Refine
Has Fulltext
- yes (123)
Is part of the Bibliography
- yes (123)
Year of publication
- 2021 (123) (remove)
Document Type
- Doctoral Thesis (123) (remove)
Keywords
- Melanom (3)
- Angst (2)
- Angststörung (2)
- Arteriosklerose (2)
- Chlamydia trachomatis (2)
- Chromatin (2)
- DNA repair (2)
- Entzündung (2)
- Fluoreszenzmikroskopie (2)
- Fuchsbandwurm (2)
- Furcht (2)
- Furchtkonditionierung (2)
- G-Protein gekoppelte Rezeptoren (2)
- GPCR (2)
- Genetik (2)
- HUWE1 (2)
- Induzierte pluripotente Stammzelle (2)
- MAP-Kinase (2)
- MYC (2)
- MYCN (2)
- Maus (2)
- Mausmodell (2)
- Megakaryozyt (2)
- Multiple Sklerose (2)
- Multiples Myelom (2)
- Neisseria meningitidis (2)
- Neuroblastom (2)
- Neurodegeneration (2)
- Plasmozytom (2)
- Resistenz (2)
- Serotonin (2)
- Sphingolipide (2)
- Stammzelle (2)
- Thrombozyt (2)
- Tissue Engineering (2)
- Transcription (2)
- TrkB (2)
- Ubiquitin (2)
- Zellskelett (2)
- cAMP (2)
- cell wall (2)
- host-pathogen interaction (2)
- neuroblastoma (2)
- neuropathy (2)
- regulation (2)
- 177Lu-OPS201 (1)
- 3D models (1)
- 3D tissue model (1)
- 3D-Gewebemodell (1)
- 5`-UTR (1)
- 68Ga-OPS202 (1)
- 7T (1)
- ACC (1)
- ADGRL3 (1)
- ADHD (1)
- AI (1)
- ATF5 (1)
- ATGL (1)
- Acetylation (1)
- Acute myeloid leukemia (AML) (1)
- Adhesion GPCR (1)
- Adipose (1)
- Adipose-Derived Stromal/Stem Cells (1)
- Adrenalin (1)
- Adrenokortikales Karzinom (1)
- Advanced Therapy Medicinal Products (ATMP) (1)
- Adventitia (1)
- Agarose (1)
- Aiolos (1)
- Airway epithelia (1)
- Alzheimerkrankheit (1)
- Angstverhalten (1)
- Anthropocene (1)
- Anti-CD52-Antikörper (1)
- Antibody (1)
- Antigen CD44 (1)
- Antioxidantien (1)
- Aortenaneurysma (1)
- Arbeitsteilung (1)
- Arid biomes (1)
- Arrestine (1)
- Artesunate (1)
- Arzneimittel (1)
- Arzneimitteldesign (1)
- Arzneimittelzulassung (1)
- Aspergillus fumigatus (1)
- Atherosklerose (1)
- Aufmerksamkeit (1)
- Aufmerksamkeitsdefizit-Syndrom (1)
- Aurora-A (1)
- Aussterbedynamik (1)
- Autoantikörper (1)
- B cells (1)
- BDNF (1)
- BRCA1 (1)
- BTB domain (1)
- Bacillus subtilis (1)
- Bahnung (1)
- Bakterienzellwand (1)
- Basal Ganglia (1)
- Basalganglien (1)
- Bildanalyse (1)
- Biodistribution (1)
- Biogenese (1)
- Biolayerinterferometrie (1)
- Biologie (1)
- Bioluminescence resonance energy transfer (1)
- Biomarker (1)
- Biomaterial (1)
- Bioprinting (1)
- Bioreactor (1)
- Bioreaktor (1)
- Bitopic Ligands (1)
- Bitopische Liganden (1)
- Blickbewegung (1)
- Blickinteraktion (1)
- Blut-Rückenmarkschranke (1)
- Bordetella (1)
- Brain (1)
- Brain-derived neurotrophic factor (1)
- C. elegans (1)
- CCR4 (1)
- CD28 (1)
- CD28-SA (1)
- CD44 (1)
- CD84 (1)
- CDK4 Inhibitor (1)
- CDK4 inhibitor (1)
- CIDP (1)
- CMT1A (1)
- Caenorhabditis elegans (1)
- Camponotus rufipes (1)
- Candida albicans (1)
- Cardiac (1)
- Cardiomyocyte (1)
- Cartilage (1)
- Cartilage Tissue Engineering (1)
- Cataglyphis (1)
- Ccr4-Not (1)
- Celecoxib (1)
- Cell-Assisted Lipotransfer (1)
- Ceramide (1)
- Charcot-Marie-Tooth 1A (1)
- Chemotaxis (1)
- Chimeric antigen receptor (CAR) (1)
- Chimpanzee (1)
- Chronobiologie (1)
- Ciliary neurotrophic factor (1)
- Click-Chemie (1)
- Co-Kultur (1)
- Co-culture system (1)
- Cochlear-Implantat (1)
- Comoé National Park (1)
- Complement system (1)
- Conditioning (1)
- Cortico-striatal projection neurons (1)
- Craniosynostosis (1)
- Cushing-Syndrom (1)
- Cuticular transpiration (1)
- Cuticular waxes (1)
- Cyclo-AMP (1)
- Cystein (1)
- Cytoskeleton (1)
- DC (1)
- DNS-Reparatur (1)
- DOT1 (1)
- DTI (1)
- Danio rerio (1)
- Decapping (1)
- Deeplearning (1)
- Deletion (1)
- Dendritische Zelle (1)
- Depletion (1)
- Depression (1)
- Diabetes mellitus (1)
- Differential scanning calorimetry (1)
- Diffusionsgewichtete Magnetresonanztomografie (1)
- Drosophila (1)
- Drug resistance (1)
- Duplikation (1)
- E2 (1)
- ECAP (1)
- ECG-gated PET (1)
- EEG (1)
- Early-Life Stress (1)
- Echinococcus multilocularis (1)
- Effect anticipation (1)
- Effektantizipation (1)
- Einzelmolekülmikroskopie (1)
- Elektrostimulation (1)
- Entwicklung (1)
- Eosinophiler Granulozyt (1)
- Epigenetic (1)
- Epigenetik (1)
- Evolution (1)
- Expansion microscopy (1)
- Expansionsmikroskopie (1)
- Experimentelle autoimmune Enzephalomyelitis (1)
- Exposition (1)
- Expositionslernen (1)
- Expositionstherapie (1)
- Extinktion (1)
- Extracellular Vesicles (1)
- FLT3 inhibitor (1)
- FMS-like tyrosine kinase 3 (FLT3) (1)
- FRET (1)
- FT-IR-Spektroskopie (1)
- Ferroptosis (1)
- Fertilization in angiosperm (1)
- Fibromyalgia (1)
- Fibromyalgie (1)
- Fitness (1)
- Flavonoide (1)
- Flippase (1)
- Flow (1)
- Fluconazol (1)
- Fluconazole (1)
- Fluorescence correlation spectroscopy (1)
- Fluss (1)
- Folliculin (1)
- Foxo1 (1)
- Fragmentscreening (1)
- Functional analyses (1)
- Furagieraktivität (1)
- Furchtgeneralisierung (1)
- Förster Resonanz Energie Transfer (1)
- G glycoprotein (1)
- G-Protein gekoppelter Rezeptor (1)
- G-protein-coupled receptors (1)
- GMP (1)
- Gaze interaction (1)
- Gefäßwand (1)
- Gefäßwand-residente Stamm- und Vorläuferzellen (1)
- Gehirn (1)
- Gen BRCA 1 (1)
- Genetics (1)
- Genexpression (1)
- Genotoxicity (1)
- Genotoxizität (1)
- Genregulation (1)
- Gewebekultur (1)
- Glia (1)
- Glioblastom (1)
- Gliom (1)
- Glucose Starvation (1)
- Glutathion (1)
- Glycinrezeptor (1)
- Glycophyten (1)
- Glykomodifizierung (1)
- Glykosylierung (1)
- Gonococcal invasion (1)
- GvHD (1)
- H2A.Z (1)
- HECTD1 (1)
- HFpEF (1)
- Halophyten (1)
- Halophytes (1)
- Heart (1)
- Heart development (1)
- Heat stress (1)
- Helicobacter pylori (1)
- Herz (1)
- Herzinfarkt (1)
- Heubacillus (1)
- High-thropughput screening (1)
- Hippo pathway (1)
- Hippocampus (1)
- Hirnhaut (1)
- Histon-Methyltransferase (1)
- Histonacetyltransferase (1)
- Histone (1)
- Histone Acetylation (1)
- Histone modification (1)
- Histone variant (1)
- Hochauflösungsmikroskopie (1)
- Hochdurchsatz-Screening (1)
- Hornhaut (1)
- Huh6 (1)
- Human Transcriptome (1)
- Human-pathogenic (1)
- Hyaliner Knorpel (1)
- Hyaluronsäure (1)
- Hydrogel (1)
- Hypoxia (1)
- Hypoxie (1)
- Hämophilie A (1)
- Höhenangst (1)
- IKZF1 (1)
- IKZF3 (1)
- IRAK2 (1)
- Ideomotor gaze control (1)
- Ideomotorik (1)
- Ideomotorische Blickkontrolle (1)
- Ikaros (1)
- Immunological synapse (1)
- Immunozyt (1)
- Immunsystem (1)
- Immunzellrekrutierung (1)
- In-vitro-Kultur (1)
- Individualisierte Medizin (1)
- Infektion (1)
- Inflammation (1)
- Inhibitor (1)
- Insektenstaaten (1)
- Insulin (1)
- Interleukin (1)
- Interleukin 5 (1)
- Interleukin-4 Antagonist (1)
- Intracellular bacteria (1)
- Intrazelluläre Bakterien (1)
- Ischemia (1)
- Karotis-Intima-Media-Dicke (1)
- Kleine Proteine (1)
- Kleinkern (1)
- Knorpel (1)
- Kofaktorbindung (1)
- Kognition (1)
- Kognitive Beeinträchtigung (1)
- Komplement (1)
- Konditionierung (1)
- Konnektivitätsschätzung (1)
- Kopienzahlvariation (1)
- Kraniosynostose (1)
- Krebsforschung (1)
- Kutikula (1)
- Kutikularwachs (1)
- Künstliche Intelligenz (1)
- LPS Biosynthese (1)
- Labeling (1)
- Langerhans cells (1)
- Lasiocarpin (1)
- Latrophilin (1)
- Lebendzellmikroskopie (1)
- Leber-Metabolismus (1)
- Leishmania (1)
- Lenalidomid (1)
- Lernen (1)
- Letalität (1)
- Ligand <Biochemie> (1)
- Lipid Raft (1)
- Lipid Transfer Protein (1)
- Lipide (1)
- Lipolysis (1)
- Lung (1)
- Lunge (1)
- M1 Muscarinic Receptor (1)
- MEK5/ERK5 (1)
- MGMT (1)
- MIZ1 (1)
- MS2-affinity purification and RNA-seq (1)
- Macrophages (1)
- Magnetresonanztomographie (1)
- Makrophage (1)
- Marketing authorisation of pharmaceuticals (1)
- Mauerbiene (1)
- Mechanical deformation (1)
- Mechanismus (1)
- Medium spiny neurons (1)
- Megakaryocyte (1)
- Meninges (1)
- Merkel cell carcinoma (1)
- Merkel cell polyomavirus (1)
- Mesenchymal stem cell (1)
- Mesenchymal stromal cells (1)
- Mesenchymzelle (1)
- Metabolism (1)
- Metabolismus (1)
- Methylierung (1)
- MgrB (1)
- Microglia (1)
- Micronucleus (1)
- Microvesicle (1)
- Mikroglia (1)
- Mitochondria (1)
- Mitochondrien (1)
- Mitochondrium (1)
- Mittelohrimplantat (1)
- Model Organism (1)
- Modellorganismus (1)
- Modellsystem (1)
- Modifizierung (1)
- Monozyt (1)
- Morbus Alzheimer (1)
- Motor learning (1)
- Motorisches Lernen (1)
- Mouse Model (1)
- Mucormykose (1)
- Multiproteinkomplex (1)
- Mundschleimhautzellen (1)
- Muscarinrezeptor (1)
- Mutagenität (1)
- Myb-MuvB complex (1)
- Myeloid (1)
- Myokardinfarkt (1)
- N-Myc (1)
- NK cell (1)
- NK-DC cross-talk (1)
- NRF2 (1)
- NTRK fusions (1)
- Nasenschleimhaut (1)
- Natürliche Killerzelle (1)
- Neisseria gonorrhoeae (1)
- Neuartige Arzneimittel (1)
- Neuralgie (1)
- Neuroanatomie (1)
- Neuroethologie (1)
- Neuroinflammation (1)
- Neuronales visuelles System (1)
- Neuropathie (1)
- Neuropathischer Schmerz (1)
- Neuropeptide (1)
- Neuroprotektivum (1)
- Neuroscience (1)
- Neutrophil (1)
- Neutrophil granulocyte (1)
- Neutrophiler Granulozyt (1)
- Nozizeption (1)
- Nucleotide excision repair (1)
- Nukleotid-Exzisions-Reparatur (1)
- O(6)-Methylguanine-DNA Methyltransferase (1)
- Obesity (1)
- Opiatrezeptor (1)
- Opioide (1)
- Oxidativer Stress (1)
- Oxygen partial pressure (1)
- P4-ATPase (1)
- PAF1c (1)
- PDE (1)
- PRR (1)
- PVM (1)
- Palbociclib (1)
- Parc National de la Comoé (1)
- Parkinson Krankheit (1)
- Parkinson's disease (1)
- Pathogenität (1)
- Pathophysiologie (1)
- Pathophysiologische Mechanismen (1)
- Pattern-Recognition-Receptors (1)
- Persistence (1)
- Pertussis (1)
- Pflanzenaussterben (1)
- Phasenvariation (1)
- Phenotypic switch (1)
- Phosphodiesterase (1)
- Pilzkörper (1)
- PinT (1)
- Plant cuticle (1)
- Plasmamembran (1)
- Plastizität (1)
- Pneumoviruses (1)
- Pollen (1)
- Positronen-Emissions-Tomografie (1)
- Preclinical studies (1)
- Primäre Ziliendyskinesie (1)
- Primärelement (1)
- Primärzellen (1)
- Progenitor (1)
- Protein purification (1)
- Protein-Protein-Interaktion (1)
- Protein-Protein-Wechselwirkung (1)
- Proteinkinase D (1)
- Proteinmodifizierung (1)
- Präklinische Studien (1)
- Präzisionsmedizin (1)
- Psychotherapie (1)
- Punktmutation (1)
- Pyrrolizidinalkaloide (1)
- QPCR (1)
- Qualitätsindikator (1)
- RNS-Interferenz (1)
- RSV (1)
- Receptor dynamics (1)
- Regenerative Medicine (1)
- Regulation (1)
- Regulatorischer T-Lymphozyt (1)
- Resilienz (1)
- Resistance (1)
- Respiratorisches System (1)
- Rezeptorblocker (1)
- Rezidiv (1)
- Rhizopus arrhizus (1)
- Ribonuclease H2 (1)
- Riboregulation (1)
- Risikofaktoren (1)
- Rossameise (1)
- Röntgenkristallographie (1)
- SLC2A3 (1)
- SMN (1)
- Salmonella (1)
- Salmonella Typhimurium (1)
- Scaffold bone implant (1)
- Schimpanse (1)
- Schizosaccharomyces pombe (1)
- Schlaganfall (1)
- Schließzellfunktion (1)
- Secretion (1)
- Sekundäre Inflammation (1)
- Selective extraction (1)
- Senecionin (1)
- Seneciphyllin (1)
- Sequenzwiederholung (1)
- Serotonindefizienz (1)
- Signaltransduktion (1)
- Sleeping Beauty Transposon System (1)
- Small RNA (1)
- Small angle X-ray scattering (1)
- Social action effects (1)
- Sociomotor gaze control (1)
- Soziale Handlungseffekte (1)
- Soziomotorische Blickkontrolle (1)
- SteC (1)
- Sterblichkeit (1)
- Stiff-Person Syndrom (1)
- Stiff-person syndrome (1)
- Stress (1)
- Striatum (1)
- Strukturbiologie (1)
- Superresolution microscopy (1)
- Survival Motor Neuron (1)
- Synaptic plasticity (1)
- Synchronitätsmessung (1)
- T cell engaging Antibody (1)
- TFIIH (1)
- TH2 Immunantwort (1)
- TLR signaling (1)
- Telomerase (1)
- Thebain (1)
- Therapie (1)
- Therapieresistenz (1)
- Thrombopoiesis (1)
- Tight junction (1)
- Tissue engineering (1)
- Toll-like-Rezeptoren (1)
- TraDIS (1)
- Transkription (1)
- Transkriptionsfaktor (1)
- Transkriptomanalyse (1)
- Transkriptomik anlyze (1)
- Transpiration <Pflanzen> (1)
- Transposon (1)
- Treg (1)
- Trend (1)
- Trophic Factors (1)
- Trypanosoma Brucei (1)
- Trypanosoma brucei (1)
- UBE2S (1)
- Ubiquitin-PA (1)
- Ubiquitin-Protein-Ligase (1)
- Ubiquitin-conjugating enzyme (1)
- Ubiquitinierung (1)
- VW-SCs (1)
- Validierung (1)
- Verhalten (1)
- Verhaltenskontrolle (1)
- Very-long-chain aliphatic (1)
- Vesikelbildung (1)
- Vessel wall (1)
- Virtuelle Realität (1)
- Visuelle Aufmerksamkeit (1)
- Vorläufer (1)
- Water stress (1)
- Wirkstoffdesign (1)
- X-ray crystallography (1)
- X-ray diffraction (1)
- Yeast (1)
- Zellzyklus (1)
- Zink-Finger-Proteine (1)
- aGPCR (1)
- adhesion-GPCR (1)
- adrenal gland (1)
- adrenergic receptor (1)
- adrenocortical carcinoma (1)
- airways (1)
- allogeneic stem cell transplantation (1)
- antimicrobial (1)
- anxiety disorders (1)
- articular cartilage progenitor cells (1)
- atopic diseases (1)
- attributable fraction (1)
- attributable risk (1)
- autoantibody (1)
- autophagocytose (1)
- autophagy (1)
- bacterial lipid rafts (1)
- behavior (1)
- beta-Arrestin2 (1)
- bicuculline (1)
- binding mode (1)
- biochemistry (1)
- biodistribution (1)
- bioimage analysis (1)
- biokinetics (1)
- biolayerinterferometry (1)
- bioreactor (1)
- blood nerve barrier (1)
- bone marrow (1)
- bumblebee*s (1)
- cancer (1)
- candida (1)
- candida albicans (1)
- cartilage (1)
- chlamydia (1)
- chondrogene Differenzierung (1)
- circadian rhythms (1)
- claudin-12 (1)
- co-culture (1)
- cochlea implant (1)
- cofactorbinding (1)
- compartments (1)
- corneal confocal microscopy (1)
- correlation (1)
- cushing's syndrome (1)
- cytokine release syndrome (1)
- cytoskeleton (1)
- dCIRL (1)
- dSTORM (1)
- developmental differentiation (1)
- diabetic cardiomyopathy (1)
- diagnostics (1)
- diastolic dysfunction (1)
- diet (1)
- disease model (1)
- distance (1)
- distribution (1)
- double-strand break repair (1)
- drug repurposing (1)
- dual RNA-seq (1)
- ecology (1)
- effector protein (1)
- electron microscopy (1)
- eosinophil (1)
- evozierte Potentiale (1)
- exposition training (1)
- expression (1)
- extinction dynamics (1)
- fabry disease (1)
- fear conditioning (1)
- fibromyalgia sydrome (1)
- foraging activity (1)
- foxtapeworm (1)
- fragment screening (1)
- functional membrane microdomains (1)
- functional studies (1)
- fungus (1)
- genotoxicity (1)
- germinative cell (1)
- gezielte Therapie (1)
- glycine receptor autoantibodies (1)
- glycophytes (1)
- guard cells (1)
- habitat (1)
- hematopoiesis (1)
- hippocampus (1)
- homoFRET (1)
- honeybee (1)
- honeybee*s (1)
- huh6 (1)
- iPSC (1)
- in vitro Kulturmodelle (1)
- in vitro Testsystem (1)
- interleukin-5 signaling (1)
- internal dosimetry (1)
- interphase gap (1)
- interpulse interval (1)
- intraoperative Ankopplungseffizienz (1)
- kinase (1)
- labeling techniques (1)
- lasiocarpine (1)
- learning (1)
- lipid rafts (1)
- mechanistische Modellierung (1)
- melanoma dedifferentiation (1)
- membrane dynamics (1)
- metabolic theory of ecology (1)
- microelectrode array (1)
- mucormycosis (1)
- myelin barrier (1)
- neuroethology (1)
- neuromodulation (1)
- neuronal visual system (1)
- neuropeptides (1)
- neutrophil (1)
- nociception (1)
- nutrients (1)
- nutrition (1)
- p97 (1)
- pain (1)
- pathophysiological mechanisms (1)
- peptide (1)
- peptide engineering (1)
- peptide-based interleukin-5 inhibitor (1)
- perception (1)
- phosphorylation (1)
- platelets (1)
- polarized epithelium (1)
- pollen (1)
- polyethism (1)
- polyradiculoneuropathy (1)
- population attributable fraction (1)
- precision medicine (1)
- preclinical PET (1)
- primary ciliary dyskinesia (1)
- primate (1)
- proboscis extension response (1)
- protease (1)
- protein kinase a (1)
- protein regulation and expression (1)
- protein-protein-interaction (1)
- psychosocial resilience (1)
- rapid evolution (1)
- reception (1)
- receptor (1)
- replication (1)
- resilience (1)
- salinen Wachstumsbedingungen (1)
- salt stress (1)
- schnelle Evolution (1)
- self-activation (1)
- senecionine (1)
- seneciphylline (1)
- shRNA (1)
- signalling (1)
- similarity (1)
- skin (1)
- skin biopsy (1)
- small RNA (1)
- small fiber pathology (1)
- small proteins (1)
- somatic resilience (1)
- somatostatin receptor antagonists (1)
- sphingolipids (1)
- ssVEP (1)
- stiff person syndrome (1)
- structural mechanism (1)
- structure analysis (1)
- super-resolution microscopy (1)
- synaptic plasticity (1)
- synaptische Plastizität (1)
- targeted therapy (1)
- tdcs (1)
- temporal information transfer (1)
- test system (1)
- therapy (1)
- time-resolved anisotropy (1)
- tool (1)
- tool-use (1)
- tracheal cytotoxin (1)
- trachomatis (1)
- transcription/replication conflicts (1)
- transcriptome data analysis (1)
- transcriptomic analysis (1)
- transient dynamics (1)
- tumour (1)
- tyrosine kinase (1)
- virulence (1)
- yeast (1)
- zeitlich Informationsübertragung (1)
- zonal Hydrogels (1)
- µ-Opioid Rezeptor (1)
- Ängstliche Depression (1)
Institute
- Graduate School of Life Sciences (123) (remove)
Sonstige beteiligte Institutionen
- Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München (1)
- Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München (1)
- Max-Delbrück-Center für molekulare Medizin, Berlin (1)
- Research Center for Infectious Diseases, University of Würzburg (1)
- Spanish National Center for Biotechnology (CNB-CSIC) (1)
- Technische Universität Darmstadt (1)
- University of Applied Sciences Aschaffenburg (1)
- University of Leipzig, Faculty of Life Sciences, Institute for Biology (1)
- Zentrum für Infektionsforschung (ZINF) Würzburg (1)
Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing.
This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration.
The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration.
In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.
Depressionen und Angststörungen sind die beiden häufigsten psychischen Erkrankungen. Für Angststörungen wurde in zahlreichen Untersuchungen die Bedeutung veränderter Muster in den basalen emotional-assoziativen Lernprozessen für die Ätiologie und Aufrechterhaltung der Erkrankung gezeigt. Hierzu zählen eine verstärkte Akquisitionsreaktion auf den konditionierten Stimulus, Defizite in der Inhibition der Furchtreaktion auf den Sicherheit signalisierenden Stimulus, Übergeneralisierung und Beeinträchtigungen in der Extinktion konditionierter Reaktionen.
Aufgrund der hohen Prävalenzen einer Komorbidität mit Depressionen rückte in den letzten Jahren zunehmend die Untersuchung der genannten Prozesse bei Depressionen in den Fokus. Hierfür konnten bisher keine einheitlichen Ergebnisse gezeigt werden.
Weiterhin wird der Subtyp der ängstlichen Depression einerseits mit hohen Prävalenzen beschrieben, andererseits zeigen Untersuchungen eine schlechtere Prognose, stärkere Einschränkungen in der Funktionalität und ein schlechteres Ansprechen auf die Therapie im Vergleich zu depressiven Patienten ohne hohes Ängstlichkeitsniveau.
In dieser Arbeit wurden die Akquisition, Generalisierung und Extinktion in einem differentiellen Konditionierungsparadigma bei schwer depressiven ängstlichen und nicht ängstlich-depressiven Patienten sowie einer gesunden Kontrollgruppe untersucht. Ängstliche und nicht ängstlich-depressive Patienten zeigten ein beeinträchtigtes Sicherheitslernen in der Akquisition und Beeinträchtigungen in der Extinktion der konditionierten Furcht. Es ergaben sich keine Unterschiede hinsichtlich der Stärke der Generalisierung zwischen Patienten und den gesunden Kontrollen und es konnten keine differenzierenden Muster zwischen den ängstlich- und den nicht ängstlich-depressiven Patienten gezeigt werden.
Zusammenfassend weisen die Ergebnisse auf Veränderungen im Furchtlernen bei Patienten mit Depressionen hin. Es konnten keine Belege für unterschiedliche Mechanismen im Furchtlernen von ängstlich- und nicht ängstlich-depressiven Patienten gefunden werden. Unsere Ergebnisse stützen somit die Klassifikation der ängstlichen Depression als Subtyp der Depression. Weiterhin weisen die Ergebnisse der beeinträchtigten Extinktion bei Patienten mit Depressionen darauf hin, dass Expositionselemente, welche bei der Therapie von Angststörungen als Verfahren der Wahl eingesetzt werden, auch bei der Behandlung von Depressionen integriert werden sollten, um so den Therapieerfolg zu verbessern.
In dieser Dissertation wird der MEK5/ERK5- Signalweg als möglicher Angriffspunkt in der zielgerichteten Melanomtherapie identifiziert. Die Adressierung von ERK5 bietet eine Alternative, um einer Resistenzentwicklung gegenüber Inhibitoren des MAPK- Signalwegs entgegenzuwirken. Das maligne Melanom ist ein hochaggressiver Tumor mit steigender Inzidenz. Zunehmende Sonnenstunden im Rahmen des Klimawandels mit erhöhter Belastung der Haut durch UV-Strahlung werden die Problematik des malignen Melanoms für den Menschen in den nächsten Jahren weiter zunehmen lassen.
Die Aktivierung des MEK5/ERK5- Signalwegs scheint eine Reaktion von Tumorzellen auf Therapiestress zu sein. Diese Aktivierung liefert den Melanomzellen einen Überlebensvorteil und verhindert ein langfristiges Therapieansprechen. ERK5 beeinflusst den Zellzyklus von Melanomzellen und ist somit möglicherweise von wichtiger Bedeutung in der Tumorgenese des malignen Melanoms.
Patienten mit NRAS- Mutation profitieren auffallend weniger von einer gezielten MEKi-Therapie als solche mit BRAF Mutation. Für ersteres Patientenkollektiv steht aktuell lediglich die Immuntherapie zur Verfügung, wodurch oft nur ein kurzes, progressionsfreies Intervall erreicht werden kann und die Patienten häufig unter schweren Nebenwirkungen leiden. Grund für die problematische Behandlung könnte das häufige Auftreten einer basalen ERK5- Aktivierung in NRAS- mutierten Melanomen sein. Diese Arbeit liefert eine positive Prognose über den Nutzen einer ERK5- Inhibition als Erweiterung des Therapieschemas. Diese These gilt auch für Melanompatienten mit einer BRAF- Mutation. Patienten, die an einem malignen Melanom erkrankt sind, weisen zu 80% eine Mutation in einem dieser beschriebenen Onkogene auf. Die Arbeit lässt darauf schließen, dass eine ERK5- Inhibition in der Therapie von beiden Gruppen erfolgreich sein könnte und somit das Leben nahezu aller Melanompatienten betrifft.
The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens.
This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly.
Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells.
CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus.
Adrenal Cushing’s Syndrome (CS) is a rare but life-threatening disease and therefore it is of great importance to understand the pathogenesis leading to adrenal CS. It is well accepted that Protein Kinase A (PKA) signalling mediates steroid secretion in adrenocortical cells. PKA is an inactive heterotetramer, consisting of two catalytic and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits are released and are able to phosphorylate their target proteins. Recently, activating somatic mutations affecting the catalytic subunit a of PKA have been identified in a sub-population of cortisol-producing adenomas (CPAs) associated with overt CS. Interestingly, the PKA regulatory subunit IIb has long been known to have significantly lower protein levels in a sub-group of CPAs compared to other adrenocortical tumours. Yet, it is unknown, why these CPAs lack the regulatory subunit IIb, neither are any functional consequences nor are the underlying regulation mechanisms leading to reduced RIIb levels known. The results obtained in this thesis show a clear connection between Ca mutations and reduced RIIb protein levels in CPAs but not in other adrenocortical tumours. Furthermore, a specific pattern of PKA subunit expression in the different zones of the normal adrenal gland is demonstrated. In addition, a Ca L206R mutation-mediated degradation of RIIb was observed in adrenocortical cells in vitro. RIIb degradation was found to be mediated by caspases and by performing mutagenesis experiments of the regulatory subunits IIb and Ia, S114 phosphorylation of RIIb was identified to make RIIb susceptible for degradation. LC-MS/MS revealed RIIb interaction partners to differ in the presence of either Ca WT and Ca L206R. These newly identified interaction partners are possibly involved in targeting RIIb to subcellular compartments or bringing it into spatial proximity of degrading enzymes. Furthermore, reducing RIIb protein levels in an in vitro system were shown to correlate with increased cortisol secretion also in the absence of PRKACA mutations. The inhibiting role of RIIb in cortisol secretion demonstrates a new function of this regulatory PKA subunit, improving the understanding of the complex regulation of PKA as key regulator in many cells.
Cellular membranes form a boundary to shield the inside of a cell from the outside. This is of special importance for bacteria, unicellular organisms whose membranes are in direct contact with the environment. The membrane needs to allow the reception of information about beneficial and harmful environmental conditions for the cell to evoke an appropriate response. Information gathering is mediated by proteins that need to be correctly organized in the membrane to be able to transmit information. Several principles of membrane organization are known that show a heterogeneous distribution of membrane lipids and proteins. One of them is functional membrane microdomains (FMM) which are platforms with a distinct lipid and protein composition. FMM move within the membrane and their integrity is important for several cellular processes like signal transduction, membrane trafficking and cellular differentiation. FMM harbor the marker proteins flotillins which are scaffolding proteins that act as chaperones in tethering protein cargo to FMM. This enhances the efficiency of cargo protein oligomerization or complex formation which in turn is important for their functionality. The bacterium Bacillus subtilis contains two flotillin proteins, FloA and FloT. They form different FMM assemblies which are structurally similar, but differ in the protein cargo and thus in the specific function.
In this work, the mobility of FloA and FloT assemblies in the membrane was dissected using live-cell fluorescence microscopy techniques coupled to genetic, biochemical and molecular biological methods. A characteristic mobility pattern was observed which revealed that the mobility of both flotillins was spatially restricted. Restrictions were bigger for FloT resulting in a decreased diffusion coefficient compared to FloA. Flotillin mobility depends on the interplay of several factors. Firstly, the intrinsic properties of flotillins determine the binding of different protein interaction partners. These proteins directly affect the mobility of flotillins. Additionally, binding of interaction partners determines the assembly size of FloA and FloT. This indirectly affects the mobility, as the endo-cytoskeleton spatially restricts flotillin mobility in a size-dependent manner. Furthermore, the extracellular cell wall plays a dual role in flotillin mobility: its synthesis stimulates flotillin mobility, while at the same time its presence restricts flotillin mobility. As the intracellular flotillins do not have spatial access to the exo-cytoskeleton, this connection is likely mediated indirectly by their cell wall-associated protein interaction partners. Together the exo- and the endo-cytoskeleton restrict the mobility of FloA and FloT.
Similar structural restrictions of flotillin mobility have been reported for plant cells as well, where the actin cytoskeleton and the cell wall restrict flotillin mobility. These similarities between eukaryotic and prokaryotic cells indicate that the restriction of flotillin mobility might be a conserved mechanism.
Investigations into the Pathogenic Antibody-Antigen-Interference of Glycine Receptor Autoantibodies
(2021)
Anti-glycine receptor (GlyR) autoantibodies belong to the novel group of autoantibodies that target neuronal cell-surface antigens (NCS), which are accompanied with various neurologic and neuropsychiatric conditions. The inhibitory ionotropic GlyR is one of the major inhibitory neurotransmitter receptors and therefore involved in maintaining homeostasis of neuronal excitation levels at brain stem and spinal cord. Anti-GlyR autoantibodies are associated with progressive encephalomyelitis with rigidity and myoclonus or stiff person syndrome. These neuromotor disorders are characterized by exaggerated startle, muscle stiffness, and painful spasms, leading to immobility and fatal outcome in some cases. It was hypothesized that imbalance of motoneuronal inhibition by functional impairment of GlyR and receptor internalization are direct consequences of antibody-antigen interference. Here, serum samples of four patients were tested for anti-GlyR autoantibodies and were used for the analysis of the functional impact on the electrophysiological properties of recombinant GlyRs, transiently expressed in HEK293 cells. Furthermore, the recognition pattern of anti- GlyR autoantibodies to human, zebrafish and chimeric GlyRα1 located the epitope to the far N-terminal region. The pathogenicity of anti-GlyR autoantibodies and thereby the autoimmunologic etiology of the disease was confirmed by passive transfer of patient serum to zebrafish (Danio rerio) larvae, that yielded an abnormal escape response – a brain stem reflex that corresponds to the exaggerated startle of afflicted patients. The phenotype was accompanied by profound reduction of GlyR clusters in spinal cord cryosections of treated zebrafish larvae. Together, these novel insights into the pathogenicity of GlyR autoantibodies confirm the concept of a novel neurologic autoimmune disease and might contribute to the development of innovative therapeutic strategies.
The past decades have witnessed the development of new pharmaceutical compounds that modulate receptor function by targeting allosteric sites. Allosteric sites are, by definition, domains topographically distinct from the orthosteric binding pocket where the natural ligand binds. Exploring the possibilities of linking orthosteric and allosteric pharmacophores in one compound to yield ‘bitopic’ compounds is a strategy derived from the “message-address” concept by Schwyzer , first applied to GPCRs by Portoghese et al. This concept explicitly underlines the orthosteric/allosteric combination, in opposite to the more general umbrella term bivalent. The broad possibilities of bitopic ligands in the pharmaceutical field are under continuous study. Bitopic compounds are promising pharmaceutical tools for taking advantage of the allosteric binding to achieve subtype selectivity while preserving high affinity at the receptor. The development of bitopic ligands, based on the idea of combining high affinity (via orthosteric sites) with high selectivity (via allosteric sites), have led to the development of highly selective bivalent ligands for GPCRs , such as for the opioid receptors , muscarinic acetylcholine receptors (mAChRs), serotonin receptors, cannabinoid receptors, and gonadotropin-releasing hormone receptors. This concept has even been extended to other receptors, for examples nicotinic receptors and other proteins, such as acetylcholinesterases and the tyrosine kinase receptors TrkA and TrkC. The reasons to pursue a bitopic ligand approach are various. An improved affinity for the target GPCR and/or an improved selectivity either at the level of receptor subtype, or at the level of signaling pathway. Another advantage of bitopic ligands over purely allosteric ligands is that the former rely on the appropriate presence of endogenous agonist tone to mediate their effects, whereas a bitopic ligand would engage the orthosteric site irrespective of the presence or absence of endogenous tone. By way of introduction to the hybrid approach, a review of the concept of hybrids compounds targeting the cholinergic system is presented in section A of this thesis. Recent updates in hybrid molecule design as a strategy for selectively addressing multiple target proteins involved in Alzheimer's disease (AD) is here reported . This represents the potential and the growing interest in hybrid compound as pharmacological tools to achieve receptor subtype selectivity and/or, to study the overall functional activity of the receptor. Until now, muscarinic acetylcholine receptors (mAChRs) have proved to be a particularly fruitful receptor model for the development and characterization of bitopic ligands. In this thesis, several examples of new muscarinic bitopic approach are reported in the results section. A study of bipharmacophoric ligands composed of the muscarinic positive allosteric modulators (BQCAderived compounds) linked with chain of various lengths to different orthosteric building blocks is reported in the result part 1. Synthesis and examination of the potential pharmacological characteristic of Oxotremorine-BQCAd compounds and Xanomeline-BQCAd hybrid derivatives are described in results parts 2 and 4, respectively. Moreover, the bitopic concept has even been extended to other proteins, such as acetylcholinesterase. In the result part 5 an overview of the new Tacrine-Xanomeline hybrids aiming to improve the inhibitory potency of the acetylcholinesterase and simultaneously to increase the cholinergic tone, via the xanomelinic portion acting on the M1 receptor is given. A new trivalent approach is presented for the first time to deepen the study of the M1 muscarinic receptor in the result part 6. Moreover, the synthesis of a new series of iperoxo-derived alkane, bis(ammonio)alkane-type and rigidified chain ligands is given in the result part 7 together with some prospects for further research.
Chlamydia trachomatis, an obligate intracellular human pathogen, is the world’s leading cause of infection related blindness and the most common, bacterial sexually transmitted disease. In order to establish an optimal replicative niche, the pathogen extensively interferes with the physiology of the host cell. Chlamydia switches in its complex developmental cycle between the infectious non-replicative elementary bodies (EBs) and the non-infectious replicative reticulate bodies (RBs). The transformation to RBs, shortly after entering a host cell, is a crucial process in infection to start chlamydial replication. Currently it is unknown how the transition from EBs to RBs is initiated. In this thesis, we could show that, in an axenic media approach, L glutamine uptake by the pathogen is crucial to initiate the EB to RB transition. L-glutamine is converted to amino acids which are used by the bacteria to synthesize peptidoglycan. Peptidoglycan inturn is believed to function in separating dividing Chlamydia. The glutamine metabolism is reprogrammed in infected cells in a c-Myc-dependent manner, in order to accomplish the increased requirement for L-glutamine. Upon a chlamydial infection, the proto-oncogene c-Myc gets upregulated to promote host cell glutaminolysis via glutaminase GLS1 and the L-glutamine transporter SLC1A5/ASCT2. Interference with this metabolic reprogramming leads to limited growth of C. trachomatis. Besides the active infection, Chlamydia can persist over a long period of time within the host cell whereby chronic and recurrent infections establish. C. trachomatis acquire a persistent state during an immune attack in response to elevated interferon-γ (IFN-γ) levels. It has been shown that IFN-γ activates the catabolic depletion of L-tryptophan via indoleamine 2,3-dioxygenase (IDO), resulting in the formation of non-infectious atypical chlamydial forms. In this thesis, we could show that IFN-γ depletes the key metabolic regulator c-Myc, which has been demonstrated to be a prerequisite for chlamydial development and growth, in a STAT1-dependent manner. Moreover, metabolic analyses revealed that the pathogen de routs the host cell TCA cycle to enrich pyrimidine biosynthesis. Supplementing pyrimidines or a-ketoglutarate helps the bacteria to partially overcome the persistent state. Together, the results indicate a central role of c-Myc induced host glutamine metabolism reprogramming and L-glutamine for the development of C. trachomatis, which may provide a basis for anti-infectious strategies. Furthermore, they challenge the longstanding hypothesis of L-tryptophan shortage as the sole reason for IFN-γ induced persistence and suggest a pivotal role of c-Myc in the control of the C. trachomatis dormancy.
Small proteins, often defined as shorter than 50 amino acids, have been implicated
in fundamental cellular processes. Despite this, they have been largely understudied throughout all domains of life, since their size often makes their identification and characterization challenging.
This work addressed the knowledge gap surrounding small proteins with a focus
on the model bacterial pathogen Salmonella Typhimurium. In a first step,
new small proteins were identified with a combination of computational and experimental approaches. Infection-relevant datasets were then investigated with
the updated Salmonella annotation to prioritize promising candidates involved in virulence.
To implement the annotation of new small proteins, predictions from the algorithm
sPepFinder were merged with those derived from Ribo-seq. These were added to the Salmonella annotation and used to (re)analyse different datasets. Information
regarding expression during infection (dual RNA-seq) and requirement for virulence (TraDIS) was collected for each given coding sequence. In parallel,
Grad-seq data were mined to identify small proteins engaged in intermolecular
interactions.
The combination of dual RNA-seq and TraDIS lead to the identification of small
proteins with features of virulence factors, namely high intracellular induction
and a virulence phenotype upon transposon insertion. As a proof of principle of
the power of this approach in highlighting high confidence candidates, two small
proteins were characterized in the context of Salmonella infection.
MgrB, a known regulator of the PhoPQ two-component system, was shown to be essential for the infection of epithelial cells and macrophages, possibly via its stabilizing effect on flagella or by interacting with other sensor kinases of twocomponent
systems. YjiS, so far uncharacterized in Salmonella, had an opposite role in infection, with its deletion rendering Salmonella hypervirulent. The mechanism underlying this, though still obscure, likely relies on the interaction with
inner-membrane proteins.
Overall, this work provides a global description of Salmonella small proteins in
the context of infection with a combinatorial approach that expedites the identification
of interesting candidates. Different high-throughput datasets available for
a broad range of organisms can be analysed in a similar manner with a focus on small proteins. This will lead to the identification of key factors in the regulation
of various processes, thus for example providing targets for the treatment of bacterial
infections or, in the case of commensal bacteria, for the modulation of the microbiota composition.
The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex.
The outcome of the innate immune response to biomaterials mainly determines whether the material will be incorporated in the body to fulfill its desired function or, when it gets encapsulated, will be rejected in the worst case. Macrophages are key players in this process, and their polarization state with either pro- (M1), anti-inflammatory (M2), or intermediate characteristics is crucial for deciding on the biomaterial’s fate. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates the proper healing and subsequent regeneration. Therefore, biomaterial-based polarization may aid in driving macrophages in the desired direction. However, the in vivo process is highly complex, and a mono-culture of macrophages in vitro displays only one part of the cellular system, but, to this date, there is a lack of established co-cultures to assess the immune response to biomaterials. Thus, this thesis aimed to establish a functional co-culture system of human macrophages and human mesenchymal stromal cells (hMSCs) to improve the assessment of the immune response to biomaterials in vitro. Together with macrophages, hMSCs are involved in tissue regeneration and inflammatory reactions and can modulate the immune response. In particular, endogenously derived hMSCs considerably contribute to the successful engrafting of biomaterials. This thesis focused on poly(ε-caprolactone) (PCL) fiber-based scaffolds produced by the technique of melt electrowriting (MEW) as biomaterial constructs. Via this fabrication technique, uniform, precisely ordered scaffolds varying in geometry and pore size have been created in-house.
To determine the impact of scaffold geometries and pore sizes on macrophages, mono-cultures incubated on scaffolds were conducted. As a pre-requisite to achieve a functional co-culture system on scaffolds, setups for direct and indirect systems in 2D have initially been established. These setups were analyzed for the capability of cell-cell communication. In parallel, a co-culture medium suitable for both cell types was defined, prior to the establishment of a step-by-step procedure for the co-cultivation of human macrophages and hMSCs on fiber-based scaffolds.
Regarding the scaffold morphologies tested within this thesis to improve M2-like polarization, box-shaped scaffolds outperformed triangular-, round- or disordered-shaped ones. Upon further investigation of scaffolds with box-shaped pores and precise inter-fiber spacing from 100 µm down to only 40 µm, decreasing pore sizes facilitated primary human macrophage elongation accompanied by their differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 µm. To the best of my knowledge, this was the first time that the elongation of human macrophages in a 3D environment has been correlated to their M2-like polarization. Thus, these results may set the stage for the design, the assessment, and the selection of new biomaterials, which can positively affect the tissue regeneration.
The cell communication of both cell types, detected via mitochondria exchange in direct and indirect co-cultures systems, took place in both directions, i.e., from hMSCs to macrophages and vice versa. Thereby, in direct co-culture, tunneling nanotubes enabled the transfer from one cell type to the respective other, while in indirect co-culture, a non-directional transfer through extracellular vesicles (EVs) released into the medium seemed likely. Moreover, the phagocytic activity of macrophages after 2D co-cultivation and hence immunomodulation by hMSCs increased with the highest phagocytic rate after 48 h being most pronounced in direct co-cultivation.
As the commonly used serum supplements for macrophages and hMSCs, i.e., human serum (hS) and fetal calf serum (FCS), respectively, failed to support the respective other cell type during prolonged cultivation, these sera were replaced by human platelet lysate (hPL), which has been proven to be the optimal supplement for the co-cultivation of human macrophages with hMSCs within this thesis. Thereby, the phenotype of both cell types, the distribution of both cell populations, the phagocytic activity of macrophages, and the gene expression profiles were maintained and comparable to the respective standard mono-culture conditions. This was even true when hPL was applied without the anticoagulant heparin in all cultures with macrophages, and therefore, heparin was omitted for further experiments comprising hPL and macrophages.
Accordingly, a step-by-step operating procedure for the co-cultivation on fiber-based scaffolds has been established comprising the setup for 3D cultivation as well as the description of methods for the analysis of phenotypical and molecular changes upon contact with the biomaterial. The evaluation of the macrophage response depending on the cultivation with or without hMSCs and either on scaffolds or on plastic surfaces has been successfully achieved and confirmed the functionality of the suggested procedures.
In conclusion, the functional co-culture system of human macrophages and hMSCs established here can now be employed to assess biomaterials in terms of the immune response in a more in vivo-related way. Moreover, specifically designed scaffolds used within the present thesis showed auspicious design criteria positively influencing the macrophage polarization towards the anti-inflammatory, pro-healing type and might be adaptable to other biomaterials in future approaches.
Hence, follow-up experiments should focus on the evaluation of the co-culture outcome on promising scaffolds, and the suggested operating procedures should be adjusted to further kinds of biomaterials, such as cements or hydrogels.
Design of novel IL-4 antagonists employing site-specific chemical and biosynthetic glycosylation
(2021)
The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases.
For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R.
This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist.
For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability.
The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab.
Während der letzten Jahrzehnte ist eine steigende Inzidenz von Infektionen durch Pilze der Ordnung Mucorales zu beobachten. Rhizopus arrhizus ist der häufigste Erreger dieser lebensbedrohlichen Infektionen, die vor allem immunsupprimierte Patienten betreffen. Aufgrund der oft schwierigen Diagnosestellung und limitierter therapeutischer Optionen liegt derzeit die Letalität von Mucormykosen zwischen 50 bis 100 %. Eine Voraussetzung für die Etablierung neuer Biomarker oder immuntherapeutischer Strategien ist ein verbessertes Verständnis der immunpathologischen Prozesse bei der Abwehr von Mucorales.
In dieser Arbeit wurden daher verschiedene Immunzellpopulationen durch ruhende und ausgekeimte Stadien von R. arrhizus stimuliert und anschließend deren proinflammatorische Immunantwort gemessen. Als Vergleich diente die proinflammatorische Immunantwort der untersuchten Immunzellen nach Stimulation mit Aspergillus fumigatus. Darüber hinaus war es Gegenstand dieser Arbeit, zu charakterisieren welche Pattern Recognition Receptors (PRRs) an der Erkennung von Mucorales durch verschiedene innate Immunzellen beteiligt sind. Zugleich wurde untersucht, ob unterschiedliche Morphotypen der Pilzspezies Auswirkungen auf die Stimulation der jeweiligen PRRs haben. Hierfür wurden Koinkubations-Experimente mit neutrophilen Granulozyten sowie Peripheral Blood Mononuclear Cells (PBMCs), Monozyten und monocyte derived dendritic cells (moDCs) mit verschiedenen Morphotypen von R. arrhizus durchgeführt. Die Rezeptoren TLR2, TLR4 und/oder Dectin-1 wurden dabei durch neutralisierende Antikörper oder RNA-Interferenz blockiert.
Ausgekeimte Stadien von A. fumigatus sowie R. arrhizus induzierten eine erhöhte ROS-Freisetzung in Neutrophilen, die durch isolierte oder kombinierte Blockade von TLR2, TLR4 und Dectin-1 abgeschwächt wurde. Ebenso wurde die Phagozytoseaktivität neutrophiler Granulozyten gegenüber R. arrhizus-Konidien durch Blockade von TLR4 und Dectin-1 deutlich reduziert.
Im Gegensatz zu A. fumigatus induzierten sowohl ruhende Konidien als auch ausgekeimte Stadien (Keimschläuche und Hyphen) von R. arrhizus eine robuste pro-inflammatorische Zytokinantwort durch moDCs. Nach Inhibition der Dectin-1 Expression durch RNA-Interferenz zeigte sich die Transkription und Sekretion von Interleukin-1β in Gegenwart aller drei untersuchten Morphotypen von
R. arrhizus deutlich vermindert (Transkription um 46 bis 68 % und Sekretion um 75 bis 79 % vermindert). Diese Ergebnisse legen nahe, dass Dectin-1 ein wichtiger Mediator bei der Einleitung der innaten Immunantwort verschiedener Zelltypen gegen R. arrhizus ist. Diese Beobachtung sollte in weiteren Studien eingehender untersucht werden, z. B. um die Eignung von Dectin-1 als Rezeptor für zelltherapeutische Ansätze wie T-Zell-Konstrukte mit chimären Antigen-Rezeptoren zu evaluieren.
Die alveoläre Echinokokkose (AE), verursacht durch das Metacestoden- Larvenstadium des Fuchsbandwurms Echinococcus multilocularis (E. multilocularis), ist eine lebensbedrohliche Zoonose der nördlichen Hemisphäre mit eingeschränkten therapeutischen Möglichkeiten. Bei der Suche nach neuen Therapeutika haben Mitogen-activated Proteinkinase (MAPK) -Kaskaden als pharmakologische Zielstrukturen aufgrund ihrer essentiellen Rolle bei der Zellproliferation und -differenzierung ein großes Potenzial. In der vorliegenden Arbeit wurden durch BLAST- und reziproke BLAST-Analysen elf potenzielle MAPK Kinase Kinasen (MAP3K), fünf potenzielle MAPK Kinasen (MAP2K) und sechs potenzielle MAPK im E. multilocularis-Genom identifiziert, die teils hoch konserviert sind und in nahezu allen Entwicklungsstadien des Parasiten exprimiert werden. Diese Erkenntnisse lassen auf ein komplexes MAPK-Signaltransduktions- system in E. multilocularis mit großer Bedeutung für den Parasiten schließen. Transkriptomdatenanalysen und Whole Mount in Situ Hybridisierung (WMISH) zeigten, dass emmkkk1 (EmuJ_000389600) als einzige MAP3K neben der Expression in postmitotischen Zellen in besonderem Maße in proliferativen Stammzellen des Parasiten exprimiert wird und somit eine wichtige Rolle bei der Differenzierung von Stammzellen spielen könnte. In Yeast-Two-Hybrid (Y2H) -Wechselwirkungsassays wurden Interaktionen von mehreren upstream- (EmGRB2) und downstream- wirkenden Signalkaskadekomponenten des JNK (EmMKK3, EmMPK3) und ERK (EmMKK3, EmMPK4) -Signalwegs gefunden. Daraus lässt sich schließen, dass EmMKKK1, analog zu seinem humanen Homolog HsM3K1, eine zentrale Rolle bei der Echinococcus-Wachstumsregulation durch Rezeptortyrosinkinasen und vielfältige weitere Funktionen im Parasiten besitzt. Anhand von Erkenntnissen an Platyhelminthes kann daher von einem großen Potenzial dieser neu charakterisierten Signalwege als chemotherapeutische Angriffspunkte ausgegangen werden, wenngleich erste RNA-Interferenz (RNAi)- und Inhibitorstudien an emmkkk1, emmpk1 und emmpk4 keine durchschlagenden Effekte auf das Überleben von Primärzellkulturen und die Bildung von Metacestodenvesikeln zeigten. Zusammenfassend konnte in der vorliegenden Arbeit mit EmMKKK1 und neuen ERK- und JNK-Signalwegen zentrale Komponenten der komplexen MAPK-Signalkaskaden in E. multilocularis identifiziert werden, die höchstwahrscheinlich einen großen Beitrag zur enormen Regenerationsfähigkeit der Echinococcus-Stammzellen leisten und vom Wirt abgeleitete Signale wie Insulin, Epidermaler Wachstumsfaktor (EGF) und Fibroblasten-Wachstumsfaktor (FGF) über EmGRB2 in Proliferationsnetzwerke des Parasiten integrieren. Arzneimittel-Screening-Assays, die auf diese Signalwege abzielen, könnten daher zu alternativen Arzneimitteln führen, die alleine oder in Kombination mit einer bestehenden Chemotherapie (Benzimidazol) die Prognose von für AE-Patienten verbessern könnten.
Adapting defensive behavior to the characteristics of a threatening situation is a fundamental function of the brain. Particularly, threat imminence plays a major role for the organization of defensive responses. Acute threat prompts phasic physiological responses, which are usually associated with an intense feeling of fear. In contrast, diffuse and potentially threatening situations elicit a sustained state of anxious apprehension. Detection of the threatening stimulus defines the key event in this framework, initiating the transition from potential to acute threat. Consequently, attention to threat is crucial for supporting defensive behavior. The functions of attention are finely tuned to the characteristics of a threatening situation. Potential threat is associated with hypervigilance, in order to facilitate threat detection. Once a threatening stimulus has been identified, attention is selectively focused on the source of danger. Even though the concepts of selective attention and hypervigilance to threat are well established, evidence for their neural correlates remain scarce. Therefore, a major goal of this thesis is to elucidate the neural correlates of selective attention to acute threat and hypervigilance during potential threat. A second aim of this thesis is to provide a mechanistic account for the interaction of fear and anxiety. While contemporary models view fear and anxiety as mutually exclusive, recent findings for the neural networks of fear and anxiety suggest potential interactions. In four studies, aversive cue conditioning was used to induce acute threat, while context conditioning served as a laboratory model of potential threat. To quantify neural correlates of selective attention and hypervigilance, steady-state visual evoked potentials (ssVEPs) were measured as an index of visuocortical responding. Study 1 compared visuocortical responses to acute and potential threat for high versus low trait-anxious individuals. All individuals demonstrated enhanced electrocortical responses to the central cue in the acute threat condition, suggesting evidence for the neural correlate of selective attention. However, only low anxious individuals revealed facilitated processing of the contexts in the potential threat condition, reflecting a neural correlate of hypervigilance. High anxious individuals did not discriminate among contexts. These findings contribute to the notion of aberrational processing of potential threat for high anxious individuals. Study 2 and 3 realized orthogonal combinations of cue and context conditioning to investigate potential interactions of fear and anxiety. In contrast to Study 1 and 2, Study 3 used verbal instructions to induce potentially threatening contexts. Besides ssVEPs, threat ratings and skin conductance responses (SCRs) were recorded as efferent indices of defensive responding. None of these studies found further evidence for the neural correlates of hypervigilance and selective attention. However, results for ratings and SCRs revealed additive effects of fear and anxiety, suggesting that fear and anxiety are not mutually exclusive, but interact linearly to organize and facilitate defensive behavior. Study 4 tested ssVEPs to more ecologically valid forms of context conditioning, using flickering video stimuli of virtual offices to establish context representations. Contrary to expectations, results revealed decreased visuocortical responses during sustained presentations of anxiety compared to neutral contexts. A disruption of ssVEP signals eventually suggests interferences by continuously changing video streams which are enhanced as a function of motivational relevance. In summary, this thesis provided evidence for the neural correlates of attention only for isolated forms of fear and anxiety, but not for their interaction. In contrast, an additive interaction model of fear and anxiety for measures of defensive responding offers a new perspective on the topography of defensive behavior.
These days, treatment of melanoma patients relies on targeted therapy with BRAF/MEK inhibitors and on immunotherapy. About half of all patients initially respond to existing therapies. Nevertheless, the identification of alternative therapies for melanoma patients with intrinsic or acquired resistance is of great importance. In melanoma, antioxidants play an essential role in the maintenance of the redox homeostasis. Therefore, disruption of the redox homeostasis is regarded as highly therapeutically relevant and is the focus of the present work.
An adequate supply of cysteine is essential for the production of the most important intracellular antioxidants, such as glutathione. In the present work, it was investigated whether the depletion of cysteine and glutathione is therapeutically useful. Depletion of glutathione in melanoma cells could be achieved by blocking cysteine supply, glutathione synthesis, and NADPH regeneration. As expected, this led to an increased level of reactive oxygen species (ROS). Surprisingly, however, these changes did not impair the proliferation and survival of the melanoma cells. In contrast, glutathione depletion led to cellular reprogramming which was characterized by the induction of mesenchymal genes and the repression of differentiation markers (phenotypic switch). This was accompanied by an increased migration and invasion potential which was favored by the induction of the transcription factor FOSL1. To study in vivo reprogramming, Gclc, the first and rate-limiting enzyme in glutathione synthesis, was knocked out by CRISPR/Cas9 in murine melanoma cells. The cells were devoid of glutathione, but were fully viable and showed a phenotypic switch, the latter only in MITF-expressing B16F1 cells and not in MITF-deficient D4M3A.781 cells. Following subcutaneous injection into immunocompetent C57BL/6 mice, Gclc knockout B16F1 cells grew more aggressively and resulted in an earlier tumor onset than B16F1 control cells.
In summary, this work demonstrates that inhibition of cysteine supply and thus, glutathione synthesis leads to cellular reprogramming in melanoma. In this context, melanoma cells show metastatic capabilities, promoting a more aggressive form of the disease.
Multiple Sklerose ist eine der häufigsten neurologischen Erkrankungen, die zu motorischen, sensiblen und vegetativen Einschränkungen führt. Häufig beginnt die Erkrankung mit einem schubförmigen Verlauf, dem eine progrediente Verschlechterung folgt. Trotzdem leiden ca. 15% der Patienten bereits von Beginn an, an einer primär progressiven Variante der MS, die bereits mit der progredienten Phase beginnt. Bis jetzt ist die Pathophysiologie nicht vollständig verstanden. Lange Zeit dachte man, dass MS primär eine reine Autoimmun-Erkrankung darstellt, aber in den letzten Jahren ergab sich die Frage, ob es vor allem in den progressiven Formen, eine zytodegenerative Komponente gibt, auf die eine sekundäre Inflammation folgt. Eine mögliche Ursache stellen hier Mutationen des PLP 1 Gens dar, die normalerweise mit leukodystrophischen Erkrankungen assoziiert sind. Es gab zwei Fallberichte, in denen von Patienten berichtet wurde, die unterschiedliche Mutationen in diesem Gen hatten und den klinischen Phänotyp einer MS zeigten. Diese Arbeit sollte nun die Auswirkungen der Mutationen, beziehungsweise einer Nullmutation des PLP 1 Gens in 18- und zum Teil 12-Monate alten Mausmutanten untersuchen. Hier konnten Myelin-Veränderungen und axonaler Schaden in immunhistochemischen Untersuchungen sowie mittels Elektronenmikroskopie und optischer Kohärenztomographie gezeigt werden. Weiter konnte eine Neuroinflammation und damit einhergehend eine zunehmende Anzahl CD8+ T-Zellen sowie einer erhöhten Anzahl an Mikroglia/Makrophagen gefunden werden. Dies ging mit vergleichsweise reduzierten Leistungen der Mutanten bei der motorischen Rotarod-Analyse einher. Interessanterweise wurde weniger neuraler Schaden in den heterozygoten Varianten gefunden, obwohl das Ausmaß der Entzündung gleichblieb. Dies könnte für eine zielgerichtete immunvermittelte Schädigung der Oligodendrozyten sprechen, die zu neuronalem Schaden führt. So konnte gezeigt werden, dass es durch Punktmutationen in einem Myelinprotein-codierendem Gen zu einer sekundären Entzündung kommen kann, die mit dem klinischen Phänotyp einer progressiven MS einhergeht. Weiter sind diese Mausmodelle ein Beispiel für eine genetische Erkrankung des ZNS, in denen die Klinik maßgeblich durch die begleitende Inflammation und nicht allein durch den genetischen Schaden verursacht wird.
The degradation of poly-adenosine tails of messenger RNAs (mRNAs) in the eukaryotic cells is a determining step in controlling the level of gene expression. The highly conserved Ccr4-Not complex was identified as the major deadenylation complex in all eukaryotic organisms. Plenty of biochemical studies have shown that this complex is also involved in many aspects of the mRNA metabolism, but we are still lacking the detailed structural information about its overall architecture and conformational states that could help to elucidate its multifunction and the way it is coordinated in the cells. Such information can also provide a basis to finding a possible way of intervention since the complex is also involved in some diseases such as cancer and cardiovascular disorders in humans. Meanwhile, the single particle Cryo-EM method has been through a “resolution revolution” recently due to the use of the newly developed direct electron detectors and has since resolved the high-resolution structures of many macromolecular protein complexes in their near-native state. Therefore, it was employed as a suitable method for studying the Ccr4-Not complex here. In this work, the Falcon 3EC direct detector mounted on the 300kV Titan Krios G3i Cryo-EM was evaluated for its practical performance at obtaining high-quality Cryo-EM data from protein samples of different molecular sizes. This served as a proof of principle for this detector’s capabilities and as a data collection guidance for studying the macromolecular complexes, such as the Ccr4-Not, when using an advanced high-performance microscope system. Next, the endogenous yeast Ccr4-Not complex was also purified via the immunoaffinity purification method and evaluated using negative staining EM to assess the conditions of the complex before proceeding to sample preparation for Cryo-EM. This has shown that the complex had an unexpected inherently dynamic property in vitro and extra optimisation procedures were needed to stabilise the complex during the purification and sample preparation. In addition, by using the label-free quantitative Mass spectrometry to examine the coimmunoprecipitated complex via different tagged subunits, it was deduced that two of the subunits (Not3/Not5) that shared some sequence similarity might compete for association with the scaffold subunit of the complex. An uncharacterised protein was also identified coimmunoprecipitating with the Caf130 subunit of the yeast complex. Cryo-EM data from the purified complex provided a low-resolution map that represents a surprisingly smaller partial complex as compared to 3D structures from previous studies, although gel electrophoresis and Mass spectrometry data have identified all of the nine subunits of the Ccr4-Not core complex in the sample. It was concluded that due to the presence of many predicted unstructured regions VI in the subunits and their dynamic composition in solution, the native complex could have been spontaneously denatured at the air/water interface during the sample preparation thus limiting the resolution of the Cryo-EM reconstruction. The purified complex was also examined for its deadenylase and ubiquitin ligase activity by in vitro assays. It was shown that the native complex has a different rate of activity and possibly also a different mode of action compared to the recombinant complexes from other species under similar reaction conditions. The Not4 E3 ligase was also shown to be active in the complex and was likely auto-ubiquitinated in the absence of a substrate. Both types of assays have also shown that the conformational flexibility does not seem to affect the enzymatic reactions when using a chemically crosslinked form of the complex for the assay, which implies that there can be other underlying mechanisms coordinating its structural and functional relationship. The findings from this work have therefore moved our understanding of the Ccr4-Not complex forward by looking at the different structural and functional behaviours of the endogenous complex, especially highlighting the obstacles in sample preparation for the native complex in high-resolution Cryo-EM. This would serve as foundation for future studies on the mechanism of this complex’s catalytic functions and also for optimising the Cryo-EM sample to generate better data that could eventually resolve the structure to a high-resolution.
The cuticle is constituted of the biopolymer cutin and intra- and epicuticular waxes. In some cases, it has epicuticular wax crystals, protruding from the epicuticular wax film. One of the most important tasks is protection against desiccation. Many investigations were conducted to find the transport limiting component of the cuticle. It is evidentially confirmed that the waxes form this barrier. These waxes are multifactorial blends made of very-long-chain aliphatic (VLCA) compounds and triterpenoids (TRP). The VLCAs were proposed to constitute the transpiration barrier to water. However, experimental confirmation was lacking so far. The present study focuses on the development of a method to selectively extract TRPs from the cuticle and the impact of the removal on the transpiration barrier.
The plants deployed in this study exhibited several features. They had no epicuticular crystals on their surfaces, were astomatous, had a rather durable and possibly isolatable cuticle. A broad range of wax compositions was covered from plants with no TRP content and low wax load like Hedera helix and Zamioculcas zamiifolia to plants with high TRP content and high wax load like Nerium oleander. The selective extraction was conducted using a sequence of solvents. TRPs were extracted almost exhaustively from CMs with the first MeOH extract. Only a minor amount of shorter chained VLCAs was obtained. The remaining waxes, consisting mostly of VLCAs and some remnant TRPs, were removed with the following TCM extract.
After the extractions, the water permeance of native cuticular membranes (CM), MeOH extracted (M) and dewaxed cuticular discs (MX) was investigated gravimetrically. Compared to the water permeance of CMs, Ms showed no or only a small increase in water conductance. MXs, however, always showed strongly increased values.
The knowledge about the wax compounds constituting the transport-limiting properties is vital for different projects. For various issues, it would be favourable to have a standardized wax mixture as an initial point of research. It could be used to develop screening procedures to investigate the impact of adjuvants on cuticular waxes or the influence of wax constituents on the properties of cuticular waxes. This work concentrated on the development of an artificial wax mixture, which mimics the physical properties of a plant leaf wax sufficiently.
As target wax, the leaf wax of Schefflera elegantissima was chosen. The wax of this plant species consisted almost exclusively of VLCAs, had a rather simple composition regarding compound classes and chain length distribution and CMs could be isolated. Artificial binary, ternary and quaternary waxes corresponding to the conditions within the plant wax were investigated using differential scanning calorimetry (DSC), X-ray diffraction (XRD) techniques and Fourier-transform infrared (FTIR) spectroscopy. Phase diagrams were mapped out for a series of binary, ternary and quaternary wax mixtures. FTIR experiments were conducted using, ternary and a quaternary artificial wax blends. The blends were chosen to represent the conditions within the wax of the adaxial CM plant wax. The FTIR experiments exhibited an increasing resemblance of the artificial wax to the plant wax (adaxial CM wax) with an increasing number of compounds in the artificial wax. The same trend was found for DSC thermograms. Thermograms of ternary and quaternary blends exhibited more overlapping peaks and occurred in a temperature range more similar to the range of the whole leaf plant wax. The XRD spectrum at room temperature showed good conformity with the quaternary blend.
The current work illustrates a method for selective extraction of TRPs from isolated CMs. It gives direct experimental proof of the association of the water permeance barrier with the VLCA rather than to the TRPs. Furthermore, the possibility to mimic cuticular waxes using commercially available wax compounds is investigated. The results show promising feasibility for its viability, enabling it to perform as a standardized initial point for further research (e.g. to examine the influence of different constituents on waxes), revealing valuable knowledge about the structure and the chemistry-function relationship of cuticular waxes.