Refine
Year of publication
Document Type
- Doctoral Thesis (422) (remove)
Language
- English (422) (remove)
Has Fulltext
- yes (422)
Is part of the Bibliography
- no (422)
Keywords
- Computational chemistry (3)
- Saccharomyces cerevisiae (3)
- zebrafish (3)
- Apoptosis (2)
- Arzneimitteldesign (2)
- Biomarker (2)
- Bungarus (2)
- Evolution (2)
- Genexpression (2)
- Hitzeschock-Proteine (2)
- Metabolic Engineering (2)
- Neuronal Differentiation (2)
- Pflanzenphysiologie (2)
- Pflanzenstress (2)
- Photosynthese (2)
- Plant stress (2)
- Proteine (2)
- Transkriptionsfaktor (2)
- Wirkstoff-Rezeptor-Bindung (2)
- Xenorhabdus (2)
- angiogenesis (2)
- conservation (2)
- microRNA (2)
- vasculogenesis (2)
- virtual screening (2)
- 3-alkylphenols (1)
- 6-methylsalicylic acid synthase (1)
- ADAM15 (1)
- ALE (1)
- Acetogenic bacteria (1)
- Ackerschmalwand (1)
- Actin-bindende Proteine (1)
- Agapetes (1)
- Agonist selection (1)
- Aktive Zone (1)
- Alignment <Biochemie> (1)
- Alpha-Bungarotoxin (1)
- Amphisphaeriales (1)
- Angiogenese (1)
- Angiogenesis (1)
- Animal Behavior (1)
- Anpassung (1)
- Aphanomyces astaci (1)
- Aquatisches Ökosystem (1)
- Aromatase (1)
- Asien (1)
- Ausbreitung (1)
- Autism Spectrum Disorder (1)
- Axoninitialsegment (1)
- Baleen whales (1)
- Bartonella henselae (1)
- Bassoon (1)
- Batten disease (1)
- Bayes-Lernen (1)
- Biochemie (1)
- Biodiversity (1)
- Biotechnologie (1)
- Biotic interactions (1)
- Bovidae (1)
- Breast cancer (1)
- Brustkrebs (1)
- Bungarus niger (1)
- Bungarus walli (1)
- CHIP (1)
- CNV 16p11.2 (1)
- CSOs (1)
- Calmodulin (1)
- Capoeta damascina (1)
- Carbon capture (1)
- Cardiac regeneration (1)
- Caspase-8 (1)
- Cerebral cortex (1)
- Chemische Verschiebung (1)
- Chlor (1)
- Chlorophyll Fluorescence (1)
- Chronobiologie (1)
- Chronobiology (1)
- Cicer arietinum (1)
- Cladistics (1)
- Cladocera (1)
- Climate Change (1)
- Climate change (1)
- Clock genes (1)
- Coenzym (1)
- Cofaktor (1)
- Colorectal Cancer (1)
- Connectomics (1)
- Conservation (1)
- Conventional T cell selection (1)
- Coronaries (1)
- Cyprinidae (1)
- DEPDC5 (1)
- DIRAS2 (1)
- DNA-binding region (1)
- Daboia russelii (1)
- De-Novo-Synthese (1)
- Deterministische Optimierung (1)
- Development (1)
- Developmental Biology (1)
- Diadinoxanthin (1)
- Dimensionsreduktion (1)
- Docking (1)
- Dornapparat (1)
- Dreidimensionale geometrische Modellierung (1)
- Drought (1)
- Echoorientierung (1)
- Echsen (1)
- Ecotoxicogenomics (1)
- Ecotoxicology (1)
- Eisen-Schwefel-Zentrum N1a (1)
- Elapidae (1)
- Elektronenmikroskopie (1)
- Emerging insect model organisms (1)
- Endocrine disrupter (1)
- Endokrin wirksamer Stoff (1)
- Endokrinologie (1)
- Endothelial cells (1)
- Endotheliale Vorläuferzellen (1)
- Endothelzellen (1)
- Entomology (1)
- Enzym (1)
- Enzymaktivität (1)
- Enzymologie (1)
- Eocene (1)
- EphrinB2 (1)
- European Rabbit (1)
- Europäisches Wildkaninchen (1)
- Evolutionary developmental biology (1)
- Excitatory balance (1)
- Extracellular matrix (1)
- FCP (Fucoxanthin-Chlorophyll bindende Proteine) (1)
- FCP (Fucoxanthin-Chlorophyll binding Protein) (1)
- Fabclavine (1)
- Flavoproteins (1)
- Fluoreszenz (1)
- Fossile Reptilien (1)
- Fossile Säugetiere (1)
- Freshwater (1)
- Freshwater Ecosystems (1)
- Functional Ecology (1)
- Functional traits (1)
- GPCR (1)
- Gal2 (1)
- Galakturonsäure (1)
- Gaussian processes (1)
- Gauß Prozesse (1)
- Gene expression (1)
- Gene trap (1)
- Genetics (1)
- Genfalle (1)
- Genome (1)
- Gentianinae (1)
- Giftvariabilität (1)
- Global Alignment (1)
- Graphentheorie (1)
- Green-River-Formation ; Fossile Vögel ; Systematik ; Messel Grube (1)
- Guanylatcyclase (1)
- HRGXE-Motiv (1)
- HWC database (1)
- Hauptkomponentenanalyse (1)
- Heart (1)
- Hinterkiemer (1)
- Hippocampal development (1)
- Hippocampus (1)
- Histone (1)
- Histonmodifikationen (1)
- Hitzeschocktranskriptionsfaktor (1)
- Hitzestress (1)
- Hominidae (1)
- Homologiemodellierung (1)
- Hydrogen storage (1)
- Hydrogen-dependent CO2 reductase (1)
- Infectivity (1)
- Infektiösität (1)
- Influenza (1)
- Inhibitor Binding Pocket (1)
- Inhibitorbindetasche (1)
- Inhibitory balance (1)
- Inhibitory interneurons (1)
- Inquisition post mortem (1)
- Intestinal Contents (1)
- Inthraszentin (1)
- Ischemia (1)
- Ischämie (1)
- Isopoda (1)
- Juvenile neuronal ceroid lipofuscinosis (1)
- Kalziumspeicher (1)
- Kapernartige (1)
- Kardiovaskuläres System (1)
- Katabolismus (1)
- Katze (1)
- Kieselalgen (1)
- Kristallstrukturanalyse (1)
- LTD (1)
- LTP (1)
- Lacertilia (1)
- Laetoli (1)
- Licht-Sammel-Komplex (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)
- Lycopersicon peruvianum (1)
- MAIT cells (1)
- MEK inhibition (1)
- MICOS complex (1)
- Macroevolution (1)
- Macrotermes (1)
- Magnetische Kernresonanz (1)
- Makuyuni (1)
- Maschinelles Lernen (1)
- Maternal Immune Activation (1)
- Melatonin (1)
- Messel (1)
- Messel-Schichten (1)
- Methyltransferase (1)
- Microbiology (1)
- Microsatelliten (1)
- Microvesicles (1)
- Mikroplastik (1)
- Mikrovesikel (1)
- Milchdrüse (1)
- Milchdrüsengewebe (1)
- Molekularbiologie (1)
- Molekulardesign (1)
- Molekülstruktur (1)
- Monoterpenoid (1)
- Monoterpenoid tolerance (1)
- Morphogenesis (1)
- Morphology (1)
- NADH-Dehydrogenase <Ubichinon> (1)
- NADH:ubiquinone oxidoreductase (1)
- NMR (1)
- Naja (1)
- Nase (1)
- Natural Products (1)
- Natural products (1)
- Naturstoffe (1)
- Negative selection (1)
- Neovascularisation (1)
- Nervensystem (1)
- Nervenzelle (1)
- Neuroanatomie (1)
- Neurodevelopmental Psychiatric Disorders (1)
- Neurohypophyse (1)
- Neuronale Differenzierung (1)
- Neuronale Plastizität (1)
- Neurotransmitter-Rezeptor (1)
- Non-canonical terpenes (1)
- Nucleus reuniens (1)
- Numencal Taxonomy (1)
- Oberes Pliozän (1)
- Opisthobranchia (1)
- Organic micropollutants (1)
- Organoids (1)
- Ortspezifische Mutagenese (1)
- Oxidativer Stress (1)
- Oxygenierung (1)
- Palaeobiology (1)
- Paläobiologie (1)
- Parkinson (1)
- Pestalotia (1)
- Pestalotiopsis (1)
- Pestizidbelastung (1)
- Pflanzenhormon (1)
- Pharmacophore (1)
- Photorhabdus (1)
- Photosynthetisches Reaktionszentrum (1)
- Photosystem I (1)
- Phylogeny (1)
- Pigmentproteine (1)
- Pimephales promelas (1)
- Pink1 (1)
- Pinnotheres (1)
- Plant regeneration (1)
- Plant regeneration; community assembly; diversity (1)
- Plants (1)
- Population genetics (1)
- Populationsgenetik (1)
- Positive selection (1)
- Postglaziale Verbreitung (1)
- PrP (1)
- Prenyl pyrophosphates (1)
- Pri-miRNA (1)
- Prion (1)
- Prionprotein (1)
- Proteomics (1)
- Pseudomonas (1)
- Pseudomonas putida (1)
- Pyramidal neurons (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)
- RBFOX1 (1)
- RNA sequencing (1)
- RNS (1)
- RNS-Bindungsproteine (1)
- Reaktionskinetik (1)
- Regeneration (1)
- Reproduction (1)
- Rezeptor (1)
- Rheumatoid Arthritis (1)
- Rhinophores (1)
- Ribosomen, rRNA Prozessierung, snoRNA, Ribosomenbiogenesefaktoren (1)
- Risk assessment (1)
- Russell´s Viper (1)
- SAGE (1)
- SILAC (1)
- SNARE (1)
- SPAD (1)
- SR proteins (1)
- STAT5 (1)
- STAT5-DNA Bindungsstellen (1)
- Salt stress (1)
- Salztress (1)
- Schistosomiaisis (1)
- Scincoidea (1)
- Scincomorpha (1)
- Screening (1)
- Signaling (1)
- Signaltransduktion (1)
- Similarity (1)
- Small RNA (1)
- Spatial navigation (1)
- Spektroskopie (1)
- Spine (1)
- Stadtökologie (1)
- Stehendes Gewässer (1)
- Stickstoffmonoxid (1)
- Stoffwechsel (1)
- Stressreaktion (1)
- Structure-based Mutagenesis Study (1)
- Structured Illumination Microscopy (1)
- Strukturaufklärung (1)
- Strukturbiologie (1)
- Super resolution (1)
- Super resolution fluorescence microscopy (1)
- SuperSAGE (1)
- Svetamycin (1)
- Symbiont evolution (1)
- Symbiosis (1)
- Synaptisches Protein (1)
- Synovial Fibroblast (1)
- Systematics (1)
- Systematik (1)
- T-cell development (1)
- Taphonomie (1)
- Taxonomy (1)
- Temporäres Gewässer (1)
- Tentakel <Zoologie> (1)
- Terpenes (1)
- Terpenoid (1)
- Testosteronreductase <Testosteron-5-alpha-Reductase> (1)
- Thermoregulation (1)
- Tierphysiologie (1)
- Tocochromanol (1)
- Tomate ; Hitzestress ; Transkriptionsfaktor (1)
- Torini (1)
- Transcription (1)
- Transcriptome (1)
- Transcriptomics (1)
- Transkription (1)
- Translational Psychiatry (1)
- Treg cells (1)
- Trichoptera (1)
- Tripterospermum (1)
- Trockenstress (1)
- Tropical montane forest (1)
- Truffle (1)
- Tumor necrosis factor alpha (1)
- Tumor-Nekrose-Faktor <alpha> (1)
- Ubichinonbindetasche (1)
- Ubiquinone Binding Pocket (1)
- Uhrengene (1)
- Umweltfaktor (1)
- Urban Ecology (1)
- Ustilaginomycotina (1)
- V1 (1)
- VEGF (1)
- Vaccinieae (1)
- Vaccinium (1)
- Vaskulogenese (1)
- Verhaltensbiologie (1)
- Vesikel (1)
- Virtual Screening (1)
- Vitality monitoring (1)
- Wasserflöhe (1)
- Wechselwirkung (1)
- Western Kenya (1)
- X-ray crystallography (1)
- Yarrowia lipolytica (1)
- Yeast (1)
- Yeast-Two-Hybrid-System (1)
- Zahnwale (1)
- Zebrafish (1)
- Zelldifferenzierung (1)
- Zellskelett (1)
- Zirbeldrüse (1)
- actin (1)
- adult neurogenesis (1)
- age (1)
- allosterischer Modulator (1)
- alpha-delta-Bungarotoxin (1)
- alternative splicing (1)
- antimicrobial resistance (1)
- apoptosis (1)
- arabinose (1)
- aroma (1)
- atherosclerosis (1)
- autologous stem cell transplantation (1)
- bacillary angiomatosis (1)
- bacteria-host interaction (1)
- bacterial infection (1)
- beta-Bungarotoxin (1)
- biodiversity (1)
- brain waves (1)
- calcium store (1)
- caspase-8 (1)
- cell biology (1)
- chemical shifts (1)
- chemical synthesis (1)
- chemotherapy (1)
- cisternal organelle (1)
- climate change (1)
- clonal dominance (1)
- clonal hematopoiesis (1)
- cobra (1)
- coevolution (1)
- color (1)
- complex of closely related species (1)
- computational chemistry (1)
- consortia (1)
- control theory (1)
- cooperation (1)
- cophylogeny (1)
- cospeciation (1)
- crosslinking-mass spectrometry (1)
- cryo-EM (1)
- de novo design (1)
- development (1)
- diabetes type 1 (1)
- diatom (1)
- differentiation (1)
- diurnal (1)
- drug design (1)
- drug discovery (1)
- dryland (1)
- ecological genetics (1)
- ecosystem services (1)
- elapid snake (1)
- electron microscopy (1)
- elephant (1)
- endothelial cell (1)
- endothelium (1)
- endothial precursor cells (1)
- envenoming (1)
- environmental DNA (1)
- environmental attitudes (1)
- environmental behavior (1)
- environmental education (1)
- environmental knowledge (1)
- enzyme assay (1)
- enzyme inhibitor (1)
- extracellular matrix (1)
- failure to diverge (1)
- fitness (1)
- fluorescence (1)
- freshwater (1)
- freshwater crayfish (1)
- fungal phylogeny (1)
- fungi (1)
- gamma oscillations (1)
- gen expression (1)
- genetische Diversität (1)
- genotype (1)
- geoecology (1)
- granule cells (1)
- habitat heterogeneity (1)
- heart development (1)
- heart failure (1)
- heat stress (1)
- hematopietic stress (1)
- hematopoietic stem cell (1)
- hematopoietic stem cells (1)
- herbivores (1)
- heterosynaptic plasticity (1)
- high-content screening (1)
- high-frequency stimulation (1)
- hippo (1)
- hippocampus (1)
- histone modifications (1)
- host-switch (1)
- human pancreatic organoids (hPOs) (1)
- human-wildlife conflict (1)
- in vivo screen (1)
- inflammation (1)
- infra-slow oscillation (1)
- kern-basiertes Lernen (1)
- kernel learning (1)
- krait (1)
- lateral line (1)
- leukemia (1)
- livelihood (1)
- long-term depression (1)
- long-term potentiation (1)
- mPFC (1)
- mTOR (1)
- macrohabitat (1)
- mammary gland tissue (1)
- mathematical modeling (1)
- mechanics (1)
- medically relevant (1)
- metabotroper Glutamatrezeptor (1)
- metabotropic (1)
- miR-181 (1)
- miRNA (1)
- microRNAs (1)
- microbiome (1)
- microsatellites (1)
- mitochondria (1)
- mitochondrial dysfunction (1)
- molecualr phylogeny (1)
- molecular phylogenetics (1)
- morphological features (1)
- mounting (1)
- mouse (1)
- movement (1)
- mtDNA (1)
- myeloid angiogenic cells (1)
- neuromodulation (1)
- neuronal plasticity (1)
- niche evolution (1)
- nitric oxide (1)
- nomadic (1)
- non-timber forest products (NTFPs) (1)
- npas4l (1)
- organotin compound (1)
- outdoor education (1)
- parathyroid hormone 2 (1)
- peroxisom proliferator aktivierter Rezeptor (1)
- peroxisome proliferator-activated receptor (1)
- pesticide (1)
- plant diversification (1)
- polyketide synthase (1)
- pronephric duct (1)
- propagating waves (1)
- protein-ligand docking (1)
- quantitative proteomics (1)
- rat (1)
- reptiles (1)
- ribosomes, Arabiodpsis thaliana, pre-rRNA processing, snoRNA, (1)
- sage downy mildew (1)
- scoring function (1)
- serine/arginine-rich proteins (1)
- shallow lakes (1)
- shroom (1)
- siderophore-dependent iron uptake (1)
- sleep (1)
- smut fungi (1)
- snake bite (1)
- social isolation (1)
- socio-economics (1)
- soluble guanylyl cyclase (1)
- somatic mutations (1)
- species delimitation (1)
- species distribution modelling (1)
- spheroid (1)
- spine apparatus (1)
- splicing (1)
- stimulus repetition (1)
- stream macroinvertebrates (1)
- structure modeling (1)
- sub-Saharan Africa (1)
- sugar uptake (1)
- surround suppression (1)
- symbiont association patterns (1)
- synaptic plasticity (1)
- transcription factors (1)
- transglutaminase 2 (1)
- transmembran (1)
- trimeric autotransporter adhesin (1)
- tsetse fly (1)
- vegetation (1)
- venomous snakes (1)
- verwilderte Hauskatzen (1)
- virtuelles Screening (1)
- wwtr1 (1)
- xylose (1)
- yap1 (1)
- yeast (1)
- zisternale Organelle (1)
- zoogeography (1)
- Ähnlichkeit (1)
- Übertragbarkeit (1)
- ökologische Genetik (1)
Institute
- Biowissenschaften (422) (remove)
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
Dissecting the complexities of mammalian heart development and regenerative capacity require thorough understanding of the underlying molecular mechanisms through the expression pattern of proteins and post-translational modifications. To obtain insights intoactivated signaling pathways that control the cellular phenotype during postnatal heart development, we generated a comprehensive map of phosphorylation sites. In total we identified 21,261 phosphorylation sites and 8985 proteins in developing mouse hearts by mass spectrometry. The in-vivo SILAC (stable isotope labeling of amino acids in cell culture) approach allowed robust quantification of phosphorylation sites and proteins, which are regulated during heart development. We found several activated pathways involved in cell cycle regulation and detected numerous kinases and transcription factors to be regulated on protein and phosphopeptide level. Most strikingly, we identified a novel mitochondrial protein, known previously as Perm1, as a highly phosphorylated factor regulated during heart development. We renamed Perm1 as MICOS complex subunit Mic85 since it shows robust physical interaction with MICOS complex subunits, including Mitofilin (Mic60), Chchd3 (Mic19), Chchd6 (Mic25) and the outer membrane protein Samm50. Moreover, Mic85 is localized to the mitochondrial inner membrane facing the intermembrane space and the dynamics of Mic85 protein expression is regulated by the ubiquitin-proteasomal system through phosphorylation of casein kinase 2 on its PEST motif. Silencing of Mic85 in cultured neonatal cardiomyocytes impairs mitochondrial morphology and compromises oxidative capacity. Our findings support a clear role for Mic85 in the maintenance of mitochondrial architecture and in its contribution to enhanced energetics during developing and adult mouse cardiomyocytes. The transgenic Mic85 knockout mouse generated with a GFP knock-in will support future in vivo investigations on the integrity of mitochondria and the function of Mic85 in cardiac development.
Across the entire animal kingdom, sociality, i.e. the tendency of individual animals to form a group with conspecifics, is a common trait. Environmental changes have to be met with corresponding, quick adaptations. For social species, the presence of conspecifics is important for survival and if social animals are deprived of access to conspecifics, this can lead to strong and lasting changes on a physiological level as well as behaviour. Gene expression changes responsible for these adaptations have so far not been understood in detail. As social isolation leads to changes on a neuronal level, it is important to investigate the gene expression changes that are induced in the brain. In this thesis, next-generation RNA-sequencing was applied to zebrafish, a well-established model organism characterized by its high degree of companionship. Within the entire brain, gene expression was analysed in zebrafish that were raised either with conspecifis or in isolation, ranging from 5 to 21 days post fertilization. Using this approach, several genes were identified that were downregulated by social isolation. In this thesis, I focused on one of these consistently downregulated genes, parathyroid hormone 2 (pth2). The expression of pth2 was demonstrated to be bidirectionally regulated by the number of conspecifics present and to be responsive to changes in the social environment within 30 minutes. Regulation of pth2 does not occur by visual or chemosensory access to conspecifcs, but is mediated by mechanosensory perception of other fish via the lateral line. In an experiment using an artificial mechanical stimulation paradigm, it was shown that the features necessary to elicit pth2 transcription closely mimick the locomotion of actual zebrafish. Other, similar stimulation paradigms are not capable to induce this transcriptional response.
The reggie protein family consists of two homologous members, reggie-1 and reggie-2, also termed flotillin-2 and flotillin-1, respectively, that are ubiquitously expressed and evolutionarily well conserved, suggesting an important but so far ill-defined function. In various cell types, both reggies have been found to be constitutively associated with lipid rafts by means of acylation modifications and oligomerization. Lipid rafts are glycosphingolipid- and cholesterol-rich membrane microdomains which have been implicated in several cellular processes including membrane transport and signal transduction through growth factor receptors. However, the molecular details of these processes are still poorly understood. With the observation that reggies colocalize with activated glycosylphosphatidylinositolanchored proteins (GPI-APs) and Fyn kinase in rafts, a role for these proteins in signaling events has been suggested. In agreement with that, we have previously shown that reggie-1 becomes multiply tyrosine phosphorylated by Src kinases in response to epidermal growth factor (EGF) stimulation, pointing to a function for reggie-1 in growth factor signaling. Furthermore, overexpression of reggie-1 enhances spreading on fibronectin substrate in a tyrosine-dependent manner, thus revealing a role for reggie-1 in regulation of actin cytoskeleton through growth factor receptors. Due to the similarity shared by reggie proteins at amino acid level and to their ability to form hetero-oligomeric complexes, the first aim of this study was to analyze the putative tyrosine phosphorylation of reggie-2 in growth factor stimulated cells. Similarly to reggie-1, reggie-2 was found to be multiply tyrosine phosphorylated by Src kinase and to exist in a molecular complex with Src, with the degree of co-immunoprecipitation dependent on the activity of Src. Recent studies from us have also shown that administration of EGF results in the endocytosis of reggie-1 from the plasma membrane into endosomes, which is in line with a proposed role for reggies in membrane trafficking processes. In order to characterize in detail the endocytic mechanism that mediates the uptake of reggie-1, the dependency of reggie-1 endocytosis on clathrin and dynamin was investigated by means of overexpressing a variant form of Eps15 or a dominant negative form of dynamin-2. In either case the translocation of reggie-1 into endosomes in response to EGF was not affected, and this, together with the results that reggie-1 colocalized with cholera toxin (CTX) but not with transferrin receptor (TfnR) during EGF signaling, indicates that reggie-1 is taken up by means of a dynaminindependent, raft-mediated pathway. These findings are very well in line with recent data showing the pathway of entry into cells of reggie-2 as a raft-mediated endocytic pathway. The endocytosis of reggie-2 in response to EGF was also analyzed in this study. Similarly to reggie-1, in growth factor stimulated cells reggie-2 underwent a translocation from the plasma membrane to endosomes where the two reggies were found to colocalize with each other, suggesting that epidermal growth factor signaling might trigger the endocytosis of reggie oligomers. In addition, colocalization with both the late endosomal marker LAMP3/CD63 and epidermal growth factor receptor (EGFR) was detected, again indicating a function for reggies in signal transduction through growth factor receptors. EGFR has been reported to localize in rafts but, although this association is thought to be functional during EGF stimulation, how segregation of EGFR into rafts modulates its endocytosis and signaling is still under debate. Since reggie oligomers have recently been suggested to define a raft subtype, a further aim of this study was to investigate whether the depletion of reggies by means of small interfering RNA could interfere with the signaling and the trafficking through EGFR. Knockdown of reggie-2 resulted in an altered tyrosine phosphorylation of EGFR in response to EGF, while the degree of ubiquitination was not affected. Less efficient phosphorylation of tyrosine residues, especially of those which are docking sites for Grb2 and Shc, led in turn to an impaired activation of p38 and ERK1/2 MAPKs. Depletion of reggie-2 did not affect the early trafficking of activated EGFRs, with receptors being endocytosed and delivered to late endosomes as efficiently as in control cells. This would be in line with the normal degree of ubiquitination observed for EGFR, as ubiquitin moieties have been proposed to represent sorting tags that ensure receptor endocytosis into early endosomes and its proper intracellular trafficking. On the contrary, after prolonged EGF stimulation, depletion of reggie-2 resulted in a decreased downregulation of both receptor-bound ligand and EGFR, and in their accumulation in intracellular vesicles, thus pointing to a role for reggie-2 in the degradative pathway. Taken all together, these data ndicate that the association of EGFR with reggie-microdomains is likely to be important for proper receptor trafficking and signaling.
Capoeta damascina (Teleostei: Cyprinidae) is one of the most common freshwater fish species, found throughout the Levant, Mesopotamia, Turkey and Iran. According to the state of knowledge prior to this study, C. damascina, which is distributed over a wide range of isolated water bodies, was not a well-defined species. It was questionable whether it represents a single species or a complex of closely related species with high intraspecific and comparatively low interspecific variability. The goal of this study was to investigate the taxonomy, systematic position of the C. damascina species complex and the phylogenetic relationships among its members, based on morphological features as well as molecular phylogeny. Samples obtained from throughout the geographic range of this species complex were subjected to comparative morphological analyses in order to define, properly diagnose and separate species within the C. damascina complex. To elucidate phylogenetic relationships among members of the C. damascina species complex, samples were subjected to genetic analyses, using two molecular markers targeting the mitochondrial cytochrome oxidase I (COI, n = 103) and the two adjacent divergence regions (D1-D2) of the nuclear 28S rRNA genes (LSU, n = 65). Based on morphological and molecular genetic data, six closely related species were recognized within the C. damascina complex: C. buhsei, C. caelestis, C. damascina, C. saadii, C. umbla and an undescribed species, Capoeta sp.1. Analyses of the morphometric and meristic data obtained in this study revealed phenotypic variability among the various populations within a species and among the different species. Such differences in morphological characters reflect genetic differences, environmentally induced phenotypic variation or both, as the meristic phenotype of fish is sometimes a consequence of environmental parameters acting on the genotype. Based on phylogenetic analyses, two main lineages were identified within the C. damascina species complex: a western lineage represented by C. caelestis, C. damascina and C. umbla and an eastern lineage represented by C. buhsei, C. saadii and Capoeta sp.1. The close phylogenetic relationships between C. damascina and C. umbla and the sharing of same haplotypes between one specimen of C. damascina from Euphrates and another of C. umbla from Tigris reflect one of three possibilites: recent speciation, mitochondrial introgression or a combination of both. The results obtained in this study indicate that speciation of the above-mentioned six taxa is quite recent and that their dispersal and present-day distribution can be related to Pleistocene events. The drying out of the Persian Gulf, probably during one of the first glacials of the Pleistocene, led the ancestor of the C. damascina species complex in Mesopotamia to reach the rivers of the Gulf and of Hormuz basins and differentiate there, giving rise to the eastern lineage (ancestor of C. buhsei, C. saadii and Capoeta sp.1). As connections presumably existed among the different river drainages and basins in Iran during the wet periods of the Pleistocene, the ancestor of C. buhsei, C. saadii and Capoeta sp.1 was subsequently able to colonize the various Iranian drainages and differentiate there, giving rise to C. buhsei, C. saadii and Capoeta sp.1. After the separation from the eastern lineage, the western lineage, represented by the ancestor of C. damascina, C. umbla and C. caelestis, most likely reached the Levant from the Tigris-Euphrates system during the Pleistocene glacials, when river connections existed in the regions of the upper courses of Ceyhan Nehri (southern Turkey) and some western affluents to the Euphrates. From Ceyhan Nehri, it dispersed into other rivers in southern Turkey during Pleistocene periods of low sea levels until it reached Göksu Nehri and evolved into C. caelestis. The sister population differentiated into C. damascina and C. umbla. Based on the results obtained in this study, it is likely that C. damascina colonized the Levant and southern Turkey during the Pleistocene glacials. This is well supported by the low genetic variability among the C. damascina populations. Direct connections existed among the river drainages in the Levant during the Pleistocene periods of low sea level, thus serving as a pathway for the dispersal of C. damascina. The results of this study provide a coherent picture of the taxonomic position, phylogenetic relationships and evolutionary history of the C. damascina species complex and explain present patterns of distribution considering paleogeographic events.
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
Nearly 170 million people are chronically infected with HCV and thus at risk of developing liver cirrhosis and hepatocellular carcinoma. Although new and effective oral antiviral drugs are available, there is still the need for a preventive vaccine. In addition, in light of the high number of patients who are chronically infected with HCV the development of a therapeutic vaccine will present a support or even an alternative to the expensive medications.
To induce HCV-specific immune responses in a vaccine model, the HBV capsid is used as a carrier to deliver HCV antigens. Due to its icosahedral structure, the HBV capsid is highly immunogenic and helps to elicit a strong B cell response against the delivered antigens. In addition, the translocation motif (TLM) from the HBV surface protein is fused to the core protein. The TLM conveys membrane-permeability to the carrier capsid, enabling antigen transfer into the cytoplasm, and thus allows immunoproteasomal processing and MHC class I-mediated presentation of the antigen. To load the capsid with foreign antigens, a strep-Tag/streptavidin system is utilized. Recombinant capsids and antigens were purified from the E. coli production system. Detailed characterization of the carrier capsid demonstrated the proper assembly, adequate thermal stability and the successful loading of the foreign antigens onto the capsid surface.
As a further step, seven different HCV-derived proteins were produced and purified for the coupling on the surface of TLM-core particles. The characterization of their immunogenicity using this system is being performed.
Using ovalbumin as a model antigen, which is coupled to the carrier capsids via strep-Tag/streptavidin binding, shows that this system is suitable to efficiently deliver antigens into the cytoplasm of antigen-presenting cells (APCs), leading to the activation of APCs. This activation was assessed by measuring the secretion of IL-6 and TNF-α, in addition to the upregulation of activation markers (CD40, CD80, CD69, and MHC class I). Upon activation, the APCs were able to activate ova-specific CD8+ T cells measured by secreted IFN-γ, which was up to 20-folds more than IFN-γ secreted upon incubation with free ovalbumin. These data indicate that the TLM-capsid is suitable to serve as a carrier to deliver foreign antigens into the cytoplasm of APCs leading to MHC class I-mediated presentation and induction of an antigen-specific CTLs response.
The development of the atrioventricular (AV) canal and the cardiac valves is tightly linked and a critically regulated process. Anomalies in components of the involved pathways can lead to congenital valve malformations, a leading cause of morbidity and mortality in neonates. Myocardial Bmp as well as endocardial Notch and Wnt signaling have been identified as critical factors for the induction of EMT during the formation of the endocardial cushions and cardiac valves. Of these, canonical Wnt signaling positively regulates endocardial proliferation and EMT but negatively regulates endocardial differentiation. Further, elevated Wnt signaling leads to the ectopic expression of myocardial Bmp ligands suggesting a high level of integration of the involved pathways and crosstalk amongst the different cardiac tissues.
Here we have identified a novel role for Id4 as a mediator between Bmp and Wnt signaling. Id4 belongs to the Id family of proteins and is known to be involved in bone and nervous system development. We found that in zebrafish, id4 is expressed in the endocardium of the AV canal at embryonic stages and throughout the atrial chamber in addition to AV canal, in adults. Using transcription activator-like effector nucleases (TALENs) we established an id4 mutant allele. Our analysis shows that id4 mutant larvae are susceptible to retrograde blood flow, and show aberrant expression of developmental valvular markers. These include expanded expression domains of markers like bmp4, cspg2a and Alcam. In contrast, valve maturation as assessed by the expression of spp1 is considerably reduced in id4 mutants. Using conditional transgenic systems, along with elegant in vivo imaging of transgenic reporter lines, we further found that id4 is a transcriptional target of Bmp signaling, and it is capable of dose dependently restricting Wnt signaling in the endocardium of the Atrioventricular Canal.
Taken together, our data identifies Id4 as a novel player in Atrioventricular Canal and valve development. We show that Id4 function is important in valve development acting downstream of Bmp signaling by restricting endocardial Wnt to allow valve maturation
Seed dispersal is a key ecosystem function for plant regeneration, as it involves the movement of seeds away from the parental plants to particular habitats where they can germinate and transition to seedlings and ultimately adult plants. Seed dispersal is shaped by a diversity of abiotic and biotic factors, particularly by associations between plants and climate and between plants and other species. Due to the ongoing loss of biodiversity and changing global conditions, such interactions are prone to change and pose a severe threat to plant regeneration. One way to address this challenge is to study associations between plant traits and abiotic and biotic factors to understand the potential impacts of global change on plant regeneration. Plant communities have long been analyzed through the lens of vegetative traits, mainly ignoring how other traits interact and respond to the environment. For instance, while associations between vegetative traits (e.g., specific leaf area, leaf nitrogen content) and climate are well studied, there are few case studies of reproductive traits in relation to trait-environment associations in the context of global change.
Thus, the overarching aim of this dissertation is to explore how trait-environment associations, with a special focus on reproductive traits, can improve our understanding of the effect that global change may have on seed dispersal, and ultimately on plant regeneration. To this end, my research focuses on studying associations between plant traits and abiotic and biotic factors along an elevational gradient in both forests and deforested areas of tropical mountains. This dissertation addresses three principal research objectives.
First, I investigate the extent to which reproductive (seed and fruit traits) and vegetative traits (leaf traits) are related to abiotic and biotic factors for communities of fleshy-fruited plants in the Ecuadorian Andes. I used multivariate analyses to test associations between four (a)biotic factors and seven reproductive traits and five vegetative traits measured on 18 and 33 fleshy fruited plant species respectively. My analyses demonstrate that climate and soil conditions are strongly associated with the distribution of both reproductive and vegetative traits in tropical tree communities. The production of “costly” vs. “cheap” seeds, fruits and leaves, i.e., the production of few rewarding fruits and acquisitive leaves versus the production of many less-rewarding fruits and conservative leaves, is primarily limited by temperature, whereas the size of plant organs is more related to variation in precipitation and soil conditions. My findings suggest that associations between reproductive and vegetative traits and the abiotic environment follow similar principles in tropical tree communities.
Second, I assess how climate and microhabitat conditions affect the prevalence of endozoochorous plant species in the seed rain of tropical montane forests in southern Ecuador. I analyzed seed rain data for an entire year from 162 traps located across an elevational gradient spanning of 2000 m. I documented the microhabitat conditions (leaf area index and soil moisture next to each seed trap) at small spatial scale as well as the climatic conditions (mean annual temperature and rainfall in each plot) at large spatial scale. After a one-year of sampling, I counted 331,838 seeds of 323 species/morphospecies. My analyses demonstrate that the prevalence of endozoochorous plant species in the seed rain increases with temperature across elevations and with leaf area index within elevations. These results show that the prevalence of endozoochory is shaped by the interplay of both abiotic and biotic factors at large and small spatial scales.
Third, I examine the potential of seed rain to restore deforested tropical areas along an elevational gradient in southern Ecuador. For this chapter, I collected seed rain using 324 seed traps installed in 18 1-ha plots in forests (nine forest plots) and in pastures (nine deforested plots) along an elevational gradient of 2000 m. After a sampling period of three months, I collected a total of 123,039 seeds of 255 species/morphospecies from both forests and pastures along the elevational gradient. I did not find a consistent decrease in the amount and richness of seed rain between forests and pastures, but I detected a systematic change in the type of dispersed seeds, as heavier seeds and a higher proportion of endozoochorous species were found in forests compared to pastures at all elevations. This finding suggests that deforestation acts as a strong filter selecting seed traits that are vital for plant regeneration.
Understanding the role that trait-environment associations play in how plant communities regenerate today could serve as a basis for predicting changes in regeneration processes of plant communities under changing global conditions in the near future. Here, I show how informative the measurement of reproductive traits and trait environment associations are in facilitating the conservation of forest habitats and the restoration of deforested areas in the context of global change.