Rudolf-Virchow-Zentrum
Refine
Has Fulltext
- yes (299)
Is part of the Bibliography
- yes (299)
Year of publication
Document Type
- Journal article (190)
- Doctoral Thesis (102)
- Preprint (5)
- Master Thesis (1)
- Report (1)
Keywords
- Thrombozyt (19)
- platelets (17)
- ischemic stroke (10)
- Maus (9)
- Thrombose (8)
- platelet (8)
- gephyrin (7)
- platelet activation (6)
- GPVI (5)
- Microscopy (5)
- Signaltransduktion (5)
- blood (5)
- glycoprotein VI (5)
- platelet aggregation (5)
- thrombo-inflammation (5)
- Biologie (4)
- DNS-Reparatur (4)
- Drosophila melanogaster (4)
- Fluoreszenzmikroskopie (4)
- Mice (4)
- Mikroskopie (4)
- Platelets (4)
- Rezeptor (4)
- Staphylococcus aureus (4)
- Taufliege (4)
- Thrombosis (4)
- X-ray crystallography (4)
- actin (4)
- expression (4)
- integrins (4)
- megakaryocytes (4)
- phosphorylation (4)
- thrombosis (4)
- Arteriosklerose (3)
- Arzneimitteldesign (3)
- Dendritische Zelle (3)
- Diabetes mellitus (3)
- Drosophila (3)
- Entzündung (3)
- Fettsäurestoffwechsel (3)
- GPCR (3)
- Genexpression (3)
- HUWE1 (3)
- Hämostase (3)
- Inflammation (3)
- Megakaryozyt (3)
- Metalloproteinasen (3)
- NRF2 (3)
- Platelet (3)
- Proteinkinase D (3)
- Rac1 (3)
- Transkriptionsfaktor (3)
- Typ 1 (3)
- Ubiquitin (3)
- cancer (3)
- cell wall (3)
- crystal structure (3)
- differentiation (3)
- escherichia coli (3)
- flow cytometry (3)
- fluorescence imaging (3)
- fluorescence microscopy (3)
- inflammation (3)
- lipid bilayer (3)
- macrophages (3)
- mechanisms (3)
- protein (3)
- thrombin (3)
- tumorigenesis (3)
- AFM (2)
- ATF4 (2)
- Agent (2)
- Aorta (2)
- Apoptosis (2)
- Blut-Hirn-Schranke (2)
- Breast-tumors (2)
- C. elegans (2)
- C/EBP (2)
- CML (2)
- CXCR4 (2)
- Caenorhabditis elegans (2)
- Calcium (2)
- Cancer (2)
- Cryo-EM (2)
- Cytomegalie-Virus (2)
- Cytoskeleton (2)
- DNA repair (2)
- Differenzierung (2)
- Einzelmolekülmikroskopie (2)
- Expansion Microscopy (2)
- FRET (2)
- Fettsäurebiosynthese (2)
- Fluorescence (2)
- Fluoreszenz (2)
- G protein-coupled receptors (2)
- GABA (2)
- GABAA receptors (2)
- Gephyrin (2)
- Helicasen (2)
- Herzhypertrophie (2)
- Hirnhautentzündung (2)
- Inhibition (2)
- Inhibitorische Synapse (2)
- Inhibitory synapse (2)
- Interleukin 6 (2)
- Invasion (2)
- Kollagen (2)
- Kristallographie (2)
- Kristallstruktur (2)
- LASP1 (2)
- Learning and memory (2)
- Lung cancer (2)
- MIZ1 (2)
- MYC (2)
- Megakaryocyte (2)
- Megakaryopoese (2)
- Meningitis (2)
- Metabolism (2)
- Molekulargenetik (2)
- Muskelzelle (2)
- Nude-mice (2)
- OSMR (2)
- Obesity (2)
- Oncostatin M (2)
- PIP2 (2)
- Peptidsynthese (2)
- Phagozytose (2)
- Phosphorylierung (2)
- Plasmamembran (2)
- Plättchen (2)
- Pneumolysin (2)
- Protein (2)
- Protein p53 (2)
- Protein p73 (2)
- Regulation (2)
- Rhabdomyosarkom (2)
- Rho GTPase (2)
- SOCE (2)
- Schlaganfall (2)
- Smad (2)
- T-Lymphozyt (2)
- T-cells (2)
- TFIIH (2)
- Therapy (2)
- Toxin (2)
- Type 1 Diabetes (2)
- Zelle (2)
- Zellmigration (2)
- Zellskelett (2)
- Zelltod (2)
- activation (2)
- artemisinin (2)
- atherosclerosis (2)
- atomic-force microscopy (2)
- binding (2)
- biosensors (2)
- blood coagulation (2)
- bone marrow (2)
- brain (2)
- breast-tumors (2)
- cell biology (2)
- cell signalling (2)
- chloroquine (2)
- coagulation (2)
- collagens (2)
- collybistin (2)
- crystallography (2)
- dSTORM (2)
- dendritic cells (2)
- diazepam (2)
- domain (2)
- drug discovery (2)
- enzymes (2)
- extracellular domain (2)
- factor XII (2)
- fluorescence resonance energy transfer (FRET) (2)
- gene expression (2)
- glycine receptor (2)
- gp130 (2)
- human (2)
- hydrodynamics (2)
- inhibitory neurotransmission (2)
- inhibitory postsynapse (2)
- interactome (2)
- ischemic penumbra (2)
- isothermal titration calorimetry (2)
- lipid bilayer membrane (2)
- matrix protein porin (2)
- melanoma (2)
- membrane (2)
- membrane potential (2)
- meningitis (2)
- metastasis (2)
- miRNS (2)
- mice (2)
- middle cerebral artery occlusion (2)
- molecular biology (2)
- mouse (2)
- mouse model (2)
- mouse models (2)
- murine (2)
- neuroinflammation (2)
- neuronal dendrites (2)
- neurons (2)
- nucleotide excision repair (2)
- nucleotide excision-repair (2)
- nude-mice (2)
- oncolytic viruses (2)
- p34 (2)
- p44 (2)
- p53 (2)
- p63 (2)
- p73 (2)
- pathway (2)
- phagocytosis (2)
- phagosome maturation (2)
- phospholipase D (2)
- platelet receptors (2)
- porin (2)
- recognition (2)
- rheumatoid arthritis (2)
- signaling (2)
- stroke (2)
- structural basis (2)
- structure based drug design (2)
- sulfur (2)
- synapses (2)
- synaptic plasticity (2)
- therapy (2)
- thermodynamics (2)
- thrombopoiesis (2)
- translocation (2)
- tumors (2)
- type VII secretion system (2)
- ubiquitin (2)
- vision (2)
- vitamin B6 (2)
- 1,25-dihydroxyvitamin D\(_{3}\) (1)
- 2-photon microscopy (1)
- 3D printing (1)
- 7,8-dihydroxyflavone (7,8-DHF) (1)
- 7-Dehydrocholesterol (1)
- 8-oxoguanine (1)
- AAA (1)
- AAA+ ATPase p97 (1)
- ADAM10 (1)
- ADP-ribosyltransferases (1)
- AGT (1)
- AKT1 (1)
- AMP-activated protein kinase (AMPK) (1)
- AMP‐activated protein kinase (1)
- ATGL (1)
- Abszission (1)
- Acetylation (1)
- Acetylierung (1)
- Ackerschmalwand (1)
- Actin (1)
- Actin binding proteins (1)
- Actin cytoskeleton-related protein (1)
- Actin-bindende Proteine (1)
- Activation (1)
- Active zone (1)
- Adapterprotein (1)
- Adipokine (1)
- Adipositas (1)
- Adrenalin (1)
- Affinity probe (1)
- Aktive Zone (1)
- Allgemeine Zelle (1)
- Anthrax Toxin (1)
- Anti-Gehirn Autoantikörper (1)
- Antikörper (1)
- Antithrombotics (1)
- Antithrombotika (1)
- Antithrombotikum (1)
- Archaea (1)
- Archaebakterien (1)
- Arp2/3 complex (1)
- Art (1)
- Arterial water (1)
- Arterielles Blut (1)
- Aspergillus fumigatus (1)
- Astrozyt (1)
- Atherosclerosis (1)
- Atriales natriuretisches Hormon (1)
- Autoantigen (1)
- Autoantikörper (1)
- Autoinhibition (1)
- Automatische Biegewinkelmessung (1)
- Autophagie (1)
- BCR‐ABL (1)
- BIN2 (1)
- BMP (1)
- BRAF (1)
- BTB domain (1)
- BV-2 (1)
- Bacillus anthracis (1)
- Bacillus subtilis (1)
- Bacterial Toxins (1)
- Bakteriengift (1)
- Basen-Exzisionsreparatur (1)
- Bcl-2 (1)
- Behavior (1)
- Beige adipocytes (1)
- Bericht (1)
- Biegewinkel (1)
- Bildbearbeitung (1)
- Bildgebung intakten Knochens (1)
- Bildverarbeitung (1)
- Bindungsprozess (1)
- Biochemie (1)
- Biochemistry (1)
- Biology (1)
- Biomarker (1)
- Black-lipid-bilayer (1)
- Blood-brain barrier (1)
- Blutgefäßsystem (1)
- Blutplättchen (1)
- Bone morphogenetic proteins (1)
- Bordetella pertussis (1)
- Brain (1)
- Burkholderia (1)
- Burkholderia pseudomallei (1)
- C2-toxin (1)
- CCR2 (1)
- CD coreceptors (1)
- CD11b+ myeloid cells (1)
- CD84 (1)
- CHAC1 (1)
- CIB1 (1)
- CLEC-2 (1)
- CLEC-2 ITAM (1)
- COVID-19 (1)
- COX-2 (1)
- COX2 expression (1)
- CVT (1)
- CXCL12-abundant reticular (CAR)-cells (1)
- CXCL4 (1)
- CXCL7 (1)
- Calcium signalling (1)
- Calcium-bindende Proteine (1)
- Carboxy-Terminus (1)
- Carcinogenese (1)
- Carcinoma (1)
- Caspr1 (1)
- Cation Homeostasis (1)
- Cell (1)
- Cell line (1)
- Cells (1)
- Central nervous system (1)
- Cerebral-ischemia (1)
- ChIP-sequencing (1)
- Chaetomium thermophilum (1)
- Chemerin (1)
- Chemerin processing (1)
- Chemotherapy (1)
- Chlamydia trachomatis (1)
- Cholesterin (1)
- Cholesterol (1)
- Chylomicron (1)
- Chylomicrons (1)
- Circadian rhythms (1)
- Clec16a (1)
- Clostridioides binary toxins (1)
- Clostridium-botulinum (1)
- Cluster (1)
- Coefficient (1)
- Collaborative Research Center (1)
- Collagen (1)
- Combination therapy (1)
- Component selectivity (1)
- Computerprogramm (1)
- Conditioning (1)
- Contactin 1 (1)
- Cortactin (1)
- Corynebacterium urealyticum (1)
- Cranial window (1)
- Crystal structure of MTR1 (1)
- Crystal-structure (1)
- Current noise-analysis (1)
- CyaA (1)
- Cyclophosphamide (1)
- Cys-loop receptor (1)
- Cytokine (1)
- DAMGO (1)
- DAPI staining (1)
- DHCR7 (1)
- DNA Repair (1)
- DNA damage (1)
- DNA metabolism (1)
- DNA origami (1)
- DNA structures (1)
- DNA-Reparatur (1)
- DNA-based nanostructures (1)
- DNA-processing enzymes (1)
- DNS-Bindung (1)
- Darm (1)
- Deletion (1)
- Dendra2 (1)
- Dendritic cells (1)
- Diabetes (1)
- Diabetes Typ 1 (1)
- Differentiation (1)
- Diphenylether (1)
- Disulfidbrücken (1)
- Diversifikation <Biologie> (1)
- Dopamine (1)
- Durchflusscytometrie (1)
- Dynamik (1)
- Dynamik von Membranrezeptoren (1)
- E1 (1)
- E2 (1)
- E3 enzyme (1)
- EHT1864 (1)
- ERK map kinease (1)
- ERK1/2 (1)
- ESAT‐6‐like secretion system (1)
- ESS (1)
- Edema factor (1)
- Einzelpartikelverfolgung (1)
- Enoyl-Reduktase (1)
- Enoyl-acyl-carrier-protein-Reductase (1)
- Entwicklung (1)
- Enzym (1)
- Enzyme Regulation (1)
- Epitop (1)
- Erlernte Hilflosigkeit (1)
- EsaA (1)
- Escherichia coli (1)
- Escherichia coli AlkA (1)
- Escherichia-coli (1)
- Experimental Biomedicine (1)
- Experimental stroke (1)
- Extracellular Vesicles (1)
- FANCM (1)
- FAS-II (1)
- FAT10ylation (1)
- FIONA (1)
- FMMs (1)
- FRAP (1)
- FRET sensors (1)
- FabI (1)
- FabV (1)
- FeS cluster (1)
- Ferroptose (1)
- Ferroptosis (1)
- Fettsäure-Synthase (1)
- Fgf-signalling (1)
- Fibroblast (1)
- Flippase (1)
- Flotillin (1)
- Fluorescence Microscopy (1)
- Fluorescence Resonance Energy Transfer (1)
- Fluorescence correlation spectroscopy (1)
- Fluorescence imaging with one nanometer accuracy (1)
- Fluorescence microscopy (1)
- Fluorescence resonance energy transfer (1)
- Fluorescent probes (1)
- Fluoreszenzsonde (1)
- Fluoreszmikroskopie (1)
- FoxO3 (1)
- Foxo1 (1)
- Fragiles X Syndrom (1)
- Frühdiagnostik (1)
- Förster Resonance Energy Transfer (1)
- Förster resonance energy transfer (1)
- G Protein-Coupled Receptors (1)
- G protein-coupled receptor (1)
- G-Protein gekoppelte Rezeptoren (1)
- G-protein coupled receptors (1)
- G-protein-coupled receptors (1)
- GABA(A) receptors (1)
- GABA-A receptor (1)
- GABAA (1)
- GBM (1)
- GFAP (1)
- GI-101A tumor xenografts (1)
- GIST (1)
- GLV-1 h153 (1)
- GLV-1H68 (1)
- GPCRs (1)
- GPVI shedding (1)
- GPX4 (1)
- GSH (1)
- GapN (1)
- Gelernte Hilflosigkeit (1)
- Genanalyse (1)
- Gene-expression (1)
- General Transcription Factor II H (1)
- Gerinnungsfaktor (1)
- Geruchswahrnehmung (1)
- Gleiten (1)
- Gliafaserprotein (1)
- Glycoprotein GPV (1)
- Glycoprotein VI (1)
- Glycoprotein hormone (1)
- Glykoprotein VI (1)
- Glykoprotein-Shedding (1)
- Glykoproteine (1)
- Glykosylasen (1)
- Gram-positive bacteria (1)
- Guanine nucleotide exchange factor (GEF) (1)
- Guanylatcyclase (1)
- Guanylyl cyclase A (1)
- Gα\(_{i1}\), Gα\(_{i2}\) and Gα\(_{i3}\) activation (1)
- H1 helix (1)
- H1-Helix (1)
- H2A histone family member X (H2AX) (1)
- HECT Ligase (1)
- HHV-6A (1)
- HIF1alpha (1)
- Head-injury (1)
- Heart (1)
- Helicase (1)
- Hemodynamic depression (1)
- Hemostasis (1)
- Hepatitis B Virus (1)
- Herpesvirus (1)
- Heubacillus (1)
- Hic-5 (1)
- High-throughput screening (1)
- Hirschsprung disease (1)
- Homöostase (1)
- Hypothalamus (1)
- Hypoxia (1)
- Hypoxie (1)
- IMA2.1 (1)
- ITAM (1)
- Identification (1)
- Immune Thrombocytopenia (1)
- Immunologische Synapse (1)
- Immunthrombozytopenie (1)
- In vivo (1)
- In vivo imaging (1)
- In-vivo (1)
- Inhibitor (1)
- Inhibitory-postsynapse (1)
- Insulin (1)
- Insulinsekretion (1)
- Integrine (1)
- Interleukin 17 (1)
- Intravascular coagulation (1)
- Intrazellulärraum (1)
- Invertebrate vision (1)
- Ion-channel (1)
- Iron-uptake (1)
- Ischemic stroke (1)
- JAQ1 (1)
- Jolly bodies (1)
- KEA (1)
- KEAP1 (1)
- KIT (1)
- Kardial Hypertrophy (1)
- Kardiovaskulär (1)
- Karzinomzellen (1)
- Kationen-Homöostase (1)
- Knochen-Morphogenese-Proteine (1)
- Knockout <Molekulargenetik> (1)
- Koagulation (1)
- Kollektive Invasion (1)
- Konditionierung (1)
- Kraftmikroskopie (1)
- Krebsforschung (1)
- Krebszelle (1)
- L-3,4-Dihydroxyphenylalanine-induced dyskinesia (1)
- LC-MS (1)
- LC3-associated phagocytosis (1)
- LC3-assoziierte Phagozytose (1)
- LIFR (1)
- Latrophilin (1)
- Learned Helplessness (1)
- Lebenszyklus (1)
- Leber-Metabolismus (1)
- Lentiviral transgenesis (1)
- Lernen und Gedächtnis (1)
- Lethal factor (1)
- Leukaemia-inhibitory factor (1)
- Lichtblattmikroskopie (1)
- Lichtscheibenmikroskopie (1)
- Lipid Rafts (1)
- Lipid bilayer-membranes (1)
- Lipide (1)
- Lipolysis (1)
- LpxC inhibitors (1)
- Lungenkrebs (1)
- Lymphom (1)
- M14 carboxypeptidasses (1)
- MAPK signaling (1)
- MAX (1)
- MGL (1)
- MRI reporter (1)
- MYCN (1)
- MYCNv (1)
- Magnetic-resonance (1)
- Malignant effusion (1)
- Maligne Transformation (1)
- Mass Spectrometry (1)
- Massenspektrometrie (1)
- Mast cells (1)
- Matrix protein porin (1)
- Mc4r (1)
- Mcl-1 (1)
- Mechanismus (1)
- Medicine (1)
- Medizin (1)
- Megakaryozytopoese (1)
- Membrane Receptor Dynamics (1)
- Membranglykoproteine (1)
- Membranrezeptor (1)
- Metabolismus (1)
- Metabotropic glutamate receptor (1)
- Metalloproteases (1)
- Metalloproteinase (1)
- Methyltransferase Ribozyme MTR1 (1)
- Microarray (1)
- Microvesicle (1)
- Mitose (1)
- Mittelkörper (1)
- Model (1)
- Molecular Biophysics (1)
- Molecular-weight heparin (1)
- Molekularbiologie (1)
- Molekulare Marker (1)
- Mouse (1)
- Mouse model (1)
- Multidrug-Resistenz (1)
- Multiphotonenmikroskopie (1)
- Multiproteinkomplex (1)
- Mushroom body (1)
- Muskelkontraktion (1)
- Myomesin (1)
- NADPH (1)
- NAP-2 (1)
- NF-KAPPA-B (1)
- NFκB-activation (1)
- NOD (1)
- Nanofabrication (1)
- Nanofabrikation (1)
- Ndrg1 (1)
- Neurobiologie (1)
- Neurofascin (1)
- Neurogenetik (1)
- Neurons (1)
- Neuroscience (1)
- Neurowissenschaften (1)
- Neutralisation <Chemie> (1)
- Nrf2 (1)
- Nuclease (1)
- Nucleasen (1)
- Nucleotide-Excision-Repair (1)
- Nukleotid-Exzisions-Reparatur (1)
- O6-alkylguanine-DNA alkyltransferase (1)
- OSC (1)
- Obscurin (1)
- Odor-feeding-time memory (1)
- Olig2 (1)
- Oncolytic vaccinia virus (1)
- Oncolytic virotherapy (1)
- OprO (1)
- OprP (1)
- Orai2 (1)
- P14ARF (1)
- P4-ATPase (1)
- PDE (1)
- PDI (1)
- PDXP inhibitors (1)
- PF4 (1)
- PKA signaling (1)
- PKD1 (1)
- PKR (1)
- PNA (peptide nucleic acid) (1)
- PTMs (1)
- PTPN22 (1)
- Parkinson Disease (1)
- Parkinson-Krankheit (1)
- Parkinsons disease (1)
- Peptidase inhibitor 16 (PI16) (1)
- Peptide (1)
- Perforine (1)
- Perfusion (1)
- Peyer's patch (1)
- Phenylalanine clamp (1)
- Phospholipase D (1)
- Phosphorylation (1)
- Pilzkörper (1)
- Plasmonic (1)
- Platelet activating Factor (1)
- Platelet activation (1)
- Platelet-Membranglykoprotein p62 (1)
- Plätchen aktivierung (1)
- Polkörper (1)
- Pore (1)
- Pore formation (1)
- Pore-formation (1)
- Porenbildung (1)
- Posttranslationale Änderung (1)
- Primärtumor (1)
- Profilierung (1)
- Protease inhibition (1)
- Protective antigen (1)
- Protein Disulfid Isomerase (1)
- Protein Kinase C (1)
- Protein Kinase D (1)
- Protein Kinase D 1 (1)
- Protein Kinase D2 (1)
- Protein kinase D1 (PKD1) (1)
- Protein kinase D3 (PKD3) (1)
- Proteinfaltung (1)
- Proteinkinase C (1)
- Proteins (1)
- Protoplasten (1)
- Ptpn22 (1)
- Purification (1)
- Quantifizierung (1)
- Quantitation (1)
- Quergestreifte Muskulatur (1)
- RARRES2 (1)
- RCK domain (1)
- RHO (1)
- RHO-associated kinease (1)
- RKIP (1)
- RNA (1)
- RNA interference (1)
- RNA polymerase II (1)
- RNA splicing (1)
- RNA structures (1)
- RNA-catalyzed RNA methylation (1)
- RNAi (1)
- RNS-Interferenz (1)
- ROCK (1)
- ROK-alpha (1)
- RSK (1)
- RTX-Toxins (1)
- Rac (1)
- Ranvier-Schnürring (1)
- Rasterkraftmikroskop (1)
- Rats (1)
- Ratte (1)
- RecQ4 (1)
- Receptor Preference (1)
- Receptor internalization (1)
- Receptor signaling (1)
- Recombinant vaccinia (1)
- Registrierung <Bildverarbeitung> (1)
- Regression (1)
- Remorin (1)
- Rheumatoide Arthritis (1)
- Rho GTPasen (1)
- Rho GTPasw (1)
- RhoGTPase (1)
- Rhodesain (1)
- Ribosom (1)
- Röntgenkristallographie (1)
- S-layer protein (1)
- S6KII RSK (1)
- SARS-CoV-2 (1)
- SDS polyacrylamide gel electrophoresis (1)
- SLC7A11 (1)
- SOC (1)
- SPT5 (1)
- SPT6 (1)
- STIM2 (1)
- SUPT5H (1)
- Salmonella-typhimurium (1)
- Schlafkrankheit (1)
- Secretion (1)
- Sepsis (1)
- Serotonin (1)
- Sezernierte Faktoren (1)
- Signaling (1)
- Single Particle Tracking (1)
- Sinusthrombose (1)
- Solid-phase peptide synthesis (1)
- Sonderforschungsbereich Transregio 240 (1)
- Spezifität (1)
- Spleen tyrosine kinase (1)
- Src family (1)
- Ssl1 (1)
- Staphylococcus aureus USA300 (1)
- Store-Operated (1)
- Streptococcus pneumoniae (1)
- Stress (1)
- Structural Biology (1)
- Struktur-basiertes Wirkstoff Design (1)
- Sugar-transport (1)
- Super-Resolution Microscopy (1)
- Super-resolution microsopy (1)
- Superhochauflösende Mikroskopie (1)
- Synapse (1)
- T cell (1)
- T cell development (1)
- T cell migration (1)
- T cell rezeptor transfer (1)
- T lymphocyte (1)
- T-Lymphozyten-Rezeptor (1)
- T-Zell-Rezeptor Transfer (1)
- T-Zellaktivierung (1)
- T-Zellhomöostase (1)
- T-Zellrezeptor (1)
- TGN1412 (1)
- TIG2 (1)
- TPp73 (1)
- TRRAP (1)
- Tagesrhythmus (1)
- Telomerase (1)
- Tfb4 (1)
- Thrombopoese (1)
- Thrombopoiesis (1)
- Thrombozytenaggregation (1)
- Thrombozytenfunktionsanalyse (1)
- Thrombozytopathie (1)
- Thrombozytopoese (1)
- Thrombus (1)
- Thrombus formation (1)
- Tissue (1)
- Tissue staining (1)
- Titin (1)
- Transcription (1)
- Transkription (1)
- Translokation (1)
- Transparent motion (1)
- Triclosan (1)
- Triton X 100 (1)
- Trypanosoma brucei (1)
- Trypanosomiasis (1)
- TspanC8 (1)
- Tuberkelbakterium (1)
- Tumorigenese (1)
- Tumorzelle (1)
- Type 1 diabetes (1)
- Type VIIb secretion system (1)
- UBA6 (1)
- UBE2S (1)
- UBE2Z (1)
- UBE3A (1)
- US22 gene family (1)
- USP (1)
- Ube2S (1)
- Ubiquitin-PA (1)
- Ubiquitin-Protein-Ligase (1)
- Ubiquitin-conjugating (E2) enzymes (1)
- Ubiquitin-conjugating enzyme (1)
- Ubiquitinierung (1)
- Ubiquitylation (1)
- Unique Selling Proposition (1)
- VACV (1)
- VEGF (1)
- VSMC (1)
- Vaccinia virus (1)
- Vascular endothelial Growth Factor (1)
- Vaskuläre Integrität (1)
- Verhalten (1)
- Verweildauer (1)
- Vesikelbildung (1)
- Visual attention (1)
- Visuelle Aufmerksamkeit (1)
- Von-Willebrand-factor (1)
- WDR5 (1)
- Warsaw breakage syndrome (1)
- Wilms tumor (1)
- Wirkstoff (1)
- X-Ray Chrystallography (1)
- X-ray Crystallography (1)
- XPD (1)
- XPD helicase (1)
- Xeroderma pigmentosum (1)
- Xiphophorus (1)
- X‐ray crystallography (1)
- Yersinia (1)
- Yersinia pestis (1)
- Zebrabärbling (1)
- Zebrafisch (1)
- Zebrafish (1)
- Zelldifferenzierung (1)
- Zellkern (1)
- Zelllinie (1)
- Zelltransport (1)
- Zellwand (1)
- Zentralnervensystem (1)
- Zielstruktur (1)
- Zinc (1)
- Zink-Finger-Proteine (1)
- Zytoskelett (1)
- abscission (1)
- accelerated atherosclerosis (1)
- accumulation (1)
- acetylsalicylic acid (1)
- actins (1)
- activating transcription factor 4 (ATF4) (1)
- active zone (1)
- acute graft-versus host disease (1)
- acute graft-versus-host disease (1)
- acute myeloid leukemia (1)
- acute slices (1)
- adaptor protein Swiprosin-1/EFhd2 (1)
- additive manufacturing (1)
- adenylate cyclase toxin (1)
- adenylyl cyclase (1)
- adhesion GPCR (1)
- adipocyte (1)
- adipocytes (1)
- adipose (1)
- adrenergic receptor (1)
- adsorption (1)
- aggregation (1)
- alkene-alkyne [2+2] photocycloaddition (1)
- alkylation damage (1)
- alloreactive T cells (1)
- allosterism (1)
- amino acid sequence (1)
- aminoquinolinium salts (1)
- ampa receptors (1)
- ancistrocladinium A (1)
- angiogenesis (1)
- animal behavior (1)
- animal models (1)
- animal-model (1)
- animals (1)
- anions (1)
- antagonists (1)
- anthrax (1)
- anti-brain autoantibodies (1)
- anti-inflammatory (1)
- antibacterial activity (1)
- antibiotics (1)
- antibody (1)
- antibody/autoantibody (1)
- antigen processing and recognition (1)
- antioxidant function (1)
- aorta (1)
- apolipoprotein E (1)
- apoptose (1)
- apoptosis (1)
- arachidonic acid metabolic network (1)
- aspergillus fumigatus (1)
- astrocytes (1)
- ataxia teleagiectasia mutated (ATM) (1)
- atheriosclerotic lesions (1)
- atomic force microscopy (1)
- atomic force microscopy (AFM) (1)
- atopic dermatitis (1)
- atrial natriuretic peptide (1)
- attention (1)
- autoantibodies (1)
- autoantibody (aAb) (1)
- autoantigen (1)
- autoimmunity (1)
- autoinhibition (1)
- axonal transport (1)
- bacteria (1)
- bacterial fatty-acid biosynthesis (1)
- bacterial meningitis (1)
- base excision repair (1)
- base pairs (1)
- bead models (1)
- behavioral conditioning (1)
- beige adipocytes (1)
- beta (1)
- beta cell function (1)
- biased signaling (1)
- binding components (1)
- binding mode (1)
- biochemical characterization (1)
- bioconjugation (1)
- biological techniques (1)
- biomarkers (1)
- biomedicine (1)
- biopharmaceuticals (1)
- biosensor (1)
- black lipid bilayer (1)
- blood brain barrier (1)
- blood flow (1)
- blood platelets (1)
- blue native polyacrylamide gel electrophoresis (1)
- bone morphogenetic protein (1)
- bone-marrow (1)
- borrelia burgdorferi (1)
- boundary cap (1)
- brain damage (1)
- breast cancer (1)
- bulky damages recognition (1)
- cAMP (1)
- cAMP signaling (1)
- calcitonin gene-related peptide (1)
- calcium (1)
- calmodulin (1)
- cancer biology (1)
- cancer metabolism (1)
- cancer therapy (1)
- cancer treatment (1)
- carboxy terminus (1)
- carcinoma (1)
- carcinoma cells (1)
- cardiac hypertrophy (1)
- cardiovascular (1)
- cardiovascular disease (1)
- cascade (1)
- cell (1)
- cell corpse clearance (1)
- cell membranes (1)
- cell migration (1)
- cell proliferation (1)
- cell staining (1)
- cell wall channel (1)
- cell walls (1)
- cell-autonomous defense (1)
- cell-cycle arrest (1)
- cell-line (1)
- cells (1)
- cellular neuroscience (1)
- cellular stress response (1)
- cellular-trafficking (1)
- cerebellum (1)
- cerebrovascular diseases (1)
- cerebrovascular disorders (1)
- channel (1)
- chemical libraries (1)
- chemokine (1)
- chemokines (1)
- chemotherapy (1)
- cholesterol (1)
- cholesterol-dependent cytolysin (1)
- cholinergic interneurons (1)
- chronic heart failure (1)
- chronic kidney disease (1)
- chronophin (1)
- chylomicron (1)
- ciliary neurotrophic factor (1)
- click-chemistry (1)
- clinical neurology (1)
- coagulation system (1)
- coffin-lowry-syndrome (1)
- collective invasion (1)
- combinatorial libraries (1)
- complex (1)
- complex stability (1)
- computational docking (1)
- conditioned response (1)
- conformational activation (1)
- conformational auto-epitope (1)
- conjugation (1)
- conservation (1)
- conserving surgery (1)
- containing GABA(A) receptors (1)
- controllability (1)
- convergent extension movements (1)
- corticosteroids and cyclophosphamide (1)
- corynebacteria (1)
- corynebacterium diphtheriae (1)
- cultured hippocampal-neurons (1)
- cyclic AMP (1)
- cyclic peptides/cyclopeptides (1)
- cyclopeptide therapy (1)
- cysteine (1)
- cytokine release (1)
- cytomegaloviren (1)
- cytomegalovirus (1)
- cytoskeleton (1)
- cytotoxic T cells (1)
- dCIRL (1)
- damage responses (1)
- damaged DNA (1)
- deficient mice (1)
- demography (1)
- dendric cells (1)
- dendritic cell (1)
- dendritic growth (1)
- dendritic spines (1)
- dentate gyrus (1)
- detergents (1)
- deubiquitinase (1)
- developmental cycle (1)
- diabetes (1)
- diacylglycerol (DAG) (1)
- diet (1)
- dimeric peptide (1)
- dimerization (1)
- direct muss spectrometric profiling (1)
- directionality (1)
- disulfide bonds (1)
- dogs (1)
- domain swapping (1)
- dominant (1)
- dopa-induced dyskinesia (1)
- dopamine (1)
- dopamine transporters (1)
- dopaminergics (1)
- double knockout mice (1)
- drosophila melanogaster (1)
- drug development (1)
- drug repurposing (1)
- drug target (1)
- dunce (1)
- ectodomain cleavage (1)
- electrohydrodynamics (1)
- electron cryo microscopy (1)
- electron cryo-microscopy (1)
- electron microscopy (1)
- elongation rate (1)
- embryos (1)
- end-stage renal failure (1)
- endocannabinoid release (1)
- endothelial cell (1)
- endothelial cell interactions (1)
- endothelial growth-factor (1)
- enoyl reductase (1)
- enoyl-ACP reductase (1)
- enoyl-ACP reductase inhibitors (1)
- envelopment (1)
- enzyme (1)
- enzyme activator (1)
- enzyme mechanism (1)
- enzyme mechanisms (1)
- epithelial-mesenchymal transition (1)
- epitope (1)
- essential genes (1)
- exchange factor collybistin (1)
- experimental autoimmune encephalomyelitis (1)
- extracellular vesicle (1)
- eye movements (1)
- eyes (1)
- factor Sox10 (1)
- factor-I (1)
- fat absorption (1)
- fatty acid biosynthesis (1)
- fatty acid synthesis (1)
- fentanyl (1)
- ferroptosis (1)
- fetal liver (1)
- fibrin (1)
- fibrinogen (1)
- field flow fractionation (1)
- fine-mapping (1)
- flippase (1)
- flotillin (1)
- fluorescence (1)
- fluorescence lifetime imaging microscopy (1)
- fluorescence resonance energy transfer (1)
- fluorescence-detected sedimentation (1)
- fluorescent probes (1)
- fluorescent protein (1)
- fosmidomycin (1)
- fostamatinib (1)
- fractalkine (1)
- fragment screening (1)
- friut fly behaviour (1)
- fusion and fission (1)
- gamma-aminobutyric-acid (1)
- gastric cancer (1)
- gel electrophoresis (1)
- gene (1)
- gene therapy (1)
- general transcription factor IIH (TFIIH) (1)
- genetic code expansion (1)
- genome integrity (1)
- genomic stability (1)
- germline mutation (1)
- glutamate (1)
- glutamate receptor (1)
- glv-1h68 (1)
- glycine (1)
- glycine receptor (GlyR) (1)
- glycine transporter 2 (1)
- glycolytic flux control (1)
- glycoprotein GPV (1)
- glycoprotein Ib (1)
- glycoprotein Ibα (1)
- glycoprotein receptor Ib (1)
- glycoprotein receptor Ibα (1)
- glycoprotein-shedding (1)
- glycosylation (1)
- glyvine uptake (1)
- gram negative bacteria (1)
- gram-negative bacteria (1)
- granule cells (1)
- growth regulator (1)
- guanine nucleotide exchange factor (GEF) (1)
- gut microbiome (1)
- hTERT (1)
- haemostasis (1)
- haloacid dehalogenase (1)
- helicase (1)
- hematopoiesis (1)
- hemodialysis (1)
- hemostasis (1)
- hemostasis, (1)
- hepatitis B core protein (1)
- hepatitis B virus (1)
- hippocampus (1)
- histologic diversity (1)
- histology (1)
- homeostasisIon channels (1)
- homoFRET (1)
- homology modeling (1)
- hormone receptors (1)
- human cells (1)
- human interleukin-4 (1)
- human sodium iodide symporter (hNIS) (1)
- humanized tumor (1)
- humans (1)
- hybrid imaging (1)
- hybridomas (1)
- hypercholeterolemia (1)
- hyperekplexia (1)
- hyperlipedemic mice (1)
- hypertonic solution (1)
- identification (1)
- image analysis (1)
- imaging and sensing (1)
- imiquimod (1)
- immune cell function (1)
- immune cell recruitment (1)
- immune evasion (1)
- immunohistochemistry techniques (1)
- immunological synapse (1)
- immunoprecipitation (1)
- in vitro (1)
- in vitro and in vivo thrombus formation (1)
- in vivo (1)
- in vivo Modelle (1)
- in vivo cell tracking (1)
- in vivo imaging (1)
- in vivo models (1)
- in-vitro (1)
- in-vitro propagation (1)
- in-vivo (1)
- inactivation (1)
- increases atherosclersosis (1)
- indoleamine 2,3-dioxygenase (1)
- indoxyl sulfate (1)
- inflammatory sites (1)
- inhibition (1)
- inhibitor residence time (1)
- inhibitors (1)
- inhibitory synapse (1)
- innate immune response (1)
- insect flight (1)
- insulin (1)
- intact bone imaging (1)
- integrated stress response (1)
- integrin α2 (1)
- interleukin-6-type cytokines (1)
- internalization (1)
- interspecies comparison (1)
- intestine (1)
- intoxication (1)
- intracranial bleeding (1)
- intrahelical lesion (1)
- intrinsic metabolism (1)
- invasion (1)
- ion channels in the nervous system (1)
- ionic selectivity (1)
- iota-toxin (1)
- kidneys (1)
- kinase Syk (1)
- kinease (1)
- kinetics (1)
- knockout (1)
- laminin receptor (1)
- latency (1)
- learning (1)
- lesion formation (1)
- leukemia-inhibitory factor (1)
- leukocyte adhesion (1)
- ligand potencies (1)
- ligases (1)
- light pulses (1)
- light-induced interstrand DNA crosslinking (1)
- lightsheet microscopy (1)
- lines (1)
- lipid asymmetry (1)
- lipid bilayer membranes (1)
- lipidomics (1)
- listeriolysin O (1)
- liver (1)
- localization (1)
- locomotion (1)
- long-term depression (1)
- long-term potentation (1)
- loop B (1)
- loss of function (1)
- low-secretion phenotype mutants (1)
- lung and intrathoracic tumors (1)
- lymph node stromal cells (1)
- lymph node transplantation (1)
- lymph nodes (1)
- lymphocyte homing (1)
- lymphoma (1)
- lysosomal degradation (1)
- mRNA (1)
- magnetic resonance imaging (1)
- marcophages (1)
- mass spectrometry (1)
- mast cells (1)
- mastectomy (1)
- mastocytosis (1)
- mathematical modeling (1)
- matrix metalloproteinase (1)
- maturation signal (1)
- maus (1)
- mechanotransduction (1)
- medicine (1)
- medium spiny neurons (1)
- megakaryopoiesis (1)
- melanoma malignancy (1)
- melt electrowriting (1)
- membrane interaction (1)
- mesenteric lymph node (1)
- metabolism (1)
- metabotropic signalling (1)
- metalloproteinase (1)
- metals (1)
- metastases (1)
- miR-17~92 (1)
- miR-30 (1)
- miRNA (1)
- miRNA expression (1)
- miRNA processing (1)
- microenvironment (1)
- microglia (1)
- microscopy (1)
- microtubules (1)
- microvascular complications (1)
- microvascular density (1)
- microvasculature (1)
- midbody (1)
- migration (1)
- mismatch recognition (1)
- mismatches (1)
- mitochondria (1)
- mitochondrial transport (1)
- mitogenic signaling (1)
- mitosis (1)
- mobilization (1)
- model (1)
- modulation of virus replication (1)
- molecular basis (1)
- molecular mass (1)
- molecular mobility (1)
- molecular structure (1)
- molybdenum cofactor biosynthesis (1)
- monoclonal antibodies (1)
- monocyte (1)
- monocytes (1)
- monovalent cation:proton antiporter-2 (CPA2) family (1)
- morphogenesis (1)
- motion (1)
- motoneuron (1)
- movement disorders (1)
- mrsk2 KO mouse (1)
- multi-drug-resistance (1)
- multiphoton microscopy (1)
- multiple myeloma (1)
- multiple sclerosis (1)
- murine cytomegalovirus (1)
- muscle (1)
- mutant p53 (1)
- mutation screening (1)
- mutation triggers (1)
- mutations (1)
- mycobacterium smegmatis (1)
- mycolic acid (1)
- myogenic differentiation (1)
- myomesin (1)
- nanoelectronics (1)
- nanolithography (1)
- nanomedicine (1)
- nanorobot (1)
- nanotechnology (1)
- naphthylisoquinoline alkaloids (1)
- neddylation (1)
- neocortex (1)
- network (1)
- neural networks (1)
- neurite outgrowth (1)
- neuroblastoma cell (1)
- neurologin-2 (1)
- neurology (1)
- neuromuscular junction (1)
- neuronal differentiation (1)
- neuropil (1)
- neurotransmitters (1)
- neurotrophic factor (1)
- neutralization (1)
- neutrophil (1)
- neutrophils (1)
- nilotinib (1)
- nitric oxide synthase (1)
- non-apoptotic programmed cell death (1)
- non-canonical autophagy (1)
- noncooperative binding (1)
- nuclear envelope (1)
- nuclear export (1)
- nuclear receptors (1)
- obesity (1)
- object segmentation (1)
- obscurin (1)
- octopamine (1)
- oncolytic viral therapy (1)
- oncolytic virotherapy (1)
- opioid peptides (1)
- opioid receptors (1)
- outer membrane proteins (1)
- oxazolone colitis (1)
- oxidation (1)
- oxidative stress (1)
- p-cresyl sulfate (1)
- p53-dependent apoptosis (1)
- p53-inducible regulator (1)
- pain behavior (1)
- pancreas (1)
- parathyroid hormone 1 receptor (1)
- pathway analysis (1)
- peptide (1)
- peptide inhibitor design (1)
- peptide inhibitor of envelopment (1)
- peptide microarray (1)
- peptides (1)
- peptidomoics (1)
- peripheral nerve (1)
- peripheral nervous system (1)
- permeability (1)
- pet dogs (1)
- phagolysosome tubulation (1)
- phosphatidic acid (1)
- phosphatidylethanolamine (1)
- phosphatidylserine (1)
- phosphoglycolate phosphatase (1)
- phospholipase C gamma 2 (1)
- phosphorylation sites (1)
- photo-cross-linking (1)
- photoconversion (1)
- pkd (1)
- planar cell polarity (1)
- plasma membrane (1)
- plasticity (1)
- platelet adhesion (1)
- platelet degranulation (1)
- platelet factor 4 (1)
- platelet inhibition (1)
- platelet receptor (1)
- platelet-neutrophil complexes (PNCs) (1)
- pneumolysin (1)
- pocket factor (1)
- podoplanin (1)
- polar body (1)
- polarization (1)
- popliteal aneurysm (1)
- pore formation (1)
- pore-forming toxin (1)
- post-translational modification (1)
- post-translational modifications (1)
- potassium homeostasis (1)
- precursor cells (1)
- presynaptic hyperekplexia (1)
- processivity (1)
- progressive encephalitis with rigidity and myoclonus (PERM) (1)
- proliferating cell nuclear antigen (PCNA) (1)
- promoter affinity (1)
- propionic acid (1)
- proteasome inhibitor resistance (1)
- proteasome subunit beta type-5 (PSMB5) (1)
- proteasome system (1)
- protective antigen (1)
- protein NEDD8 (1)
- protein RSK2 (1)
- protein disulfide isomerase (1)
- protein folding (1)
- protein kinase (1)
- protein kinase C (1)
- protein kinase D1 (1)
- protein kinase D2/PKD2/PRKD2 (1)
- protein processing (1)
- protein structures (1)
- protein synthesis shut down (1)
- protein transport (1)
- protein ubiquitination (1)
- protein-DNA interaction (1)
- protein-DNA interactions (1)
- protein-protein interaction (1)
- protein-protein interaction (PPI) (1)
- proteins (1)
- protein–protein interaction (1)
- proteome (1)
- proteomics (1)
- protochlamydia amoebophila (1)
- protoplasts (1)
- pseudomonas aeruginosa (1)
- psoriasis (1)
- puberty (1)
- purification (1)
- pyridoxal phosphatase (1)
- pyridoxal phosphatase (PDXP) (1)
- radiation-therapy (1)
- radii (1)
- radioiodine therapy (1)
- rat spinal-cord (1)
- reaction mechanisms (1)
- receptor (1)
- receptor clustering (1)
- receptor signaling (1)
- receptor-beta subunits (1)
- recombinant tissue-type plasminogen activator (1)
- recruitment (1)
- regulation (1)
- regulatory T cells (1)
- regulatory circuit downstream (1)
- renal cancer (1)
- repair (1)
- repair and replication (1)
- replication (1)
- replication fork (1)
- resolvin (1)
- responses (1)
- restoration (1)
- restricition enzymes (1)
- rhabdomyosarcoma (1)
- rhabdomyosarcoma development (1)
- scaffold protein (1)
- scaffolding protein gephyryrin (1)
- screening (1)
- secondary lung tumors (1)
- secreted factors (1)
- selective channel (1)
- seminoma (1)
- senescence (1)
- sensitive amine oxidase (1)
- sensory cues (1)
- sensory physiology (1)
- serotonin (1)
- serum 25-hydroxyvitamin D (1)
- sex addiction (1)
- shedding (1)
- side chain properties (1)
- signal peptides (1)
- signal transduction (1)
- signal transduction pathway (1)
- signaling microdomain (1)
- signaling network (1)
- signalling pathways (1)
- silver staining (1)
- single channel analysis (1)
- single channel recording (1)
- single molecule (1)
- single strand blocking (1)
- site (1)
- size exclusion chromatography (1)
- skin autofluorescence (1)
- sleeping sickness (1)
- sliding (1)
- sodium-iodide symporter (1)
- sorafenib (1)
- spatiotemporal thrombus (1)
- species specificity (1)
- spinal-cord (1)
- spinco ultracentrifuge (1)
- splenic function (1)
- stage-i (1)
- startle (1)
- stem cells (1)
- stiff-person syndrome (SPS) (1)
- stomach (1)
- storage-pool diseases (1)
- store-operated calcium entry (1)
- stranded DNAs (1)
- stress response (1)
- structural biology (1)
- structural disruption (1)
- structural mechanism (1)
- structure-activity relationships (1)
- structure-based drug design (1)
- subcutaneous human tumors (1)
- subpopulation (1)
- subsets (1)
- substrate recognition (1)
- substrate specificity (1)
- subthreshold IKK activation (1)
- subunit (1)
- sulfates (1)
- sulfides (1)
- sulfites (1)
- super resolution microscopy (1)
- super-resolution microscopy (1)
- super-resolution microscopy (SRM) (1)
- surgery (1)
- swiss model (1)
- synapse (1)
- synapse formation (1)
- synaptic development (1)
- system (1)
- systemic micro-inflammation oxidative stress (1)
- tMCAO (1)
- telomerase (1)
- temporal range (1)
- temporal-lobe epilepsy (1)
- tetraspanin (1)
- thiol starvation (1)
- thiosulfates (1)
- thromboxane (1)
- thymine (1)
- thyroid-cancer (1)
- time-correlated single photon counting (TCSPC) (1)
- time-resolved anisotropy (1)
- titin (1)
- torque (1)
- torque meter (1)
- toxin (1)
- toxins (1)
- trafficking (1)
- trans-Golgi network (1)
- transactivation (1)
- transcription (1)
- transcription factors (1)
- transcriptome (1)
- transient middle cerebral artery occlusion (1)
- transient receptor potential channels (1)
- translesion synthesis (1)
- transmission (1)
- trypanosomes (1)
- tuberculosis (1)
- tumor microenvironment (1)
- tumour-necrosis factors (1)
- type 1 diabetes (1)
- type I interferon (1)
- tyrosine-protein kinase (1)
- ubiquitin ligase (1)
- ubiquitylation (ubiquitination) (1)
- ultrastructure (1)
- undamaged DNA (1)
- uracil binding (1)
- variocosities (1)
- vascular adhesion protein-1 (1)
- vascular system (1)
- vaskuläre glatte Muskelzelle (1)
- vasp (1)
- velocity (1)
- vibration (1)
- virotherapy (1)
- virus reactivation (1)
- walking (1)
- x-ray crystallography (1)
- xeroderma-pigmentosum (1)
- µ-Opioid receptor (1)
- Übergewicht (1)
- β cell (1)
- β1-adrenoceptor/β1-adrenergic receptor (1)
- β3 adrenergic receptor (1)
- β3 adrenergic receptor (ADRB3) (1)
Institute
- Rudolf-Virchow-Zentrum (299)
- Graduate School of Life Sciences (70)
- Institut für Experimentelle Biomedizin (30)
- Theodor-Boveri-Institut für Biowissenschaften (30)
- Institut für Pharmakologie und Toxikologie (21)
- Neurologische Klinik und Poliklinik (16)
- Institut für Molekulare Infektionsbiologie (13)
- Lehrstuhl für Biochemie (12)
- Medizinische Klinik und Poliklinik II (12)
- Comprehensive Cancer Center Mainfranken (8)
Sonstige beteiligte Institutionen
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Center for Nanosystems Chemistry (CNC), University of Würzburg (1)
- Eberhard Karls Universität Tübingen (1)
- Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, USA (1)
- MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V., Am Hubland, D-97074 Würzburg (1)
- Research Center for Infectious Diseases, University of Wuerzburg, Wuerzburg 97080, Germany (1)
- Rudolf-Virchow-Zentrum für Experimentelle Biomedizin der Universität Würzburg (1)
Introduction
Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex.
Methods
Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45–60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100–200 μm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis.
Results
Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging.
Conclusions
Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury.
Background
Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options.
Methods
In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma.
Results
We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment.
Conclusions
Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.
Pro-migratory signals mediated by the tumor microenvironment contribute to the cancer progression cascade, including invasion, metastasis and resistance to therapy. Derived from in vitro studies, isolated molecular steps of cancer invasion programs have been identified but their integration into the tumor microenvironment and suitability as molecular targets remain elusive. The purpose of the study was to visualize central aspects of tumor progression, including proliferation, survival and invasion by real-time intravital microscopy. The specific aims were to monitor the kinetics, mode, adhesion and chemoattraction mechanisms of tumor cell invasion, the involved guidance structures, and the response of invasion zones to anti-cancer therapy. To reach deeper tumor regions by optical imaging with subcellular resolution, near-infrared and infrared excited multiphoton microscopy was combined with a modified dorsal skinfold chamber model. Implanted HT-1080 fibrosarcoma and B16/F10 and MV3 melanoma tumors developed zones of invasive growth consisting of collective invasion strands that retained cell-cell contacts and high mitotic activity while invading at velocities of up to 200 μm per day. Collective invasion occurred predominantly along preexisting tissue structures, including blood and lymph vessels, collagen fibers and muscle strands of the deep dermis, and was thereby insensitive to RNAi based knockdown and/or antibody-based treatment against β1 and β3 integrins, chemokine (SDF-1/CXCL12) and growth factor (EGF) signaling. Therapeutic hypofractionated irradiation induced partial to complete regression of the tumor main mass, yet failed to eradicate the collective invasion strands, suggesting a microenvironmentally privileged niche. Whereas no radiosensitization was achieved by interference with EGFR or doxorubicin, the simultaneous inhibition of β1 and β3 integrins impaired cell proliferation and survival in spontaneously growing tumors and strongly enhanced the radiation response up to complete eradication of both main tumor and invasion strands. In conclusion, collective invasion in vivo is a robust process which follows preexisting tissue structures and is mainly independent of established adhesion and chemoattractant signaling. Due to its altered biological response to irradiation, collective invasion strands represent a microenvironmentally controlled and clinically relevant resistance niche to therapy. Therefore supportive regimens, such as anoikisinduction by anti-integrin therapy, may serve to enhance radio- and chemoefficacy and complement classical treatment regimens.
Thrombus formation at sites of vascular lesions is a dynamic process that requires a defined series of molecular events including the action of platelet adhesion/activation receptors, intracellular signal transduction, cytoskeletal rearrangements and activation of plasma coagulation factors. This process is essential to limit post-traumatic blood loss but may also contribute to acute thrombotic diseases such as myocardial infarction and stroke. With the help of genetically modified mice and the use of specific protein inhibitors and receptordepleting antibodies, the work presented in this thesis identified novel mechanisms underlying thrombus formation in hemostasis and thrombosis. In the first part of the study, it was shown that von Willebrand Factor (vWF) binding to glycoprotein (GP)Iba is critical for the formation of stable pathological thrombi at high shear rates, suggesting GPIba as an attractive pharmacological target for antithrombotic therapy. The subsequent analysis of recently generated phospholipase (PL)D1-deficient mice identified this enzyme, whose role in platelet function had been largely unknown, as a potential target protein downstream of GPIba. This was based on the finding that PLD1- deficient mice displayed severely defective GPIba-dependent thrombus stabilization under high shear conditions in vitro and in vivo without affecting normal hemostasis. The second part of the thesis characterizes the functional relevance of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing collagen receptor GPVI and the recently identified hemITAM-coupled C-type lectin-like receptor 2 (CLEC-2) for in vivo thrombus formation. Genetic- and antibody-induced GPVI deficiency was found to similarly protect mice from arterial vessel occlusion in three different thrombosis models. These results confirmed GPVI as a promising antithrombotic target and revealed that antibody-treatment had no obvious off-target effects on platelet function. Similarly, immunodepletion of CLEC-2 by treating mice with the specific antibody INU1 resulted in markedly impaired thrombus growth and stabilization under flow in vitro and in vivo. Furthermore, it could be demonstrated that double-immunodepletion of GPVI and CLEC-2 resulted in severely decreased arterial thrombus formation accompanied by dramatically prolonged bleeding times. These data revealed an unexpected redundant function of the two receptors for in vivo thrombus formation and might have important implications for the potential development of anti-GPVI and anti-CLEC-2 antithrombotic agents. The third part of the thesis provides the first functional analysis of megakaryocyte- and platelet-specific RhoA knockout mice. RhoA-deficient mice displayed a defined signaling defect in platelet activation, leading to a profound protection from arterial thrombosis andand ischemic brain infarction, but at the same time also strongly increased bleeding times. These findings identified the GTPase as an important player for thrombus formation in hemostasis and thrombosis. Based on the previous proposal that the coagulation factor (F)XII might represent an ideal target for safe antithrombotic therapy without causing bleeding side effects, the last part of this thesis assesses the antithrombotic potential of the newly generated FXIIa inhibitor rHAInfestin- 4. It was found that rHA-Infestin-4 injection into mice resulted in virtually abolished arterial thrombus formation but no change in bleeding times. Moreover, rHA-Infestin-4 was similarly efficient in a murine model of ischemic stroke, suggesting that the inhibitor might be a promising agent for effective and safe therapy of cardio- and cerebrovascular diseases.
Interleukin-6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) are members of the IL-6-type cytokine family that is characterised by sharing the common receptor subunit gp130. While the involvement of these polypeptides in cell differentiation, cell survival, proliferation, apoptosis, inflammation, haematopoiesis, immune response and acute phase reaction has already been demonstrated, the description of their role in development and progression of cardiac hypertrophy is still rather limited. A model has been postulated that declares the transient expression of IL-6-type cytokines as protective, while a continuous cardiac secretion of these proteins seems to be rather harmful for the heart. Within the first part of the study (results 4.1, 4.2 and 4.3) it was shown that OSM induces hypertrophy of primary neonatal rat cardiomyocytes (NRCM), just as its related cytokines LIF, CT-1 and hIL-6/hsIL-6R (hsIL-6R, human soluble IL-6 receptor). Regarding the hypertrophic potentials the LIFR/gp130 utilising cytokines (hLIF, hOSM and hCT-1) are stronger inducers than the OSMR/gp130 utilising mOSM. Human IL-6/hsIL-6R which signals via a gp130 homodimer has the weakest hypertrophic effect. The thorough analysis of typical signalling pathways initiated by IL-6-type cytokines revealed that STAT3 phosphorylation at Y705 seems to be the most important hypertrophy promoting pathway. In addition and in contrast to published work, we clearly demonstrate that classical IL-6 signalling (upon pure IL-6 treatment) has no hypertrophic effect on cardiomyocytes, because they lack sufficient amounts of the membrane-bound IL-6R. This is also true for neonatal rat cardiac fibroblasts (NRCFB). Since these cells can also influence cardiac hypertrophy, signalling pathways and target genes were additionally examined in NRCFB in response to OSM, LIF and IL-6/sIL-6R. One of the key findings of this thesis is the selective change in expression of cytokines and receptors of the IL-6 family in both cell types upon IL-6-type cytokine stimulation. A striking difference between NRCM and NRCFB is the fact that the target gene induction in NRCM is of similar duration upon mOSM and hIL-6/hsIL-6R treatment, while hIL-6/hsIL-6R is capable of promoting the induction of OSMR and IL-6 significantly longer in NRCFB. By searching for transcription factors or intermediate cytokines which could be responsible for this difference, a strong correlation between increased Il6 transcription and amount of mRNA levels for C/EBPβ and C/EBPδ was observed in response to IL-6/sIL-6R stimulation. Interestingly, mOSM also mediates the induction of C/EBPβ and δ, but the initiation is significantly less efficient than in response to IL-6/sIL-6R. Therefore, we assume that mOSM stimulation fails to reach threshold values required for a prolonged IL-6 secretion. Since we additionally observe a slight IL-6R mRNA upregulation in NRCFB, we assume that the combination of IL-6, LIF, C/EBPβ, C/EBPδ and IL-6R expression might be responsible for the observed different kinetics with which IL-6 and OSM stimulate NRCFB. In addition to the aforementioned proteins, members of the renin-angiotensin system seem to support the IL-6-type cytokine mediated hypertrophy. Since it has already been shown that angiotensin II vice versa induces IL-6 expression in NRCM and NRCFB, this enhanced expression of AT1α and ACE could be of crucial interest for the hypertrophy supporting phenotype. The second part of the presented work dealt with the characterisation of the receptor complexes of rat OSM. The central question of this analysis was, whether rOSM, just like mOSM, only binds the type II (OSMR/gp130) receptor complex or is able to utilise the type II and type I (LIFR/gp130) receptor complex. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant, and generation of stably transfected Ba/F3 cells expressing the newly cloned rat OSMR/gp130 or LIFR/gp130 receptor complex) we can clearly show that rat OSM surprisingly utilises both, the type I and type II receptor complex. Therefore it closely mimics the human situation. Furthermore, rOSM displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the JAK/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, the results obtained in the last section of this thesis clearly suggest that rat disease models would allow evaluation of the relevance of OSM for human biology much better than murine models.
Large-scale anatomical and functional analyses of the connectivity in both invertebrate and mammalian brains have gained intense attention in recent years. At the same time, the understanding of synapses on a molecular level still lacks behind. We have only begun to unravel the basic mechanisms of how the most important synaptic proteins regulate release and reception of neurotransmitter molecules, as well as changes of synaptic strength. Furthermore, little is known regarding the stoichiometry of presynaptic proteins at different synapses within an organism. An assessment of these characteristics would certainly promote our comprehension of the properties of different synapse types. Presynaptic proteins directly influence, for example, the probability of neurotransmitter release as well as mechanisms for short-term plasticity. We have examined the strength of expression of several presynaptic proteins at different synapse types in the central nervous system of Drosophila melanogaster using immunohistochemistry. Clear differences in the relative abundances of the proteins were obvious on different levels: variations in staining intensities appeared from the neuropil to the synaptic level. In order to quantify these differences, we have developed a ratiometric analysis of antibody stainings. By application of this ratiometric method, we could assign average ratios of presynaptic proteins to different synapse populations in two central relays of the olfactory pathway. In this manner, synapse types could be characterized by distinct fingerprints of presynaptic protein ratios. Subsequently, we used the method for the analysis of aberrant situations: we reduced levels of Bruchpilot, a major presynaptic protein, and ablated different synapse or cell types. Evoked changes of ratio fingerprints were proportional to the modifications we had induced in the system. Thus, such ratio signatures are well suited for the characterization of synapses. In order to contribute to our understanding of both the molecular composition and the function of synapses, we also characterized a novel synaptic protein. This protein, Drep-2, is a member of the Dff family of regulators of apoptosis. We generated drep-2 mutants, which did not show an obvious misregulation of apoptosis. By contrast, Drep-2 was found to be a neuronal protein, highly enriched for example at postsynaptic receptor fields of the input synapses of the major learning centre of insects, the mushroom bodies. Flies mutant for drep-2 were viable but lived shorter than wildtypes. Basic synaptic transmission at both peripheral and central synapses was in normal ranges. However, drep-2 mutants showed a number of deficiencies in adaptive behaviours: adult flies were locomotor hyperactive and hypersensitive towards ethanol-induced sedation. Moreover, the mutant animals were heavily impaired in associative learning. In aversive olfactory conditioning, drep-2 mutants formed neither short-term nor anaesthesia-sensitive memories. We could demonstrate that Drep-2 is required in mushroom body intrinsic neurons for normal olfactory learning. Furthermore, odour-evoked calcium transients in these neurons, a prerequisite for learning, were reduced in drep-2 mutants. The impairment of the mutants in olfactory learning could be fully rescued by pharmacological application of an agonist to metabotropic glutamate receptors (mGluRs). Quantitative mass spectrometry of Drep-2 complexes revealed that the protein is associated with a large number of translational repressors, among them the fragile X mental retardation protein FMRP. FMRP inhibits mGluR-mediated protein synthesis. Lack of this protein causes the fragile X syndrome, which constitutes the most frequent monogenic cause of autism. Examination of the performance of drep-2 mutants in courtship conditioning showed that the animals were deficient in both short- and long-term memory. Drep-2 mutants share these phenotypes with fmrp and mGluR mutants. Interestingly, drep-2; fmrp double mutants exhibited normal memory. Thus, we propose a model in which Drep-2 antagonizes FMRP in the regulation of mGluR-dependent protein synthesis. Our hypothesis is supported by the observation that impairments in synaptic plasticity can arise if mGluR signalling is imbalanced in either direction. We suggest that Drep-2 helps in establishing this balance.
Unter Verwendung fluoreszenzmarkierter Remorine der taxonomischen Gruppe 1b wurden Nanodomänen in Arabidopsis Plasmamembranen (PM) unter Verwendung hoch auflösender Laser Scanning-Systeme sichtbar gemacht. In diesen kompartimentierten Membranbereichen lagerten sich Sterol-abhängige Remorine aus verschiedenen Pflanzen-familien zusammen und zeigten dort Kolokalisation. Dies wurde statistisch belegt durch hohe Pearson und Spearman Korrelationskoeffizienten. Remorine konnten schließlich als pflanzliche Markerproteine für kompartimentierte Membranbereiche etabliert werden. Die Nanodomänen zeigten zu keinem Zeitpunkt laterale Bewegungen in der PM und scheinen sowohl von zytoskelettären Strukturen als auch von Komponenten der Zellwand stabilisiert zu werden. Möglicherweise spielen transmembrane Tetraspanine sowie GPI-verankerte SKU5-Proteine eine Rolle bei der stabilen Verankerung. Für zwei native Arabidopsis Remorine wurden posttranslationale Modifikationsstellen aufgedeckt, die der Anheftung dieser hydrophilen Proteine an die PM dienen. Weiterhin scheinen gleichartige Remorine miteinander zu interagieren. Beispielsweise waren im Zytosol lokalisierte Remorin-Mutanten bei einer gleichzeitigen Expression der entsprechenden Vollängenproteine erneut an der PM zu finden. Für die Remorine wurde postuliert, dass sie mit anderen Proteinen interagieren und dabei makromolekulare Strukturen ausbilden. Den Remorinen könnte daher eine Aufgabe bei der molekularen Organisation pflanzlicher Membrandomänen zukommen, indem sie ein filamentartiges Netzwerk innerhalb distinkter Domänen ausbilden, das möglicherweise zur Stabilität und Aufrechterhaltung dieser spezialisierten Bereiche beiträgt. Unter Einbeziehung der STED-Mikroskopie wurde eine empirische Größenverteilung von 97±4nm Durchmesser für PM-ständige Domänen in Arabidopsis ermittelt. Hinsichtlich der physiologischen Relevanz konnte gezeigt werden, dass die Domänen eine Rolle bei der ABA-vermittelten, kalziumabhängigen Regulation des Anionenkanals SLAH3 einnehmen. SLAH3 wird durch kalziumabhängige Kinasen aus der CDPK-Familie aktiviert, im Speziellen durch CPK21 und CPK23. Beide Kinasen werden durch die ABA-sensitiven Phosphatasen ABI1 und ABI2 reguliert. Die spezifisch stattfindenden Interaktionen zwischen SLAH3 und CPK21, sowie zwischen CPK21 und ABI1 waren auf Nanodomänen beschränkt und wurden durch die Methodik der bimolekularen Fluoreszenzkomplementation erstmals in planta nachgewiesen, mit Remorinen der taxonomischen Gruppe 1b als etablierte Markerproteine für Membrandomänen.
Investigation on Distinct Roles of Smad Proteins in Mediating Bone Morphogenetic Proteins Signals
(2011)
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily and play important roles in numerous biological events in the development of almost all multi-cellular organisms. Dysregulated BMP signaling is the underlying causes of numerous heritable and non-heritable human diseases including cancer. The vast range of biological responses induced by BMPs converges on three closely related Smad proteins that convey intracellular signals from BMP receptors to the nucleus. The specificity of BMP signaling has been intensively investigated at the level of ligand-receptor interactions, but how the different Smad proteins contribute to differential signals elicited by BMPs remains unclear. In this work, we investigated the BMP/Smad signaling in different aspects. In search for an appropriate fluorescence reporter in zebrafish, we compared different photo-switchable proteins and found EosFP the best candidate this model system for its fast maturation and fluorescence intensity. We modified and created appropriate vectors enabling Tol2-transposon based trangenesis in zebrafish, with which transgenic zebrafish lines were generated. We combined fluorescence protein tagging with high resolution microscopy and investigate the dynamics of Smad proteins in model system zebrafish. We observed that Smad5 undergoes nucleo-translocation as BMP signal transmitter during zebrafish gastrulation. We explored the Smad involvement during myogenic-to-osteogenic conversion of C2C12 cell line induced by BMP4. We created transient loss-of-function of Smads by siRNA-mediated knockdowns and analyzed the effects on these coupled yet distinct procedures by quantitative real-time PCR and terminal marker staining. We found that different Smad-complex stoichiometry might be responsible for distinct cellular signals elicited by BMPs.
Platelet activation and adhesion results in thrombus formation that is essential for normal hemostasis, but can also cause irreversible vessel occlusion leading to myocardial infarction or stroke. The C-type lectin-like receptor 2 (CLEC-2) was recently identified to be expressed on the platelet surface, however, a role for this receptor in hemostasis and thrombosis had not been demonstrated. In the current study, the involvement of CLEC-2 in platelet function and thrombus formation was investigated using mice as a model system. In the first part of the thesis, it was found that treatment of mice with a newly generated monoclonal antibody against murine CLEC-2 (INU1) led to the complete and highly specific loss of the receptor in circulating platelets (a process termed “immunodepletion”). CLEC-2-deficient platelets were completely unresponsive to the CLEC-2-specific agonist rhodocytin, whereas activation induced by all other tested agonists was unaltered. This selective defect translated into severely decreased platelet aggregate formation under flow ex vivo; and in vivo thrombosis models revealed impaired stabilization of formed thrombi with enhanced embolization. Consequently, CLEC-2 deficiency profoundly protected mice from occlusive arterial thrombus formation. Furthermore, variable bleeding times in INU1-treated mice indicated a moderate hemostatic defect. This reveals for the first time that CLEC-2 significantly contributes to thrombus stability in vitro and in vivo and plays a crucial role in hemostasis and arterial thrombosis. Thus, CLEC-2 represents a potential novel anti-thrombotic target that can be functionally inactivated in vivo. This in vivo down-regulation of platelet surface receptors might be a promising approach for future anti-thrombotic therapy. The second part of the work investigated the effect of double-immunodepletion of the immunoreceptor tyrosine-based activation motif (ITAM)- and hemITAM-coupled receptors, platelet glycoprotein (GP) VI and CLEC-2, on hemostasis and thrombosis using a combination of the GPVI- and CLEC-2-specific antibodies, JAQ1 and INU1, respectively. Isolated targeting of either GPVI or CLEC-2 in vivo did not affect expression or function of the respective other receptor. However, simultaneous treatment with both antibodies resulted in the sustained loss of GPVI and CLEC-2 signaling in platelets, while leaving other activation pathways intact. In contrast to single deficiency of either receptor, GPVI/CLEC-2 double-deficient mice displayed a dramatic hemostatic defect. Furthermore, this treatment resulted in profound impairment of arterial thrombus formation that far exceeded the effects seen in single-depleted animals. Importantly, similar results were obtained in Gp6-/- mice that were depleted of CLEC-2 by INU1-treatment, demonstrating that this severe bleeding phenotype was not caused by secondary effects of combined antibody treatment. These data suggest that GPVI and CLEC-2 can be independently or simultaneously down-regulated in platelets in vivo and reveal an unexpected functional redundancy of the two receptors in hemostasis and thrombosis. Since GPVI and CLEC-2 have intensively been discussed as potential anti-thrombotic targets, these results may have important implications for the development of novel, yet save anti-GPVI or anti-CLEC-2-based therapies.
Evaluation of a pathophysiological role of the interleukin-6-type cytokine oncostatin M (OSM) for human diseases has been complicated by the fact that mouse models of diseases targeting either OSM or the OSM receptor (OSMR) complex cannot fully reflect the human situation. This is due to earlier findings that human OSM utilizes two receptor complexes, glycoprotein 130 (gp130)/leukemia inhibitory factor receptor (LIFR) (type I) and gp130/OSMR (type II), both with wide expression profiles. Murine OSM on the other hand only binds to the gp130/OSMR (type II) receptor complex with high affinity. Here, we characterize the receptor usage for rat OSM. Using different experimental approaches (knock-down of the OSMR expression by RNA interference, blocking of the LIFR by LIF-05, an antagonistic LIF variant and stably transfected Ba/F3 cells) we can clearly show that rat OSM surprisingly utilizes both, the type I and type II receptor complex, therefore mimicking the human situation. Furthermore, it displays cross-species activities and stimulates cells of human as well as murine origin. Its signaling capacities closely mimic those of human OSM in cell types of different origin in the way that strong activation of the Jak/STAT, the MAP kinase as well as the PI3K/Akt pathways can be observed. Therefore, rat disease models would allow evaluation of the relevance of OSM for human biology.
It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His6-tag to different proteins increased their binding affinity to the protective antigen (PA) PA63-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB7/8-family of toxins. His6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to 270 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA63. Our results revealed that addition of His6-tags to several factors increase their binding properties to PA63 and enhance the property to intoxicate cells.
Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.
Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds – covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.
Platelet activation induces cytoskeletal rearrangements involving a change from discoid to spheric shape, secretion, and eventually adhesion and spreading on immobilized ligands. Small GTPases of the Rho family, such as Rac1 and Cdc42, are known to be involved in these processes by facilitating the formation of lamellipodia and filopodia, respectively. This thesis focuses on the role Rac1 and Cdc42 for platelet function and formation from their precursor cells, the megakaryocytes (MKs), using conditional knock-out mice. In the first part of the work, the involvement of Rac1 in the activation of the enzyme phospholipase (PL) C2 in the signaling pathway of the major platelet collagen receptor glycoprotein (GP) VI was investigated. It was found that Rac1 is essential for PLC2 activation independently of tyrosine phosphorylation of the enzyme, resulting in a specific platelet activation defect downstream of GPVI, whereas signaling of other activating receptors remains unaffected. Since Rac1-deficient mice were protected from arterial thrombosis in two different in vivo models, the GTPase might serve as a potential target for the development of new drugs for the treatment and prophylaxis of cardio- and cerebrovascular diseases. The second part of the thesis deals with the first characterization of MK- and platelet-specific Cdc42 knock-out mice. Cdc42-deficient mice displayed mild thrombo-cytopenia and platelet production from mutant MKs was markedly reduced. Unexpectedly, Cdc42-deficient platelets showed increased granule content and release upon activation, leading to accelerated thrombus formation in vitro and in vivo. Furthermore, Cdc42 was not generally required for filopodia formation upon platelet activation. Thus, these results indicate that Cdc42, unlike Rac1, is involved in multiple signaling pathways essential for proper platelet formation and function. Finally, the outcome of combined deletion of Rac1 and Cdc42 was studied. In contrast to single deficiency of either GTPase, platelet production from double-deficient MKs was virtually abrogated, resulting in dramatic macrothrombocytopenia in the animals. Formed platelets were largely non-functional leading to a severe hemostatic defect and defective thrombus formation in double-deficient mice in vivo. These results demonstrate for the first time a functional redundancy of Rac1 and Cdc42 in the hematopoietic system.
XPD is a 5‘-3‘ helicase of the superfamily 2. As part of the transcription factor IIH it functions in transcription initiation and nucleotide excision repair. This work focus on the role of XPD in nucleotide excision repair. NER is a DNA repair pathway unique for its broad substrate range. In placental mammals NER is the only repair mechanism able to remove lesions induced by UV-light. NER can be divided into four different steps that are conserved between pro- and eukaryotes. Step 1 consists of the initial damage recognition, during step 2 the putative damage is verified, in step 3 the verified damage is excised and in the 4th and final step the resulting gap in the DNA is refilled. XPD was shown to be involved in the damage verification step. It was possible to solve the first apo XPD structure by a MAD approach using only the endogenous iron from the iron sulfur cluster. Based on the apo XPD structure several questions arise: where is DNA bound? Where is DNA separated? How is damage verification achieved? What is the role of the FeS cluster? These questions were addressed in this work. Hypothesis driven structure based functional mutagenesis was employed and combined with detailed biochemical characterization of the variants. The variants were analyzed by thermal unfolding studies to exclude the possibility that the overall stability could be affected by the point mutation. DNA binding assays, ATPase assays and helicase assays were performed to delineate amino acid residues important for DNA binding, helicase activity and damage recognition. A structure of XPD containing a four base pair DNA fragment was solved by molecular replacement. This structure displays the polarity of the translocated strand with respect to the helicase framework. Moreover the properties of the FeS cluster were studied by electron paramagnetic resonance to get insights into the role of the FeS cluster. Furthermore XPD from Ferroplasma acidarmanus was investigated since it was shown that it is stalled at CPD containing lesions. The data provide the first detailed insight into the translocation mechanism of a SF2B helicase and reveal how polarity is achieved. This provides a basis for further anlayses understanding the combined action of the helicase and the 4Fe4S cluster to accomplish damage verification within the NER cascade.
Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing Escherichia coli
(2011)
Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes
Type 1 diabetes is an autoimmune disease that leads to the destruction of insulin-producing pancreatic beta cells and consequently to hyperglycemia. In the last 60 years, the prevalence of type 1 diabetes has been increasing constantly and is predicted to continue rising. About 80% of the disease risk is attributable to the genetic variation. Thanks to genome wide association studies the number of known disease-associated polymorphisms climbed from five to 53 in the last 10 years. As these studies reveal possible candidate genes but not underlying mechanisms we strove to take the next step and explore the association of two genes suggested by these studies with type 1 diabetes. As a method of choice we decided to use lentiviral RNAi in non obese diabetic (NOD) mice, a widely-used model for type 1 diabetes, introducing a shRNA directed against the target message into the genome of this mouse strain via a lentivirus. This allowed us to study the partial loss-of-function of the target gene within the context of diabetes, directly seeing its effect on autoimmune mechanisms. In this thesis we examined two different genes in this manner, Ctla4 and Clec16a. A type 1 diabetes associated polymorphism in the CTLA4 gene had been found to alter the splicing ratio of its variants soluble CTLA-4 (sCTLA-4) and full length CTLA-4, the associated allele producing less sCTLA-4 than the protective allele. We mimicked this effect by specifically targeting the sCtla4 mRNA via lentiviral RNAi in the NOD model. As a result we could confirm the reduction of sCTLA-4 to accelerate type 1 diabetes development. Furthermore we could show a function of sCTLA-4 in regulatory T cells, more specifically at least partly in their ability to modulate costimulation by antigen presenting cells. The second candidate gene, Clec16a was targeted with the shRNA in a way that was designed to knock down most splice variants. As the gene function and the effect of the associated SUMMARY 10 polymorphism was unknown, we reasoned this method to be feasible to investigate its role in type 1 diabetes. The knockdown of Clec16a in NOD mice resulted in an almost complete protection from diabetes development that could be attributed to T cells dysfunction. However, as expression patterns and a study of the Drospophila orthologue suggested a possible role of CLEC16A in antigen presentation we also examined antigen presenting cells in the thymus and periphery. Although we did not detect any effect of the knockdown on peripheral antigen presenting cells, thymic epithelial cells were clearly affected by the loss of CLEC16A, rendering them more activated and shifting the ratio of cortical to medullary epithelial cells in favor of cortical cells. We therefore suggest a role of CLEC16A in the selection of T cells, that needs, however, to be further investigated. In this thesis we provided a feasible and fast method to study function of genes and even of single splice variants within the NOD mouse model. We demonstrate its usefulness on two candidate genes associated with type 1 diabetes by confirming and unraveling the cause of their connection to the disease.
PTPN22 encodes the lymphoid tyrosine phosphatase Lyp that can dephosphorylate Lck, ZAP-70 and Fyn to attenuate TCR signaling. A single-nucleotide polymorphism (C1858T) causes a substitution from arginine (R) to tryptophan (W) at 620 residue (R620W). Lyp-620W has been confirmed as a susceptible allele in multiple autoimmune diseases, including type 1 diabetes (T1D). Several independent studies proposed that the disease-associated allele is a gain-of-function variant. However, a recent report found that in human cells and a knockin mouse containing the R620W homolog that Ptpn22 protein degradation is accelerated, indicating Lyp-620W is a loss-of-function variant. Whether Lyp R620W is a gain- or loss-of-function variant remains controversial. To resolve this issue, we generated two lines (P2 and P4) of nonobese diabetic (NOD) mice in which Ptpn22 can be inducibly silenced by RNAi. We found long term silencing of Ptpn22 increased spleen cellularity and regulatory T (Treg) cell numbers, replicating the effect of gene deletion reported in the knockout (KO) B6 mice. Notably, Ptpn22 silencing also increased the reactivity and apoptotic behavior of B lymphocytes, which is consistent with the reduced reactivity and apoptosis of human B cells carrying the alleged gain-of-function PTPN22 allele. Furthermore, loss of Ptpn22 protected P2 KD mice from spontaneous and Cyclophosphamide (CY) induced diabetes. Our data support the notion that Lyp-620W is a gain-of-function variant. Moreover, Lyp may be a valuable target for the treatment of autoimmune diseases.
Single-molecule microscopy is one of the decisive methodologies that allows one to clarify cellular signaling in both spatial and temporal dimentions by tracking with nanometer precision the diffusion of individual microscopic particles coupled to relevant biological molecules. Trajectory analysis not only enables determination of the mechanisms that drive and constrain the particles motion but also to reveal crucial information about the molecule interaction, mobility, stoichiometry, all existing subpopulations and unique functions of particular molecules. Efficacy of this technique depends on two problematic issues the usage of the proper fluorophore and the type of biochemical attachment of the fluorophore to a biomolecule. The goal of this study was to evolve a highly specific labeling method suitable for single molecule tracking, internalization and trafficking studies that would attain a calculable 1:1 fluorophore-to-receptor stoichiometry. A covalent attachment of quantum dots to transmembrane receptors was successfully achieved with a techinque that amalgamates acyl carrier protein (ACP) system as a comparatively small linker and coenzyme A (CoA)-functionalized quantum dots. The necessity of optimization of the quantum dot usage for more precise calculation of the membrane protein stoichiometries in larger assemblies led to the further study in which methods maximizing the number of signals and the tracking times of diverse QD types were examined. Next, the optimized techniques were applied to analyze behavior of interleukin-5 β-common chain receptor (IL-5Rβc) receptors that are endogenously expressed at low level on living differentiated eosinophil-like HL-60 cells. Obtained data disclosed that perused receptors form stable and higher order oligomers. Additionally, the mobility analysis based on increased in number (>10%) uninterrupted 1000-step trajectories revealed two patterns of confined motion. Thereupon methods were developed that allow both, determination of stoichiometries of cell surface protein complexes and the acquisition of long trajectories for mobility analysis. Sequentially, the aforementioned methods were used to scrutinize on the mobility, internalization and recycling dynamics characterization of a G protein-coupled receptor (GPCRs), the parathyroid hormone receptor (PTHR1) and several bone morphogenetic proteins (BMPs), a member of the TGF-beta superfamily of receptors. These receptors are two important representatives of two varied membrane receptor classes. BMPs activate SMAD- and non-SMAD pathways and as members of the transforming growth factor β (TGF-β) superfamily are entailed in the regulation of proliferation, differentiation, chemotaxis, and apoptosis. For effective ligand induced and ligand independent signaling, two types of transmembrane serine/threonine kinases, BMP type I and type II receptors (BMPRI and BMPRII, respectively) are engaged. Apparently, the lateral mobility profiles of BMPRI and BMPRII receptors differ markedly, which determinate specificity of the signal. Non-SMAD signaling and subsequent osteoblastic differentiation of precursor cells particularly necessitate the confinement of the BMP type I receptor, resulting in the conclusion that receptor lateral mobility is a dominative mechanism to modulate SMAD versus non-SMAD signaling during differentiation. Confined motion was also predominantly observed in the studies devoted to, entailed in the regulation of calcium homeostasis and in bone remodeling, the parathyroid hormone receptor (PTHR1), in which stimulation with five peptide ligands, specific fragments of PTH: hPTH(1–34), hPTHrP(107–111)NH2; PTH(1–14); PTH(1–28) G1R19, bPTH(3–34), first four belonging to PTH agonist group and the last to the antagonist one, were tested in the wide concentration range on living COS-1 and AD293 cells. Next to the mobility, defining the internalization and recycling rates of the PTHR1 receptor maintained in this investigation one of the crucial questions. Internalization, in general, allows to diminish the magnitude of the receptor-mediated G protein signals (desensitization), receptor resensitization via recycling, degradation (down-regulation), and coupling to other signaling pathways (e.g. MAP kinases). Determinants of the internalization process are one of the most addressed in recent studies as key factors for clearer understanding of the process and linking it with biological responses evoked by the signal transduction. The internalization of the PTH-receptor complex occurs via the clathrin-coated pit pathway involving β-arrestin2 and is initiated through the agonist occupancy of the PTHR1 leading to activation of adenylyl cyclase (via Gs), and phosphatidylinositol-specific phospholipase Cβ (via Gq). Taken together, this work embodies complex study of the interleukin-5 β-common chain receptor (IL-5Rβc) receptors, bone morphogenetic proteins (BMPs) and the parathyroid hormone receptor with the application of single-molecule microscopy with the newly attained ACP-quantum dot labeling method and standard techniques.
Cross-striated muscles enable higher animals to perform directed movements and to create mechanical force. The cells of heart and skeletal muscles consist of myofibrils, serial arrays of the smallest contractile subunits, the sarcomeres. Main components of the sarcomeres are the thin and thick filaments, large protein assemblies consisting of mainly actin (thin filaments) and myosin (thick filaments), whose energy-dependent interaction is responsible for the contraction of sarcomeres and so of the whole muscle. The thin filaments are anchored in the sarcomere bordering Z-discs, while the thick filaments are anchored in the M-bands, traverse structures in the sarcomere center. Electron-microscopic studies revealed that the M-bands consist of regular, lattice-like structures that appear to cross-link the thick filaments. A number of proteins could be identified by immune-fluorescence and biochemical binding studies to be present and interact with each other in the M-bands. These data have been integrated into preliminary models of the M-bands. Detailed knowledge of how these proteins interact with each other in the center of the sarcomeres is, however, largely missing. The current study focuses on the structural characterization of the interactions between the titin, myomesin-1, obscurin and obscurin-like 1 (OBSL1), modular filamentous proteins interacting with each other in the M-bands. The high-resolution crystal structure of the titin M10 – OBSL1 Ig1 complex was solved. The structure and additional biophysical data show that titin and OBSL1 as well as titin and obscurin form stable binary complexes through the formation of a small intermolecular ß-sheet. In contrast to previously characterized intermolecular assemblies of sarcomeric proteins, this sheet is formed between parallel non- homologous ß-strands of the interaction partners. The investigation of disease-related variants of the M10 domain by biophysical methods did not allow to draw unambiguous conclusions on a direct connection between impaired OBSL1/obscurin binding and disease development. Two out of four known M10 variants have effects on the correct domain folding and so interfere with the ability to bind obscurin/OBSL1. The two other known variants displayed however only minor effects on fold and binding affinities. It should therefore be further elucidated whether a direct connection between impaired complex formation and disease development exists. -I- Abstract A direct interaction between titin and myomesin-1 could not be confirmed in vitro. Possible explanations for the different results are discussed. While the consequences of the inability of both proteins to interact are unclear, the further characterization of the putative interacting parts of titin and myomesin-1 led to the discovery of two new potential sites of self-assembly on M-band titin and myomesin-1. The crystal structure of titin M4 showed that this domain can form dimeric assemblies through the formation of a disulfide bridge and an intermolecular metal binding site between residues that are unique to this domain. On myomesin-1, in addition to the described C-terminal interaction site, a potential second site of self-assembly was found in its central Fn3-domain segment. The interacting site was mapped to the predicted Fn3 domain My5. The crystal structure of the domain in its dimeric form showed that the interaction is mediated by a mechanism that has previously not been observed in sarcomeric proteins. Two My5 interact with each other by the mutual exchange of an N-terminal ß-strand which complements the Fn3 fold on the binding partner. This type of interaction can be interpreted as misfolding. However, the position of the interacting domain and its mode of interaction allowed the postulation of a model of how myomesin-1 could be integrated in the M-bands. This model is in good agreement with the electron-microscopic appearance of the M-bands.