Institutes
Refine
Year of publication
Document Type
- Doctoral Thesis (311)
- Article (282)
- Preprint (38)
- Contribution to a Periodical (25)
- Book (18)
- Report (2)
Language
- English (521)
- German (154)
- Multiple languages (1)
Has Fulltext
- yes (676)
Is part of the Bibliography
- no (676)
Keywords
- RNA (12)
- SARS-CoV-2 (10)
- NMR spectroscopy (9)
- inflammation (9)
- photochemistry (9)
- NMR (8)
- Biochemistry (7)
- Cell biology (7)
- E2 enzyme (6)
- TRACT (6)
- X-ray crystallography (6)
- oligomerization (6)
- ubiquitination (6)
- Alzheimer’s disease (5)
- COVID19-NMR (4)
- DNA (4)
- DNA-PAINT (4)
- ISGylation (4)
- Mass spectrometry (4)
- fluorescence (4)
- neurodegeneration (4)
- optogenetics (4)
- photolabile protecting groups (4)
- rhodopsin (4)
- structural biology (4)
- Antimicrobial resistance (3)
- Biophysical chemistry (3)
- C. elegans (3)
- Cancer (3)
- Non-structural protein (3)
- PBPK (3)
- RNA structures (3)
- Schmerz (3)
- Solution NMR spectroscopy (3)
- Solution NMR-spectroscopy (3)
- acetylcholine (3)
- autophagy (3)
- drug discovery (3)
- hippocampus (3)
- machine learning (3)
- mass spectrometry (3)
- membrane proteins (3)
- microdialysis (3)
- nuclear receptor (3)
- oligonucleotides (3)
- pain (3)
- polypharmacology (3)
- solid-state NMR (3)
- 5-lipoxygenase (2)
- 5′-UTR (2)
- 6-shogaol (2)
- ABC transporter (2)
- Arzneimittel (2)
- Bacterial biofilms (2)
- Bacterial structural biology (2)
- Baird's rule (2)
- Biotechnology (2)
- Chemical tools (2)
- Chromones (2)
- Covid19-NMR (2)
- Cryoelectron microscopy (2)
- DFT (2)
- EGFR (2)
- EPR (2)
- Endothelial cells (2)
- Enzyme mechanisms (2)
- G-Quadruplex (2)
- G-quadruplexes (2)
- GABARAP (2)
- GPCR (2)
- Host-biofilm interactions (2)
- ISGlyation (2)
- Inflammation (2)
- Innate immune response (2)
- LC3 (2)
- MLL (2)
- Mechanisms of disease (2)
- Medicinal chemistry (2)
- Membrane Proteins (2)
- Membrane proteins (2)
- Metabolism (2)
- NR4A2 (2)
- NanoBRET (2)
- PROTAC (2)
- Permeation and transport (2)
- Persistent wound infections (2)
- Pharmazeutische Technologie (2)
- Pharmazie (2)
- Photochemie (2)
- Photolabile Schutzgruppen (2)
- Precipitation inhibition (2)
- Protein drugability (2)
- RNA modification (2)
- RNA processing (2)
- Rietveld refinement (2)
- SL1 (2)
- SiGe alloys (2)
- Solution-state NMR (2)
- Structural biology (2)
- Supersaturation (2)
- Ubiquitin (2)
- Wirkstofffreisetzung (2)
- Zingiber officinale Roscoe (2)
- accessibility switch (2)
- actin (2)
- adenylyl cyclases (2)
- ankyrin (2)
- aptamers (2)
- bioequivalence (2)
- boron (2)
- boryl anions (2)
- boswellic acids (2)
- click chemistry (2)
- cluster compounds (2)
- computational chemistry (2)
- cyclic nucleotide-gated channels (2)
- deep learning (2)
- dorsal root ganglia (2)
- epigenetics (2)
- erythrocyte (2)
- excited state aromaticity (2)
- farnesoid X receptor (2)
- fluorescent probes (2)
- fluorine (2)
- fragment screening (2)
- germanium (2)
- glucose (2)
- guanylyl cyclases (2)
- inward proton pump (2)
- lactate (2)
- malaria (2)
- microbial rhodopsin (2)
- microscopy (2)
- microtubule-targeting agents (2)
- mitochondrial respiration (2)
- molecular dynamics (2)
- non-alcoholic steatohepatitis (2)
- phosphorylation (2)
- photoisomerization (2)
- photolabile Schutzgruppen (2)
- protein structure (2)
- reactive oxygen species (2)
- rearrangements (2)
- receptor tyrosine kinases (2)
- silicon (2)
- similarity measures (2)
- single-particle tracking (2)
- skin infection model (2)
- spinal cord (2)
- substituent effects (2)
- subvalent compounds (2)
- super-resolution microscopy (2)
- tRNA splicing (2)
- time-resolved spectroscopy (2)
- transcription factor (2)
- tumor microenvironment (2)
- ultrafast spectroscopy (2)
- vascular endothelial cells (2)
- virulence factor (2)
- 1,3-diamine (1)
- 10-Hydroxyaloins A/B (1)
- 140Ce (1)
- 19F (1)
- 19F NMR shifts for fluoroarenes (1)
- 2'-deoxyguanosine riboswitch (1)
- 2-Photonen (1)
- 2D NMR spectroscopy (1)
- 2′-O-methylation (1)
- 3D electron diffraction (1)
- 3D reconstruction and image processing (1)
- 4-fluoroamphetamine (1)
- 5'-UTR (1)
- 5-lipoxygenase; (1)
- 9-HODE (1)
- 9-borafluorene (1)
- ABC Transporter (1)
- ABC proteins, ribosome recycling (1)
- ABC transporters (1)
- ABCE1 (1)
- ADAR (1)
- AF4 (1)
- AF4–MLL (1)
- AMP-activated kinase (AMPK) (1)
- ATG4 (1)
- ATG8 (1)
- ATP binding (1)
- ATPases (1)
- ATTO 390 (1)
- Ab initio calculations (1)
- Absorption modeling (1)
- Acute leukemia, chromosomal translocation, MLL, KMT2A, AF4-MLL, reciprocal fusion protein, MLL-r leukemia, (1)
- Acute myeloid leukemia (AML) (1)
- Adeno-associated virus vector (1)
- Adenosine A1 receptor (1)
- Age (1)
- Airport emissions (1)
- Akute lymphoblastische Leukämie (1)
- Alkylation of RNA (1)
- Aloe (1)
- Aloe lateritia ENGLER (1)
- Alterung (1)
- Alzheimer's disease (1)
- Analytical chemistry (1)
- Angiogenesis (1)
- Anthranoids (1)
- Anthraquinones (1)
- Anthrones (1)
- Antibiotic resistance (1)
- Antigen carrier (1)
- Antigen processing and presentation (1)
- Antimuscarinic and Papaverine-Like Activity (1)
- Antisense agents (1)
- Apoptosis (1)
- Aptamer (1)
- Aptamere (1)
- Arc protein (1)
- Armadillo repeat protein (1)
- Arzneimittelanalogon (1)
- Asphodelaceae (1)
- Assignment (1)
- Atg8 (1)
- Aurora kinase (1)
- Autophagy (1)
- B-cell lymphoma (1)
- BACE1 (1)
- BET proteins (1)
- BH3 mimetics (1)
- BRG1 (1)
- Bacterial Signal Transduction (1)
- Bacterial biofilm (1)
- Binding Kinetics (1)
- Binding kinetic (1)
- Bindungsanalyse (1)
- BioID (1)
- Biochemische Analyse (1)
- Bioenabling formulations (1)
- Bioenergetics (1)
- Bioequivalence (1)
- Biological techniques (1)
- Biologie (1)
- Biomaterials – proteins (1)
- Biophysics (1)
- Biophysik (1)
- Biopysikalische Chemie (1)
- Biowaiver (1)
- Bispecific (1)
- Black Lipid Membrane (1)
- Brain (1)
- Broken symmetry (1)
- Bärnighausen tree (1)
- B−B bonds (1)
- B−H bonds (1)
- CAR-immunotherapy (1)
- CEST (1)
- CNN (1)
- CNP (1)
- COVID-19 (1)
- CPVT (1)
- CRBN (1)
- CRISPR/Cas9 (1)
- CXCL13 (1)
- CXCR5 (1)
- Caenorhabditis elegans (1)
- Calcium (1)
- Calstabin2/FKBP12.6 (1)
- Cancer models (1)
- Capsule Synthesis (1)
- Carbanions (1)
- Cardiovascular disease (1)
- Catalysis (1)
- Cell division (1)
- Cell-free Protein Expression (1)
- Cell-free Protein Synthesis (1)
- Cell-free protein synthesis (1)
- Cellular microbiology (1)
- Cellular neuroscience (1)
- Cellular suicide switch (1)
- Ceramide Synthase (1)
- ChIP-Seq (1)
- Chaperones (1)
- Chemical characterization (1)
- Chemical modification (1)
- Chemie (1)
- Chemistry (1)
- Chikungunya (1)
- Chloroxospezies (1)
- Chrimson (1)
- Clinical relevant dissolution specification (1)
- Colitis assoziate colorectal cancer (1)
- Colorectal cancer (1)
- Complex I (1)
- Complex II (1)
- Computational biology and bioinformatics (1)
- Controlled freezing/thawing (1)
- Covid19-nmr (1)
- Cryo-concentration (1)
- Cryo-electron Tomography (1)
- Crystal and Molecular Structure (1)
- Curcuminoid concentrations (1)
- Cy5.5 (1)
- Cyclization (1)
- CysLTR1 (1)
- Cysteine‐Rich Domain (CRD) (1)
- DDR (1)
- DEAD-Box Helicase (1)
- DFT+MBD calculations (1)
- DNA G-quadruplex (1)
- DNA conjugate (1)
- DNA damage response (1)
- DNA methylation (1)
- DNA pulldown (1)
- DNA recognition (1)
- DNA-protein-interactions (1)
- DNP (1)
- DNP in solids (1)
- Daclatasvir (1)
- Degrader design (1)
- Designed ankyrin repeat protein (1)
- Desinfektionsmittel (1)
- DiI (1)
- Diffuse large B-cell lymphoma (1)
- Dipol (1)
- Directional freezing (1)
- Diseases (1)
- Dissolution (1)
- Dissolution nanoparticles (1)
- Disulfide bond (1)
- Disulphide Bridges (1)
- Drug Delivery (1)
- Drug delivery systems (1)
- Drug discovery (1)
- Drug screening (1)
- Drug substance bottles (1)
- Dynamics (1)
- E3 Ligase (1)
- EPR spectroscopy (1)
- ERK3 (1)
- ERQC (1)
- Efflux system (1)
- Electron Paramagnetic Resonance (1)
- Electron microscopy (1)
- Electrospinning (1)
- Elektrochemie (1)
- Enantioselektive Katalyse (1)
- Endothelial cell (1)
- Energie (1)
- Enthalpy (1)
- Entwicklungsbiologie (1)
- FAIRE-Seq (1)
- FBDD (1)
- FBS (1)
- FPR (1)
- Faltungsdynamik (1)
- Fatty acid synthases (1)
- Ferroptosis (1)
- Festkörperstruktur (1)
- Flash photolysis (1)
- Flavin homeostasis (1)
- Fluorene derivatives (1)
- Food effect (1)
- Fortpflanzung (1)
- Fragment (1)
- Freezing and thawing (1)
- Functional aspects of cell biology (1)
- Fusion proteins (1)
- G protein-coupled receptors (1)
- G-protein-coupled receptors (1)
- G-quadruplex binders (1)
- G2A receptor (1)
- GC-MS (1)
- GIIb/IIIa-receptor (1)
- Gastrointestinaltrakt (1)
- GdIr2Si2 (1)
- Gene therapy (1)
- Genetic engineering (1)
- Genetics (1)
- Genexpression (1)
- Genome engineering (1)
- Genregulation (1)
- Geschichte der Pharmazie (1)
- Glucose (1)
- Glutathione (1)
- Guanylatzyklasen (1)
- Gβγ subunit (1)
- H/ACA-RNP (1)
- HCV (1)
- HIV protease inhibitors (1)
- Haloferax volcanii (1)
- HeLa cells (1)
- Helicase (1)
- Hepatitis B virus (1)
- Herpes Simplex Virus Type 1 (HSV-1) (1)
- Heterocycles (1)
- High Performance Liquid (1)
- Histidine Kinases (1)
- Homeoviscous adaptation (1)
- Homonataloins A and B (1)
- Hydrodynamik (1)
- IAP (1)
- INS (1)
- ITGA9 (1)
- Image processing (1)
- Immune evasion (1)
- Immunochemistry (1)
- Immunology (1)
- Immunseren (1)
- In situ hybridization (1)
- In vitro wound models (1)
- Infectious diseases (1)
- Inflammatory Bowel Disease (1)
- Influenza (1)
- Innovation (1)
- Instrumentation & Devices (1)
- Ion transport (1)
- Ischämie (1)
- Isolation, separation and purification (1)
- Isothermal Titration Calorimetry (1)
- Jet engine oil (1)
- Juckreiz (1)
- Jurkat (1)
- K48-linked (1)
- K63-linked (1)
- KCNT1 (1)
- KMT2A (1)
- KR2 (1)
- Kaliumkanal (1)
- Kaliumkanal-Aktivatoren (1)
- Kd determination (1)
- Ketonkörper (1)
- Kinase (1)
- Kinase Inhibitors (1)
- Kinase inhibitors (1)
- Kinetik (1)
- Knochenmarksmikromilieu (1)
- Knotless phytochrome (1)
- LASS (1)
- LC-MS-based lipid mediator analysis (1)
- LC3/GABARAP (1)
- LILBID-MS (1)
- LIMK1 (1)
- LIMK2 (1)
- LINC00607 (1)
- LINE-1 (1)
- LIR (1)
- LIR motif (1)
- LIR motifs (1)
- Lehramt (1)
- Leukaemia (1)
- Leukemia (1)
- Leukocytes (1)
- Lewis acid (1)
- Ligands (1)
- Lipid Environment (1)
- Lipid packing (1)
- Lipidinteraktion (1)
- Lipoprotein Structure (1)
- Liposomes (1)
- M1 Ubiquitination (1)
- MACS (1)
- MAPK6 (1)
- MET (1)
- MET receptor (1)
- MFS-type tripartite system (1)
- MHC I (1)
- MLH1 (1)
- MLL-r acute leukemia (1)
- MODY (1)
- Macrodomain (1)
- Makrophagen (1)
- Massenspektrometrie (1)
- Mechanistic (1)
- Medication Appropriatness Index (1)
- Membrane Protein (1)
- Membrane fluidity (1)
- Membrane homeostasis (1)
- Membranes (1)
- Mesoporous silica (1)
- Meta effect (1)
- Metabolic Regulation (1)
- Metabolismus (1)
- Mice (1)
- Microbial Rhodopsins (1)
- Microbiology (1)
- Microdialysis (1)
- Mitochondria (1)
- Mitochondrial dysfunction (1)
- Mitosis (1)
- Modification (1)
- Molecular Interactions (1)
- Molecular Neuroscience (1)
- Molecular biology (1)
- Molecular glue (1)
- Monolage (1)
- Multi-domain proteins (1)
- Multidrug Transporters (1)
- Multimedikation / Polypharmazie (1)
- Multimorbidität (1)
- Multiphoton microscopy (1)
- Mycobacterium tuberculosis (1)
- N-glycoproteome (1)
- NADPH oxidase (1)
- NASH (1)
- NMR Solution Structure (1)
- NMR shift calculation (1)
- NMR solution structure (1)
- NMRI (1)
- NOESY (1)
- NPR2 (1)
- NR4A3 (1)
- NS5A (1)
- Nanocarriers (1)
- Nanodiscs (1)
- Nanotechnologie (1)
- Natural product (1)
- Natural products (1)
- Neuropathic pain (1)
- New E3-ligase ligands (1)
- Nox (1)
- Nox inhibition (1)
- Nucleic acid-binding domain (1)
- Nucleolar protein 14 (1)
- Nukleinsäuren (1)
- Nukleäre Rezeptoren (1)
- OTULIN (1)
- Oligonukleotid (1)
- Oncogenes (1)
- Oncoprotein activation (1)
- Ophthalmologie (1)
- Optogenetics (1)
- Organische Synthese (1)
- Organokatalyse (1)
- Orphan nuclear receptor (1)
- Outer Membrane Protein RcsF (1)
- Overhauser effect (1)
- Oxanthrone Glucosyls (1)
- Oxidative phosphorylation (1)
- PAC-1 (1)
- PBPK modelling (1)
- PBPK/PD (1)
- PC12APPsw (1)
- PDF-Global-Fit (1)
- PELDOR (1)
- PELDOR / DEER (1)
- PELDOR/DEER spectroscopy (1)
- PIM kinases (1)
- PLGA nanoparticles (1)
- PRIMUM-Studie (1)
- PXRD (1)
- Pain behavior (1)
- Paramyxoviruses (1)
- Parkinson’s disease (1)
- Partial agonist (1)
- Patch Clamp (1)
- Patch-clamp (1)
- Peptide nucleic acids (1)
- Pharmacology (1)
- Pharmazeutische Chemie (1)
- Phenalen, Heteroatom-Dotiert (1)
- Phosphoproteome (1)
- Photocages (1)
- Photochemistry (1)
- Photoisomerization (1)
- Photolabile protecting groups (1)
- Photoschalter (1)
- Photosynthesis (1)
- Physical chemistry (1)
- Physiologically Based Pharmacokinetic Modelling (1)
- Phytochrome (1)
- Pigment Green 7 (1)
- Pneumoviruses (1)
- Polyketide synthases (1)
- Polyketides (1)
- Polypeptid transport-associated domains (1)
- Post-Hartree-Fock methods (1)
- Potassium channels (1)
- Pre‐Ligand Assembly Domain (PLAD) (1)
- Process characterization (1)
- Process time (1)
- Proteasome inhibitor (1)
- Protein Kinase (1)
- Protein biosynthesis (1)
- Protein engineering (1)
- Protein folding (1)
- Protein synthesis (1)
- Proteins (1)
- Proteomics (1)
- Proteostasis (1)
- Pruritus (1)
- Pseudocontact shift (1)
- Pseudouridine (1)
- Pseudouridylation (1)
- Pulverdiffraktometrie (1)
- Puromycin (1)
- QbD (1)
- Quality control (1)
- Quantenpunkte (1)
- Quinonoids (1)
- R values (1)
- RMN (1)
- RNA PT (1)
- RNA cleavage (1)
- RNA degradation (1)
- RNA dynamics (1)
- RNA modifications (1)
- RNA synthesis (1)
- RNA therapy (1)
- RNA, biochemistry, mass spectrometry (1)
- RNA-Seq (1)
- RNA-binding proteins (1)
- RND (1)
- RXR (1)
- RXR-Selektivität (1)
- RXR-Subtypen (1)
- Rab27a (1)
- Radiotherapy (1)
- Regulation der Genexpression (1)
- Regulation of lipid metabolism (1)
- Release testing (1)
- Repair (1)
- Repression <Genetik> (1)
- Retinoid X Rezeptoren Subtypen (1)
- Retrotransposition (1)
- Rh(II) catalysis (1)
- Rhabdomyosarcoma (1)
- Rhamnus purshianus D. C. (1)
- RhlB (1)
- Rhodopsine (1)
- Ribonucleoproteins (1)
- Ribosome Recycling (1)
- Ribosome biogenesis (1)
- RyR2 (1)
- Ryanodin (1)
- Röntgenstrukturanalyse (1)
- S107 (1)
- SAR (1)
- SEDDS (1)
- SERCA (1)
- SHAPE analysis (1)
- SL5a (1)
- SL5b (1)
- SL5b + c (1)
- SL5c (1)
- SMEDDS (1)
- SNEDDS (1)
- SNP (1)
- SOFI (1)
- SPM (1)
- STED microscopy (1)
- STM (1)
- Saccharomyces cerevisiae (1)
- Schleimpilze (1)
- Screening (1)
- Selectivity (1)
- Semi-empiricalcalculations (1)
- Sexualität (1)
- Sila-Substitution (1)
- Silacyclobutenes (1)
- Silafullerane (1)
- Silane (1)
- Silole (1)
- Silylation (1)
- Single-Molecule Localization Microscopy (SMLM) (1)
- Slack (1)
- Smac mimetic (1)
- Small molecules (1)
- Sodium ion pump (1)
- Solid Oral Dosage Forms (1)
- Solid dispersion (1)
- Solubility (1)
- Spektroskopie (1)
- Sphingolipids (1)
- Sphingosinkinase 2 (1)
- Spinach (1)
- Spinlabel (1)
- Stem-cell research (1)
- Stereochemistry (1)
- Stress response (1)
- Stroke (1)
- Structural Biology (1)
- Structure refinement (1)
- Subtomogram averaging (1)
- T-cell (1)
- TBK1 (1)
- TERRA RNA (1)
- TICT (1)
- TSEN (1)
- Target validation (1)
- Taspase1 (1)
- Temperature mapping (1)
- Tetrelbindung (1)
- TgF344-AD (1)
- Theoretical chemistry (1)
- Thyroidhormone (1)
- Tiermodell (1)
- Time-resolved spectroscopy (1)
- Tissue distribution (1)
- TmRh2Si2 (1)
- Transcription factors (1)
- Transcriptional regulation (1)
- Transient absorption (1)
- Transport Drugs (1)
- Transporters (1)
- Transposon systems (1)
- Tumour-suppressor proteins (1)
- Two-component System (1)
- UBA5 (1)
- UBL (1)
- UFIM (1)
- UFM1 (1)
- ULK4 (1)
- UV/Vis spectroscopy (1)
- Ubiquitin chains (1)
- Ubiquitinome (1)
- Ultrafine particles (UFPs) (1)
- Ultrakurzzeit-Spektroskopie (1)
- Unc-51-like kinase (1)
- VHL (1)
- Viologene (1)
- Vioprolide A (1)
- Viral entry (1)
- Virologie (1)
- Virus-host Interaction (1)
- Wasserlöslichkeit (1)
- Wound dressings (1)
- Wound infection (1)
- X-ray structure analysis (1)
- Xenocoumacin (1)
- ZIKV (1)
- Zolpidem (1)
- Zolpidem; Food-effect, PBBM, PBPK (1)
- Zweiphotonen-Anregung (1)
- Zyklisierung (1)
- absolute quantification of RNA modifications (1)
- ab initio calculations (1)
- accessory proteins (1)
- actin cytoskeleton dynamics (1)
- acute leukemia (1)
- acyclovir (1)
- adaptor mediated recruitment (1)
- additive manufacturing (1)
- adenosine–arginine conjugate (1)
- aging (1)
- alternative matrices (1)
- ambiguous structures (1)
- amorphous solid dispersions (1)
- amyloid beta (1)
- amyloid precursor protein (1)
- amyloidogenesis (1)
- analgesia (1)
- analytical chemistry (1)
- angiogenesis-related cell functions (1)
- anilinopyrimidine (1)
- antibiotic resistance (1)
- antigen presentation (1)
- antigen processing (1)
- antisense technology (1)
- antiviral response (1)
- antiviral signaling (1)
- apoptosis (1)
- aptamer (1)
- arachidonic acid (AA) (1)
- arrhythmia model (1)
- astrogliosis (1)
- atropisomers (1)
- attention module (1)
- atypical MAPK kinase (1)
- automated assignment (1)
- axon bifurcation (1)
- axon branching (1)
- azo compounds (1)
- baicalein (1)
- barstar (1)
- benchmark standards (1)
- benzazepinone (1)
- benzothiazoles (1)
- big data (1)
- bilin-binding photoreceptors (1)
- bio-enabling formulations (1)
- bioavailability (1)
- bioconjugation (1)
- biomarker (1)
- biophysics (1)
- bioprinting (1)
- biorelevant dissolution (1)
- bio‐enabling formulations (1)
- bisubstrate inhibitors (1)
- body-on-a-chip (1)
- bond activation (1)
- boron heterocycles (1)
- breast milk (1)
- brightness (1)
- buchu (1)
- c-Myc (1)
- cGKI (1)
- cGMP signalling (1)
- cMYC (1)
- caged compounds (1)
- calsequestrin (1)
- cancer (1)
- cancer associated fibroblasts (1)
- canine cancer (1)
- capture (1)
- carbazole ligands (1)
- carbon direct detection (1)
- catalytic mechanism (1)
- cell cycle (1)
- cell migration (1)
- cell stress (1)
- cell-free expression (1)
- cell-free protein synthesis (1)
- cellular uptake and localization (1)
- ceramide synthase (1)
- cerium (1)
- cerumen (1)
- channelrhodopsin (1)
- charge transfer (1)
- chemical shift assignment (1)
- chemiluminescence (1)
- chemistry education (1)
- chemoenzymatic synthesis (1)
- chemogenomics (1)
- cholesterol (1)
- chronobiology (1)
- circular dichroism (1)
- classical Hodgkin lymphoma (1)
- clustering (1)
- co-crystal structure (1)
- coarse-grained simulations (1)
- cofilin (1)
- colon (1)
- combinatorial approaches (1)
- commercialization (1)
- comparative oncology (1)
- complex I (1)
- complex IV (1)
- computer-aided diagnosis (1)
- conductivity (1)
- confocal Raman microscopy (1)
- confocal microscopy (1)
- connection to nature (1)
- continuous rotation (1)
- copper phthalocyanine (1)
- correlated electrons (1)
- coumarin (1)
- coumarin 6 (1)
- covalent inhibitors (1)
- creams (1)
- cross-correlation functions (1)
- cross-section (1)
- cryo-electron microscopy (1)
- crystal structure (1)
- curcumin (1)
- cyanines (1)
- cycloaddition (1)
- cysteine reactivity (1)
- cysteine-redox regulation (1)
- cytokine (1)
- data curation (1)
- data projection (1)
- data science (1)
- de novo design (1)
- dendritic spines (1)
- denoising (1)
- de novo design (1)
- diacylglycerol kinase (1)
- dialysis (1)
- diazo compounds (1)
- diboranes (1)
- differential scanning fluorimetry (1)
- diffusion states (1)
- digestion artifact (1)
- dissolution (1)
- dopamine D2S/D3 receptor (1)
- drug abstinence (1)
- drug development (1)
- drug metabolism and distribution (1)
- drug release (1)
- drug resistance (1)
- drug targets (1)
- drug therapy (1)
- drug-likeness (1)
- dynamic nuclear polarization (1)
- dynamics (1)
- efflux pump (1)
- eicosanoid (1)
- electron beam induced deposition (1)
- electron crystallography (1)
- electron transfer system (1)
- electronic nose (1)
- elegans (1)
- enamides (1)
- end maturation (1)
- endolysosomal system (1)
- endonuclease (1)
- endoplasmic reticulum (1)
- endothelial cells (1)
- energy barriers (1)
- enolase-1 (1)
- environmental education (1)
- enzyme catalysis (1)
- enzyme kinetics (1)
- epitranscriptome (1)
- epitranscriptomics (1)
- ethnobotany (1)
- etravirine (1)
- evolution (1)
- exosomes (1)
- extracellular matrix (1)
- extract (1)
- famotidine (1)
- fatty acid (1)
- fatty acid/biosynthesis (1)
- filopodia (1)
- flavolignan (1)
- fluidity (1)
- fluorescent labeling (1)
- fluorophore (1)
- flux growth (1)
- folding landscapes (1)
- food effect (1)
- fragment-based design (1)
- fragment-based drug design (1)
- fragment-based screening (1)
- frankincense (1)
- free mRNA (1)
- fulgide (1)
- gallein (1)
- gapmers (1)
- gene expression (1)
- genetic code expansion (1)
- glucose transport (1)
- glutathione (1)
- glutathionylation (1)
- glycerophospholipid (1)
- glycolysis (1)
- green gap (1)
- guanidine riboswitch (1)
- guanidine-II riboswitch (1)
- hTERT (1)
- hairpin polyamides (1)
- heart-on-a-chip (1)
- hepatische Ketogenese (1)
- hepatocyte nuclear factor 4α (1)
- heteronuclear detection (1)
- hierarchical clustering (1)
- high content imaging (1)
- high dimensional complex data (1)
- high-throughput sequencing (1)
- histamine (1)
- histone deacetylase (1)
- histone modifications (1)
- homogeneous catalysis (1)
- human milk (1)
- human neurons (1)
- human pain models (1)
- human pharmacology (1)
- hxt0 strain (1)
- hydromethylthionine (1)
- hypoxia (1)
- iPSc (1)
- immune system (1)
- in silico tools (1)
- in vitro activities (1)
- in vivo neuroimaging (1)
- in-vitro–in-vivo correlation (1)
- infant health (1)
- information literacy (1)
- interaction partners (1)
- interest in animals (1)
- interface engineering (1)
- intracellular internalization (1)
- intrinsically disordered region (1)
- inflammatory pain (1)
- ion beam induced deposition (1)
- ionization (1)
- isopropanol (1)
- isothermal titration calorimetry (ITC) (1)
- isotope labeling (1)
- itch (1)
- kidney-on-a-chip (1)
- kinase (1)
- kinase inhibitors (1)
- kinetics (1)
- labelfree quantitative mass spectrometry (1)
- lattice-energy minimization (1)
- leucocytes (1)
- leukocyte adhesion (1)
- leukotriene (1)
- leukotriene A4 hydrolase (1)
- lichtaktivierbare Werkzeuge (1)
- ligand design (1)
- ligases (1)
- light control (1)
- light-induced polymerization (1)
- lipid (1)
- lipid based formulation (1)
- lipid metabolism (1)
- lipid suspension (1)
- lipidic cubic phase (1)
- lipidomics (1)
- liposomes (1)
- lipoxin (1)
- lipoxygenase (1)
- lipoxygenase inhibitor (1)
- liquid chromatography–mass spectrometry (1)
- liver fibrosis (1)
- liver-on-a-chip (1)
- long non-coding RNA (1)
- longitudinale Medikationsveränderung (1)
- lung cancer (1)
- lung tumor heterogeneity (1)
- lung-on-a-chip (1)
- lysosome (1)
- lysosomes (1)
- mRNA surveillance (1)
- macromolecular machines (1)
- macrophage (1)
- magnetic exchange coupling constants (1)
- magnetic resonance imaging (1)
- magnetism (1)
- main group elements (1)
- maternal diet (1)
- mature milk (1)
- meat adulteration (1)
- medicinal chemistry (1)
- medicinal plant (1)
- memantine (1)
- membrane anchor; substrate-binding protein dependent secondary transport; TRAP-associated extracytoplasmic immunogenic (TAXI); tripartite ATP-independent periplasmic (TRAP) (1)
- membrane dynamics (1)
- membrane fluidity (1)
- membrane mimics (1)
- membrane organization (1)
- membrane protein complexes (1)
- membrane receptors (1)
- membranes/fluidity (1)
- memory consolidation (1)
- metabolic sydrome (1)
- metabolomics (1)
- metallo β lactamases (1)
- metastasis (1)
- mice (1)
- micro-physiological systems (1)
- microRNA (1)
- microsomal prostaglandin E2 (1)
- mitochondria (1)
- mixed valence compounds (1)
- modeling & simulation (1)
- modeling and simulation (1)
- molecular docking (1)
- molecular electronics (1)
- molecular machines (1)
- molecular switch (1)
- molekulare Werkzeuge (1)
- multi-objective optimization (1)
- multi-target drug (1)
- multi-task learning (1)
- multicolor imaging (1)
- multidrug resistance protein 4 (1)
- multiple sclerosis (1)
- multiresistant bacteria (1)
- multispectral flow cytometry (1)
- multitarget drugs (1)
- multitasking protein (1)
- n_TOF (1)
- nanodiscs (1)
- nanomedicine (1)
- natural compound synthesis (1)
- nerve injury (1)
- neural network (1)
- neurodegenerative disorders (1)
- neuromuscular system (1)
- neuropathic pain (1)
- neuropathy (1)
- neurotropic virus (1)
- neutron (1)
- nitrogen direct detection (1)
- nitroindoles (1)
- nitrosylation (1)
- nociception (1)
- non-alcoholic fatty liver disease (1)
- non-canonical amino acid (1)
- non-coding RNAs (1)
- non-invasive imaging (1)
- non-natural nucleotide (1)
- nonstructural proteins (1)
- nuclear magnetic resonance (NMR) (1)
- nuclear magnetic resonance spectroscopy (1)
- nuclear receptor related-1 (1)
- nuclear receptor-related 1 (1)
- nucleobases (1)
- nucleophilic boron (1)
- nucleophilic substitution (1)
- nucleoside analysis (1)
- nucleosynthesis (1)
- oligonucleotide (1)
- oncogene promoters (1)
- one-dimensional convolutional neural network (1)
- one-pot reaction (1)
- optogenetic (1)
- organs-on-a-chip (1)
- osteopontin (1)
- overtaking innovation (1)
- oxidative folding (1)
- p38 (1)
- pSer domain (1)
- pair distribution function analysis (1)
- pair distribution function refinement (1)
- pair distribution functions (1)
- pancreatic cancer (1)
- peptide antibiotics (1)
- peptide loading complex (1)
- peptide transport (1)
- performance tests (1)
- peripheral injury (1)
- peroxisome prolifer ators-activated receptor-γ (1)
- phage lysis proteins (1)
- pharmacokinetics (1)
- pharmacokinetics/ pharmacodynamics (PK/PD), (1)
- pharmacological activity (1)
- pharmacophore model (1)
- phase separation (1)
- phenotypic screening (1)
- phospholipids/phosphatidylglycerol (1)
- photo-tethering (1)
- photoacid (1)
- photobleaching (1)
- photocage (1)
- photocages (1)
- photocaging (1)
- photocycle (1)
- photoschalbare (1)
- photospaltbare Linker (1)
- photoswitch (1)
- photoswitches (1)
- phototherapeutic window (1)
- phytochemical composition (1)
- phytochromes (1)
- platelets (1)
- polymers (1)
- polyubiquitin (1)
- potassium (1)
- potassium transporter (1)
- powder data (1)
- precipitation inhibitor (1)
- precursor synthesis (1)
- privileged structures (1)
- problem-based learning (1)
- proline isomerization (1)
- prostaglandin (1)
- prostaglandin E (1)
- prostaglandins (1)
- protein X-ray crystallography (1)
- protein capture (1)
- protein compartmentalization (1)
- protein engineering (1)
- protein folding (1)
- protein kinase inhibitor (1)
- protein motif (1)
- protein oxidation (1)
- protein tyrosine phosphatase (1)
- protein-protein interactions (1)
- proteins (1)
- proteostasis stress (1)
- proton transfer (1)
- pseudokinase (1)
- pseudouridylation (1)
- purification of protein complexes (1)
- quantenpunkte (1)
- quantum yield (1)
- quercetin (1)
- quinacridone (1)
- radical reactions (1)
- random forest regressor (1)
- real-time NMR spectroscopy (1)
- receptor condensates (1)
- receptor cross-interaction (1)
- receptor dynamics (1)
- red light (1)
- redox chemistry (1)
- regulatory science (1)
- release testing (1)
- resmetirom (1)
- resolution of inflammation (1)
- resolvin (1)
- respiratory chain (1)
- resveratrol (1)
- retinal (1)
- retinoid X receptor (1)
- rhabdomyosarcoma (1)
- rhodamine 123 (1)
- ribosomal exit tunnel (1)
- ribosome-associated quality control (1)
- riboswitch (1)
- ribozymes (1)
- rivastigmine (1)
- s-process (1)
- screening system (1)
- selbst-anordnende (1)
- self-assembled monolayers (1)
- self-initiated photografting and photopolymerization (1)
- semisolids (1)
- siRNAs (1)
- signal transduction (1)
- silibinin (1)
- silybin (1)
- single particle cryo-EM (1)
- single-molecule FRET (1)
- single-molecule imaging (1)
- single-molecule localization microscopy (1)
- single-particle cryoEM (1)
- single-trajectory analysis (1)
- site-directed spin labeling (1)
- site-specific labeling (1)
- skin delivery (1)
- skin-on-a-chip (1)
- smFRET (1)
- small protein (1)
- small-molecule inhibitor (1)
- smooth muscle cells (1)
- sodium alkoxide (1)
- solid-phase synthesis (1)
- solid-state NMR spectroscopy (1)
- soluble epoxide hydrolase (1)
- solution-state NMR (1)
- solvate (1)
- somatosensory system (1)
- soraphen (1)
- spiro compounds (1)
- splice-modulating oligonucleotides (1)
- stability of fluorescent label (1)
- stearic acid (1)
- stereoselective synthesis (1)
- streptozotocin (1)
- stress dependent RNA modification dynamics (1)
- structural proteins (1)
- structure determination (1)
- structure determination from powder data (1)
- structure-activity relationships (1)
- sugar transport assays (1)
- sugar transport inhibitors (1)
- sulfamide (1)
- super-SMEDDS (1)
- super-SNEDDS (1)
- supercomplex (1)
- supersaturating drug delivery systems (1)
- supersaturation (1)
- surface modification (1)
- surface-initiated polymerization (1)
- surfactant (1)
- swelling (1)
- synaptic transmission (1)
- synaptic vesicle recycling (1)
- synthase-1 (1)
- t(4;11) leukemia (1)
- tRNA (1)
- target variability (1)
- temperature jump (1)
- tetrapyrrole-binding photoreceptors (1)
- therapeutic equivalence (1)
- therapy (1)
- thermal stability (1)
- thiol inhibitors (1)
- thyroid hormone receptor (1)
- tissue distribution (1)
- tissue-plasma-ratio (1)
- toll-like receptor (1)
- topical formulations (1)
- total scattering technique (1)
- total scattering techniques (1)
- trafficking (1)
- transcription (1)
- transient complex (1)
- tumor (1)
- type 2 diabetes (1)
- ubiquitin-conjugating enzymes (1)
- umpolung (1)
- validation (1)
- vascular biology (1)
- vascular biology/endothelial cells (1)
- venetoclax (1)
- viral immune escape (1)
- virtual labeling (1)
- virus (1)
- vitamin B12 (1)
- voltage-gated proton channels (1)
- voltage-sensing domains (1)
- voltage-sensing phosphatases (1)
- weakly supervised learning (1)
- x-ray crystallography (1)
- yeast fatty acid synthase (1)
- zinc finger (1)
- zolpidem (1)
- zoo education (1)
- µ-protein (1)
Institute
- Biochemie, Chemie und Pharmazie (676)
- Präsidium (43)
- Medizin (35)
- Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) (31)
- Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) (30)
- Biowissenschaften (23)
- MPI für Biophysik (15)
- Physik (12)
- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES) (5)
- Geowissenschaften / Geographie (4)
Acinetobacter baumannii is a worldwide opportunistic pathogen responsible for nosocomial infections. One of the main factors contributing to multidrug resistance in A. baumannii is the upregulation of various chromosomally encoded or acquired efflux pumps, which expel toxic compounds out of the cells with high efficiency.
The resistance-nodulation-cell division (RND)-type efflux pump gene deletion strains ∆adeAB, ∆adeFG or ∆adeIJ and the major facilitator superfamily (MFS) chloramphenicol efflux pump gene deletion strain ∆craA of A. baumannii ATCC 19606 were created and a differential gene expression study was conducted via RT-qPCR. The expression of efflux pump genes adeB, adeG, adeJ, craA, and the outer membrane protein ompA were examined in the absence and presence of chloramphenicol. No significant up- or downregulation of these genes for any of these deletion strains in comparision to the wild-type strain in absence of the drug chloramphenicol.
In contrast, craA was significantly up-regulated in A. baumannii exposed to chloramphenicol, emphasizing the importance of CraA in chloramphenicol resistance. CraA is widely present in clinical isolates of A. baumannii. It is homologous to the well-studied multiple-drug efflux transporter MdfA from Escherichia coli (61% similarity), but surprisingly reported to be acting as a specific chloramphenicol transporter of A. baumannii (Roca et al., 2009).
The drug susceptibility assay done with A. baumannii ATCC 19606 ΔcraA showed that CraA could confer resistance towards phenicols (chloramphenicol, thiamphenicol, and florfenicol), which was in line with the previous report. CraA was heterologously overproduced in E. coli BW25113 ∆emrE∆mdfA and its substrate specificity was determined by drug susceptibility assays and whole cell fluorescent dye uptake experiments. We observed that the substrate specificity of craA overexpressed in E. coli was more diverse and resembling that of the E. coli MdfA homolog. Apart from resistance towards phenicols (chloramphenicol, thiamphenicol, and florfenicol), CraA also confer resistance towards monovalent cationic drugs (benzalkonium, TPP+, and ethidium), long dicationic drugs (dequalinium and chlorhexidine), fluoroquinolones (norfloxacin and ciprofoxacin) and anticancer drugs (mitomycin C). We showed that CraA is a drug/H+ antiporter by ACMA quenching in inverted CraA or CraA variant containing membrane vesicles.
To address the molecular determinants for multidrug binding and transport, 45 mostly single Ala-substitution variants of CraA were created. These include substitution variants for membrane-embedded proton-titratable residues (E38, D46, and E338) and residues predicted to be important for binding and transport of drug, as inferred from docking experiments on basis of a MdfA-derived CraA model. The combined results indicated a high degree of functional similarities between MdfA and CraA. The conserved titratable residues E26 and D34 (E38 and D46 in CraA) are important for transport in both these homologs. The CraA variant E38A is inactive against all tested drugs, but D46A is only inactive for some drugs, suggesting that only E38 is involved in H+-transport.
Another focus of this thesis is the three tetracycline transporters of A. baumannii strain AYE, TetA, TetG and TetA(A). Susceptibility assays involving tetracycline, minocycline, doxycycline and the last-resort antibiotic tigecycline were conducted on E. coli BW25113 ∆emrE∆mdfA overexpressing these transporters. TetA(A) was excluded from further study due to toxicity of the cells caused by protein overexpression. Both TetA and TetG confer resistance against tetracycline, minocycline and doxycycline. Although tigecycline was reported not to be recognized by tetracycline efflux pumps, we surprisingly found that TetA is able to transport tigecycline. The role of TetA in tigecycline efflux in A. baumannii was confirmed by conducting tigecycline susceptibility assays on A. baumannii.
We speculate that TetA embedded in the inner membrane acts in cooperation with RND-type tripartite systems that span the inner and outer membrane to extrude tigecycline from the periplasm across the outer membrane. A. baumannii ATCC 19606 ∆adeAB were indeed sensitive to tigecycline in comparison to wild-type strain. Deletion of adeIJ also leads to sensitivity to tigecycline, but less so compared to the DadeAB phenotype, while A. baumannii ATCC 19606 ∆adeFG did not show any difference compared to wild-type strain in tigecycline susceptibility. Differential gene expression analysis of the RND efflux pumps (adeB, adeG and adeJ) and tetA of A. baumannii strain AYE showed that the expression of tetA expression is significantly upregulated when tigecycline is present in the growth medium.
We conclude that craA encodes a broad-spectrum efflux pump rather than a specific chloramphenicol transporter. In A. baumannii, the synergistic effects with the outer membrane and/or the presence of other transporters could result in the discrepancy observed. Thus, the possibility of CraA in conferring multidrug resistance should not be overlooked, especially when it is up-regulated under antibiotic stress conditions.
Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.
Mast cells are long-lived tissue-resident leukocytes, located most abundantly in the skin and mucosal surfaces. They belong to the first line of defence of the body, protecting against invading pathogens, toxins and allergens. Their secretory granules are densely packed with a plethora of mediators, which can be released immediately upon activation of the cell. Next to their role in IgE-mediated allergic diseases and in promoting inflammation, potential anti-inflammatory functions have been assigned to mast cells, depending on the biological setting. The aim of this thesis was to contribute to a better understanding of the role of mast cells during the resolution of a local inflammation. Therefore, in a first of step a suitable model of a local inflammation had to be identified. Since comparison of the two Toll-like receptor (TLR)-agonists zymosan and lipopolysaccharide (LPS), which are most commonly used to locally induce inflammation, revealed a systemic response after LPS-injection and a local inflammation after zymosan-injection, the TLR2 agonist zymosan was chosen for the subsequent experiments. Multi epitope ligand cartography (MELC) combined with statistical neighbourhood analysis showed that mast cells are located in an anti-inflammatory microenvironment next to M2 macrophages during resolution of inflammation, while neutrophils and M1 macrophages are located in the zymosan-filled core of the inflammation. Furthermore, infiltrating neutrophils during peak inflammation and an increasing population of macrophages phagocytosing neutrophils during resolution of inflammation could be observed. MELC as well as flow cytometry analysis of mast cell-deficient mice revealed a decreased phagocytosing activity of macrophages in the absence of mast cells. As an untargeted approach to identify mast cell-derived mediators induced by zymosan, mRNA sequencing of bone marrow-derived mast cells (BMMCs) was performed. Gene ontology term analysis of the sequencing data revealed the induction of the type I interferon (IFN) pathway as the dominant response. Contradicting previous studies, I could validate the production of IFN-β by mast cells in response to zymosan and LPS in vitro. Furthermore IFN-β expression by mast cells was also detected in vivo. In accordance with previous studies regarding other cell types the release of IFN-β by mast cells depends on endosomal signaling. The potential of IFN-β to enhance the phagocytosing activity of macrophages has been demonstrated recently. Besides IFN-β, various other mediators with reported enhancing effects on macrophage phagocytosis were also induced by zymosan in BMMCs, including Interleukin (IL)-1β, IL-4, IL-13, and Prostaglandin (PG) E2. Thus, either one of these mediators alone or a combination of them could promote macrophage phagocytosis.
In conclusion, I herein present mast cells as a novel source for IFN-β induced by non-viral TLR ligands and demonstrate their enhancing effect on macrophage phagocytosis, thereby contributing to the resolution of inflammation.
Resistant microbes are a growing concern. It was estimated that about 33,000 of people die because of the infections caused by multidrug resistant bacteria each year in Europe (ECDC, 2018, https://www.ecdc.europa.eu/). Bacteria can acquire resistance against toxic compounds via different mechanisms and intrinsic active efflux is one of the first mechanisms deployed by bacterial cells. The membrane-localized efflux pumps catalysing this reaction, extract toxic compounds from the interior of the cell and transport these to the outside, thereby maintaining sub-lethal toxin levels in the cytoplasm, periplasm and membranes. Gram-negative three-component efflux pumps, analysed in this study, are composed of an inner membrane protein, a member of the Resistance-Nodulation cell Division (RND) superfamily, an Outer Membrane Factor (OMF) protein and a Membrane Fusion Protein (MFP) that connects the two afore mentioned components into an active efflux pump. The pumps described in this work, AcrAB-TolC and EmrAB-TolC, are drug efflux pumps belonging to the RND and MFS superfamilies, respectively, while CusCBA is an efflux pump that belongs to the RND heavy metal efflux family. Another efflux pump that was used as a model for the design of an in vitro assay for the silver ion transport studies, CopA, belongs to the P-type ATPase superfamily. All pumps analysed in this study are part of the resistance system of Escherichia coli, which is a highly clinically relevant pathogen.
In order to examine the AcrAB-TolC, CopA and CusA efflux pumps, the individual components were separately produced in E. coli, purified to monodispersity and reconstituted in large unilamellar vesicles, LUVs. Means for the optimized production and adequate conditions for efficient reconstitution were presented in this study. The activity of AcrB in LUVs was detected using fluorescence quenching of the dye 8-hydroxy-1,3,6 pyrenetrisulfonate (pyranine), which is incorporated inside the proteoliposomes and is sensitive to the pH changes in its surrounding. The inactive AcrB variant with a substitution in the proton relay network, D407N, showed no activity in proteoliposomes, which correlates with the measurements done in empty liposomes. When AcrA was co-reconstituted with AcrB D407N proteoliposomes it did not restore protein activity. To test the assembly of the AcrAB-TolC pump out of its single components, an in vitro assay was established where the complex assembly was tested with AcrAB- and TolC-containing liposomes. These experiments showed putative AcrAB-TolC formation in the presence or absence of a pump substrate, taurocholate, as well as in the presence of the pump inhibitor, MBX3132. The assembly appeared stable over time and results were invariant in the presence or absence of a pH gradient across the AcrAB-containing membrane.
After determination of the ATPase activity of the P-type ATPase, CopA, in detergent micelles, the protein was reconstituted in LUVs. Quenching of the Ag+-sensitive dye Phen Green SK (PGSK), present on the inside of the CopA-containing proteoliposomes, was observed in presence of ATP and Ag+. Under the same conditions, but in absence of Ag+-ions, quenching was reduced by 80 % after 300 seconds. No PGSK-quenching was observed in control liposomes in the presence of ATP and Ag+. The additional presence of sodium azide led to minimal reduction of the PGSK-quenching as expected since sodium azide is not an inhibitor of P-type ATPases, but the quenching rate was similar to that of the same experimental condition with control liposomes.
The RND superfamily member CusA, as part of the tripartite CusCBA efflux pump, has been proposed to sequester Ag+ or Cu+ from either the cytoplasmic or periplasmic side of the inner membrane. The periplasmic transport of silver ions was implied from an in vitro assay where the quenching of a pH sensitive dye, 9-amino-6-chloro-2-methoxyacridine (ACMA), indicates acidification of the lumen of the proteoliposomes containing CusA when an inwardly directed pH was imposed. The same experiment with the CusA D405N variant, which was previously reported to be an inactive variant, also led to ACMA quenching, although at a slightly lower rate. Under application of an inwardly directed pH and a (negative inside), CusA-containing proteoliposomes showed a strong quenching of the incorporated PGSK dye, suggesting strong Ag+ influx.
The Major Facilitator Superfamily-(MFS-) type EmrAB-TolC pump has an analogous structural setup as the RND-type AcrAB-TolC pump. To examine the efflux of one of its substrates, carbonyl - cyanide m-chlorophenylhydrazone (CCCP), a plate-based susceptibility assay was used. The presence of the EmrAB-TolC pump confers lower susceptibility levels towards CCCP in E. coli, compared to cells not expressing the pump or cells expressing only the MFS component, indicating that EmrAB-TolC extrudes CCCP.
The work done in this study opens up a path towards investigation of drug and metal resistance in vitro. The methodologies to obtain proteoliposomal samples of multicomponent efflux pumps and subsequent measurements of drug/metal ion and H+ fluxes, as well as the determination of pump assembly are crucial for the future research on pump catalysis and transport kinetics. The in vivo drug-plate assays done in this work provide initial insights for future investigations of the drug susceptibility of E. coli expressing the MFS-type tripartite efflux pumps.
Die Funktion nukleärer Rezeptoren (NR) beruht auf einem empfindlichen Zusammenspiel zwischen ihren Domänen, Coregulatoren und Liganden. Die meisten Rezeptoren binden die DNA als Homo- oder Heterodimere und transregulieren die Gentranskription in Folge von Ligandenbindung. Klassische Assay-Systeme, die sich auf die Untersuchung der NR-Funktion oder auf die Charakterisierung von Substanzen richten, bilden nur die Coregulator-Rekrutierung zu isolierten NR-Ligandenbindungsdomänen (LBDs) ab und vernachlässigen dabei die NR:NR-Interaktion. Damit klammern sie die NR:NR-Wechselwirkung aus, obwohl die Rekrutierung von Cofaktoren durch allosterischen Crosstalk mit der Oligomerisierung verbunden ist. Dies war die Motivation dafür, Assay-Systeme zu entwickeln, welche die Untersuchung von NR-Interaktionen,
insbesondere der NR-Dimerisierung, und deren Modulation durch verschiedene Arten von Liganden ermöglichen. Im Rahmen dieser Doktorarbeit wird ein vielfältiges modulares Set von Assays für die Untersuchung der NR-Dimerisierung und NR-Coregulator-Rekrutierung vorgestellt und deren Anwendbarkeit auf eine Vielzahl von NRs demonstriert. Die Verwendung einer
rekrutierungsunfähigen RXRα-Variante mit einer mutierten AF-2-Domäne ermöglichte den spezifischen Nachweis der Coaktivatorrekrutierung durch PPARγ im Kontext des Heterodimers mit seinem obligatorischen Dimerpartner RXRα. Außerdem konnte gezeigt werden, dass die Aktivierung der RXRα LBD mit ihrem Agonisten SR11237 zu einer Destabilisierung des RXRα-Homodimers, aber zu einer Förderung der Bildung des Heterodimers mit der PPARγ LBD führte.
Ein zentrales Ergebnis war das Phänomen, dass der Einbau von PPARγ in das Heterodimer zu einem erheblichen Anstieg an Affinität gegenüber Coaktivatoren führt, auch in Abwesenheit von Liganden. Somit fördert die RXRα-Aktivierung die Coaktivator-Rekrutierung von PPARγ indirekt durch eine Verschiebung der Oligomerisierungspräferenz von RXRα in Richtung des Heterodimers. Zusätzlich wurde die Wirkung von Tetrac, einem nicht-klassischen Schilddrüsenhormon, auf PPARγ und RXRα untersucht und dessen Aktivierungsvermögen gegenüber beiden Rezeptoren mit einer deutlich vervielfachten Wirkung auf das Heterodimer demonstriert. Mit Hilfe des neu etablierten Cofaktor-Rekrutierungsscreens konnte die Dynamik
zwischen dem Nurr1 NR und 29 kanonischen Coregulatoren, von denen einige ligandenabhängig hohe Affinitäten zum Rezeptor aufwiesen, beleuchtet werden. Diese Interaktionen wurden
bidirektional durch eine Reihe von strukturell unterschiedlichen nicht-steroidalen Antirheumatika moduliert, die auch die Affinitäten sowohl des Nurr1-Homodimers als auch des Heterodimers mit der RXRα LBD beeinflussen konnten. Die Nurr1-Dimere zeigten zudem auch eine hohe Empfindlichkeit gegenüber dem Endocannabinoid Anandamid. Zusätzlich zu PPARγ, RXRα und Nurr1 wurden erste Schritte zur Untersuchung der TLX NR-Funktion unternommen. Unter Anwendung der entwickelten Assays konnte die Heterodimerbildung der TLX und der RXRα LBD
beschrieben und die ligandenabhängige Rekrutierung des Corepressors SMRT beobachtet werden.
Zusammenfassend beschreibt diese Arbeit einen Satz von Werkzeugen für die Untersuchung von ligandenabhängiger NR-Coregulator-Interaktion und Oligomerisierung. Auf diese Weise trug sie zu einer umfassenderen Identifizierung und Charakterisierung von NR-Liganden bei und stellt eine valide Basis für die weitere Assayentwicklung und Ligandendesign dar.
The deubiquitinase USP32 regulates non-proteolytic ubiquitination in the endosomal-lysosomal system
(2021)
The regulation of essential cellular processes requires tightly controlled and directed transport of proteins and membranes. The highly dynamic endosomal and lysosomal system forms the key network for exchange and trafficking of molecules with its early endosomes, recycling endosomes, late endosomes, lysosomes, and additionally autophagosomes.
In this system, the small GTPase Rab7 has an essential role at the late endosomal stage regulating vesicle transport, tethering, and fusion, and retromer mediated receptor recycling back to the trans-Golgi network (TGN). Thus, Rab7 is also important for autophagosomes and lysosomes.
Lysosomes do not only represent the end point of the degradation pathway with several feeder pathways. But these organelles are also a dynamic signaling hub for a variety of metabolic processes. The ever-important regulator of cellular biosynthetic pathways mTORC1 dynamically associates with lysosomes where it is activated. mTORC1 activation is a complex multi-step process where a series of signaling events converge in dependence of amino acid levels thereby enabling interactions between the lysosomal v-ATPase, Ragulator complex (consisting of LAMTOR1-5), and Rag GTPases.
Ubiquitin signals are involved in almost all cellular processes. With this, their regulatory mechanism is also described for the endosomal-lysosomal system as well as mTORC1 signaling. Deubiquitinases (DUBs) release conjugated ubiquitin from proteins and thereby maintain the dynamic state of the cellular ubiquitinome.
The ubiquitin-specific protease 32 (USP32) is a poorly characterized DUB with only emerging cellular function. However, its predicted domain structure includes two unique domains within the entire DUB family. It has been linked to the development of breast cancer and small cell lung cancer. Furthermore, overexpressed GFP-USP32 was localized at the TGN, and a global mass spectrometry-based DUB interactome study suggested an interaction with the retromer complex. Based on these data, USP32 was a very interesting candidate to study its cellular function in this PhD project.
To investigate the function without disease background, a polyclonal USP32 knockout (USP32KO) RPE1 cell line was generated using the CRISPR/Cas9 technology. First experiments revealed different protein expression levels in various cell lines, and a subcellular localization of USP32 at membranes of the Golgi and lysosomal compartments. In a subsequent SILAC-based ubiquitinome analysis potential substrates of USP32 were identified. Interestingly, various proteins of the endosomal-lysosomal system were detected with enriched non-proteolytic ubiquitination upon USP32 depletion.
The further characterization of Rab7 as USP32 substrate confirmed the USP32-sensitive ubiquitination of Rab7 at lysine (K) residues 191 and 194. The ubiquitination in USP32KO cells did not change the subcellular localization of Rab7, but enhanced the interaction with the effector protein RILP. This implied that Rab7 was either more active or RILP had higher affinity to ubiquitinated Rab7. The subsequent results verified this theory. The retromer mediated recycling of CI-M6PR back to the TGN was faster or more efficient in USP32-depleted cells.
Accompanying this, levels of hydrolases were enriched in lysosomes isolated from USP32KO cells. Notably, USP32 had no direct effect on expression level or assembly of the retromer complex itself.
The observed lysosomal phenotypes connected another identified substrate to the function of USP32 in the endosomal-lysosomal system: LAMTOR1. LAMTOR1 is a component of the Ragulator complex and thus involved in the activation of mTORC1 at the lysosomal surface. Similar as for Rab7, the first experiments to characterize LAMTOR1 as USP32 substrate confirmed the USP32-sensitive ubiquitination at K20 independent of amino acid availability. However, ubiquitination of LAMTOR1 decreased its lysosomal localization in untreated and amino acid starved USP32KO cells. The following label-free interactome study detected a reduced interaction of LAMTOR1 and subunits of the lysosomal v-ATPase upon loss of USP32. This resulted in a shifted subcellular localization of mTOR (subunit of mTORC1) away from lysosomes. Furthermore, direct substrates of mTORC1 were less or slower re-phosphorylated after long amino acid starvation and re-activation of mTORC1 in USP32KO cells indicating a reduced mTORC1 activity.
Both USP32-dependent regulations of Rab7 and LAMTOR1/Ragulator converged in enhanced autophagic processes analyzed by increased LC3 levels upon amino acid starvation and USP32 depletion.
In summary, the presented thesis described the diverse role of USP32 in the endosomal and lysosomal system, and contributes to the understanding of novel ubiquitin signals in this context.
RNA ist vor allem als Vermittler von Erbinformationen bekannt. Doch neben der Translation in Proteine ist sie auch maßgeblich an regulatorischen Prozessen in der Zelle beteiligt. So kommen in vielen Organismen Argonautenproteine vor, die zusammen mit microRNA einen Komplex bilden, der in der Lage ist, mRNA zu spalten oder auf andere Weise deren Translation zu unterdrücken. Da die Deregulierung von microRNA bei verschiedenen Krankheiten wie Krebs, Parkinson oder Alzheimer auftritt, wurden in dieser Arbeit Alkylanzien entwickelt, die zur besseren Inhibierung von microRNA beitragen sollen.
Als Alkylierungsmittel wurden ortho-Chinonmethide verwendet, die zunächst in geschützter Form synthetisiert wurden und nach Aktivierung mit einer Nukleobase reagieren können. Für die Erkennung der miRNA-Sequenz wurden diese zu einem Konjugat mit Peptid-Nukleinsäuren (PNAs) verbunden. Es wurden zwei Arten von Chinonmethid-Präkursoren hergestellt: Mit o Nitrobenzyl photolabil geschützte, die sich mit Licht der Wellenlänge 365 nm aktivieren lassen, und über ein Disulfid geschützte, die mithilfe eines Reduktionsmittels aktiviert werden. Die photolabil geschützten Derivate lassen sich damit gezielt örtlich und zeitlich aktivieren. Vom reduktiv aktivierbaren Präkursor wurden drei Derivate mit sterisch unterschiedlichen Resten am Disulfid (Benzyl-, Isopropyl- oder tert-Butyl-Rest) hergestellt, die einen Einfluss auf die Kinetik der Entschützung haben. Diese Derivate können nach Eintritt in eine Zelle durch die dort vorherrschende hohe Glutathion-Konzentration aktiviert werden, während sie extrazellulär unreaktiv sind.
Zunächst wurde die Kinetik eines photolabil geschützten Konjugats ohne RNA untersucht. Hier kommt es nach Bestrahlung zur Selbstalkylierung, bei der die Nukleobasen der PNA angegriffen werden. Bei 37 °C erfolgte dies mit einer Halbwertszeit von 0.43 h unter Annahme einer Reaktion 1. Ordnung. Die Kinetik der Alkylierung der komplementären RNA ließ sich durch zwei parallel ablaufende Reaktionen 1. Ordnung abbilden. Die Schnelle hatte eine Halbwertszeit von 0.42 h und die Langsame 11 h mit einer Ausbeute von 73 % nach 168 h. Bei Bestrahlung des Konjugats und erst anschließender Zugabe der RNA wurde ebenfalls eine Halbwertszeit von 11 h bei einer einzelnen Reaktionen 1. Ordnung erhalten. Dies lässt sich mit der Reversibilität mancher Reaktionsprodukte erklären. Die schnelle Reaktion entspricht der direkten Reaktion des Chinonmethids mit der RNA, die langsame entsteht durch Umlagerung von reversiblen Addukten.
Die Analyse der RNA-Alkylierung erfolgte mithilfe von denaturierender Polyacrylamid-Gelelektrophorese, bei der in Abhängigkeit der Gel-Temperatur scheinbar unterschiedliche Kinetiken gemessen wurden. Dies ist ebenfalls eine Folge der Reversibilität. Bei 57 °C kann ein Teil der Bindungen zwischen RNA und den Konjugaten brechen und es wird am Anfang der Reaktion eine geringere Ausbeute gemessen als bei 25 °C Geltemperatur. Die Ausbeute nach 168 h änderte sich jedoch nicht, da im Verlauf der Reaktion die reversiblen Addukte in irreversible umgewandelt werden.
Mit miRNA-20a als Ziel wurden mit einem 10mer Konjugat zunächst nur 13 % Ausbeute nach 72 h und mit einem 15mer Konjugat 41 % nach 75 h erreicht. Durch internen Einbau des Chinonmethid-Präkursors in die PNA, sodass es einem Adenosin der RNA gegenübersteht, konnte die Ausbeute auf 75 % nach 72 h gesteigert werden, da Adenosin bevorzugt alkyliert wird.
Bei den reduktiv aktivierbaren Chinonmethid-Präkursoren waren alle synthetisierten Konjugate in Puffer ohne Glutathion (GSH) stabil. Die Reihenfolge der Reaktionsgeschwindigkeit der Disulfidspaltung war bei 0.5 mM und 10 mM GSH: Benzyl > Isopropyl > tert-Butyl. Die Halbwertszeit bei 10 mM GSH betrug weniger als 5 min (Benzyl-Konjugat) bis 2 h (t Butyl Konjugat). Jedoch bildeten sich mit allen Konjugaten bei 10 mM GSH auch Addukte mit GSH.
Die Reaktivitätsreihenfolge blieb bei der Alkylierung von RNA erhalten. Allein das Benzyl-Konjugat erreichte bei einer GSH-Konzentration von 0.5 mM schon die gleiche Reaktionsgeschwindigkeit wie das photolabil geschützte Chinonmethid. Bei 10 mM GSH erreichten die Derivate zwar nach wenigen Stunden ihre maximale Ausbeute, diese betrug jedoch nur 23 % (tert-Butyl-Konjugat) bis 43 % (Benzyl-Konjugat), da die Chinonmethide auch durch GSH als Nukleophil abgefangen werden.
Mit einem Konjugat, das ein photolabiles Chinonmethid sowie Biotin trägt, wurde ein Fluoreszenzpulldown mit Cy5-markierter RNA durchgeführt. Hier zeigte die bestrahlte Probe eine deutlich höhere Fluoreszenz (6.8x), als eine unbestrahlte Vergleichsprobe. Bei einem Pulldown-Versuch mit miRNA-20a bzw. mit RISCs aus HeLa-Zelllysat konnte das Argonautenprotein jedoch nicht eindeutig mittels Westernblot nachgewiesen werden.
Anhand des reduktiv aktivierbaren Benzyl-Konjugats konnte gezeigt werden, dass sich das Konjugat in Zelllysat zersetzt und nur ein Teil zu Addukten mit Nukleobasen reagiert. Die Ursache wurde in der hydrolyselabilen Abgangsgruppe gesehen, sodass weitere photolabil geschützte Derivate mit Dimethylamino-, Trimethylammonium-, Pivaloylester- und Benzoylestergruppe synthetisiert wurden. Von diesen war nur das Benzoylester-Konjugat in der Lage, RNA mit 72 % Ausbeute nach 48 Stunden zu alkylieren. Zudem war es für mindestens 1 h in Zelllysat stabil.
Polyketides are highly valuable natural products, which are widely used as pharmaceuticals due to their beneficial characteristics, comprising antibacterial, antifungal, immunosuppressive, and antitumor properties, among others. Their biosynthesis is performed by large and complex multiproteins, the polyketide synthases (PKSs). This study solely focuses on the class of type I PKSs, which arrange all their enzymatic domains on one or more polypeptides. Despite their high medical value, little is known about mechanistic details in PKSs.
One central domain is the acyl transferase (AT), which is present in all PKSs and channels small acyl substrates into the enzyme. More precisely, the AT loads the substrates onto the essential acyl carrier protein (ACP), which subsequently shuttles the substrates and all intermediates for condensation and modification to additional domains to build the final polyketide.
Some PKSs use their domains several times during biosynthesis and work iteratively – these are called iterative PKSs. Others feature several sets of domains, each being used only once during biosynthesis – these PKSs are called modular PKSs. All PKSs or PKS modules consist of minimum three essential domains to connect the acyl substrates. Three modifying domains are optional and can enlarge the minimal set. According to the domain composition, the acyl substrate is fully reduced, partly reduced, or not reduced at all. This variation of modifying domains accounts for the huge structural and therefore functional variety of polyketides.
Even though the structure of fatty acids is not exactly reminiscent of polyketides, their biosynthetic pathways are closely related. Fatty acid biosynthesis is carried out by fatty acid synthases (FASs), which share many similarities with PKSs. Both megasynthases feature the same domains, performing the same reactions to connect and modify small acyl substrates. In contrast to PKSs, FASs always contain one full set of modifying domains which is used iteratively, leading to fully reduced fatty acids.
The present thesis extensively analyzes the AT of different PKSs in its substrate selectivity, AT-ACP domain-domain interaction, and enzymatic kinetic properties. The following key findings are revealed through comparison: 1.) ATs of PKSs appear slower than the ones of FASs, which may reflect the different scopes of biosynthetic pathways. Fatty acids as essential compounds in all organisms are needed in high amounts for physiological functions, whereas polyketides as secondary metabolites only require basal concentrations to take effect. 2.) The slower ATs from modular PKSs do not load non-native substrates even in absence of the native substrates. This is different to the faster ATs from iterative PKSs and FASs, which indicates high substrate specificity solely for the ATs from modular PKSs and emphasizes their role as gatekeepers in polyketide synthesis. 3.) The substrate selectivity can emerge in either the first or the second step of the AT-mediated ACP loading and is not assured by a hydrolytic proofreading function.
Moreover, a mutational study on the AT-ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) shows that single surface point mutations can influence AT-mediated reactions in a complex manner. Data reveals high enzyme kinetic plasticity of the AT-ACP interaction, which was also recently demonstrated for the interaction in a type II FAS.
Based on these findings, the mammalian FAS is engineered towards a modular PKS-like as- sembly line with the long-term goal to rationally synthesize new products. Basically, three important aspects need to be considered: 1.) AT’s loading needs to be splitted in specific loading of a priming substrate by a priming AT and in specific loading of an elongation substrate by an elongation AT. 2.) FAS-based elongation modules need to be designed with varying domain compositions for introducing functional groups in the product. 3.) Covalent and non-covalent linkers need to be designed for connection of priming and elongation modules.
This study focuses on the first aspect, splitting loading of priming and elongation substrates. An elongation substrate-specific AT is installed in the mammalian FAS via domain swapping. Since ATs from modular PKSs were proven to be substrate specific, these are used to exchange the mammalian FAS AT. This work demonstrates that it is extremely challenging to create stable and functional chimeras, but first essential steps are taken. Proper domain boundaries for AT swapping are established and a stable chimera with 70 % wild type AT activity is created. However, this chimera is only of limited value for application in an elongation module due to the intrinsic slow turnover rate of the wild type AT. Using another PKS AT, a stable elongation module is designed and analyzed in its activity in combination with a priming module. These experiments demonstrate that the loading of priming substrates are successfully suppressed in the elongation module, but nonetheless only minor turnover rates are detected in the assembly line.
...
Organ-on-a-chip technology has the potential to accelerate pharmaceutical drug development, improve the clinical translation of basic research, and provide personalized intervention strategies. In the last decade, big pharma has engaged in many academic research cooperations to develop organ-on-a-chip systems for future drug discoveries. Although most organ-on-a-chip systems present proof-of-concept studies, miniaturized organ systems still need to demonstrate translational relevance and predictive power in clinical and pharmaceutical settings. This review explores whether microfluidic technology succeeded in paving the way for developing physiologically relevant human in vitro models for pharmacology and toxicology in biomedical research within the last decade. Individual organ-on-a-chip systems are discussed, focusing on relevant applications and highlighting their ability to tackle current challenges in pharmacological research.
In den letzten 20 Jahren haben sich zunehmend Schülerlabore an Universitäten und Forschungszentren etabliert, um naturwissenschaftliche Kompetenzen von Schülern/innen aufzubauen und zu fördern. Das wachsende Feld der Schülerlaborforschung zeigt allerdings auf, dass die Eingangsvoraussetzungen, mit denen die Schüler/innen an der naturwissenschaftlichen Lernumgebung teilnehmen, einen starken Einfluss auf die Annahme sowie die Entwicklung von interessens-, selbstkonzept- und motivationsbezogenen Persönlichkeitsmerkmalen haben können. Diese Erkenntnisse lenken den Blick auf die Entwicklungsumwelt der Familien, in der die Eltern von Geburt an auf die kindliche Persönlichkeitsentwicklung und Lernprozesse einwirken. Die Integration des vielversprechenden familiären Kontexts in eine naturwissenschaftliche Lernumgebung wird seit 2008 anhand des Eltern-Kind-Projekts KEMIE® an der Ruhr-Universität Bochum umgesetzt, indem Eltern-Kind-Paare gemeinsam an alltagsnahen naturwissenschaftlichen Phänomenen experimentieren. Seit 2016 wird KEMIE® zudem an der Goethe-Universität Frankfurt am Main im Goethe-Schülerlabor Chemie durchgeführt.
Am Beispiel des Frankfurter KEMIE®-Projekts will die vorliegende Arbeit das Potential von Eltern-Kind-Interaktionen für naturwissenschaftliche Lernprozesse untersuchen. Mithilfe der Grounded-Theory-Methodologie wurde das zentrale Forschungsdesiderat der Eltern-Kind-Interaktion über die Projektjahre 2016/17 und 2017/18 herausgestellt sowie mögliche Erhebungsmethoden pilotiert. Im Projektjahr 2018/19 konnte dieses schließlich in einer Mixed-Methods-Studie multiperspektivisch beforscht werden. Dafür wurden Interviews, Beobachtungen und Pre-Posttest-Daten von 46 teilnehmenden Eltern-Kind-Paaren sowie weitere Kontrollgruppendaten von 202 Frankfurter Familien erhoben.
Um zunächst die naturwissenschaftliche Lernumgebung zu beschreiben, wurden die vorherrschenden Einflussfaktoren identifiziert, die zugehörigen Phänomene beschrieben sowie die prozess- und personenbezogenen Verhaltensmuster herausgestellt. Über letztere können die Eltern-Kind-Interaktionen in das SELE-Modell elterlicher Unterstützungsmaßnahmen eingeordnet werden, wobei sich das Ermöglichen von Selbsttätigkeit als herausragendes Merkmal für die naturwissenschaftliche Lernumgebung manifestiert. Zudem zeigt sich, dass das Projekt positive Effekte auf naturwissenschaftsbezogene Werteorientierungen der Kinder sowie deren Interesse an den Naturwissenschaften hat.
Anhand der Entwicklung eines induktiven Beobachtungsschemas, konnten die Familien schließlich über die Verhaltensmerkmale der Eltern-Kind-Interaktion gruppiert und Zusammenhänge zu sowohl den Vorbedingungen als auch den Konsequenzen für die psychischen Dispositionen der Kinder aufgedeckt werden. Erste Erkenntnisse dieser explorativen Erhebung zeigen, dass das akademische Selbstkonzept und die naturwissenschaftsbezogenen Werteorientierungen der Eltern und Kinder die Verhaltensmuster in der naturwissenschaftlichen Umgebung determinieren. Auf der anderen Seite können die Kinder gerade hinsichtlich dieser beiden Faktoren am meisten profitieren. Dabei können diese positiven Effekte auf eine Regulation seitens der Eltern, das gemeinsame Ausführen von Tätigkeiten sowie den Einbezug in die Gestaltung der Lernumgebung zurückgeführt werden.
Osteopontin levels in human milk are related to maternal nutrition and infant health and growth
(2021)
Background: Osteopontin (OPN) is a glycosylated phosphoprotein found in human tissues and body fluids. OPN in breast milk is thought to play a major role in growth and immune system development in early infancy. Here, we investigated maternal factors that may affect concentrations of OPN in breast milk, and the possible associated consequences for the health of neonates. Methods: General characteristics, health status, dietary patterns, and anthropometric measurements of 85 mothers and their babies were recorded antenatally and during postnatal follow-up. Results: The mean concentration of OPN in breast milk was 137.1 ± 56.8 mg/L. Maternal factors including smoking, BMI, birth route, pregnancy weight gain, and energy intake during lactation were associated with OPN levels (p < 0.05). Significant correlations were determined between body weight, length, and head circumference, respectively, and OPN levels after one (r = 0.442, p = < 0.001; r = −0.284, p = < 0.001; r = −0.392, p = < 0.001) and three months (r = 0.501, p = < 0.001; r = −0.450, p = < 0.001; r = −0.498, p = < 0.001) of lactation. A negative relation between fever-related infant hospitalizations from 0–3 months and breast milk OPN levels (r = −0.599, p < 0.001) was identified. Conclusions: OPN concentrations in breast milk differ depending on maternal factors, and these differences can affect the growth and immune system functions of infants. OPN supplementation in infant formula feed may have benefits and should be further investigated.
Die Kommunikation von Zellen mit ihrer Umgebung wird durch Rezeptorproteine arrangiert, die sich in der Plasmamembran befinden. Membranrezeptoren werden durch die Bindung von extrazellulären Liganden, Pathogenen oder Zell-Zell-Interaktionen aktiviert, wodurch die Bildung eines aktiven Zustands gefördert wird, der eine intrazelluläre Reaktion einleitet. Eine Beschreibung auf molekularer Ebene, wie sich Membranrezeptoren in Proteinanordnungen organisieren und wie diese Proteinanordnungen eine spezifische funktionelle Aufgabe ausführen, ist der Ausgangspunkt für das Verständnis der molekularen Mechanismen, die Gesundheit und Krankheit zugrunde liegen.
Die Fluoreszenzmikroskopie gibt Aufschluss über die Lage von Proteinen in Zellen, und mit der Einführung der höchstauflösenden Mikroskopie wurde der Nachweis einzelner Proteingruppierungen möglich. Eine Einschränkung der meisten Methoden der höchstauflösenden Mikroskopie ist, dass einzelne Komponenten einer Proteingruppierung optisch nicht aufgelöst werden können, was an der geringen Größe und dichten Packung der Bestandteile im Vergleich zur erreichbaren räumlichen Auflösung liegt. Eine Lösung, die für Einzelmolekül-Lokalisierungsmethoden gezeigt wurde, besteht darin, zusätzliche experimentelle Informationen in die Analyse zu implementieren, also „die Aufl sungsgrenze der höchstauflösenden Mikroskopie zu umgehen". Bei der Einzelmolekül-Bildgebung kann diese zusätzliche Information zum Beispiel die Kinetik von mehrfachen und wiederkehrenden
Emissionsereignissen sein, die bei einzelnen Fluorophoren beobachtet werden, was als "Blinken" bezeichnet wird. Das Ziel dieser Arbeit war die Entwicklung einer höchstauflösenden Fluoreszenzmikroskopiemethode zur Detektion von Proteinmonomeren und -dimeren in der Plasmamembran von Zellen durch die Verwendung der kinetischen Information.
Im ersten Teil dieser Arbeit wurden photoschaltbare fluoreszierende Proteine als Reporter verwendet, deren photoschaltbare Kinetik mit kinetischen Gleichungen analysiert wurden.
Synthetische, genetische und zelluläre Referenzproteine wurden konstruiert und dienten als Kalibrierungsreferenzen für monomere und dimere Proteine.
Im zweiten Teil dieser Arbeit wurde das kinetische Modell, das zur Annäherung des Häufigkeitshistogramms von Blinkereignissen einzelner Fluorophore verwendet wird, auf Oligomere höherer Ordnung erweitert. Ein Vergleich mit einem zuvor entwickelten Modell zeigte, dass das erweiterte Modell genauere Ergebnisse für Oligomere höherer Ordnung und Mischungen verschiedener Oligomere liefert. Zusätzlich wird die Anwesenheit von unerkannten Oligomeren berücksichtigt. Die erweiterte Theorie bietet somit die Grundlage, um größere Oligomere und Mischungen unterschiedlicher Stöchiometrie mit besserer Genauigkeit zu untersuchen.
Im dritten Teil dieser Arbeit wurde eine Methode zur stöchiometrischen endogenen Markierung von Proteinen verwendet, um zwei Rezeptortyrosinkinasen, MET und EGFR, mit einem photoschaltbaren fluoreszierenden Protein zu markieren. Das Vorkommen von monomerem und dimerem MET-Rezeptor wurde auf der Plasmamembran von HEK293T- Zellen mittels quantitativer höchstauflösender Mikroskopie bestimmt. Der Diffusionskoeffizient und der Diffusionsmodus des MET-Rezeptors in lebenden HEK293T-Zellen wurden mit
Einzelpartikelverfolgung gemessen. Dieser Teil der Arbeit zeigte, dass die Kombination von CRISPR/Cas12a-gestützter endogener Markierung und Einzelmolekül-Lokalisierungsmikroskopie ein leistungsfähiges Werkzeug zur Untersuchung der molekularen Organisation und Dynamik von Membranproteinen ist.
Im vierten Teil dieser Arbeit wurde die Einzelmoleküldatenanalyse durch ein Softwaretool beschleunigt, das eine automatisierte und unvoreingenommene Detektion von Einzelmolekül-Emissionsereignissen ermöglicht. Der Anteil von Monomeren und Dimeren von fluoreszierenden Reportern wurde durch die Implementierung eines neuronalen Netzwerks bestimmt (die Software wurde von Alon Saguy geschrieben; Gruppe von Prof. Yoav Shechtman, Technion, Israel). Der oligomere Zustand der monomeren und dimeren Referenzproteine CD86 und CTLA-4 wurde erfolgreich bestimmt. Die automatisierte Detektion einzelner Proteingruppierungen ermöglichte die Analyse von MET-mEos4b in einzelnen Zellen, wodurch die Heterogenität zwischen den Zellen bestimmt und das Expressionsniveau des Rezeptors mit der Dimerisierung korreliert werden konnte.
Zusammenfassend wurden in dieser Arbeit Ergebnisse zu elementaren Aspekten hin zu einer molekularen Quantifizierung von Proteinzahlen mittels Einzelmolekül-
Lokalisationsmikroskopie generiert, die fluoreszierende Reporter, stöchiometrische Markierung von zellulären Proteinen und Bildanalyse umfassen. Das Potential dieser
Entwicklungen wurde anhand der Beobachtung der Liganden-induzierten Verschiebung von monomeren zu dimeren MET-Rezeptoren in einzelnen HEK293T-Zellen gezeigt.
Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4.
Pancreatic cancer is a common malignant tumor with a high incidence and mortality rate. The prognosis of patients with pancreatic cancer is considerably poor due to the lack of effective treatment in clinically. Despite numerous studies have revealed that baicalein, a natural product, is responsible for suppressing multiple cancer cells proliferation, motility and invasion. The mechanism by which baicalein restraining pancreatic cancer progression remains unclear. In this study, we firstly verified that baicalein plays a critical role in inhibiting pancreatic tumorigenesis in vitro and in vivo. Then we analyzed the alteration of microRNAs (miRNAs) expression levels in Panc-1 cells incubated with DMSO, 50 and 100 μM baicalein by High-Throughput sequencing. Intriguingly, we observed that 20 and 39 miRNAs were accordingly up- and down-regulated through comparing Panc-1 cells exposed to 100 μM baicalein with the control group. Quantitative PCR analysis confirmed that miR-139-3p was the most up-regulated miRNA after baicalein treatment, while miR-196b-5p was the most down-regulated miRNA. Further studies showed that miR-139-3p induced, miR-196b-5p inhibited the apoptosis of Panc-1 cells via targeting NOB1 and ING5 respectively. In conclusion, we demonstrated that baicalein is a potent inhibitor against pancreatic cancer by modulating the expression of miR-139-3p or miR-196b-5p.
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
Background and Purpose: The cyclic nucleotides cAMP and cGMP are ubiquitous second messengers regulating numerous biological processes. Malfunctional cNMP signalling is linked to diseases and thus is an important target in pharmaceutical research. The existing optogenetic toolbox in Caenorhabditis elegans is restricted to soluble adenylyl cyclases, the membrane-bound Blastocladiella emersonii CyclOp and hyperpolarizing rhodopsins; yet missing are membrane-bound photoactivatable adenylyl cyclases and hyperpolarizers based on K+ currents.
Experimental Approach: For the characterization of photoactivatable nucleotidyl cyclases, we expressed the proteins alone or in combination with cyclic nucleotide-gated channels in muscle cells and cholinergic motor neurons. To investigate the extent of optogenetic cNMP production and the ability of the systems to depolarize or hyperpolarize cells, we performed behavioural analyses, measured cNMP content in vitro, and compared in vivo expression levels.
Key Results: We implemented Catenaria CyclOp as a new tool for cGMP production, allowing fine-control of cGMP levels. We established photoactivatable membrane-bound adenylyl cyclases, based on mutated versions (“A-2x”) of Blastocladiella and Catenaria (“Be,” “Ca”) CyclOp, as N-terminal YFP fusions, enabling more efficient and specific cAMP signalling compared to soluble bPAC, despite lower overall cAMP production. For hyperpolarization of excitable cells by two-component optogenetics, we introduced the cAMP-gated K+-channel SthK from Spirochaeta thermophila and combined it with bPAC, BeCyclOp(A-2x), or YFP-BeCyclOp(A-2x). As an alternative, we implemented the B. emersonii cGMP-gated K+-channel BeCNG1 together with BeCyclOp.
Conclusion and Implications: We established a comprehensive suite of optogenetic tools for cNMP manipulation, applicable in many cell types, including sensory neurons, and for potent hyperpolarization.
We investigated the folding kinetics of G-quadruplex (G4) structures by comparing the K+-induced folding of an RNA G4 derived from the human telomeric repeat-containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real-time NMR spectroscopy. While DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs. DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with 19F bound to C2′ in arabino configuration adopt exclusively anti conformations for χ. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.
The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.
Extracellular signal-regulated kinase 3 (ERK3), known also as mitogen-activated protein kinase 6 (MAPK6), is an atypical member of MAPK kinase family, which has been poorly studied. Little is known regarding its function in biological processes, yet this atypical kinase has been suggested to play important roles in the migration and invasiveness of certain cancers. The lack of tools, such as a selective inhibitor, hampers the study of ERK3 biology. Here, we report the crystal structure of the kinase domain of this atypical MAPK kinase, providing molecular insights into its distinct ATP binding pocket compared to the classical MAPK ERK2, explaining differences in their inhibitor binding properties. Medium-scale small molecule screening identified a number of inhibitors, several of which unexpectedly exhibited remarkably high inhibitory potencies. The crystal structure of CLK1 in complex with CAF052, one of the most potent inhibitors identified for ERK3, revealed typical type-I binding mode of the inhibitor, which by structural comparison could likely be maintained in ERK3. Together with the presented structural insights, these diverse chemical scaffolds displaying both reversible and irreversible modes of action, will serve as a starting point for the development of selective inhibitors for ERK3, which will be beneficial for elucidating the important functions of this understudied kinase.
Lead-optimization strategies for compounds targeting c-Myc G-quadruplex (G4) DNA are being pursued to develop anticancer drugs. Here, we investigate the structure-activity- relationship (SAR) of a newly synthesized series of molecules based on the pyrrolidine-substituted 5-nitro indole scaffold to target G4 DNA. Our synthesized series allows modulation of flexible elements with a structurally preserved scaffold. Biological and biophysical analyses illustrate that substituted 5-nitroindole scaffolds bind to the c-Myc promoter G-quadruplex. These compounds downregulate c-Myc expression and induce cell-cycle arrest in the sub-G1/G1 phase in cancer cells. They further increase the concentration of intracellular reactive oxygen species. NMR spectra show that three of the newly synthesized compounds interact with the terminal G-quartets (5′- and 3′-ends) in a 2 : 1 stoichiometry.