyrosine kinases and interact dependent on their class with either GPI-linked proteins (efrin A) or with transmembrane proteins (efrin B). It has been shown that Eph receptors, which bind to efrin A ligands, signal in a forward, reverse and/or bidirectional manner through tyrosine phosphorylation (Murai et al, 2003).A lot of work has been done on the role Eph receptor using knockout mutants but little is known about its ligands. For that reason we screened for ephrinb2 stop mutants to knock down both orthologs in zebrafish.We found an efnb2a mutant which has an early stop mutation at amino acid 86 and an efnb2b stop mutation at position 78. The single mutants are homozygous viable and don’t show any phenotype. For further investigations we crossed these 2 fish lines and could show morphological changes from day 4 onwards in homozygous double mutants. The homozygous double mutants are lethal.These data and the similar expression pattern of the efrin b2 ligands in various brain regions suggest that the efnb2a and efnb2b act in a redundant way.Further we would like to examine the effects of the double mutants in the developing brain on a genetically level. To address that question we will do stainings with brain markers like ntl, pax2.1 and krox20 to show whether the formation of boundaries like the MHB or the rhombomeres is still defined.

Annekathrin Kraenkel

Technische Universität DresdenBIOTEChnology Center Dresdenannekathrin.kraenkel@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/brand/

Genome and Protein Engineering

103

74 Expression of the vast majority of genes in embryon- ic stem cells does not depend on MLL complex-medi- ated H3K4 methylation Andrea Kranz, Anita S. Bledau, Konstantinos Anastassiadis, Francis Stewart

H3K4me3 is an important epigenetic mark for ES cell identity. Unlike H3K9 methylation, H3K4 methylation is a post-translational modification that is exclusively associated with actively transcribed genes. Mammals have at least six H3K4 methyltransferases that occur as three pairs of sister genes, namely, the two fly trithorax orthologues Mll1 and Mll2, the two fly trithorax-related orthologues Mll3 and Mll4 and the two yeast Set1 orthologues Setd1a and Setd1b. These enzymes are working in large protein complexes, which are composed of conserved subunits, which are common for all six. In addition there are subunits, which are unique. Concordant with the expected roles of H3K4 methylation in gene expression all six genes appear to be widely expressed throughout development. We have established mouse knockout lines as well as conditional mouse ES cell lines for all six genes. According to their ascribed roles deletion of each of the enzymes is either embryonic or perinatal lethal disfavoring redundant mechanisms. However conditional deletion of Mll1-4 in embryonic stem cells retains viability, self-renewal and ESC maintenance demonstrated by alkaline phosphatase staining amongst others. To identify potential target genes we performed RNA expression profiling for Mll1, Mll2 and Mll4 so far. Our analyses have revealed that only a few hundred genes in ES cells require MLL family members to maintain the appropriate levels of gene expression. Expression of the majority of stemness genes seems to be independent of MLL complex-mediated H3K4 methylation instead induction of lineage specific genes is highly dependent on these enzymatic complexes.

Dr. Andrea Kranz

Technische Universität DresdenBIOTEChnology Center Dresdenandrea.kranz@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Genome and Protein Engineering

104

75 Protein Tagging and Gene KO strategies for Systems Biology

Ashish Gupta,

Systems Biology approaches call for a robust system to carry out proteomic and regulomic investigations on a large scale to decipher the underlying regulatory networks. However, such aims are frequently thwarted by the lack of high grade antibodies against many less-worked-on or novel proteins. Similarly, the loss-of-function studies are affected by less than optimal efficiencies of RNAi knockdown.

Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. Generation of BAC transgenes through recombineering tools presents a way to express a chosen tagged protein at reasonable physiological levels, with all regulatory elements in their native configurations. Besides, recombineering presents a number of advantages over conventional engineering methods, including the ability to place target sites for site-specific recombinases at chosen positions. Site-specific recombinases are essential tools for altering the mouse genome.

Here we have applied these advantages to establish two methods: (i) protein tagging for BAC (bacterial artificial chromosome) transgenesis and/or gene targeting, including the evaluation of the tagged protein expression; (ii) generation of conditional alleles that employ Cre, Flp, and Dre recombinase target sites.

Ashish Gupta

Technische Universität DresdenBIOTEChnology Center Dresdenashish.gupta@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/stewart/

Genome and Protein Engineering

105

76 Notum expression and its effect on fgf8 gradient and signalling in Danio reiro. Shradha Das

The tissue patterning in developing vertebrates is significantly dependant on morphogen gradients like Wingless, Hedgehog, Fibroblast growth factor and bone morphogentic protein. Notum bearing its origin in nōton and primarily used for reference to the dorsal part of the thoracic segment in fly was later identified as a hydrolase (homologous to plant pectinacetylesterase). It has been shown to play a critical role in the moulding of the Wingless (Wg) morphogen gradient and signalling in Drosophila melanogaster. HSPG’s (Dally and Dlp), intermediating between Notum and Wg, have been proven to be cleaved at GPI anchors by Notum thus influencing the Wg gradient. The Fgf gradient and signalling has a substantial influence on the embryonic development in Danio reiro. The role of Notum and its homolgues in impacting the Fgf gradient and signalling in zebrafish has not been studied so far. We aim at establishing the expression patterns of the three Notum homologues in the embryonic stages of zebrafish and investigate the effect on the Fgf8 gradient by over-expression studies. Further, we would like to examine if the homologues comepnsate for each other or their action is mutually exclusive. Knockdown studies will additionally prove if there could be a mutant phenotype. Studies so far have shown that Notum3 (chr. 3) has a broad expression pattern ranging from shield stage to 24 hour post fertilization (hpf). The noteworthy observation was the expression at the margin in the shield stage (similar to that of Fgf8) and in the diencephalic region in front of the MHB (in contrast to Fgf8 expression at MHB). Significant expression in the tailbud is indicative of its possible role in other signalling pathways. Notum1 (chr. 6) was observed to be expressed in the 24 hpf embryos at the ventral somites which could primarily suggest its non-involvement in Fgf8 signalling that will be confirmed with over-expression studies. The possibility of Notum acting downstream to Fgf8 was disproved by Fgf8 inhibitor studies. In theory, we expect Notum to cleave Glypicans which would result in a wider spread of the Fgf gradient and thus a broader signalling range. The effect on signalling can be studied by examining the expression patterns of Fgf downstream genes like sprouty 4 (spry4), spry2 and pea3. Further goals include probing the interaction between Notum and Glypicans through Fluorescence Correlation Spectroscopy and thus building the complete picture.

Shradha Das

Technische Universität DresdenBIOTEChnology Center Dresdenshradha.das@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/brand/

Biopysical Technologies

106

8. Biophysical Technologies

107

77 When cell extracts diffract: Probing the mechanical behavior and structure of mammalian membranes Suzanne Balko, Tonya Kuhl, Chad E. Miller, Bruce J. German, Gillies Laura, Jarek Majewski, Vishal Trivedi, Walter Stockinger, Axel Nohturfft

Phagosomes are intracellular vesicles whose primary functions are the digestion of internalized particles and proteins. Most mammalian cells are capable of phagocytosis/endocytosis to some degree, with certain cell types having evolved to be particularly adept at this process, e.g. phagocytes/macrophages. It has been shown that infectious pathogens, such as tuberculosis, ebola virus and legionella enter cells via the phagocytic/endocytic pathway[1-5]. In addition, certain drugs are taken up via the endocytic pathway [6-8] and diseases are caused by malfunction of the organelles comprising the endocytic pathway, e.g. lysosomes and Tay-Sachs disease [9, 10]. Therefore, a better understanding of the physical properties of the phagolysosome system may greatly enhance drug development and medical treatments for a wide variety of illnesses. During phagocytosis/endocytosis, one of the primary fates of phagosomes is to fuse with lysosomes to facilitate digestion/decomposition. Because the phagosome membrane is fusogenic, it is imperative to understand the membrane composition and behavior to study phagosome biological function. Therefore, we have focused on studying the physical properties of phagosomal membranes in vitro. Raw phagosomes were studied in vitro using fluorescence microscopy and the composition, structure and mechanical properties of phagosome extracts were probed via Langmuir isotherm, x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD). We present the first report of phase behavior from extracts of mammalian cell membrane.

References:[1] Goren, M. B., et. al., PNAS 73 (7) 2510-2514, 1976.[2] MacMicking, J. D., et. al., Science 302 (5645) 654-659, 2003. [3] Vandal, O. H., et. al., Nature Medicine 14 849-854, 2008.[4] Saeed, M. F., et. al., PLOS Pathogens 6 (9) 1622-1633, 2010.[5] Horwitz, M. A., et. al., Journal of Experimental Medicine 158 (6) 2108-2126, 1983.[6] Palu, G., et. al., Antiviral Research 16 (1) 115-119, 1991.[7] Wagner, E., et. al., Adv. Drug Delivery Rev. 14 (1) 113-135, 1994.[8] Rippley, R. K., et. al., Biophys. J. 69 (3) 825-839, 1995.[9] Neufeld, E. F., Ann. Rev. Biochem. 60 257-280 1991.[10] Platt, F. M. et. al., Science 276 (5311) 428-431, 1997

Dr. Suzanne Balko

Max Bergmann Center of BiomaterialsLeibniz-Institute for Polymer Research (IPF)balko@ipfdd.dehttp://www.ipfdd.de/Research-Division-Biofunctional-Polymer.34.0.html?&L=1

Biophysical Technologies

108

78 Getting closer to the nature of specific bonds: Dynamic force spectroscopy on the binding of anti- bodies and tau peptides

Carolin Wagner, David Singer, Olaf Ueberschär, Tim Stanger, Ralf Hoffmann, Friedrich Kremer

Optical tweezers-assisted dynamic force spectroscopy (DFS) is employed to investigate specific receptor/ligand bindings on the level of single binding events. Here, the specific binding of two anti-human tau monoclonal antibodies (mAbs), HPT-110 and HPT-104, to synthetic tau-peptides with different phosphorylation pattern is analyzed. The specificity of HPT-110 to the tau-peptide containing a phosphorylation at Ser235 and of HPT-104 to the tau-peptide containing a phosphorylation at Thr231 is confirmed. Additionally, our approach allows for a detailed characterization of the unspecific interactions that are observed between HPT-104 and the peptide phosphorylated only at Ser235 and between HPT-110 and the peptide phosphorylated only at Thr231. By analyzing the measured rupture-force distributions it is possible to separate unspecific from specific interactions. Thereby for the latter characteristic parameters like the lifetime of the bond without force, the characteristic length and the free energy of activation are determined. The results are in accordance with conventional ELISA tests but offer a much more refined insight [1].

References:[1] C. Wagner et al., Soft Matter (2011), DOI:10.1039/C0SM01414A

Carolin Wagner

University LeipzigFaculty of Physics and Geo SciencesInstitute of Experimental Physics 1carolin.wagner@physik.uni-leipzig.dehttp://www.uni-leipzig.de/~mop/

Biophysical Technologies

109

79 Real-time 3-dimensional particle tracking with subnanometer accuracy at 5,000 frames per second Alexander Huhle, Sihwa Joo, Daniel Klaue, Ralf Seidel

Tracking the position of single spherical micrometer-sized particles in all 3 dimensions is crucial for modern force sensing techniques, such as optical and magnetic tweezers. It can be realized using either position sensitive devices in combination with focused laser illumination or a camera and wide-field illumination. While camera-based detection is very simple to implement and offers the attractive possibility to determine the positions of many particles in parallel, real-time tracking rates have so far been limited to several tens of frames per second due to the high computational effort of the employed software routines. Here we demonstrate 3 dimensional real-time tracking at 5,000 framesper second with sub-nanometer accuracy using a fast CMOS camera for image acquisition and employing GPU based computing. Computationally demanding parts of the tracking algorithm are carried out in the GPU that is specialized for highly parallelized execution. Tracking of the lateral particle positions is obtained by cross-correlating theimage with its mirror image. The axial position is obtained from the radial intensity profile of the particles diffraction pattern when imaged in overfocus. High tracking rates are crucial to overcome the shot-noise limitations of camera-based detection at the second time scale and to resolve fast, dynamic processes.

Dr. Alexander Huhle

Technische Universität DresdenBIOTEChnology Center Dresdenalexander.huhle@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/seidel/

Biophysical Technologies

110

80 DNA unwinding mechanism of a RecQ helicase from Arabidopsis thaliana investigated with magnetic tweezers

Daniel Klaue, Daniela Kobbe, Holger Puchta, Ralf Seidel

Helicases are ATP hydrolysis driven molecular motors that processively unwind double stranded DNA. In this study we investigate RecQ2 helicase from Arabidopsis thaliana (AtRecQ2) which plays an important role in genomic maintenance. We use high resolution magnetic tweezers in order to probe the unwinding of a DNA hairpin by this enzyme in real time under different external forces. We find an unwinding rate of 7-9 bp/s which is slow compared to many prokaryotic helicases. Applied forces between 5 and 12 pN only weakly affect this parameter, while the AT versus GC content of the unwound DNA has a significant impact. The weak force- but the relatively strong sequence dependence of DNA unwinding are in disagreement with a passive ratchet unwinding mechanism. High-resolution measurements reveal that AtRecQ2 unwinds the DNA in 3-4 bp steps. Beyond the behavior of AtRecQ2 during unwinding we analyze its behavior on single-strand DNA. The data shows that the enzyme moves randomly on single-strand DNA with a preference in 3´ to 5´ direction. We interpret this as a weak contact to single-strand DNA what is supported by the observation that even on a stretched hairpin configuration the enzyme is capable of repetitive shuttling, i.e. the instantaneous restart of DNA unwinding after a terminated event. Based on this we hypothesize that AtRecQ2 permanently switches strands in order to keep a target hairpin constantly open.

Daniel Klaue

Technische Universität DresdenBIOTEChnology Center Dresdendaniel.klaue@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/seidel/

Biophysical Technologies

111

81 Scanning evanescent fields in TIRF microscopy using a single point-like light source and a DNA worm gear Hergen Brutzer, Friedrich Schwarz, Ralf Seidel

Total internal reflection fluorescence (TIRF) microscopy is an elegant technique that limits the dimension of the excitation volume along the z-direction to the hundred nanometer-scale. The method makes use of the evanescent field arising when light is totally internally reflected at the boundary to a medium of lower refractive index. Often the penetration depth of this exponentially decaying field is left undetermined limiting the reproducibility in different experiments. We directly measure this quantity by using a quantum dot as a point-like light source and a Holliday junction as a drive to move the fluorescent probe with nanometer precision along the z-direction. The junction serves as a worm drive, which couples rotation into translational movement, while the DNA pitch serves as an intrinsic ruler. The junction is forced to migrate by adding negative turns to the DNA stretched perpendicular to the surface using magnetic tweezers. This causes the quantum dot, which is attached upstream of the junction, to decrease its height above the surface by 3.4 nm per turn. Thus it can be moved continuously through the excitation field while monitoring its height-dependent fluorescent signal. Since the quantum dot is a point-like light source, the intensity decay of the evanescent field can be obtained by dividing the signal recorded in TIRF illumination by the one recorded in conventional epi-illumination without further corrections.

Hergen Brutzer

Technische Universität DresdenBIOTEChnology Center Dresdenhergen.brutzer@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/seidel/

Biophysical Technologies

112

82 Monitoring of bioelectrical properties of murine embryonic stem cell-derived 3D cell cultures for in vitro drug testing

Silvia Vinz, Randy Kurz, Andrea A. Robitzki

In drug development, new potential active pharmaceutical ingredients have to be tested for severe side effects before they can be applied to humans. For these studies cell culture models and animal models are in use. But standard monolayer or suspension cell cultures show only low complexity and represent a very artificial cellular environment limiting the predictive power of such studies. Therefore, three-dimensional cell cultures as a system of intermediate complexity that better reflects the behaviour of the cells in vivo, could optimize in vitro drug testing to reduce animal testing. Embryonic stem cells can be used to generate 3D structures to investigate effects on their growth and viability to assess the embryotoxic potential of drug candidates. For this purpose the impedance measurement is used as a non-invasive, label-free, real-time detection system for cellular alterations of the 3D structures in a specially designed microcavity array. Additionally, embryonic stem cells can be differentiated to many different cell types in cell culture, so they can also be used to investigate drug effects on their differentiation as well as on the differentiated cells. Especially an undesired influence on the electrophysiology of cardiac cells is an important exclusion criterion in drug development. With the microcavity array extracellular field potentials of spontaneously contracting stem cell-derived 3D cardiomyocyte structures can be recorded to detect suchlike effects. So the stem cell-derived 3D cultures together with the microcavity array provide versatile tools for in vitro drug testing.

Silvia Vinz

University LeipzigCenter for Biotechnology and Biomedicine (BBZ)Molecular biological-biochemical Processing Technologysilvia.vinz@bbz.uni-leipzig.dehttp://www.uni-leipzig.de/~dmpt/

Biophysical Technologies

113

83 High resolution optical coherence tomography for in vivo monitoring of retinal degeneration in rodents Peter Cimalla, Anke Burkhardt, Julia Walther, Aline Hoefer, Dierk Wittig, Richard Funk, Edmund Koch

Rodent models play an important role for the understanding of the mechanisms underlying retinal diseases like age-related macular degeneration (AMD). Optical coherence tomography (OCT) as a noninvasive imaging modality allows in vivo cross-sectional visualization of the retinal microstructure and therefore facilitates longitudinal animal studies. In this work, a novel self-developed OCT system is applied which enables simultaneous spectral domain imaging in two near-infrared wavelength bands centered at 800 nm and 1250 nm. The system is illuminated by a broadband supercontinuum laser and allows three-dimensional imaging with high axial resolution better than 3.6 μm and 5.3 μm in tissue at 800 nm and 1250 nm, respectively. A fan-shaped scanning pattern and a contact lens are applied to obtain optical access to the eye’s fundus. The imaging speed amounts to 12,800 axial scans per second resulting in a video image rate of 25 fps. In a first in vivo study, retinal monitoring was performed in RCS (royal college of surgeons) rats with gene-related degeneration of the photoreceptor cells which serve as a model for AMD. The animals were treated by intravitreous injection of mesenchymal stem cells to investigate the suitability of stem cell therapy for retinal degeneration treatment. In general, the OCT images show a good visibility of the retinal microstructure which enables precise thickness measurement of individual intraretinal layers at 800 nm. Additional simultaneous imaging at 1250 nm allows depth-enhanced tissue visualization revealing sub-choroidal structures that are not visible at 800 nm. Furthermore, the combination of both wavelength bands enables image quality improvement by speckle noise reduction. The degeneration-induced rapid morphological changes inside the retina and the stem cell growth in the vitreous could successfully be followed over four weeks. These results are encouraging for further time-course studies concerning the development of the retina related to disease progression and treatment response.

Peter Cimalla

Technische Universität DresdenMedical FacultyClinical Sensoring and Monitoringpeter.cimalla@tu-dresden.dehttp://www.tu-dresden.de/medksm/index.htm

Biophysical Technologies

114

84 Fast and Precise Optical Tracking of Motor Proteins and Motor Systems using Gold Nanoparticles

René Schneider, Stefan Diez

Fast and precise imaging of biological processes has become of increasing interest in the field of microscopy-based molecular biology in the recent years. Motivated by an increasing number of newly developed nanoparticles with novel optical properties, the limitations of conventional organic dyes, namely the limited number of emitted photons and photobleaching, can be overcome. Furthermore, the nanosecond lifetime of fluorescent molecules leads to a limited number of detected photons in each time interval and therefore to a minimal integration time necessary for high precision tracking experiments.Gold nanoparticles (GNPs) are promising candidates due to their high scattering cross section and their simple surface chemistry. The applicability of GNPs in order to improve temporal as well as spatial resolution for the study of bio-molecular motor systems is investigated using camera-based optical detection systems utilizing an innovative widefield total-internal reflection (TIR) microscopy setup.Here, promising results for localizing GNP-labelled Kinesin-1 motors in a stepping assay configuration will be presented.

René Schneider

Technische Universität DresdenCenter of Innovation Competence B CUBErene.schneider@mpi-cbg.dehttp://www.mpi-cbg.de/research/research-groups/stefan-diez.html

Biophysical Technologies

115

85 Under-filling trapping objectives optimizes the use of the available laser power in optical tweezers Mohammed Mahamdeh, Citlali Pérez Campos, Erik Schäffer

For optical tweezers, especially when used in biological studies, optimizing the trapping efficiency reduces photo damage or enables the generation of larger trapping forces. One important, yet not-well understood, tuning parameter is how much the laser beam needs to be expanded before coupling it into the trapping objective. Here, we measured the trap stiffness for 0.5-2 μm-diameter microspheres for various beam expansions. We show that the highest overall trapping efficiency is achieved by slightly under-filling a high-numerical aperture objective when using microspheres with a diameter corresponding to about the trapping-laser wavelength in the medium. The optimal filling ratio for the lateral direction depended on the microsphere size, whereas for the axial direction it was nearly independent. Our findings are in agreement with Mie theory calculations and suggest that apart from the choice of the optimal microsphere size, slightly under-filling the objective is key for the optimal performance of an optical trap.

Mohammed Mahamdeh

Technische Universität DresdenBIOTEChnology Center Dresdenmohammed.mahamdeh@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/schaeffer/

Biophysical Technologies

116

86 3D imaging of lung tissue structure and dynamics using non-invasive optical techniques

Maria Gärtner, Sven Meissner, Christian Schnabel, Peter Cimalla, Hannah Nickles, Lilla Knels, Wolfgang Kübler, Edmund Koch

Thorough understanding of lung dynamics on a microscale has become an urgent need nowadays for the access of reliable parameters for protective artificial ventilation. One approach to this is the simulation of lung behavior on the alveolar level by using realistic geometrical and molecular data.In our study we demonstrate the ability of optical coherence tomography (OCT) as a non-invasive, non-contact, three-dimensional, high-speed imaging modality with micrometer resolution to observe lung parenchyma during artificial ventilation in vivo. Alveolar volume changes can be extracted from the geometrical tissue representation provided by the OCT. To further enhance the imaging depth, from 220 μm to 650 μm, and to reduce optical artifacts caused by the index mismatch between the tissue and the air within the alveoli, the concept of liquid ventilation was additionally introduced. A ventilation apparatus, switching from air to liquid ventilation, serves as a measuring system for the access of respiratory parameters of small animals.Beside all its advantages, OCT unfortunately lacks information about molecular constituents embedded in the sample due to its non-specific tissue interaction. Combination with confocal fluorescence microscopy together with the application of specifically binding fluorophors (e.g. sulforhodamine B for elastin), expands the yield of information. Using this system for simultaneous image acquisition, elastic fibers can be localized in relation to the structural data given by the OCT.With these approaches the investigation of lung behavior at an alveolar level for distinct ventilatory strategies becomes feasible. The obtained structural and molecular information can be used as a framework for realistic lung modeling. Future work will focus on the assessment of in vivo dynamics as well as a comparison between artificial ventilation and spontaneous breathing.

Maria Gärtner

Technische Universität DresdenMedical FacultyClinical Sensoring and Monitoringmaria.gaertner@tu-dresden.dehttp://www.tu-dresden.de/medksm/index.htm

Biophysical Technologies

117

87 Mechanical measurements reveal high bending but low twisting rigidity of 3D DNA-origami Dominik Kauert, Tim Liedl, Ralf Seidel

DNA-origami is a recently developed method to design and assemble DNA nanostructures of arbitrary shape and property. The understanding of their mechanical behavior is crucial to develop a toolbox of these nanostructures for a broad range of applications. We used magnetic tweezers that support also direct torque measurements to determine the bending and torsional rigidities of DNA multi-helix bundles assembled by the origami method. In particular we investigated 4-helix bundles of 480nm and 6-helix bundles of 400nm length. To analyze the measurements Monte Carlo simulations and numerical finite-elementmodeling was applied. The bending rigidity was found increased about15-fold and 40-fold for 4-helix bundles and 6-helix bundles compared to double-strand DNA, respectively. In contrast, the torsional rigidity increased only 4-fold and 6-fold for 4-helix bundles and 6-helix bundles. We also show the importance of a rigid attachment of the multi-helical structures, necessary to effectively use their rigid properties.

Dominik Kauert

Technische Universität DresdenBIOTEChnology Center Dresdendominik.kauert@biotec.tu-dresden.dehttp://www.biotec.tu-dresden.de/research/seidel/

Biopysical Technologies

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