Biologische Hochschulschriften (Goethe-Universität)
Refine
Year of publication
Document Type
- Doctoral Thesis (848) (remove)
Has Fulltext
- yes (848) (remove)
Is part of the Bibliography
- no (848)
Keywords
- Gentherapie (7)
- NMR-Spektroskopie (6)
- gene therapy (6)
- Elektrophysiologie (5)
- Molekularbiologie (5)
- RNA (5)
- Schmerz (5)
- Apoptosis (4)
- Arzneimitteldesign (4)
- Carotinoide (4)
- Computational chemistry (4)
- Cytochromoxidase (4)
- Docking (4)
- Immunologie (4)
- Inhibition (4)
- Metabolic Engineering (4)
- Mitochondria (4)
- Mitochondrium (4)
- Proteine (4)
- Altern (3)
- Ameisen (3)
- Angiogenese (3)
- Biodiversität (3)
- Biotechnologie (3)
- Cochlea (3)
- DPOAE (3)
- Elektronentransfer (3)
- Evolution (3)
- Genexpression (3)
- Genregulation (3)
- HIV (3)
- Heterologe Genexpression (3)
- Krebs (Medizin) (3)
- Membranprotein (3)
- Mongolische Rennmaus (3)
- Paracoccus denitrificans (3)
- Photosynthese (3)
- Phylogenie (3)
- Proteinfaltung (3)
- RNS (3)
- Saccharomyces cerevisiae (3)
- Screening (3)
- Signaltransduktion (3)
- Strukturaufklärung (3)
- Transkriptionsfaktor (3)
- Virtual Screening (3)
- West Africa (3)
- Westafrika (3)
- Wirkstoff-Rezeptor-Bindung (3)
- Yarrowia lipolytica (3)
- Zellkultur (3)
- Zellzyklus (3)
- angiogenesis (3)
- apoptosis (3)
- cell cycle (3)
- microRNA (3)
- vasculogenesis (3)
- zebrafish (3)
- 5-Lipoxygenase (2)
- Allergie (2)
- Apoptose (2)
- Aptamer (2)
- Archaea (2)
- Archaebakterien (2)
- Arzneimittel (2)
- Belize (2)
- Biochemie (2)
- Bioinformatik (2)
- Biomarker (2)
- Biomechanik (2)
- Biosynthese (2)
- Brustkrebs (2)
- C. elegans (2)
- Chemie und Pharmazie (2)
- Drug Design (2)
- Drug design (2)
- Eisenzeit (2)
- Electron transfer (2)
- Elektronenmikroskopie (2)
- Endothelin (2)
- Entzündung (2)
- Enzym (2)
- Epigenetik (2)
- Fluoreszenz (2)
- Frankfurt <Main> / Universität / Fachbereich Biochemie (2)
- G-Protein gekoppelte Rezeptoren (2)
- G-Protein-gekoppelter Rezeptor (2)
- GPCR (2)
- Gentransfer (2)
- Guanylatcyclase (2)
- Hitzeschock-Proteine (2)
- Hitzestress (2)
- Hyperalgesie (2)
- Hören (2)
- Hörrinde (2)
- In silico-Methode (2)
- Iron Age (2)
- Ischämie (2)
- Kaliumkanal (2)
- Kinetik (2)
- Konformationsänderung (2)
- Lentiviren (2)
- Licht-Sammel-Komplex (2)
- Ligand (2)
- MHC Klasse I (2)
- Magnetische Kernresonanz (2)
- Melatonin (2)
- Membranproteine (2)
- Messung (2)
- Methanbakterien (2)
- Mikroplastik (2)
- Mittelhirn (2)
- Molekulardesign (2)
- Molekulardynamik (2)
- Molekulare Bioinformatik (2)
- Molekülstruktur (2)
- NADH-Dehydrogenase <Ubichinon> (2)
- NADH:ubiquinone oxidoreductase (2)
- NMR (2)
- NMR spectroscopy (2)
- Neuronal Differentiation (2)
- Neuronales Netz (2)
- Onkologie (2)
- Pflanzenstress (2)
- Photolabile Schutzgruppen (2)
- Pichia pastoris (2)
- Plant stress (2)
- Proteomanalyse (2)
- Protonentransfer (2)
- Retrotransposition (2)
- Rotkehlchen (2)
- Schmerzforschung (2)
- Schülerlabor (2)
- Small RNA (2)
- Spektroskopie (2)
- Stress (2)
- Systematik (2)
- Südostasien (2)
- Thermus thermophilus (2)
- Tierphysiologie (2)
- Translokation (2)
- Tropischer Regenwald (2)
- Ubihydrochinon-Cytochrom-c-Reductase (2)
- Umweltfaktor (2)
- Verbreitung (2)
- Verhalten (2)
- Verhaltensbiologie (2)
- Virtuelles Screening (2)
- Wirkstoffdesign (2)
- Xenorhabdus (2)
- Yeast-Two-Hybrid-System (2)
- Zellskelett (2)
- ageing (2)
- auditory cortex (2)
- caging (2)
- conservation (2)
- endothelial cell (2)
- endothelium (2)
- extracellular matrix (2)
- fungi (2)
- gene expression (2)
- hearing (2)
- immunology (2)
- inflammation (2)
- microtubule-targeting agents (2)
- mitochondria (2)
- molecular biology (2)
- mongolian gerbil (2)
- photolabile Schutzgruppe (2)
- sRNA (2)
- transcription factors (2)
- vascular endothelial cells (2)
- virtual screening (2)
- yeast (2)
- Ökotoxikologie (2)
- 19F NMR shifts for fluoroarenes (1)
- 2-Hydroxylase (1)
- 2-Photonen (1)
- 2-hydroxylase (1)
- 3-alkylphenols (1)
- 5-lipoxygenase (1)
- 6-methylsalicylic acid synthase (1)
- A-Typ-ATPasen (1)
- A1AO ATPase (1)
- ABC-Transporter (1)
- ADAM10 (1)
- ADAM15 (1)
- ADAMTS-13 (1)
- AF4 (1)
- AFLPs (1)
- ALE (1)
- ATP-Binding Cassette Transporter (ABC) (1)
- AVA (1)
- Absorptionsspektroskopie (1)
- Acetogenic bacteria (1)
- Acetylcholin (1)
- Actin-bindende Proteine (1)
- Acylimin Sulfonylimin (1)
- Adaptation (1)
- Adenom (1)
- Adrenodoxine (1)
- Aedes albopictus (1)
- Agapetes (1)
- Ageing (1)
- Agelas (1)
- Aktives Zentrum (1)
- Aktuelle Motivation (1)
- Akute lymphatische Leukämie (1)
- Aldosteron (1)
- Aldosteronantagonist (1)
- Alignment <Biochemie> (1)
- Alkaloide (1)
- Allergy (1)
- Allosterischer Effektor (1)
- Alzheimer's disease (1)
- Alzheimer-Krankheit (1)
- Ameisenpflanzen (1)
- Aminobuttersäure <gamma-> (1)
- Aminosäurensequenz (1)
- Amphisphaeriales (1)
- Amplitudenmodulation (1)
- Amyloid (1)
- Anabolismus (1)
- Ananasgewächse (1)
- Angewandte Botanik (1)
- Angiogenesis (1)
- Animal Behavior (1)
- Anisotropy (1)
- Anpassung (1)
- Anthrakologie (1)
- Anthropologie (1)
- Anthropometrie (1)
- Antikörperdetektion (1)
- Antisense-Oligonucleotide (1)
- Aphanomyces astaci (1)
- Apolipoprotein E (1)
- Aptamere (1)
- Aquatisches Ökosystem (1)
- Archäobotanik (1)
- Aromatase (1)
- Artbildung (1)
- Artensterben (1)
- Artificial kidney (1)
- Arzneimittel / Zulassung (1)
- Arzneimittelanalogon (1)
- Arzneimittelprüfung (1)
- Ascidien (1)
- Asian Tiger Mosquito (1)
- Asiatische Tigermücke (1)
- Assembly (1)
- Ataxin-2 (1)
- Atmungskette (1)
- Atoll (1)
- Aufreinigung (1)
- Ausbreitung (1)
- Autism Spectrum Disorder (1)
- Autoproteolyse (1)
- Außerschulisches Lernen (1)
- Axoninitialsegment (1)
- Azide (1)
- B-Lymphozyt (1)
- B-Zell-Lymphom (1)
- BH3 mimetics (1)
- BH3-Mimetika (1)
- BMI (1)
- BRG1 (1)
- Baculovirussystem (1)
- Baleen whales (1)
- Bartonella henselae (1)
- Batten disease (1)
- Bayes-Lernen (1)
- Bildungswahrscheinlichkeit (1)
- Bindestelle (1)
- Bioakkumulation (1)
- Bioartificial kidney (1)
- Bioassay-guided fractionation (1)
- Biochemische Analyse (1)
- Biochemistry (1)
- Biodiversity (1)
- Bioenergetik (1)
- Biogeographie (1)
- Biogeography (1)
- Biokonzentration (1)
- Biologie (1)
- Biologische Uhr (1)
- Biomagnifikation (1)
- Biomolekül (1)
- Biotechnologische Industrie (1)
- Biotechnology (1)
- Biotest (1)
- Biotic interactions (1)
- Bivalven-Vergesellschaftung (1)
- Black Lipid Membrane (1)
- Blech- und Metallwarenindustrie (1)
- Blitzlicht (1)
- Blitzlicht-Photolyse (1)
- Blood-Brain Barrier (1)
- Blut (1)
- Blut-Hirn-Schranke (1)
- Blutgefäßsystem (1)
- Bodycomposition (1)
- Bodyfat (1)
- Borrelia burgdorferi (1)
- Boswelliasäuren (1)
- Bovidae (1)
- Bovines Herpesvirus 1 (1)
- Breast Cancer (1)
- Breast cancer (1)
- Bremsen (1)
- Brevibacterium flavum (1)
- Brevibacterium linens (1)
- Brieftaube ; Orientierungsverhalten ; Flugdatenregistriergerät ; GPS <Satellitengeodäsie> (1)
- Broken symmetry (1)
- Bromeliaceae (1)
- Bungarus (1)
- Bungarus niger (1)
- Bungarus walli (1)
- Burkina Faso (1)
- C peptide (1)
- C-Peptid (1)
- CDK1 Kinase (1)
- CHO (1)
- CIDNP (1)
- CLPXP-Protease (1)
- CNGA3 (1)
- CNGB1 (1)
- CNV 16p11.2 (1)
- CRASP-Proteine (1)
- CRISPR/Cas9 (1)
- CSOs (1)
- Cadherine ; Proteine ; Struktur-Aktivitäts-Beziehung (1)
- Caenorhabditis elegans (1)
- Calcium activated potassium channels (1)
- Calcium-aktivierte (1)
- Calmodulin (1)
- Candida albicans ; Antimykotikum (1)
- Capoeta damascina (1)
- Capsaicin (1)
- Carbendazim (1)
- Carbon capture (1)
- Carcinogenese (1)
- Cardiac regeneration (1)
- Cardiovascular disease (1)
- Caribbean (1)
- Carnivora (1)
- Carotinoidbiosynthese (1)
- Caspase-8 (1)
- Cell-free Protein Synthesis (1)
- Cellular suicide switch (1)
- Central Nervous System (1)
- Cerebral cortex (1)
- Chagas (1)
- Chalydomonas (1)
- Channel Protein (1)
- Channelrhodopsin (1)
- Cheminformatics (1)
- Cheminformatik (1)
- Chemische Verschiebung (1)
- Chemische Ökologie (1)
- Chinchilla (1)
- Chinese hamster ovary (1)
- Chinoloxidase ba3 (1)
- Chironomus riparius (1)
- Chlamydomonas (1)
- Chlor (1)
- Chlorophyll Fluorescence (1)
- Chrimson (1)
- Chromatin (1)
- Chronischer Schmerz (1)
- Chronobiologie (1)
- Chronobiology (1)
- Cicer arietinum (1)
- Cladocera (1)
- Clathrin-vermittelte Endozytose (1)
- Climate (1)
- Climate Change (1)
- Climate change (1)
- Clock genes (1)
- Cluster-Analyse (1)
- Coccoidea (1)
- Coenzym (1)
- Cofaktor (1)
- Coiled coil (1)
- Colorectal Cancer (1)
- Columba livia (1)
- Connectomics (1)
- Conservation (1)
- Copper (1)
- Corbicula (1)
- Coronaries (1)
- Cortisol im Speichel (1)
- Cryptochrom (1)
- Crystallography (1)
- Ctenidae (1)
- Culicidae (1)
- Cupiennius salei (1)
- Curcumin (1)
- Cyclo-AMP (1)
- Cyclo-GMP (1)
- Cyclooxygenase-2 (1)
- Cyclooxygenasen (1)
- Cyprinidae (1)
- Cytochrom c Oxidase (1)
- Cytochrome c Oxidase (1)
- Cytochrome c oxidase (1)
- Cytomegalievirus (1)
- Cytoplasmatische Vererbung (1)
- DASPMI (1)
- DFT (1)
- DIRAS2 (1)
- DNA Methylation (1)
- DNA-Methylierung (1)
- DNA-binding region (1)
- DNS-Sequenz (1)
- DNS-Sonde (1)
- DNS-Synthese (1)
- DR5 (1)
- Daboia russelii (1)
- Daphnia (1)
- Datenbank (1)
- De novo Design (1)
- De-Novo-Synthese (1)
- Death-receptor (1)
- Decapping (1)
- Degeneration (1)
- Delaunay-Triangulierung (1)
- Deoxymannojirimycin (1)
- Depolymerisierung (1)
- Deskriptor (1)
- Detergenz (1)
- Deterministische Optimierung (1)
- Deutschland / Abwasserverordnung (1)
- Development (1)
- Developmental Biology (1)
- Diadinoxanthin (1)
- Dictyostelium discoideum (1)
- Didaktik Neurowissenschaften (1)
- Differentielle Genexpression (1)
- Digitalisierung (1)
- Dimensionsreduktion (1)
- Dipol (1)
- Direkteinleiter (1)
- Dispersal (1)
- Diversität (1)
- Docking protein (1)
- Dolastatinderivate ; Konformationsanalyse ; Tubuline ; Proteinbindung ; NMR-Spektroskopie ; Hammerkopf-Ribozym ; Übergangszustand ; Phospholamban (1)
- Domatien (1)
- Dominanzspektrum (1)
- Dornapparat (1)
- Dreidimensionale NMR-Spektroskopie (1)
- Dreidimensionale geometrische Modellierung (1)
- Drought (1)
- Druckerzeugungsmechanismen (1)
- Drug Delivery (1)
- Drug Targeting (1)
- Druggability (1)
- Dynamik (1)
- EAAT3 (1)
- EAAT4 (1)
- EGFR (1)
- EMCP (1)
- EMSA (1)
- EPR spectroscopy (1)
- Echoorientierung (1)
- Ecology (1)
- Ecotoxicogenomics (1)
- Ecotoxicology (1)
- Ectodomain Shedding (1)
- Efferentes System (1)
- Eintrittsinhibitor (1)
- Einzelpartikelelektronenmikroskopie (1)
- Eisen- (1)
- Eisen-Schwefel-Zentrum N1a (1)
- Electron Paramagnetic Resonance (1)
- Electron microscopy (1)
- Electrophysiology (1)
- Electrospinning (1)
- Elektrisches Remodeling (1)
- Elektrokardiogramm (1)
- Elektronenspinresonanzspektroskopie (1)
- Elektrospray-Ionisation (1)
- Emerging insect model organisms (1)
- Enchytraeidae (1)
- Enchytraeidae-Artengemeinschaft (1)
- Endocrine disrupter (1)
- Endokrin wirksamer Stoff (1)
- Endokrinologie (1)
- Endothel (1)
- Endothelial cell (1)
- Endothelial cells (1)
- Endotheliale Vorläuferzellen (1)
- Endothelin B Rezeptor (1)
- Endothelin Receptor B (1)
- Endothelin-Rezeptor (1)
- Endothelprogenitorzellen (1)
- Endothelzelle (1)
- Endothelzellen (1)
- Endsteinzeit (1)
- Entomologie (1)
- Entomology (1)
- Enzymaktivität (1)
- Enzyme (1)
- Enzymologie (1)
- EphrinB2 (1)
- Eplerenon (1)
- Erdmagnetfeld (1)
- Erdmagnetismus (1)
- Erithacus rubecula (1)
- Ernährungsweise (1)
- Escherichia coli (1)
- Ethnobotanik (1)
- Ethnoökologie (1)
- European Rabbit (1)
- European Robins (1)
- European robin (1)
- Europäisches Wildkaninchen (1)
- Evolutionary developmental biology (1)
- Excitation (1)
- Excitatory balance (1)
- Exozytose (1)
- Export (1)
- Extracellular matrix (1)
- Extrakt (1)
- Extremhabitat (1)
- Exzitation (1)
- Exzitotoxizität (1)
- FCP (Fucoxanthin-Chlorophyll bindende Proteine) (1)
- FCP (Fucoxanthin-Chlorophyll binding Protein) (1)
- FHL-1 (1)
- FRET (1)
- FVIII (1)
- Fabclavine (1)
- Faktor H (1)
- Felsentaube ; Ortsgedächtnis (1)
- Ferroptosis (1)
- Flavobacterium (1)
- Flavobacterium spec P99-3 (1)
- Flavoproteins (1)
- Fledermäuse (1)
- Floristische Kartierung (1)
- Fluorene derivatives (1)
- Fluorescence Lifetime (1)
- Fluorescence labelling (1)
- Fluoreszenz <Motiv> (1)
- Fluoreszenz-Resonanz-Energie-Transfer (1)
- Fluoreszenzmarkierung (1)
- Fluoreszenzspektrometer (1)
- Fluoreszenzspektroskopie (1)
- Fluorid cleavable linker (1)
- Fluorid spaltbarer Linker (1)
- Fluss (1)
- Flussnapfschnecke (1)
- Follikuläre dendritische Zellen (1)
- Foraminiferen (1)
- Force Field Development (1)
- Formvergleich (1)
- Fraßschaden (1)
- Freilandstudie (1)
- Freshwater (1)
- Freshwater Ecosystems (1)
- Fucoxanthin-Chlorophyll-Protein (1)
- Fulani (1)
- Fulbe (1)
- Functional Ecology (1)
- Functional traits (1)
- Funktion (1)
- Fusionsinhibitor (1)
- G-protein coupled receptor (1)
- GABA (1)
- GABA Transporter Typ 1 (1)
- GC-MS (1)
- GEF (1)
- GLUT4 (1)
- GSVs (1)
- Gal2 (1)
- Galakturonsäure (1)
- Gaussian processes (1)
- Gauß Prozesse (1)
- Gehölze (1)
- Gehölzfrüchte (1)
- Gemeinschwämme (1)
- Gene expression (1)
- Gene therapy (1)
- Gene trap (1)
- Genetics (1)
- Genetische Variabilität (1)
- Genfalle (1)
- Genklonierung (1)
- Genom (1)
- Genome (1)
- Genome engineering (1)
- Gentianinae (1)
- Geoinformationssystem (1)
- Gephyrin (1)
- Gerinnungsfaktor VIII (1)
- Gerontologie (1)
- Giraffe (1)
- Glatter Krallenfrosch (1)
- Glioblastom (1)
- Global Alignment (1)
- Global change (1)
- Glycinrezeptor (1)
- Glykoprotein GP 41 (1)
- Glykoproteine5550 !085487139!{{Chemische Synthese}}; Enzymkatalyse (1)
- Glyzinrezeptor (1)
- Graphentheorie (1)
- Grün fluoreszierendes Protein (1)
- HC-Pro (1)
- HIV infection (1)
- HIV-Infektion (1)
- HPLC (1)
- HRGXE-Motiv (1)
- HWC database (1)
- Habituation (1)
- Halobacterium salinarium (1)
- Halobacterium salinarum (1)
- Haloferax volcanii (1)
- Halophile (1)
- Halophile Bakterien (1)
- Hauptkomponentenanalyse (1)
- Heart (1)
- Hefe (1)
- Hefeartige Pilze (1)
- Helix <alpha-> (1)
- Hematopoetic differentiation (1)
- Hemicellulose (1)
- Herpesvirus (1)
- Hexachlorbenzol (1)
- Hinterhorn <Rückenmark> (1)
- Hinterkiemer (1)
- Hippocampal development (1)
- Hippocampus (1)
- Hirntumor (1)
- Histon-Deacetylierung (1)
- Histone (1)
- Histone Deacetylation (1)
- Histonmodifikationen (1)
- Hodgkin-Lymphom (1)
- Homeobox (1)
- Homeodomänenproteine ; Stat1 (1)
- Homologiemodellierung (1)
- Homöobox (1)
- Honigbiene (1)
- Hydrogen storage (1)
- Hydrogen-dependent CO2 reductase (1)
- Hydrogenasen (1)
- Hydrolasen (1)
- Hämatopoese (1)
- Höhle (1)
- Hörschädigung (1)
- Hörzelle (1)
- IKK epsilon (1)
- IMAC (1)
- Immortalisierung (1)
- Immunadsorption (1)
- Immunkrankheit (1)
- Immunology (1)
- Immunreaktion (1)
- Immunrekonstitution (1)
- Immunseren (1)
- Immunsuppression (1)
- Import (1)
- Indikator (1)
- Indirekteinleiter (1)
- Induced Fit (1)
- Industrieabwasser (1)
- Infectivity (1)
- Infektiösität (1)
- Inflammation (1)
- Influenza (1)
- Inhibitor Binding Pocket (1)
- Inhibitor-binding (1)
- Inhibitorbindetasche (1)
- Inhibitory balance (1)
- Inhibitory interneurons (1)
- Innenohr (1)
- Innovation (1)
- Inquisition post mortem (1)
- Integrale Membranproteine (1)
- Interaktionsanalyse (1)
- Interferon (1)
- Inthraszentin (1)
- Intrazellulärer Transport (1)
- Ionenkanal (1)
- Ionenspezifität (1)
- Ischemia (1)
- Isolierung <Chemie> (1)
- Isopoda (1)
- Isoprenoide (1)
- JAK/STAT-Signalweg (1)
- Java (1)
- Juckreiz (1)
- Juvenile neuronal ceroid lipofuscinosis (1)
- Kalium Kanäle (1)
- Kalmar <Art> ; Diisopropylfluorophosphatase (1)
- Kalziumspeicher (1)
- Kampfsport (1)
- Kanalprotein (1)
- Karate (1)
- Kardiovaskuläres System (1)
- Karibik (1)
- Katabolismus (1)
- Katalase (1)
- Kation ; Organische Verbindungen ; Elektrophysiologie (1)
- Katze (1)
- Kern-Zytoplasma-Transport (1)
- Kernhülle ; Hitzeschock-Proteine ; Proteine ; Wechselwirkung (1)
- Kernpilze (1)
- Ketonkörper (1)
- Kichererbse (1)
- Kieselalgen (1)
- Kinabalu (1)
- Kinematik (1)
- Klassifikation (1)
- Klimarekonstruktion (1)
- Knockout <Molekulargenetik> (1)
- Koaktivatoren (1)
- Kohlenstoffisotop (1)
- Kohlenstoffkreislauf (1)
- Konkurrenz (1)
- Konstitutionstypen (1)
- Kopplungskonstanten (1)
- Korrelation (1)
- Krebsforschung (1)
- Kristallographie (1)
- Kristallzüchtung (1)
- Kronendach (1)
- Kryo-Elektronenkristallographie (1)
- Kryoelektronenmikroskopie (1)
- Kryokonservierung (1)
- Kuala Lumpur <Region> ; Bambus ; Ameisen ; Biozönose (1)
- Kulturlandschaft (1)
- Kupfer (1)
- Kupfer-ATPase (1)
- Kupferstoffwechsel (1)
- Körperfett (1)
- Künstliche Niere (1)
- LINC00607 (1)
- LINE-1 (1)
- LTD (1)
- LTP (1)
- Labyrinth <Anatomie> (1)
- Lactalbumin (1)
- Landschaftsentwicklung (1)
- Landwirtschaft (1)
- Laserin (1)
- Later Stone Age (1)
- Lebensmittelallergie (1)
- Lebensweise (1)
- Lebenszykluseffekte (1)
- Lentivirale Vektoren (1)
- Licht (1)
- Lichtabhängigkeit (1)
- Lichtsammelkomplexe (1)
- Ligand (Biochemie) (1)
- Ligand <Biochemie> (1)
- Light sheet-based fluorescence microscopy (1)
- Light-sheet microscopy (1)
- Limonene-3-hydroxylase (1)
- Line Notation (1)
- Lineage Through Time (1)
- Linearisierung (1)
- Linker (1)
- Lipid Environment (1)
- Lipidmembran (1)
- Lipoxygenase <5-> (1)
- Lycopersicon peruvianum ; Hitzestress ; Transkriptionsfaktor ; Proteintransport ; Hitzeschocktranskriptionsfaktor (1)
- Lymphknoten (1)
- Lysosomal storage disease (1)
- Lysosomale Speicherkrankheit (1)
- Lysozym (1)
- M87o (1)
- MALDI (1)
- MCAK (1)
- MD Simulation (1)
- MEA (1)
- MEK inhibition (1)
- MHC (1)
- MICOS complex (1)
- MLL (1)
- Macroevolution (1)
- Macrotermes (1)
- Magnesium (1)
- Magnetkompass (1)
- Magnetkompassorientierung (1)
- Magnetokardiographie (1)
- Magnetperzeption (1)
- Magnetrezeption (1)
- Magnetrezeptormolekül (1)
- Maillard reaction (1)
- Maillard-Reaktion (1)
- Makromolekulare Chemie (1)
- Makromolekül (1)
- Makroreste (1)
- Malaysia (1)
- Malaysia ; Leptogenys ; Schwarmverhalten ; Pheromon ; Verhalten (1)
- Manteltiere (1)
- Markierungsgen (1)
- Maschinelles Lernen (1)
- Massenspektrometrie (1)
- Maternal Immune Activation (1)
- Maus ; Recombinasen (1)
- Mediale olivo-cochleäre Efferenz (1)
- Mediterranean (1)
- Medizintechnik (1)
- Meerschweinchen (1)
- Mehrdimensionale NMR-Spektroskopie (1)
- Membran (1)
- Membrane Potential (1)
- Membrane Proteins (1)
- Membranfusion (1)
- Membranlipide (1)
- Messenger-RNS ; Translokation ; Peptide ; Wechselwirkung ; Leukämie (1)
- Meta effect (1)
- Metabolon (1)
- Methanogen (1)
- Methanol (1)
- Methylobacterium (1)
- Methyltransferase (1)
- Microarray (1)
- Microbielle rhodopsine (1)
- Microbiology (1)
- Microsatelliten (1)
- Microvesicles (1)
- Migration (1)
- Mikrobiom (1)
- Mikroplastik, Boden, Reifenabrieb, Analytik (1)
- Mikroskopie (1)
- Mikrovesikel (1)
- Milchdrüse (1)
- Milchdrüse ; Entwicklung ; Genregulation (1)
- Milchdrüsengewebe (1)
- Mitochondriale DNS (1)
- Mitochondrien (1)
- Mitose (1)
- Mitteleuropa (1)
- Mittelmeerraum (1)
- Molarenevolution (1)
- Molecular Evolution (1)
- Molecular dynamic simulation (1)
- Molekulare Evolution (1)
- Molekulargenetik (1)
- Moleküldesign (1)
- Moleküldynamiksimulation (1)
- Molekülkomplex (1)
- Monoclonal Antibodies (1)
- Monographie (1)
- Monoklonaler Antikörper (1)
- Monolage (1)
- Monoterpenoid (1)
- Monoterpenoid tolerance (1)
- Monozyklase (1)
- Monozyt (1)
- Morphogenesis (1)
- Morphology (1)
- Moschcowitz-Syndrom (1)
- Motivation (1)
- Motorprotein (1)
- Mulitvariate Statistik (1)
- Multi-domain proteins (1)
- Multielektroden (1)
- Multikomponentenreaktion (1)
- Multiproteinkomplex (1)
- Multispezies-Testsysteme (1)
- Multivariate Modellierung (1)
- Muscheln (1)
- Mutante (1)
- Mutualismus (1)
- MySQL (1)
- Myrmekophytie (1)
- Myxol (1)
- NCoA proteins (1)
- NCoA-Proteine (1)
- NES (1)
- NLS (1)
- NMDA (1)
- NMDA-Antagonist (1)
- NMDA-Rezeptor (1)
- NMR shift calculation (1)
- NMR-Spectroscopy (1)
- NMR-spectroscopy (1)
- Naja (1)
- Nanodiscs (1)
- Nanoparticle (1)
- Nanopartikel (1)
- Nase (1)
- Natriumglutamat <Natrium-L-glutamat> (1)
- Natural Products (1)
- Natural products (1)
- Naturheilkunde (1)
- Naturstoff (1)
- Naturstoffe (1)
- Nekrose (1)
- Neovascularisation (1)
- Neozoen (1)
- Nervensystem (1)
- Nervenzelle (1)
- Nestbau (1)
- Netzhaut (1)
- Neuroanatomie (1)
- Neurodevelopmental Psychiatric Disorders (1)
- Neuronale Differenzierung (1)
- Neuronale Plastizität (1)
- Neurosimulation (1)
- Neurotransmitter-Rezeptor (1)
- Niche (1)
- Nichtsteroidales Antiphlogistikum (1)
- Niere (1)
- Nierentubulus (1)
- Non-canonical terpenes (1)
- Normalkoordinatenanalyse (1)
- Novel Object Test (1)
- Nuclear factor kappa B (1)
- Nucleinsäuren (1)
- Nucleus reuniens (1)
- Nuklearfaktor Kappa B (1)
- Nukleotidzucker (1)
- OAE (1)
- ORF1 (1)
- Offener Leserahmen (1)
- Ohrenqualle ; Phylogenie ; Sequenzanalyse <Chemie> ; Artbildung ; Tiergeographie (1)
- Ohrenqualle ; Strobilation ; Umweltüberwachung (1)
- Oligonukleotid (1)
- Omp85 (1)
- Omp85-Protein (1)
- Ontogenese (1)
- Oozyten von Xenopus laevis (1)
- Opisthobranchia (1)
- Optical electrophysiology (1)
- Optische Spektroskopie (1)
- Optogenetics (1)
- Optogenetik (1)
- Orexin (1)
- Organic micropollutants (1)
- Organische Synthese (1)
- Organogenese (1)
- Organoids (1)
- Orientation (1)
- Orientierung (1)
- Ortsgedächtnis (1)
- Ortspezifische Mutagenese (1)
- Oxidativer Stress (1)
- Oxoferrylzustand (1)
- Oxygenierung (1)
- P-bodies (1)
- PAM (1)
- PELDOR / DEER (1)
- PGE2 (1)
- PGE2 Cancer Tumorigenesis (1)
- PSGP (1)
- Paarverteilungsfunktion (1)
- Pain (1)
- Palladium-Katalyse (1)
- Pan paniscus (1)
- Papierindustrie (1)
- Paprika ; Desaturasen ; Genexpression ; Paprika (1)
- Paracoccus denitrificans ; Cytochrom c1 (1)
- Paracoccus denitrificans ; Cytochromoxidase ; Protonentransfer ; Elektronentransport (1)
- Parallelverarbeitung (1)
- Parkinson (1)
- Paruroctonus mesaensis (1)
- Patch-Clamp-Methode (1)
- Patch-clamp (1)
- Paul-Ehrlich-Institut (1)
- Peddigrohr (1)
- Peptidbeladungskomplex (1)
- Peptide (1)
- Peptide Loading Complex (PLC) (1)
- Perania nasuta (1)
- Perlhirse (1)
- Permeasen (1)
- Persischer Golf ; Krabben ; Systematik ; Tiergeographie ; Mittelkrebse (1)
- Persönlichkeit (1)
- Pestalotia (1)
- Pestalotiopsis (1)
- Pestizidbelastung (1)
- Pflanzen ; Katalase ; Heterologe Genexpression (1)
- Pflanzenameisen (1)
- Pflanzenfressende Insekten (1)
- Pflanzenhormon (1)
- Pflanzenkartierung (1)
- Pflanzenphysiologie (1)
- Pflanzensoziologie (1)
- Phaeodactylum tricornutum (1)
- Pharmacophore (1)
- Pharmazeutische Chemie (1)
- Pharmazie (1)
- Phasenverschiebung (1)
- Photocages (1)
- Photoisomerization (1)
- Photolabile protecting groups (1)
- Photorhabdus (1)
- Photosynthetisches Reaktionszentrum (1)
- Photosystem I (1)
- Phylogeny (1)
- Phytochrome (1)
- Phytoen (1)
- Pichia ciferrii (1)
- Pigmentproteine (1)
- Pimephales promelas (1)
- Pink1 (1)
- Pinnotheres (1)
- Plant physiology (1)
- Plant regeneration (1)
- Plant regeneration; community assembly; diversity (1)
- Plants (1)
- Plasmamembran ; Calcium-ATPasen ; Calmodulin ; Peptide ; Wechselwirkung ; Dreidimensionale NMR-Spektroskopie (1)
- Pleistozän (1)
- Podospora anserina (1)
- Poecilia (1)
- Polymere (1)
- Polypeptid transport-associated domains (1)
- Population genetics (1)
- Populationsgenetik (1)
- Positive reinforcement training (1)
- Post-Targeting Funktionen (1)
- Postglaziale Verbreitung (1)
- Posttranslationale Modifikationen (1)
- Potyviren (1)
- PrP (1)
- Prenyl pyrophosphates (1)
- Primär aktive Transporter (1)
- Prion (1)
- Prionprotein (1)
- Processing bodies (1)
- Prognose (1)
- Proliferation (1)
- Promoter (1)
- Promotor (1)
- Promotor <Genetik> (1)
- Prostaglandine (1)
- Prostgandin E Synthasen (1)
- Proteasom (1)
- Proteasomeninhibitoren (1)
- Protein Arginin Methyltransferase (1)
- Protein associated with myc (1)
- Protein flexibility (1)
- Protein-Protein Interaktion (1)
- Protein-Protein-Wechselwirkung (1)
- Protein-Sortierung (1)
- Protein-Tyrosin-Kinase (1)
- Protein-Tyrosin-Phosphatase (1)
- Proteine ; Ligand <Biochemie> ; Wechselwirkung ; NMR-Spektroskopie (1)
- Proteine; Posttranslationale Änderung; Elektrospray-Ionisation; Massenspektrometrie (1)
- Proteinexpression (1)
- Proteinflexibilität (1)
- Proteinregulation (1)
- Proteinsekretion (1)
- Proteintransport (1)
- Proteintyrosinphosphatase (1)
- Proteoliposomen (1)
- Proteomics (1)
- Proteorhodopsin (1)
- Proteostasis (1)
- Proton transfer (1)
- Protonenpumpe (1)
- Pseudomonas (1)
- Pseudomonas putida (1)
- Pseudorezeptoren (1)
- Pseudotypisierung (1)
- Pulmonale Hypertonie (1)
- Puromycin (1)
- Pyramidal neurons (1)
- Pyrrolimidazolalkaloide (1)
- Qinghai-Tibet Plateau (1)
- Quercus (1)
- Quercus frainetto Ten. (Ungarische Eiche) (1)
- Quercus ilex L. (Steineiche) (1)
- Quercus pubescens Willd. (Flaumeiche) (1)
- Quercus robur L. (Stieleiche) (1)
- Quercus rubra L. (Roteiche) (1)
- Quinolinate Phosphoribosyltransferase (1)
- Quinon-Fumarat-Reduktase (1)
- R peptide (1)
- R-Peptid (1)
- RAPDs (1)
- RBFOX1 (1)
- RDCs (1)
- REM-Schlaf (1)
- RNA interference (1)
- RNA-Interferenz (1)
- RNA-folding (1)
- RNS-Bindungsproteine (1)
- RNS-Interferenz (1)
- RUNX1 (1)
- Radical-Pair-Mechanism (1)
- Radikal <Chemie> (1)
- Radikalpaar (1)
- Radikalpaar-Mechanismus (1)
- Radikalpaar-Prozess (1)
- Radioliganden (1)
- Raf <Biochemie> (1)
- Ras (1)
- Reaktionskinetik (1)
- Receptor-based (1)
- Reconstitution (1)
- Regeneration (1)
- Regulation (1)
- Regulation der Genexpression (1)
- Reinigung (1)
- Rekonstitution (1)
- Remote sensing (1)
- Renal tubule (1)
- Renin-Angiotensin-System (1)
- Repetitive DNS (1)
- Reproduction (1)
- Retina (1)
- Retroviren (1)
- Revision (1)
- Rezeptor (1)
- Rezeptorbasiert (1)
- Rezeptorinternalisierung (1)
- Rheumatoid Arthritis (1)
- Rhinophores (1)
- Rhodnius prolixus (1)
- Rhodopsin (1)
- Rhodopsine (1)
- Ribosomen, rRNA Prozessierung, snoRNA, Ribosomenbiogenesefaktoren (1)
- Ribozym (1)
- Rind (1)
- Risikoanalyse (1)
- Risikomanagement (1)
- Risk assessment (1)
- Rotang-Palme (1)
- Rotoren (1)
- Russell´s Viper (1)
- Ruthenium (1)
- Ruthenium-Laserflash-Spektroskopie (1)
- Rückenmark (1)
- Rückzugsreflex (1)
- SAGE (1)
- SCA2 (1)
- SCO2 (1)
- SILAC (1)
- SIMPrint (1)
- SPAD (1)
- SR proteins (1)
- STAT5 (1)
- STAT5-DNA Bindungsstellen (1)
- STM (1)
- Sahel ; Unkraut ; Pflanzensoziologie ; Burkina Faso (1)
- Salt stress (1)
- Salztress (1)
- Sarkoplasmatisches Retikulum ; Calcium ; ATP ; Wechselwirkung (1)
- Sauerstoffisotop (1)
- Sauerstoffradikal (1)
- Savanna (1)
- Scaffold Hopping (1)
- Schadstoffbelastung (1)
- Schallintensität (1)
- Schistosomiaisis (1)
- Schleimpilze (1)
- Schmierläuse (1)
- Schutz (1)
- Schwefelwasserstoff (1)
- SecYEG (1)
- Sediment (1)
- Sehzelle (1)
- Sekretion (1)
- Sekundenherztod (1)
- Sekundärmetabolite (1)
- Sensorrhodopsin (1)
- Septische Granulomatose (1)
- Sequenzierung durch Synthese (1)
- Shapecomparison (1)
- Signaling (1)
- Signalpeptide (1)
- Signalverarbeitung (1)
- Similarity (1)
- Simulation (1)
- Sklerochronologie (1)
- Somatochart (1)
- Somatologie (1)
- Soziobiologie (1)
- Sp1 (1)
- Spatial navigation (1)
- Species distribution modelling (1)
- Sphingolipide (1)
- Sphingosin (1)
- Sphingosin-1-Phosphat (1)
- Sphingosinkinase 2 (1)
- Spinach (1)
- Spine (1)
- Spinlabel (1)
- Spironolacton (1)
- Sprung (1)
- Stadtökologie (1)
- Stammzellen (1)
- Stammzelltransplantation (1)
- Stat1 (1)
- Stat3 Gliom Curcumin (1)
- Stechmücken (1)
- Stehendes Gewässer (1)
- Stickstoffmonoxid (1)
- Stofftransport <Biologie> (1)
- Stoffwechsel (1)
- Streptomyces coelicolor (1)
- Stress response (1)
- Stressreaktion (1)
- Structure-based Mutagenesis Study (1)
- Structured Illumination Microscopy (1)
- Struktur (1)
- Struktur-Aktivitäts-Beziehung (1)
- Strukturanalyse (1)
- Strukturbiologie (1)
- Sudden cardiac death (1)
- Sumoylation (1)
- Sumoylierung (1)
- Super resolution (1)
- Super resolution fluorescence microscopy (1)
- SuperSAGE (1)
- Survivin (1)
- Svetamycin (1)
- Symbiont evolution (1)
- Symbiose (1)
- Symbiosis (1)
- Synapse (1)
- Synovial Fibroblast (1)
- Systematics (1)
- Säugetiere ; Melatonin ; Biosynthese ; Regulation (1)
- Südostasien; Macaranga; Bestäubung; Blasenfüße; Mutualismus; Crematogaster; Südostasien; Macaranga; Fortpflanzung; Bestäubung; Blasenfüße; Kastration; Ameisen (1)
- Süßwasser (1)
- T-Lymphozyt (1)
- T-Zell-Leukämie (1)
- T-Zellen (1)
- TAP (1)
- TIGAR (1)
- TKTL1 (1)
- TRAIL (1)
- TRPV1 (1)
- Tabanidae (1)
- Talent (1)
- Talentsuche (1)
- Taphonomie (1)
- Targeted drug delivery (1)
- Taschenoberfläche (1)
- Tau-Protein (1)
- Taube (1)
- Taunus (1)
- Taxonomie (1)
- Taxonomy (1)
- Technologieakzeptanz (1)
- Telomerase (1)
- Temperatur (1)
- Temperaturabhängigkeit (1)
- Temporäres Gewässer (1)
- Tentakel <Zoologie> (1)
- Terbutryn (1)
- Termiten (1)
- Terpenes (1)
- Terpenoid (1)
- Testosteronreductase <Testosteron-5-alpha-Reductase> (1)
- TetR (1)
- Tetrablemmidae (1)
- Tetramerisation (1)
- Tetrazyklinrepressor (1)
- Thermophile (1)
- Thermophile Bakterien (1)
- Thermoregulation (1)
- Thiosulfat Sulfurtransferase (1)
- Thrombotic thrombocytopenic purpura (1)
- Thrombozyten (1)
- Time-averaging (1)
- Tissue Engineering (1)
- Tocochromanol (1)
- Todesrezeptor (1)
- Tomate (1)
- Tomate ; Hitzestress ; Transkriptionsfaktor (1)
- Tonhöhe (1)
- Torini (1)
- Toxizität (1)
- Transcription (1)
- Transcriptional activity (1)
- Transcriptome (1)
- Transcriptomics (1)
- Transduktion B Zellen (1)
- Transformation (1)
- Transient absorption (1)
- Transkription (1)
- Transkription <Genetik> (1)
- Transkriptionsfaktoren (1)
- Transkriptomanalyse (1)
- Translation <Genetik> (1)
- Translational Psychiatry (1)
- Translocation (1)
- Transplantation (1)
- Transponierbares Element (1)
- Transport (1)
- Transporter associated with antigen processing (TAP) (1)
- Transporter assoziiert mit Antigen-Prozessierung (1)
- Transposon systems (1)
- Triatominae (1)
- Trichoptera (1)
- Trichostatin A (1)
- Tripterospermum (1)
- Trockenstress (1)
- Tropical montane forest (1)
- Truffle (1)
- Trypanosoma cruzi (1)
- Tubastrin (1)
- Tumor (1)
- Tumor necrosis factor alpha (1)
- Tumor-Nekrose-Faktor <alpha> (1)
- Tumorsuppressor-Gen (1)
- Tumorwachstum (1)
- Typ 4 Pilus (1)
- Typ I Interferon Rezeptor (1)
- Tyrosin (1)
- UDP-Glucose-Dehydrogenase (1)
- UDP-glucose dehydrogenase (1)
- UL49.5 (1)
- UV-VIS-Spektroskopie (1)
- UVB (1)
- Ubichinonbindetasche (1)
- Ubiquinon-Cytochrome c-Reductase (1)
- Ubiquinone Binding Pocket (1)
- Ubiquitin chains (1)
- Uhrengene (1)
- Umweltchemikalie (1)
- Umweltdaten (1)
- Umweltgefährdung (1)
- Umweltgeochemie (1)
- Umweltrisikoabschätzung (1)
- Umwelttoxikologie (1)
- Umweltwahrnehmung (1)
- Unterart; Pollen; Sammeln; Verhalten (1)
- Untranslated Region (1)
- Urban Ecology (1)
- Ustilaginomycotina (1)
- V1 (1)
- VEGF (1)
- VSV (1)
- Vaccinieae (1)
- Vaccinium (1)
- Varicellovirus (1)
- Varizellen-Virus (1)
- Vaskularisation (1)
- Vaskulogenese (1)
- Vegetationsgeschichte (1)
- Verbreitungsökologie (1)
- Verzögerte Fluoreszenz (1)
- Vesikel (1)
- Viral Infection (1)
- Viral entry (1)
- Virologie (1)
- Virtual screening (1)
- Virusinfektion (1)
- Viskoelastizität (1)
- Vitality monitoring (1)
- Vogelzug (1)
- Voltage Clamp (1)
- Voltage Imaging (1)
- Volumenverschiebungen (1)
- Von Willebrand Faktor (1)
- Von Willebrand factor (1)
- Vorhofflimmern (1)
- Voronoi-Diagramm (1)
- Vögel (1)
- Wasserflöhe (1)
- Wasserstoffionenkonzentration (1)
- Wasserstoffperoxid (1)
- Wechselwirkung (1)
- Western Kenya (1)
- Whole Effluent Assessment (1)
- Wilson Disease Protein (1)
- Wirbellose (1)
- Wirtspflanzen (1)
- X-ray crystallography (1)
- Xylose (1)
- Yeast (1)
- YidC (1)
- ZIKV (1)
- ZYMV (1)
- Zahn-Wellens test (1)
- Zahn-Wellens-Test (1)
- Zahnwale (1)
- Zeaxanthin (1)
- Zebrafish (1)
- Zeit-Frequenz-Analyse (1)
- Zeitauflösung (1)
- Zeitverarbeitung (1)
- Zell-basierte Assaysysteme (1)
- Zellaufnahme (1)
- Zelldifferenzierung (1)
- Zelltod (1)
- Zellulares neuronales Netz (1)
- Zellwand (1)
- Zentralnervensystem (1)
- Zirbeldrüse (1)
- Zoologie (1)
- Zugvögel (1)
- Zuwachs (1)
- Zweiphotonen-Anregung (1)
- Zyklisierung (1)
- Zytokinrezeptor (1)
- accessory subunit (1)
- actin (1)
- acyl carrier protein, polyketide synthases, Curacin cluster (1)
- adult neurogenesis (1)
- aggregopathy (1)
- aktuelles Interesse (1)
- akute Toxizität (1)
- aldosterone (1)
- allosterischer Modulator (1)
- alternative splicing (1)
- ant-plants (1)
- anthracology (1)
- anti-inflammatory (1)
- antimicrobial resistance (1)
- aptamer (1)
- arabinose (1)
- archaeobotany (1)
- aroma (1)
- atrial fibrillation (1)
- atrophy (1)
- auditory Midbrain (1)
- autoproteolysis (1)
- bacillary angiomatosis (1)
- bacteria-host interaction (1)
- bacterial infection (1)
- bacterial two hybrid system (1)
- bakterielles Two Hybrid System (1)
- bats (1)
- bc1-complex (1)
- benthic (1)
- benthisch (1)
- bioassay (1)
- biodiversity (1)
- bioenergetics (1)
- bioinformatic (1)
- biomechanics (1)
- birds (1)
- bivalve assemblage (1)
- black lipid membrane (1)
- blood (1)
- boswellic acids (1)
- brain waves (1)
- cAMP (1)
- calcium store (1)
- carotenoid biosynthesis (1)
- caspase-8 (1)
- cell biology (1)
- cell death (1)
- cell migration (1)
- cell targeting (1)
- cell uptake (1)
- cell wall precursor (1)
- cell-based assay-systems (1)
- cell-free (1)
- chemical shifts (1)
- chemical synthesis (1)
- chemistry education (1)
- chronische Toxizität (1)
- cisternal organelle (1)
- climate change (1)
- climate reconstruction (1)
- coactivators (1)
- coagulation factor VIII (1)
- cobra (1)
- cochlear amplifier (1)
- cochleärer Verstärker (1)
- coevolution (1)
- color (1)
- complex of closely related species (1)
- computational chemistry (1)
- conformation (1)
- consortia (1)
- cooperation (1)
- cophylogeny (1)
- cospeciation (1)
- crosslinking-mass spectrometry (1)
- cryo-EM (1)
- cryo-eletron crystallography (1)
- cryptochrome (1)
- cultivation (1)
- cultural landscape (1)
- cyclic nucleotide-gated ion channel (1)
- cyclooxygenase (1)
- cyclooxygenase-2 (1)
- cytochrome c oxidase (1)
- cytokine (1)
- cytokine receptor (1)
- dMM (1)
- de novo design (1)
- depolymersation (1)
- detergent (1)
- development (1)
- diabetes type 1 (1)
- diatom (1)
- differentiation (1)
- display Bibliotek (1)
- display library (1)
- distribution (1)
- diurnal (1)
- diversity (1)
- domatia (1)
- drug design (1)
- drug discovery (1)
- dryland (1)
- dynamics (1)
- ecological genetics (1)
- ecosystem services (1)
- elapid snake (1)
- electrical remodeling (1)
- electron microscopy (1)
- electron transfer (1)
- electrophysiologie (1)
- electrophysiology (1)
- elektrochemisches Protonenpotential (1)
- elephant (1)
- enantioselektive Synthese (1)
- endothelin receptor (1)
- endothial precursor cells (1)
- entry inhibitor (1)
- envenoming (1)
- environmental DNA (1)
- environmental attitudes (1)
- environmental behavior (1)
- environmental education (1)
- environmental knowledge (1)
- environmental perception (1)
- environmental stressors (1)
- enzymatically cleavable Linker (1)
- enzymatisch spaltbarer Linker (1)
- enzyme assay (1)
- enzyme inhibitor (1)
- epigenetic regulation (1)
- ethnobotany (1)
- ethnoecology (1)
- export (1)
- failure to diverge (1)
- feeding habit (1)
- fitness (1)
- flora (1)
- fluorescence (1)
- follicular dendritic cells (1)
- food allergy (1)
- food quality (1)
- frequency-time analysis (1)
- freshwater (1)
- freshwater crayfish (1)
- fucoxanthin-chlorophyll-protein (1)
- full-thickness skin model (1)
- functional (1)
- functional coupling (1)
- fungal phylogeny (1)
- fusion inhibitor (1)
- gamma oscillations (1)
- gen expression (1)
- generation probability (1)
- genetical engineering (1)
- genetransfer (1)
- genotype (1)
- geoecology (1)
- geographic information system (GIS) (1)
- gephyrin (1)
- gerbil (1)
- gerontology (1)
- glatte Gefäßmuskelzellen (1)
- glioma (1)
- glucocorticoid (1)
- glutamate transporter (1)
- glycerophospholipid (1)
- glycine rezeptor (1)
- gp41 (1)
- granule cells (1)
- hGPR63 (1)
- hS1P5-Rezeptor (1)
- habitat heterogeneity (1)
- hair cell (1)
- hearing loss (1)
- heart development (1)
- heat stress (1)
- hematopoietic stem cell (1)
- hemicellulose (1)
- hen egg white lysozyme (1)
- hepatische Ketogenese (1)
- herbivores (1)
- herpesvirus (1)
- heterosynaptic plasticity (1)
- high-content screening (1)
- high-frequency stimulation (1)
- hippo (1)
- hippocampus (1)
- histone modifications (1)
- homeobox (1)
- homeobox A9 (1)
- homeodomain proteins (1)
- host-switch (1)
- human pancreatic organoids (hPOs) (1)
- human-wildlife conflict (1)
- hyperalgesia (1)
- immortalization (1)
- import (1)
- in vivo screen (1)
- indirect discharger (1)
- industrial effluents (1)
- information literacy (1)
- infra-slow oscillation (1)
- inhibition (1)
- integral membrane proteins (1)
- intensity (1)
- intermediate state (1)
- intracellular transport (1)
- intramolecular protein interaction (1)
- intramolekulare Proteininteraktion (1)
- katalytischer Zyklus (1)
- kern-basiertes Lernen (1)
- kernel learning (1)
- kinetics (1)
- konstitutive Aktivität (1)
- krait (1)
- lange Signalpeptide (1)
- lateral line (1)
- left ventricular hypertrophy (1)
- lentiviral vector (1)
- lentiviral vectors (1)
- lentivirale Vektoren (1)
- lentivirale siRNA Transduktion (1)
- lentiviraler Vektor (1)
- leukocyte adhesion (1)
- lichtaktivierbare Nukleinsäure (1)
- life cycle effects (1)
- life habit (1)
- light dependent magnetic compass (1)
- light-harvesting complexes (1)
- lightactivatable nucleic acids (1)
- lipid metabolism (1)
- livelihood (1)
- long non-coding RNA (1)
- long signal peptide (1)
- long-term depression (1)
- long-term potentiation (1)
- mPFC (1)
- mPGES-1 (1)
- mRNA-Abbau (1)
- mRNA-Speicherung (1)
- mTOR (1)
- macrohabitat (1)
- macroremains (1)
- magnetic compass orientation (1)
- magnetic exchange coupling constants (1)
- magnetoreception (1)
- mammary gland tissue (1)
- mapping (1)
- mechanics (1)
- medically relevant (1)
- membrane anchor; substrate-binding protein dependent secondary transport; TRAP-associated extracytoplasmic immunogenic (TAXI); tripartite ATP-independent periplasmic (TRAP) (1)
- membrane fluidity (1)
- membrane fusion (1)
- membrane protein (1)
- metabolism (1)
- metabotroper Glutamatrezeptor (1)
- metabotropic (1)
- metastasis (1)
- miRNA (1)
- miRNS (1)
- microRNA-17-92 cluster (1)
- microRNAs (1)
- microbiome (1)
- microsatellites (1)
- migratory birds (1)
- mitochondrial dysfunction (1)
- mitosis (1)
- mitotic spindle (1)
- modeling (1)
- molecualr phylogeny (1)
- molecular beacons (1)
- molecular phylogenetics (1)
- molecular structure (1)
- monocyclase (1)
- monocytes (1)
- morphological features (1)
- motor protein (1)
- mounting (1)
- mouse (1)
- movement (1)
- mtDNA (1)
- multidimensional nmr spectroscopy (1)
- multielectrode array (1)
- mutants (1)
- mutualism (1)
- myeloid angiogenic cells (1)
- myrmecophytes (1)
- neuromodulation (1)
- neuronal plasticity (1)
- niche evolution (1)
- nicht kodierende RNA (1)
- nicht-native Proteine (1)
- nitric oxide (1)
- nociception (1)
- nomadic (1)
- non coding RNA (1)
- non-native proteins (1)
- non-timber forest products (NTFPs) (1)
- npas4l (1)
- nucleotid-sugars (1)
- olivo-cochlear efferents (1)
- olivo-cochleäre Efferenzen (1)
- ontogenesis (1)
- organische Verbindungen (1)
- organotin compound (1)
- outdoor education (1)
- overtaking innovation (1)
- oxygen radical (1)
- p38 MAPK (1)
- p53 (1)
- p63 (1)
- pH-indicator dye (1)
- parathyroid hormone 2 (1)
- patch-clamp technique (1)
- pearl millet (1)
- peroxisom proliferator aktivierter Rezeptor (1)
- peroxisome proliferator-activated receptor (1)
- pesticide (1)
- photlabile protecting group (1)
- photolabile Schutzgruppen (1)
- photooxidativer Stress (1)
- photoschalbare (1)
- photospaltbare Linker (1)
- pitch (1)
- plant diversification (1)
- plant species diversity (1)
- plant-ants (1)
- platelets (1)
- pocket surface (1)
- polyketide synthase (1)
- potassium channel (1)
- potyvirus (1)
- pre-steady state (1)
- preparative cell-free expression (1)
- problem-based learning (1)
- promoter (1)
- pronephric duct (1)
- propagating waves (1)
- prostaglandin synthases (1)
- proteasomeinhibitors (1)
- protein expression (1)
- protein flexibilty (1)
- protein folding (1)
- protein sorting (1)
- protein-ligand docking (1)
- protein-protein interaction (1)
- proteome (1)
- proteome analysis (1)
- proton transfer (1)
- pseudoreceptors (1)
- pseudotyping (1)
- pulmonary hypertension (1)
- purification (1)
- quantenpunkte (1)
- quantitative proteomics (1)
- radical-pair process (1)
- radioligand (1)
- rat (1)
- rationale Stammentwicklung (1)
- repetitive Tonpulse (1)
- repetitive tone pips (1)
- reptiles (1)
- residual dipolar couplings (1)
- respiratory chain (1)
- retina (1)
- retrotransposition (1)
- retroviral vectors (1)
- retrovirale Vektoren (1)
- retrovirus (1)
- reversible Terminatoren (1)
- reversible terminator (1)
- ribosomes, Arabiodpsis thaliana, pre-rRNA processing, snoRNA, (1)
- rna interference (1)
- sRNA-Bindung (1)
- sRNA-binding (1)
- sage downy mildew (1)
- scFv (1)
- sclerochronology (1)
- scoring function (1)
- secretion (1)
- selbst-anordnende (1)
- sequencing by synthesis (1)
- serine/arginine-rich proteins (1)
- shallow lakes (1)
- shroom (1)
- siRNA (1)
- siderophore-dependent iron uptake (1)
- signal transduction (1)
- simulation (1)
- single particle electron microscopy (1)
- skin equivalent (1)
- sleep (1)
- small RNA (1)
- smooth muscle cell (1)
- smut fungi (1)
- snake bite (1)
- social isolation (1)
- socio-economics (1)
- soluble guanylyl cyclase (1)
- species delimitation (1)
- species distribution modelling (1)
- spheroid (1)
- sphingolipids (1)
- sphingosine (1)
- spinal cord (1)
- spine apparatus (1)
- splicing (1)
- stabile Isotope (1)
- stable isotopes (1)
- stem cells (1)
- stimulus repetition (1)
- stream macroinvertebrates (1)
- structure determination (1)
- structure modeling (1)
- sub-Saharan Africa (1)
- subgenera (1)
- subzelluläre Fraktionierung (1)
- sugar uptake (1)
- surround suppression (1)
- survivin (1)
- symbiont association patterns (1)
- synapse (1)
- synaptic plasticity (1)
- synaptic transmission (1)
- synaptic vesicle recycling (1)
- t cell leukemia (1)
- t(4;11) (1)
- targeted cell entry (1)
- tau protein (1)
- temperature (1)
- time-averaging (1)
- time-processing (1)
- time-resolved absorption spectroscopy (1)
- time-resolved fluorescence (1)
- transcriptome (1)
- transduction B cells (1)
- transglutaminase 2 (1)
- transient absorption (1)
- transiente Absorption (1)
- transkription factor (1)
- transmembran (1)
- tree crops (1)
- trimeric autotransporter adhesin (1)
- trnL-Intron (1)
- trnL-trnF & trnT-trnL Intergenischer Spacer (1)
- tsetse fly (1)
- type I interferon receptor (1)
- tyrosine radical (1)
- unfolded proteins (1)
- unterrichtliche Vor- und Nachbereitung (1)
- varicellovirus (1)
- vegetation (1)
- vegetation history (1)
- venomous snakes (1)
- verwilderte Hauskatzen (1)
- virtuelles Mikroskop (1)
- virtuelles Screening (1)
- woody vegetation (1)
- wwtr1 (1)
- xCGD (1)
- xylose (1)
- yap1 (1)
- zeitaufgelöste Fluoreszenz (1)
- zellfrei (1)
- zellfreie Expression (1)
- zielgerichteter Zelleintritt (1)
- zisternale Organelle (1)
- zoogeography (1)
- zyklisch Nukleotid-gesteuerter Ionenkanal (1)
- Ähnlichkeit (1)
- Übertragbarkeit (1)
- ökologische Genetik (1)
Institute
- Biowissenschaften (540)
- Biochemie und Chemie (168)
- Biochemie, Chemie und Pharmazie (78)
- Pharmazie (32)
- Institut für Ökologie, Evolution und Diversität (20)
- Georg-Speyer-Haus (6)
- Geowissenschaften (4)
- Medizin (3)
- Physik (3)
- keine Angabe Institut (3)
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
This work focused on the biosynthesis and characterization of esterified lipid mediators. Lipid mediators were generally thought to exert their effects as free molecules, and their esterification was regarded as a storage mechanism. However, more recent studies indicate that esterified lipid mediators are a distinct class of mediators. When this thesis started back in 2017, the idea of esterified lipids as a new class of mediators was relatively new so that respective compounds were either quite expensive or not commercially available at all. Therefore, a biosynthetic approach had to be established first to enable the study of the new lipid mediator class. Within the cell, esterified lipids are produced by activation and subsequent incorporation of polyunsaturated fatty acids. These steps are enzymatically catalyzed by members of the acyl-CoA synthetase family and the lysophosphatidylcholine acyltransferase family, respectively. Therefore, the enzymes acyl-CoA synthetase long-chain family member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 2 (LPCAT2) were selected for a biosynthetic approach due to their broad substrate acceptance.
In a first attempt, recombinant protein expression in E. coli was studied. While the expression and purification of C-terminally His6x-tagged ACSL4 resulted in a pure and active protein, the expression of LPCAT2 turned out quite troublesome. Although several expression and purification parameters were varied, including purification tags, buffer compositions, and chromatography strategies, successful purification of LPCAT2 was not achieved.
Instead, a second approach was studied. This time, stably transfected cells overexpressing ACSL4 and/or LPCAT2 were generated from the human embryonal kidney (HEK) 293T cell line. Stably transfected cell lines were characterized on protein level and regarding their oxylipin profile. After confirming the overexpression and functionality of the enzymes, lipoxygenases (LOs) were co-expressed in a doxycycline-inducible manner to prevent premature cell death due to increased oxidative stress. As a result, LO product formation was enhanced and enabled the investigation of specific oxylipins. Since increased lipid peroxidation is also a key component of the ferroptosis cell death mechanisms, cell lines were investigated towards their cell viability. Indeed, expression of ACSL4 and/or LPCAT2 promoted cell death when treated with the ferroptosis inducers erastin or RSL3, even in the absence of LO expression. Furthermore, analysis by laser scanning confocal microscopy revealed that the localization of 15-LO1 was altered in the presence of LPCAT2, similar to treatment with RSL3 in vector control cells.
In conclusion, a stable overexpression system of ACSL4 and/or LPCAT2 was successfully established in HEK293T cells, which enabled the synthesis and characterization of esterified oxylipins. Interestingly, characterization of the cell lines revealed a correlation with the cell death mechanism ferroptosis. Although the expression of ACSL4 has already been reported as a biomarker for ferroptosis, this is the first time that a potential connection of LPCAT2 with ferroptosis was demonstrated. As a result, this may provide new therapeutic options for ferroptosis-related pathologies such as neurodegeneration, autoimmune diseases, or tumorigenesis.
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
Impact of pectin dietary supplementation on experimental food allergy via gut microbiota modulation
(2023)
In recent years, dietary fibers gained focus in regard of their immune-modulatory effects and the potentially beneficial effect on allergies. The dietary fiber and prebiotic pectin is able to promote growth and activity of beneficial bacteria and thereby induce modulation of different immune responses. However, structurally different types of pectin might promote different immune-modulatory responses and to date the optimal pectin type for induction of beneficial health effects is not identified. Furthermore, it is still unclear, whether pectins provide a beneficial effect on certain allergies, such as food allergy.
Having this in consideration, this study examined the immune-modulatory effects of structurally different pectins on naive as well as peach allergic mice. Furhtermore, the impact of dietary pectin supplementation on composition and diversity of the murine gut microbiota was determined.
This study showed that dietary pectin intervention was able to suppress allergy-related Th2 responses considering humoral and cellular immune responses. Only apple-derived high-methoxyl pectin revealed an impact on total IgA levels and affected the microbial richness. Furthermore, it is not known whether the effects observed with the two pectins are caused by modulations of the bacterial composition or induced at least partly by direct interaction with the immune cells. Further studies are required to fully understand the mechanisms underlying the immune-modulatory capacities of different pectins.
Finally, the obtained results generated evidence that dietary pectin intervention can beneficially modulate the immune response in healthy mice and – at least partially – suppress allergy-related immune responses in a model of food allergy, depending on the structural characteristics of the used pectin.
The prefrontal cortex (PFC) is considered the cognitive center of the mammalian brain. It is involved in a variety of cognitive functions such as decision making, working memory, goal-directed behavior, processing of emotions, flexible action selection, attention, and others (Fuster, 2015). In rodents, these functions are associated with the medial prefrontal cortex (mPFC). Experiments in mice and rats have shown that neurons in the mPFC are necessary for successful performance of many cognitive tasks. Moreover, measurements of neural activity in the mPFC show excitation or inhibition in different cells in relation to specific aspects of the tasks to be solved. To date, however, it is largely unknown whether prefrontal neurons are stably activated during the same behaviors within a task and whether similar aspects are represented by the same neurons in different tasks. In addition, it is unclear how specifically neurons are activated, for example, whether cells that are activated in response to reward are activated in a different task without reward in a different situation or remain inactive. To address these questions, we recorded the same neurons in the mPFC of mice over the course of several weeks while the animals performed various behaviors.
To do this, we expressed GCaMP6 in pyramidal neurons in the mPFC of mice. A small lens was implanted in the same location and a miniature microscope ("miniscope") was used to record neural activity. Later the extracted neurons got aligned based on their shape and position across multiple days and sessions. The mice performed five different behavioral tests while neural activity was measured: A spatial working memory test in a T-maze, exploration of the elevated plus maze (EPM), a novel object recognition (NO) test including free open field (OF) exploration, a social interaction (SI) test and discriminatory auditory fear conditioning (FC). Each task was repeated at least twice to check for stable task encoding across sessions. Behavioral performance and neural correlates to specific task events were similar to earlier studies across all tasks. We utilized generalized linear models (GLM) to determine which behavioral variables most strongly influence neural activity in the mPFC. The position of the mouse in the environment was found to explain most of the variance in neural activity, together with movement speed they were the strongest predictors of neural activity across all tasks. Reward time points in the working memory test, the conditioned stimulus after fear conditioning, or head direction in general were also strongly encoded in the mPFC.
Many of the recorded neurons showed a stable spatial activity profile across multiple sessions of the same task. Similarly, cells that coded for position in one task tended to code for position in other tasks. Not only did the same cells code for position across multiple tasks, but cells also coded for movement speed and head direction. This indicates that at least these general behavioral variables are each represented by the same neurons in the mPFC. Interestingly, the stability of position or speed coding did not depend on the time between two sessions, but only on whether it was within the same or across different tasks. Within the same task, stability was slightly higher than across different tasks.
To find out whether task-specific behavioral aspects were also stably encoded in the mPFC, difference scores as the difference in neural activity between two task aspects like left- and right-choice trials or exposed and enclosed locations were calculated. Many cells encoded these aspects stably across different sessions of each task. Both the left-right differences in the different phases of the working memory test, the open-closed-arm differences in the elevated plus maze, the different activity between center and corners in the open field, the social target-object differences in the social interaction test, and the differences between the two tones during fear conditioning were all stably encoded across the population of mPFC cells. Only the distinction between the novel and the familiar object during object recognition was not stably encoded, but also the preference for the novel object was not present in the second session of novel object exploration.
There was also an overlap in coding for different aspects within a task across multiple sessions. For example, cells stably encoded left-right differences in the T-maze between different sessions as a function of walking direction across different phases of working memory, an aspect that we could already show within one session (Vogel, Hahn et al., 2022). During fear conditioning, the same cells showed a discrimination between CS+ and CS- that also responded to the start of CS+.
Consistency in the neurons activity across different tasks was also found, but only between tasks with similar demands, the elevated plus-maze and free exploration of the open field. Cells that were more active in the open arms also showed more activity in the center of the open field and vice versa. This could be an indicator that the cells were coding for anxiety or exposure across those tasks, indicating that neurons in the mPFC also stably encode general task aspects independent of the specific environment. However, it remains unclear what exactly these neurons encode; in the case of a general fear signal, one would also expect activation during fear conditioning which could not be found.
Overall, we found that neurons in the mPFC of mice encoded multiple general behavioral variables across multiple tasks and task-specific variables were encoded stably within each of the tested tasks. However, we found little task-specific variables that were systematically encoded by the same neurons with the exception being the elevated plus-maze and open field exploration, two tasks with similar features.
Life and biological resilience rely on the execution of precise gene expression profiles. A key mechanism to ensure cellular homeostasis is the regulation of protein synthesis. Recent studies have unveiled an intrinsic regulatory capacity of ribosomes, previously considered mere executors of mRNA translation. Neurons in particular finely regulate protein synthesis, at both global and local levels. This sustains their complex morphology and allows them to rapidly transmit, integrate, and respond to external stimuli. In this thesis, I investigated the neuronal ribosome and how subcellular environments and physiological perturbations shape it, by profiling its molecular composition, functional interconnections, and cellular distribution.
First, I used genetic engineering, biochemical purification, and mass spectrometry, to characterize in an unbiased manner the translation machinery specifically from excitatory and inhibitory neurons of the mouse cortex. I found that neuronal ribosomes commonly interact with RNA-binding proteins, components of the cytoskeleton, and proteins associated with the endoplasmic reticulum and vesicles. In line with the requirement for local protein synthesis in the distal parts of neurons, we observed that neuronal ribosomes preferentially interact with proteins involved in cellular transport. Remarkably, I observed a strong association between ribosomes and pre-synaptic vesicles, which suggests a potential regulatory interaction between local translation and neuronal activity.
Intriguingly, I and others have observed mRNAs encoding for core ribosomal proteins (RPs) among the genes most enriched in neuronal processes. This observation challenges two historical assumptions of ribosome biology: (1) new RPs are incorporated only into newly forming ribosomes, and (2) this incorporation occurs only in the nucleus and perinuclear region. In my PhD, I aimed to directly test these two assumptions and if proven wrong ask whether and why neurons would localize RP mRNAs far from their known assembly site.
Employing a combination of metabolic labeling and highly sensitive mass spectrometry techniques, I discovered that a subset of RPs rapidly and dynamically binds on and off mature ribosomes. Strikingly, this incorporation does not depend on the supply of new ribosomes from the nucleus. Therefore, my data refuted the assumption that ribosomes are built and degraded as a unit and revealed a more dynamic view of these machines, which can actively exchange core components. In particular, I found that the association of certain exchanging RPs is influenced by location (e.g., cell body versus neurites) and cellular state (e.g., post-oxidative stress). Neurons may use this mechanism to repair and/or specialize their protein synthesis machinery in a rapid and context-dependent manner.
Finally, I asked whether some steps of ribosome biogenesis could also take place in distal processes. Although most steps of ribosome assembly occur within the nucleus, the final stages of maturation are known to occur in the cytosol. By combining several imaging and biochemical approaches, I found that cytosolic (but not nuclear) pre-ribosomal particles are present in neuronal processes. Through the incorporation of new RPs into these immature particles, neurons may be able to locally “turn on” previously incompetent ribosomes. This may enable regions near synapses to enhance and customize their translational capacity, independently of the central pool of ribosomes from the cell body. Indeed, I observed that synaptic plasticity induces a maturation of cytosolic pre-ribosomes.
In summary, this thesis shows how neuronal ribosomes can sense cellular states, respond by adjusting their core composition, and in doing so influence the local capacity for protein synthesis. By overturning long-held assumptions in ribosome biology, this work highlights new molecular mechanisms of gene expression and enriches our understanding of the rapid and dynamic strategies cells employ to operate, thrive, and adaptively respond to environmental changes.
Precise regulation of gene expression networks is required to develop and maintain a healthy organism before and after birth and throughout adulthood. Such networks are mostly comprised of regulatory proteins, but meanwhile many long non-coding transcripts (lncRNAs) are shown to participate in these regulatory processes. The functions and mechanisms of these lncRNAs vary greatly, however they are often associated with transcriptional regulation. Three lncRNAs, namely Sweetheart RNA (Swhtr), Fetal-lethal noncoding developmental regulatory RNA / Foxf1 adjacent non-Coding developmental regulatory RNA (Fendrr) and lncFsd2, were studied in this work to demonstrate the variety of cellular and biological processes that require lncRNA-mediated fine-tuning, in regard to the cardiopulmonary system.
Swhtr was found to be expressed exclusively in cardiomyocytes and became critical for regeneration after myocardial injury. Mice lacking Swhtr did not show issues under normal conditions, but failed to undergo compensatory hypertrophic remodeling after injury, leading to increased mortality. This effect was rescued by re-expressing Swhtr, demonstrating importance of the RNA. Genes dependent on Swhtr during cardiac stress were found to likely be regulated by NKX2-5 through physical interaction with Swhtr. Fendrr was found to be expressed in lung and interacted with target promoters through its RNA:dsDNA binding domain, the FendrrBox, which was partially required for Fendrr function. Fendrr, together with activated WNT signaling, regulated fibrosis related target genes via the FendrrBox in fibroblasts. LncFsd2, an ubiquitously expressed lncRNA, showed possible interaction with the striated muscle specific Fsd2, but its exact function and regulatory role remain unclear in muscle physiology. Immunoprecipitation and subcellular fractionation experiments suggest that lncFsd2 might be involved in nuclear retention of Fsd2 mRNA, thus fine-tuning FSD2 protein expression. These investigations have shed light on the roles of these lncRNAs in stress responses, fibrosis-related gene regulation, and localization processes, advancing our understanding of cardiovascular and pulmonary maintenance, reaction to injury, and diseases. The diverse and intricate roles of these three lncRNAs highlight how they influence various cellular processes and disease states, offering avenues for exploring lncRNA functions in different biological contexts.
Mitochondria perform essential energetic, metabolic and signalling functions within the cell. To fulfil these, the integrity of the mitochondrial proteome has to be preserved. Therefore, each mitochondrial subcompartment harbours its own system for protein quality control. However, if the capacity of mitochondrial chaperones and proteases is overloaded, mitochondrial misfolding stress (MMS) occurs. Upon this stress condition, mitochondria communicate with the nucleus to increase the transcription of nuclear encoded mitochondrial chaperones and proteases. This proteotoxic stress pathway was termed the mitochondrial unfolded protein response (UPRmt) aiming at restoring protein homeostasis. Despite being discovered over 25 years ago, the signalling molecules released by stressed mitochondria as well as the corresponding receptor and transcription factor remain poorly understood. With this study, we aimed at characterising the underlying signalling events and mechanisms of how mitochondria react to misfolded proteins. First, we aimed to establish different methods to induce MMS that triggers the transcriptional induction of mitochondrial chaperones and proteases detected by quantitative polymerase chain reaction. We were able to induce UPRmt signalling by overexpression of an aggregation-prone protein and by knock-down or inhibition of mitochondrial protein quality control components. To study the signalling in a time-resolved manner, we focused on the usage of the mitochondrial HSP90 inhibitor GTPP and the mitochondrial LONP1 protease inhibitor CDDO.
Early time point RNA sequencing analysis of cells stressed with GTPP or CDDO revealed upregulated genes in response to oxidative stress. Indeed, measurements of mitochondrial superoxide with the fluorescent dye MitoSOX showed increased levels of reactive oxygen species (ROS) upon MMS induction. In contrast, there was no induction of mitochondrial chaperones and proteases when combining MMS with antioxidants. Compartment-specific targeting of the hydrogen peroxide sensor HyPer7 revealed increased ROS levels in the intermembrane space and matrix of mitochondria, followed by elevated ROS levels in the cytosol at later time points. The importance of cytosolic ROS for the signalling was supported by preventing UPRmt induction with an inhibitor blocking the outer mitochondrial membrane pore. Thus, ROS were identified as an essential UPRmt signal.
To understand which cytosolic factor is modified by ROS, redox proteomics was performed. Here, reversible changes on cysteine residues of the HSP40 co-chaperone DNAJA1 were observed upon MMS. Consequently, transcriptional induction of UPRmt genes was abolished by DNAJA1 knock-down. To understand the function of DNAJA1 during UPRmt signalling, quantitative interaction proteomics upon MMS revealed an increased binding to mitochondrial proteins and its interaction partner HSP70. Immunoprecipitation confirmed a ROS-dependent interaction between HSP40 and HSP70. Increased binding to mitochondrial proteins represented a cytosolic interaction of DNAJA1 with mitochondrial precursor proteins, whose accumulation was confirmed by western blot. Moreover, a fluorescent protein targeted to mitochondria accumulated in the cytosol during GTPP treatment, confirming a reduced import efficiency upon MMS. Preventing the accumulation of precursors by a translation inhibitor or depletion of a general mitochondrial transcription factor resulted in reduced UPRmt activation. Thus, DNAJA1 is essential for UPRmt signalling, since its oxidation by mitochondrial ROS and its enhanced recruitment to mitochondrial precursors allows the integration of both MMS-induced signals.
To link these findings to an increased transcription of mitochondrial chaperones and proteases, we screened for transcription factors accumulating in the nucleus upon MMS by cellular fractionation mass spectrometry. We demonstrated that specifically HSF1 accumulates in nuclei of cells stressed with GTPP or CDDO. Depletion of HSF1 by knock-down or knock-out resulted in the abrogation of the UPRmt-specific transcriptional response. HSF1 activation was visualised by nuclear accumulation on western blot, a process inhibited by ROS and precursor suppression. Moreover, DNAJA1 depletion prevented HSF1 activation. Ultimately, we proved by immunoprecipitation that the inhibitory interaction between HSF1 and HSP70 is reduced upon MMS.
Thus, we conclude that MMS increases mitochondrial ROS that are released into the cytosol. In addition, the import efficiency is reduced upon MMS, resulting in the accumulation of non-imported mitochondrial precursor proteins in the cytosol. Both signals are recognised via DNAJA1 oxidation and substrate binding. The concurrent recruitment of HSP70 to DNAJA1 results in the loss of the inhibitory HSP70-HSF1 interaction. Thus, active HSF1 can migrate to the nucleus to initiate transcription of mitochondrial chaperones and proteases. These findings are in accordance with observations in yeast, where mistargeted mitochondrial proteins activate cellular stress responses. Our results highlight a surprising interconnection and dependence of the mitochondrial and the cytosolic proteostasis network, in which the UPRmt is activated by a combination of two mitochondria-specific proteotoxic stress signals.
This dissertation constitutes a series of successive research papers, starting with the characterization of various optogenetic tools up to the establishment of purely optical electrophysiology in living animals.
Optogenetics has revolutionized neurobiology as it allows stimulation of excitable cells with exceptionally high spatiotemporal resolution. To cope with the increasing complexity of research issues and accompanying demands on experimental design, the broadening of the optogenetic toolbox is indispensable. Therefore, one goal was to establish a wide variety of novel rhodopsin-based actuators and characterize them, among others, with respect to their spectral properties, kinetics, and efficacy using behavioral experiments in Caenorhabditis elegans. During these studies, the applicability of highly potent de- and hyperpolarizers with adapted spectral properties, altered ion specificity, strongly slowed off-kinetics, and inverted functionality was successfully demonstrated. Inhibitory anion channelrhodopsins (ACRs) stood out, filling the gap of long-sought equivalent hyperpolarizing tools, and could be convincingly applied in a tandem configuration combined with the red-shifted depolarizer Chrimson for bidirectional stimulation (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing, BiPOLES). A parallel study aimed to compare various rhodopsin-based genetically encoded voltage indicators (GEVIs) in the worm: In addition to electrochromic FRET-based GEVIs that use lower excitation intensity, QuasAr2 was particularly convincing in terms of voltage sensitivity and photostability in C. elegans. However, classical optogenetic approaches are quite static and only allow perturbation of neural activity. Therefore, QuasAr2 and BiPOLES were combined in a closed-loop feedback control system to implement the first proof-of-concept all-optical voltage clamp to date, termed the optogenetic voltage clamp (OVC). Here, an I-controller generates feedback of light wavelengths to bidirectionally stimulate BiPOLES and keep QuasAr’s fluorescence at a desired level. The OVC was established in body wall muscles and various types of neurons in C. elegans and transferred to rat hippocampal slice culture. In the worm, it allowed to assess altered cellular physiology of mutants and Ca2+-channel characteristics as well as dynamical clamping of distinct action potentials and associated behavior.
Ultimately, the optogenetic actuators and sensors implemented in the course of this cumulative work enabled to synergistically combine the advantages of imaging- and electrode-based techniques, thus providing the basis for noninvasive, optical electrophysiology in behaving animals.
Bioactive small molecules are used in many research areas as important tools to uncover biological pathways, interpret phenotypic changes, deconvolute protein functions and explore new therapeutic strategies in disease relevant cellular model systems. To unlock the full potential of these small molecules and to ensure reliability of results obtained in cellular assays, it is crucial to understand the properties of these small molecules. These properties encompass their activity and potency on their designated target(s), their selectivity towards unintended off-targets and their phenotypic effects in a cellular system. Approved drugs often engage with multiple targets, which can be beneficial for some applications such as treatment of cancer where several pathways need to be inhibited for treatment efficacy. However, targeting multiple key proteins in diverse pathways also increases the possibility for unspecific or unwanted side effects. For many drugs the entire target space that they modulate is not known. This makes it difficult to use these drugs for target deconvolution or functional assays with the aim to understand the underlying biological processes. In contrast to drugs, for mechanistic studies, a good alternative are chemical tool compounds so called chemical probes that are usually exclusively selective as well as chemogenomic compounds, that inhibit several targets but have narrow selectivity profiles. Because they are mechanistic tools, chemical tool compounds must meet stringent quality criteria and they are therefore well characterized in terms of their potency, selectivity and cellular on-target activity. To ensure that an observed phenotypic effect caused by a compound can be attributed to the described target(s), it is essential to study also properties of chemical tools leading to unspecific cellular effects. There are a variety of unspecific effects that can be caused by physiochemical compound properties that can interfere with phenotypic assays as well as functional compound evaluations. One of these effects is low solubility causing toxicity or intrinsic fluorescence potentially interfering with assay readouts. But unanticipated cellular responses can also arise from unspecific binding, accumulation in cellular compartments or damage caused to organelles such as mitochondria or the cytoskeleton that can result in the induction of diverse forms of cell death.
In this study, we investigated the influence of a variety of small molecules on distinct cell states, by establishing and validating high-content imaging assays, which we called Multiplex assay. This assay portfolio enabled us to detect different cellular responses using diverse fluorescent reporters, such as the influence of a compound on cell viability, induction of cell death programs and modulation of the cell cycle. Additionally, general compound properties such as precipitation and intrinsic fluorescence were simultaneously detected. The assay is adaptable to assess other cellular properties of interest, such as mitochondrial health, changes in cytoskeletal morphology or phospholipidosis. A significant advantage of the assay is that we are using live cells, so we can capture dynamic cellular changes and fluctuations that can be crucial for the understanding of cellular responses.
Der Hirntumor Glioblastom (GBM) ist aufgrund seines infiltrativen Wachstums, der hohen intra- und intertumoralen Heterogenität, der hohen Therapieresistenz als auch aufgrund der sogenannten gliomartigen Stammzellen sehr schwer zu behandeln und führt fast immer zu Rezidiven. Da es in den letzten Jahrzehnten kaum Fortschritte in der Behandlung des GBMs gab, bis auf die Therapie mit Tumortherapiefeldern, wird weiterhin nach alternativen Zelltodtherapien geforscht, wie zum Beispiel dem Autophagie-abhängigen Zelltod. Der Autophagie-abhängige Zelltod ist durch einen erhöhten autophagischen Flux gekennzeichnet und obwohl die Autophagie, als auch selektive Formen wie die Lysophagie und Mitophagie, normalerweise als überlebensfördernde Mechanismen gelten, konnten viele Studien eine duale Rolle in der Tumorentstehung, -progression und -behandlung aufzeigen, die vor allem vom Tumortyp und stadium abhängt. Um die zugrunde liegenden Mechanismen des durch Medikamente induzierten Autophagie-abhängigen Zelltods im GBM weiter zu entschlüsseln, habe ich in meiner Dissertation verschiedene Substanzen untersucht, die einen Autophagie-abhängigen Zelltod induzieren.
In einer zuvor in unserem Labor durchgeführten Studie konnte gezeigt werden, dass das Antipsychotikum Pimozid (PIMO) und der Opioidrezeptor-Antagonist Loperamid (LOP) einen Autophagie-abhängigen Zelltod in GBM Zellen induzieren können. Darauf aufbauend habe ich die Fähigkeit zur Induktion des Autophagie-abhängigen Zelltods in weiteren Zellmodellen validiert. Dies bestätigte einen erhöhten autophagischen Flux nach PIMO und LOP Behandlung, während der Zelltod als auch der autophagische Flux in Autophagie-defizienten Zellen reduziert war. In weiteren Versuchen konnte ich die Involvierung der LC3-assoziierten Phagozytose (LAP), ein Signalweg der auf die Funktion einiger autophagischer Proteine angewiesen ist, ausschließen. Weiterhin konnte ich eine massive Störung des Cholesterin- und Lipidstoffwechsels beobachten. Unter anderem akkumulierte Cholesterin in den Lysosomen gefolgt von massiven Schäden des lysosomalen Kompartiments und der Permeabiliserung der lysosomalen Membran. Dies trug einerseits zur Aktivierung überlebensfördernder Lysophagie als auch der Zell-schädigenden „Bulk“-Autophagie bei. Letztendlich konnte aber die erhöhte Lysophagie die Zellen nicht vor dem Zelltod retten und die Zellen starben einen Autophagie-abhängigen lysosomalen Zelltod. Da die Eignung von LOP als Therapie für das GBM aufgrund der fehlenden Blut-Hirn-Schranken Permeabilität und von dem Antipsychotikum PIMO aufgrund teils schwerer Nebenwirkungen eingeschränkt ist, habe ich mich im weiteren Verlauf meiner Dissertation mit einer Substanz mit einem anderen Wirkmechanismus beschäftigt.
Der Eisenchelator und oxidative Phosphorylierungs (OXPHOS) Inhibitor VLX600 wurde zuvor berichtet mitochondriale Dysfunktion und Zelltod in Kolonkarzinomzellen zu induzieren. Allerdings hat meines Wissens nach bisher noch keine Studie die therapeutische Eignung von VLX600 für das GBM untersucht. Hier zeige ich eine neuartige Autophagie-abhängige Zelltod-induzierende Fähigkeit von VLX600 für GBM Zellen, da der Zelltod signifikant in Autophagie-defizienten Zellen aber nicht durch Caspase-Inhibitoren gehemmt wurde und der autophagische Flux erhöht war. Darüber hinaus konnte ich die Hemmung der OXPHOS und die Induktion von mitochondrialem Stress in GBM Zellen bestätigen und weiterhin aufzeigen, dass VLX600 nicht nur die mitochondriale Homöostase stört, sondern auch zu einer BNIP3-BNIP3L-abhängigen Mitophagie führt, die wahrscheinlich durch HIF1A reguliert wird aber keinen erkennbaren Nettoeffekt auf den von VLX600 induzierten Zelltod hat. Demnach induziert VLX600 letale „Bulk“-Autophagie in den hier verwendeten Zellmodellen. Darüber hinaus konnte ich zeigen, dass die Eisenchelatierung durch VLX600 eine große Rolle für den von VLX600-induzierten Zelltod spielt aber auch für die Mitophagie Induktion, Histon Lysin Methylierung und den ribosomalen Stress. Letztendlich ist es wahrscheinlich ein Zusammenspiel all dieser Faktoren, die zur Zelltodinduktion durch VLX600 führen und interessanterweise werden Eisenchelatoren bereits in präklinischen und klinischen Studien für Krebstherapien untersucht. Dabei könnten gewisse metabolische Eigenschaften verschiedener Tumorzellen die Sensitivität von Wirkstoffen, die auf den Metabolismus wirken wie VLX600, beeinflussen was in zukünftigen Studien beachtet werden sollte um den bestmöglichsten Therapieerfolg zu erzielen. Zusammenfassend unterstützt meine Dissertation die duale Rolle der Autophagie, die stark vom jeweiligen Kontext abhängt und befürwortet die weitere Forschung von Substanzen, die einen Autophagie-abhängigen Zelltod induzieren, für das GBM.
Anthropogenic activities have a major impact on our planet and rapidly drive biodiversity loss in ecosystems at a global scale. Particularly over the last century, rising CO2 emissions significantly raised global temperatures and increased the intensity and frequency of droughts and heatwaves. Additionally, agricultural land use and fossil fuel combustion contribute to the continuous release of nitrogen (N) and phosphorus (P) into ecosystems worldwide through extensive fertilization and deposition from the atmosphere. It is important to understand how these rapid changes affect the evolution of plant populations and their adaptive potential. Adaptation by natural selection (i.e., adaptive evolution) within a few generations is an essential process as a response to rapid environmental changes. Rapid evolution of plant populations can be detected by using the so-called resurrection approach. Here, diaspores (i.e., seeds) from a population are collected before (ancestors) and after (descendants) a potential selection pressure (e.g., consecutive years of drought or changes in nutrient supply). Comparing phenotypes of ancestors and descendants in a common environment such as an outside garden, greenhouse, or climate chamber, may then reveal evolutionary changes. Ideally, plants are first grown in a common environment for an intermediate refresher generation to reduce parental and storage effects.
The aim of this thesis was to investigate the occurrence of adaptive evolution in natural plant populations in response to rapidly changing environments over the past three decades. I conducted three experiments using the resurrection approach to generate comprehensive data on the adaptive processes that acted on three plant populations from three different species over the last three decades. Furthermore, I filled knowledge gaps in plant evolutionary ecology and conceptually developed the resurrection approach further.
In Chapter I, I performed a novel approach by testing for adaptive evolution in natural plant populations using the resurrection approach in combination with in-situ transplantations. I cultivated seedlings from ancestors (23 – 26 years old) and contemporary descendants of three perennial species (Melica ciliata, Leontodon hispidus and Clinopodium vulgare) from calcareous grasslands in the greenhouse and In Chapter III, I assessed the reproducibility of phenotypic differences between genotypes among three different growth facilities (climate chamber, greenhouse, and outdoor garden). I also evaluated differences in phenotypic expression between plants grown after one vs. two intermediate generations (i.e., refresher generations). I performed this experiment within the framework of the resurrection approach and compared ancestors and descendants of the same population of Leontodon hispidus.
I observed very strong differences among plants growing in the different growth facilities. I found a significant interaction between the growth facility and the temporal origin (ancestors vs. descendants): descendants had significantly larger rosettes than ancestors only in the greenhouse and they flowered significantly later than ancestors exclusively in the climate chamber. I did not find significant differences between intermediate generations within the growth facilities. Overall, Chapter III shows that the use of a particular experimental system can dictate the presence and magnitude of phenotypic differences. This implies that absence of evidence is not evidence of absence when it comes to investigating genetically based trait differentiation among plant origins (in space or time). Experimental systems should be carefully designed to provide meaningful conditions, ideally mimicking the environmental conditions of the population’s origins. Finally, growing a second intermediate generation did not impact the genetic differences of ancestors and descendants within the environments, supporting the idea that only one intermediate generation may be sufficient to reduce detectable parental and storage effects.
The resurrection approach allows a better understanding of rapid plant adaptation, but some limitations deserve to be highlighted. I only studied one population per species, and Chapters II and III only focus on one population of L. hispidus, which is also hampering generalizations, as adaptive potential can vary greatly among populations of the same species. I only compared the ancestral genotypes to one descendant sample with a long time span in between (26 – 28 years), which makes it hard to pinpoint the selection agents that caused the genetic differentiation among the sampling years. Hence, closely monitoring biotic and abiotic factors of the studied populations between the ancestral and descendant sampling in future studies, would make identifying the responsible selection pressures more precise. I also recommend sampling multiple populations over consecutive years to improve the robustness of results and make generalizations more approachable.Furthermore, combining the resurrection approach with other methods such as in-situ transplantations will be valuable to offset the limitation that adaptations cannot be proven under artificial conditions (e.g., in the greenhouse).
The nucleus reuniens drives hippocampal goal‑directed trajectory sequences for route planning
(2023)
Goal-directed spatial navigation requires accurate estimates of one’s position and destination, as well as careful planning of a route between them to avoid known obstacles in the environment. Despite its general importance across species, the neural circuitry supporting the ability for route planning remains largely unclear. Previous studies described that place cells in the hippocampal CA1 encode the animal's next movement direction (Wood et al., 2000; Ito et al., 2015) and upcoming navigational routes (Pfeiffer & Foster, 2013). However, it has been shown that part of the CA1 activity representing the animal’s future behaviors is not necessarily generated in the hippocampus, but is derived from the medial prefrontal cortex (PFC) via the nucleus reuniens of the thalamus (RE) (Ito et al., 2015). Notably, the importance of the PFC in navigation has been demonstrated in several studies, including the recent finding of a goal map in the orbitofrontal cortex (Basu et al., 2021). Therefore, I hypothesized that information flow from the PFC to CA1 via the RE plays a key role in route planning.
To assess the animals' route planning ability, I designed a new navigation task in which a rat has to navigate to a fixed target location from various starting positions in an arena. Furthermore, by adding an L-shaped wall in the maze and removing all light sources in the experimental room, this task forced the animals to plan a wall-avoiding route without relying on direct sensory perceptions. I confirmed that rats could learn this task successfully, memorizing the wall location and taking a smooth wall-avoidance route. To test the role of the RE, I inactivated RE neurons by expressing the inhibitory opsin SwiChR++, which resulted in a significant deficit in the animal’s route planning ability, taking a longer non-smooth path to the destination. By contrast, this manipulation did not affect navigation performance when a straight goal-directed route was available, suggesting a specific role of the RE in route planning. I further found that DREADDs-mediated inactivation of neurons in the bilateral hippocampi resulted in a similar deficit in route planning ability, implying cooperation between the RE and the hippocampus.
I finally examined the activity of hippocampal CA1 neurons with and without RE inactivation. While neurons in the hippocampus exhibited brief trajectory sequences corresponding to the animal’s subsequent goal-directed journey, I found that this goal-directed bias of trajectory events was significantly reduced by RE inactivation, likely associated with route-planning deficits in these animals.
Altogether, this dissertation demonstrates the role of the RE from both behavioral and neural coding perspectives, identifying a pivotal circuit element supporting the animal’s route-planning ability.
Methods using environmental DNA to explore and analyze biodiversity from previously unexplored habitats and ecosystems have become increasingly popular in recent years. This is particularly due to the potential reduction in necessary taxonomic expertise, the opportunity to assess microorganismal communities, and decreased time investments required to cover large spatial extents. In forests, the surface of tree bark is an important habitat for epiphytic diversity. Because of the large surface area rich in micro-niches, the seasonal stability of the substrate, and the longevity of trees, tree bark surfaces provide an ideal habitat for many species. Yet, we lack a comprehensive understanding of their communities and the environmental drivers behind the community assembly. These missing links hinder the exploration of the forest microbiome as a whole and limits our understanding of functions of a large forest habitat and its connections to other forest microbiomes. With a holistic eDNA metabarcoding approach, encompassing samples of three major taxonomic groups (e.g. bacteria, fungi, and green algae), as well as simultaneous collections from multiple forest habitats we can contribute to closing these gaps and increase our knowledge of the forest microbiome.
My dissertation is set within the framework of the Biodiversity Exploratories and was conducted in four parts: I. the establishment of an eDNA metabarcoding workflow to reveal the local diversity of the bark surface microbiome; II. the upscaling of the method to large geographic and environmental gradients to uncover the drivers of the microbiome; III. the integration of soil and bark samples to investigate compositional differences in two important forest habitats; IV. the evaluation of eDNA metabarcoding as a tool for biodiversity assessments of lichen diversity in forests.
In the first part, I developed a simple, cost-effective and fast sampling strategy to acquire eDNA samples from the bark of trees in forest ecosystems. Using readily available medical-specimen-collection swabs I sampled bark surfaces of individual trees in Central German forests and used metabarcoding to amplify marker genes of green algae, fungi and bacteria. From the sequencing reads I calculated the first diversity estimates of the major organismal groups of bark surface microbiomes from Central European forests. Overall the methodology produced reliable results, allowing for an expanded sampling in the second part.
In the second part of the dissertation, I expanded the sampling based on the results of part one. I collected bark surface samples from the three regions of the Biodiversity Exploratories covering large spatial and environmental gradients representative for Central European forests. The collection included composite samples from 150 plots and over 750 trees. Utilizing measurements of climatic and forest structure variables provided by the Biodiversity Exploratories, as well as my own community data, I identified the biotic and abiotic drivers behind alpha and beta diversity of the bark surface microbiome.
In the third part, I studied the differences between the bark surface as an unexplored and the soil as an example of a well characterized forest microbiome. Using only the fungal part of the large sampling campaign and soil samples obtained from the same plots at the same time, I assessed the commonalities and differences of the micro-communities of these distinct forest niches. Furthermore, I included two coniferous and one deciduous tree species to examine, if the effect of tree species, previously shown for soil microbiomes, also holds true for the bark surface.
In the last part of my dissertation, I used eDNA in a more applied way as a tool in biodiversity assessments of lichenized fungi. I compared the results from eDNA metabarcoding to an expert floristic mapping conducted in the same plots in 2007/2008. I assigned functional guilds to the fungal taxa obtained in the large sampling campaign and used a subset that was assigned as lichenized fungi.
In conclusion, I showed that eDNA metabarcoding is a valuable tool to reveal the unknown diversity of microorganisms in forest ecosystems. In particular, my results advance our understanding of the bark surface microbiome, an underexplored habitat within forests. The tightly linked interactions of the three major microbial groups underline that studies need to take holistic approaches across multiple taxonomic groups to deepen our understanding of processes governing the assembly of microbiomes. Results from my dissertation may serve as a foundation to inform hypotheses addressing the functions of forest microbiomes. The massive diversity data collected may also contribute to closing the gap in our understanding of macro-organisms and micro-organisms with respect to diversity distributions and patterns of richness, and serve as a baseline for predictions of biodiversity responses under future anthropogenic change.
Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Die Zahl der gramnegativen Bakterien auf der WHO-Liste der Antibiotikaresistenzen hat in den letzten Jahrzehnten erheblich zugenommen. Schätzungen zufolge wird die Antibiotikaresistenz bis 2050 tödlicher sein als Krebs. Die äußere Membran gramnegativer Bakterien ist aufgrund ihres wichtigsten Strukturbestandteils, des Lipopolysaccharids (LPS), sehr anpassungsfähig an Umweltveränderungen. Das LPS macht gramnegative Bakterien von Natur aus resistent gegen viele Antibiotika und führt somit zu Antibiotikaresistenz. Der bakterielle ATP-bindende Kassettentransporter (ABC-Transporter) MsbA spielt eine entscheidende Rolle bei der Regulierung der bakteriellen Außenmembran, indem er das Kern-LPS durch ATP-Hydrolyse über die Innenmembran von gramnegativen Bakterien flockt. Darüber hinaus fungiert diese Floppase als Efflux-Pumpe, indem sie Medikamente durch die innere Membran transportiert, was sie zu einem interessanten Ziel für Medikamente macht. Vor kurzem wurden zwei verschiedene Klassen von MsbA-Inhibitoren entdeckt: (1) Tetrahydrobenzothiophene (TBT), die den LPS-Transport aufheben, und (2) Chinolinderivate, die sowohl die ATP-Hydrolyse als auch die LPS-Translokation blockieren. Darüber hinaus hat die Bestimmung der 3D-Struktur von MsbA durch Rontgen- und Kryo-EM mehrere interessante Zustände der Floppase ergeben. Die Kernspinresonanzspektroskopie ist eine hervorragende biophysikalische Methode zur Ergänzung der vorhandenen 3D-Strukturdaten. Insbesondere ermöglicht die Festkörper-NMR die Untersuchung von Membranproteinen in einer nativen Umgebung (z. B. in einer Lipiddoppelschicht). In der Vergangenheit hat unser Labor mithilfe der Festkörper-NMR einige detaillierte Mechanismen von MsbA aufgedeckt. Trotz der zahlreichen Fortschritte bei der Untersuchung der ABC-Transporterprotein-Superfamilie ist der spezifische Prozess der Substrattranslokation von MsbA noch immer unbekannt. Es wird angenommen, dass dieser Translokationsprozess über die Kopplungshelices (CHs) erfolgt, die sich zwischen der Transmembranregion (TMD) und der Nukleotidbindungsdomäne (NBD) befinden. Nukleotid-Bindungsdomäne (NBD). Zu diesem Zweck wird dem Zusammenspiel zwischen der TMD und der NBD über die CHs besondere Aufmerksamkeit gewidmet, mit dem Ziel, den Prozess der Substrattranslokation mithilfe von funktionellen Assays und Festkörper-NMR zu verstehen. Bei letzterem wurden spezifische Reporter in die CHs eingeführt, um Konformationsänderungen in 2D-spektroskopischen Daten zu verfolgen. Darüber hinaus wurde zeitaufgelöste NMR eingesetzt, um die Auswirkungen verschiedener Substrate in der TMD während der ATP-Hydrolyse in der NBD sichtbar zu machen. Die einzigartigen Reporter in den CHs haben Konformationsänderungen in bestimmten katalytischen Zuständen gezeigt. Darüber hinaus scheinen verschiedene Substrate die Kinetik der ATP-Hydrolyse zu beeinflussen. Die Ergebnisse zeigten, dass einige Substrate einen bevorzugten katalytischen Zustand innerhalb des ATP-Hydrolyse Zyklus aufweisen, der möglicherweise einen gekoppelten oder ungekoppelten Kinasemechanismus hat. Diese Ergebnisse könnten verschiedene Einblicke in die molekulare Struktur potenzieller neuer Antibiotika liefern.