570 Biowissenschaften; Biologie
Refine
Has Fulltext
- yes (139)
Is part of the Bibliography
- yes (139)
Year of publication
- 2019 (139) (remove)
Document Type
- Journal article (78)
- Doctoral Thesis (60)
- Preprint (1)
Keywords
- Tissue Engineering (7)
- Candida albicans (3)
- Genexpression (3)
- In vitro (3)
- Signaltransduktion (3)
- cancer (3)
- leukemic cells (3)
- metagenomics (3)
- 3D Tumormodell (2)
- 3D tissue model (2)
- Annotation (2)
- Bioinformatik (2)
- Blutgerinnung (2)
- Chlamydia trachomatis (2)
- Chronobiologie (2)
- Drosophila (2)
- Drosophila melanogaster (2)
- Ecology (2)
- Fluoreszenzmikroskopie (2)
- Herzmuskelzelle (2)
- Implantat (2)
- Maus (2)
- Mikroskopie (2)
- RNA-seq (2)
- Regenerative Medizin (2)
- Serotonin (2)
- Taufliege (2)
- Vaskularisierung (2)
- Verhalten (2)
- apoptosis (2)
- colorectal cancer (2)
- cytotoxicity (2)
- dSTORM (2)
- evolution (2)
- infection (2)
- ischemic stroke (2)
- microbiome (2)
- optimal drug combination (2)
- rheumatoid arthritis (2)
- ubiquitin (2)
- (hem)ITAM signaling (1)
- 3D (1)
- 3D Modell (1)
- 3D model (1)
- 3D remote sensing (1)
- 3D tumour model (1)
- 3D-Modell (1)
- ABP1 (1)
- ADHD (1)
- ALAN (1)
- AMP-activated kinases (1)
- APOBEC3G (1)
- AUX1 (1)
- Absorbed Doses (1)
- Accurate (1)
- Acids (1)
- Ackerschmalwand (1)
- Adaptation (1)
- Adherens junction (1)
- Administered Activities (1)
- Aggregation (1)
- Algorithmus (1)
- Alkaloid (1)
- Alpine habitats (1)
- Alzheimerkrankheit (1)
- Aminerge Nervenzelle (1)
- Amygdala (1)
- Ancistrocladaceae (1)
- Aneugene (1)
- Angiogenese (1)
- Animales Nervensystem (1)
- Anthropocene (1)
- Anti-infective (1)
- Antigen CD19 (1)
- Apidae (1)
- Apis mellifera (1)
- Apolipoprotein E (1)
- Arabidopsis (1)
- Aspergillus fumigalus (1)
- Associative learning (1)
- Autoaggressionskrankheit (1)
- Automated Image Analysis (1)
- Automatisierung (1)
- Automatisierung der Analyse (1)
- Autophagie <Physiologie> (1)
- Autophagosom (1)
- Autophagozytose (1)
- B-MYB (1)
- BRAF-mutant (1)
- BRAF-mutiert (1)
- Barrier (1)
- Bees (1)
- Behaviour (1)
- Behavioural ecology (1)
- Berberine (1)
- Bildanalyse (1)
- Bildverarbeitung (1)
- Biokinetics (1)
- Biologika (1)
- Biomarker (1)
- Biomaterial (1)
- Biomedical engineering (1)
- Biomedicine (1)
- Biophysics (1)
- Bioreaktor (1)
- Brustkrebs (1)
- C-60 fullerene (1)
- C60 fullerene (1)
- CAR T-Zelltherapie (1)
- C\(_{60}\) fullerene (1)
- Cadherine (1)
- Caenorhabditis elegans (1)
- Callyspongia siphonella (1)
- Cancer Cell (1)
- Cdh13 (1)
- Cell migration (1)
- Cell stainin (1)
- Cellular neuroscience (1)
- Central nervous system (1)
- Channelrhodopsin (1)
- Chondrogenesis (1)
- Chronobiology (1)
- Circular dichroism (1)
- Click Chemie (1)
- CoQ10 (1)
- Cofaktor (1)
- Colonization (1)
- Computational and Systems Biology (1)
- Computer modelling (1)
- Computersimulation (1)
- Confocal microscopy (1)
- Conservation (1)
- Cortex (1)
- CpG (1)
- Cranial sutures (1)
- Cytokine (1)
- Cytologie (1)
- Cytosol (1)
- DC gate (1)
- DLS and AFM measurements (1)
- DNA damage (1)
- DNA-Schäden (1)
- DNS-Doppelstrangbruch (1)
- DNS-Schädigung (1)
- Dendritic cell (1)
- Dendritische Zelle (1)
- Densovirus (1)
- Developmental biology (1)
- Dezellularisierung (1)
- Diagnostic Imaging Exams (1)
- Dickdarmtumor (1)
- Dnaschaden (1)
- Dopamin (1)
- Dopamine (1)
- Dosimetry (1)
- Doxorubicin (1)
- Drug discovery (1)
- Drug targets (1)
- Drug testing (1)
- Dynamics (1)
- E8 symmetry (1)
- EMT (1)
- ERK signaling (1)
- Echinococcosis (1)
- Echinococcus (1)
- Eierstockkrebs (1)
- Einzelmolekülmikroskopie (1)
- Electron Microscopy (1)
- Elektronenmikroskopie (1)
- Embryonic induction (1)
- Embryos (1)
- Endodermis (1)
- Endogene Rhythmik (1)
- Enhancer elements (1)
- Environmental impact (1)
- Epigenetic (1)
- Evolutionary developmental biology (1)
- Evolutionary emergence (1)
- Extracellular matrix (1)
- Eye development (1)
- Eye irritation (1)
- Fear conditioning (1)
- Fgf-signalling (1)
- Flowering plants (1)
- Flowers (1)
- Fluorescence spectroscopy (1)
- Fremdkörpermodell (1)
- G-quadruplex (1)
- Ga-68-labelled Peptides (1)
- Gedächtnis (1)
- Gene by Environment (1)
- Gene expression analysis (1)
- Gene silencing (1)
- Genetik (1)
- Genom (1)
- Genome Annotation (1)
- Genotoxicitiy (1)
- Genotoxizität (1)
- Germline (1)
- Gestational diabetes (1)
- Gestationsdiabetes (1)
- Gewebemodell (1)
- Gliazelle (1)
- Glioblastom (1)
- Glykane (1)
- Haut (1)
- HeLa cells (1)
- Health (1)
- Helicasen (1)
- Hfq (1)
- Hindbrain (1)
- Hippo pathway (1)
- Hippocampus (1)
- Hochauflösende Fluoreszenzmikroskopie (1)
- Host Genome Integrity (1)
- Host-parasite interaction (1)
- Human Herpesvirus 6 (1)
- Humanes Herpesvirus 6 (1)
- Hurwitz theorem (1)
- Hypothalamus (1)
- Hypoxia (1)
- Hypoxie (1)
- Immunologe (1)
- Immunoprecipitation (1)
- Immuntherapie (1)
- Induzierte pluripotente Stammzelle (1)
- Inf-TRAP-Seq (1)
- Inhibitor (1)
- Insect flight (1)
- Insulin (1)
- Invertebrate herbivory (1)
- Isolation (1)
- KDELR2 (1)
- Kardiomyozyt (1)
- Kernspintomografie (1)
- Klassifizierung (1)
- Klastogene (1)
- Knochenimplantat (1)
- Knochenregeneration (1)
- Knorpelbildung (1)
- Kongo (1)
- Kraniosynostose (1)
- Krebs (1)
- Krebs <Medizin> (1)
- LC-HRESIMS (1)
- Leaf traits (1)
- Lee Smolin (1)
- Leichte kognitive Beeinträchtigung (1)
- Lernen (1)
- Lernverhalten (1)
- Light sheet microscopy (1)
- Limb development (1)
- Llullaillaco Volcano (1)
- Lung (1)
- Lunge (1)
- Lungenkrebs (1)
- Lungentumor (1)
- Lysosom (1)
- M14 carboxypeptidasses (1)
- MMB complex (1)
- MRI (1)
- Maculinea butterfly (1)
- Magnetic Resonance Imaging (1)
- Mamma carcinoma (1)
- Mammakarzinom (1)
- Markierungen synaptischer Proteine (1)
- Masern-Virus (1)
- Mass spectrometry (1)
- Massenspektrometrie (1)
- Mc4r (1)
- Measles virus (1)
- Megakaryozyt (1)
- Melanom (1)
- Melanoma (1)
- Meniskus (1)
- Meniskusimplantat (1)
- Metabolomics (1)
- Method development (1)
- Methodenentwicklung (1)
- Methylation (1)
- Microbiology and Infectious Disease (1)
- Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) (1)
- Mitochondria (1)
- Mitose (1)
- Mobile genetic element (1)
- Modell (1)
- Molekulare Zielstrukturen (1)
- Molekulargenetik (1)
- Molekülsystem (1)
- Motor behaviour (1)
- Multiples Myelom (1)
- Myb-MuvB (1)
- Myrmecology (1)
- Myrmica ant non-equilibrium dynamics (1)
- NIR-Spektroskopie (1)
- Nahrungserwerb (1)
- Naphthylisochinolinalkaloide (1)
- Naphthylisoquinoline (1)
- Neisseria gonorrhoeae (1)
- Neisseria meningitidis (1)
- Neural circuits (1)
- Neurodevelopmental Disorder (1)
- Neurogenese (1)
- Neurons (1)
- Next Generation Sequencing (NGS) (1)
- Non-coding RNA (1)
- Nuclear Medicine (1)
- Object recognition (1)
- Optimal foraging (1)
- Optogenetics (1)
- Optogenetik (1)
- Outer membrane proteins (1)
- PDF neurons (1)
- PI3K/mTOR inhibierung (1)
- Parvovirus (1)
- Paternal age and BMI effects (1)
- Patterns and drivers of invertebrate herbivory (1)
- Patterns and drivers of species diversity of phytophagous beetles (1)
- Patterns and drivers of species richness and community biomass of large mammals (1)
- Pediatric Nuclear Medicine (1)
- Pediatric Patients (1)
- Phosphatasen (1)
- Phosphoglykolat-Phosphatase (1)
- Phosphoglykolatphosphatase (1)
- Physiologie (1)
- Phytophthora (1)
- Phytosphingosine (1)
- Pigmentdispergierender Faktor (1)
- Piriformospora indica (1)
- Plant signalling (1)
- Plants (1)
- Plasmamembranorganisation (1)
- Plasmozytom (1)
- Polysaccharide (1)
- Prefrontal cortex (1)
- Premna (1)
- ProQ (1)
- Protein crosslinking (1)
- Protein folding (1)
- Protein interactions (1)
- Proteininteraktionen (1)
- Proteinquervernetzungen (1)
- Proteomics Analysis of Complexes (1)
- Proteotype (1)
- Proteus vulgaris (1)
- Präfrontaler Cortex (1)
- Pseudomonas syringae (1)
- Quantifizierung (1)
- Quantitative Mikroskopie (1)
- R package (1)
- REDD1 (1)
- RNA Sequencing (1)
- RNA metabolism (1)
- RNA protein interactions (1)
- RNA secondary structures (1)
- RNA sequencing (1)
- RNA-Seq (1)
- RNA-seq transcriptome (1)
- RNAi (1)
- RNAlater (1)
- RNS (1)
- Radiation Protection (1)
- Radiation-associated Cancer Risk (1)
- Raphe (1)
- Registrierung <Bildverarbeitung> (1)
- Reiz (1)
- Rescue behaviour (1)
- Research Article (1)
- Resistenz (1)
- Rezeptoren (1)
- Rhizodermis (1)
- Risk Assessment (1)
- SREBP (1)
- SSR42 (1)
- Scarabaeidae (1)
- Schlaganfall (1)
- Schmalwand <Arabidopsis> (1)
- Self-renewal (1)
- Sex chromosome (1)
- Sex determination (1)
- Sexual development and function (1)
- Signalweg (1)
- Skull (1)
- Small RNA (1)
- Small interfering RNAs (1)
- Solution-state NMR (1)
- Somites (1)
- Species delimitation (1)
- Species richness (1)
- Sphingobasen (LCB, LCB-P) (1)
- Sphingobases (1)
- Sphingolipide (1)
- Sphingosine-1-phosphate (1)
- Sphingosine-1-phosphats (1)
- Spred-Proteine (1)
- Stammzelle (1)
- Staphylococcus aureus (1)
- Stechameisen (1)
- Stem cell (1)
- Stem cells (1)
- Stem-cell biotechnology (1)
- Stoffwechsel (1)
- Strains (1)
- Structural elucidation (1)
- Subtercola vilae (1)
- Suspensionskultur (1)
- Syap1 knockout (1)
- Synaptische Vesikel (1)
- TLR3 (1)
- TMEM16F (1)
- TNFR1 (1)
- TRAIL (1)
- TWEAK (1)
- Tc-99m-MAG3 Scans (1)
- Therapeutisches System (1)
- Therapie (1)
- Therapiesimulation (1)
- Thrombo-inflammation (1)
- Thrombosis (1)
- Thrombozyt (1)
- Tiermodell (1)
- Tissue engineering (1)
- Todesdomäne (1)
- Trailmaking Test (1)
- Transcription (1)
- Transcriptomic (1)
- Translation (1)
- Transposable element (1)
- Tumor (1)
- UBXD1 (1)
- UV–Vis (1)
- Ubiquitin (1)
- Ultrashort echo time - UTE (1)
- Ustilago maydis (1)
- Vascularized (1)
- Vaskularisation (1)
- Verticillium (1)
- Vesikel (1)
- Wirkstofftestung (1)
- Wundheilung (1)
- Wurzel (1)
- Wurzelzellschichten (1)
- YAP (1)
- ZFAND1 (1)
- Zebrafish (1)
- Zell Migration (1)
- Zellbiologie (1)
- Zellkultur (1)
- Zellmigration (1)
- Zellschichtspezifische Expression (1)
- Zellteilung (1)
- Zelltod (1)
- Zentralzylinder (1)
- Zinc (1)
- accessory medulla (1)
- accumulation (1)
- acetate (1)
- adalimumab (1)
- adapterprotein (1)
- alternative splicing (1)
- altitudinal gradients (1)
- aminergic neurons (1)
- anakinra (1)
- aneugens (1)
- animal physiology (1)
- antibacterial (1)
- antibiofilm (1)
- anticancer (1)
- antitrypanosomal (1)
- artifacts (1)
- artificial light at night (1)
- autoimmune disease (1)
- automatisierte Bildanalyse (1)
- autophagocytosis (1)
- autophagosome (1)
- autophagy (1)
- auxin (1)
- bacteria (1)
- bacterial pathogen (1)
- behavioral plasticity (1)
- bioinformatics tool (1)
- bioink (1)
- biological rhythm (1)
- biological scaffolds (1)
- biological techniques (1)
- biomarker (1)
- biomarker signature (1)
- biomaterial ink (1)
- bioreactor (1)
- biotic interaction (1)
- blood–brain barrier (1)
- bone (1)
- brain (1)
- brain endothelial cell (1)
- burnt-wood (1)
- calcium (1)
- cancer metabolism (1)
- cancer therapy (1)
- carabid beetles (1)
- cardiac tissue (1)
- cardiomyocytes (1)
- cell biology (1)
- cell signalling (1)
- cell therapy (1)
- cell-type specific (1)
- channelrhodopsins (1)
- chlamydia (1)
- chlorophyll fluorescence imaging (1)
- circRNA (1)
- circadian clock (1)
- circadian rhythm (1)
- circular transcriptome sequencing (1)
- classification (1)
- clastogens (1)
- click chemistry (1)
- clinical trial (1)
- closed-loop systems (1)
- co-culture (1)
- coagulation system (1)
- cold adaptation (1)
- comparative genomics (1)
- competition (1)
- composition (1)
- conservation (1)
- cosmology (1)
- crystal growth (1)
- crystallization (1)
- cytokinesis (1)
- cytotoxic (1)
- dead-wood enrichment (1)
- decellularization (1)
- definition (1)
- dendritic cells (1)
- designer cell (1)
- developmental forms (1)
- direct muss spectrometric profiling (1)
- disease modelling (1)
- diversity gradients (1)
- domain-specific language (1)
- dopamine (1)
- doxorubicin (1)
- drivers and patterns of diversity and herbivory (1)
- drug delivery (1)
- drug release (1)
- drug resistance evolution (1)
- earlywood (1)
- ecology (1)
- ecosystem service (1)
- efficient intervention points (1)
- elementary body (1)
- enbrel (1)
- endocytosis (1)
- enercy-richness hypothesis (1)
- energy homeostasis (1)
- enhancer (1)
- enzyme mechanism (1)
- etanercept (1)
- evolutionary genetics (1)
- expansion microscopy (1)
- external stimuli (1)
- extinction dynamics (1)
- fertility (1)
- fibre length (1)
- flash freezing (1)
- fluxosome (1)
- food resources (1)
- forest ecology (1)
- forest fire (1)
- forest management (1)
- friut fly behaviour (1)
- function (1)
- functional analysis (1)
- fungal rhodopsins (1)
- gangliosides and lipid rafts (1)
- gastrointestinal tract (1)
- gene expression analysis (1)
- gene network (1)
- genetic engineering (1)
- genetic recombination (1)
- genome analysis (1)
- genome annotation (1)
- genomics (1)
- glia cells (1)
- global change (1)
- green fluorescence protein (GFP) (1)
- ground-dwelling predators (1)
- growth (1)
- growth ring width (1)
- hemostasis (1)
- heuristics (1)
- hiPSC aggregation (1)
- hochauflösende Fluoreszenzmikroskopie (1)
- honeybee (1)
- honeybees (1)
- iPSC (1)
- ichthyology (1)
- imaging (1)
- imaging PAM (1)
- immunity (1)
- immunocompetent skin (1)
- immunotherapy (1)
- immunotherapy of cancer (1)
- implant (1)
- in situ microscopy (1)
- in vitro (1)
- indole-3-acetic acid (1)
- induced pluripotent stem cells (1)
- infection biology (1)
- inflation (1)
- insect abundance (1)
- insect collection (1)
- integrative management strategy (1)
- intervention point analyzing (1)
- knockout (1)
- kolorektales Karzinom (1)
- land sharing (1)
- land use (1)
- latewood (1)
- learning (1)
- lignan (1)
- localization microscopy (1)
- lowland beech forests (1)
- mRNA (1)
- machine learning (1)
- macrophages (1)
- mating (1)
- mating preference (1)
- measles virus (1)
- mechanisms of disease (1)
- mechanistic modelling (1)
- medaka (1)
- megakaryopoiesis (1)
- melatonin (1)
- memory (1)
- meningitis (1)
- meniscus implant (1)
- meta-analysis (1)
- metabolic adaptation (1)
- metabolic flux (1)
- metabolic modeling (1)
- metabolic modelling (1)
- metabolite profiling (1)
- metabolomic (1)
- metabolomic profiling (1)
- metals (1)
- metaproteomics (1)
- methods (1)
- miR-26 (1)
- microbial rhodopsins (1)
- microbiota (1)
- mitosis (1)
- mitotic gene expression (1)
- mitotic genes (1)
- molecular biology (1)
- multi-photon microscopy (1)
- mutants (1)
- nanocomplex (1)
- native populations (1)
- natural pest control (1)
- neuronal (1)
- next generation sequencing (1)
- next-generation sequencing (1)
- noncoding RNA (1)
- noncovalent complex (1)
- noncovalent nanocomplex (1)
- nuclear envelope (1)
- nuclear export (1)
- oncogenes (1)
- oncology (1)
- oncolysis (1)
- oncolytic vaccinia virus (1)
- oncolytic virus (1)
- optimal drug targeting (1)
- optimal pharmacological modulation (1)
- optimal treatment strategies (1)
- optogenetics (1)
- oxindole alkaloids (1)
- p21-activated kinase Mbt/PAK4 (1)
- p53 (1)
- p97 (1)
- parasexual recombination (1)
- parasite biology (1)
- patch-clamp (1)
- peptidomoics (1)
- pharmacophore map (1)
- phenolic compounds (1)
- phosphorylation (1)
- photodynamic chemotherapy (1)
- piRNA (1)
- pit membrane diameter (1)
- plants (1)
- plant–pathogen interaction (1)
- plasma membrane depolarization (1)
- plasma membrane organization (1)
- platelets (1)
- pollination (1)
- population genetics (1)
- post-fire management (1)
- post-translational modification (1)
- posttranscriptional control (1)
- programmed cell death (1)
- programmierter Zelltod (1)
- protected forests (1)
- protein processing (1)
- proteomics (1)
- puberty (1)
- resonance theory (1)
- restriction factors (1)
- reticulate body (1)
- risk factors (1)
- sample storage (1)
- saproxylic organisms (1)
- secreted effectors (1)
- sentinel prey (1)
- short neuropeptide F (1)
- signalling (1)
- single-molecule tracking (1)
- skin model (1)
- sleep (1)
- small RNA (1)
- small intestinal submucosa scaffold (1)
- small-cell lung cancer (1)
- sodium (1)
- soil fauna (1)
- species richness (1)
- species‐area hypothesis (1)
- sphingolipids (1)
- sporidia (1)
- stem Cells (1)
- stem cells (1)
- storage-pool diseases (1)
- stromal vascular fraction (1)
- structure (1)
- structured illumination microscopy (1)
- super-resolution fluorescence microscopy (1)
- super-resolution microscopy (1)
- superresolution (1)
- suppressor cells (1)
- suppressor mutation (1)
- survival analysis (1)
- synapses (1)
- synergistic effect (1)
- synthetic biology (1)
- systematic affiliation (1)
- systematic drug targeting (1)
- targeted therapies (1)
- targeted therapy (1)
- temperature‐mediated resource exploitation hypothesis (1)
- temperature‐richness hypothesis (1)
- therapy simulation (1)
- thrombosis (1)
- time lag (1)
- tisindoline (1)
- tissue engineering (1)
- transcription (1)
- transcriptome (1)
- transcriptomics (1)
- transient dynamics (1)
- tree cavities (1)
- trypanosomes (1)
- tumour (1)
- tumour-necrosis factors (1)
- tyloses (1)
- ubiquitin ligase (1)
- ubiquitylation (ubiquitination) (1)
- unmanaged broadleaved forests (1)
- uptake (1)
- vaccinia (1)
- vascularization (1)
- vertical and radial variation (1)
- vessel lumen diameter (1)
- vessel wall resident stem cells (1)
- virotherapy (1)
- virus (1)
- whole-genome sequencing (1)
- wood anatomy (1)
- wound healing (1)
- yH2AX-Foci (1)
- zielgerichtete Behandlung (1)
- zielgerichtete Therapien (1)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (68)
- Graduate School of Life Sciences (33)
- Julius-von-Sachs-Institut für Biowissenschaften (10)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (9)
- Rudolf-Virchow-Zentrum (8)
- Fakultät für Biologie (4)
- Institut für Hygiene und Mikrobiologie (4)
- Institut für Molekulare Infektionsbiologie (4)
- Institut für Pharmakologie und Toxikologie (4)
- Institut für Virologie und Immunbiologie (3)
Sonstige beteiligte Institutionen
Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskulären Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverzügliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gewährleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu ermöglichen und eine automatisierte Lösung für ähnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros für ImageJ, eines für die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte ermöglicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskulärer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalität der Software bewiesen werden. Die Analyse der neuromuskulären Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen.
Im zweiten Teil der Arbeit wurden neuromuskulärer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans über mehrere Monate ohne Nahrungsaufnahme überleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core“-Vesikel (CCV) und der „dense-core“-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning“-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erhöhte Anzahl von „dense-core“-Vesikeln, sowie eine veränderte Lokalisation der DCV in Dauerlarven festgestellt werden.
Im dritten Teil dieser Arbeit wurde untersucht ob die für synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierfür eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss.
Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.
Knowledge on how the timing of flowering is related to plant fitness and species interactions is crucial to understand consequences of phenological shifts as they occur under climate change. Early flowering plants may face advantages of low competition for pollinators and disadvantages of low pollinator abundances and unfavourable weather conditions. However, it is unknown how this trade-off changes over the season and how the timing affects reproductive success. On eight grasslands we recorded intra-seasonal changes in pollinators, co-flowering plants, weather conditions, flower visitation rates, floral longevity and seed set of Pulsatilla vulgaris. Although bee abundances and the number of pollinator-suitable hours were low at the beginning of the season, early flowers of P. vulgaris received higher flower visitation rates and estimated total number of bee visits than later flowers, which was positively related to seed set. Flower visitation rates decreased over time and with increasing number of co-flowering plants, which competed with P. vulgaris for pollinators. Low interspecific competition for pollinators seems to be a major driver for early flowering dates. Thus, non-synchronous temporal shifts of co-flowering plants as they may occur under climate warming can be expected to strongly affect plant-pollinator interactions and the fitness of the involved plants.
Climate warming has the potential to disrupt plant-pollinator interactions or to increase competition of co-flowering plants for pollinators, due to species-specific phenological responses to temperature. However, studies focusing on the effect of temperature on solitary bee emergence and the flowering onset of their food plants under natural conditions are still rare. We studied the effect of temperature on the phenology of the two spring bees Osmia cornuta and Osmia bicornis, by placing bee cocoons on eleven grasslands differing in mean site temperature. On seven grasslands, we additionally studied the effect of temperature on the phenology of the red-list plant Pulsatilla vulgaris, which was the first flowering plant, and of co-flowering plants with later flowering. With a warming of 0.1°C, the abundance-weighted mean emergence of O. cornuta males advanced by 0.4 days. Females of both species did not shift their emergence. Warmer temperatures advanced the abundance-weighted mean flowering of P. vulgaris by 1.3 days per 0.1°C increase, but did not shift flowering onset of co-flowering plants. Competition for pollinators between P. vulgaris and co-flowering plants does not increase within the studied temperature range. We demonstrate that temperature advances plant flowering more strongly than bee emergence suggesting an increased risk of pollinator limitation for the first flowers of P. vulgaris.
Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.
The central nervous system (CNS) barriers are highly specialized cellular barriers that promote brain homeostasis while restricting pathogen and toxin entry. The primary cellular constituent regulating pathogen entry in most of these brain barriers is the brain endothelial cell (BEC) that exhibits properties that allow for tight regulation of CNS entry. Bacterial meningoencephalitis is a serious infection of the CNS and occurs when bacteria can cross specialized brain barriers and cause inflammation. Models have been developed to understand the bacterial – BEC interaction that lead to pathogen crossing into the CNS, however, these have been met with challenges due to these highly specialized BEC phenotypes. This perspective provides a brief overview and outlook of the in vivo and in vitro models currently being used to study bacterial brain penetration, and opinion on improved models for the future.
Neurodevelopmental disorders, including attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are disorders of mostly unknown etiopathogenesis, for which both genetic and environmental influences are expected to contribute to the phenotype observed in patients. Changes at all levels of brain function, from network connectivity between brain areas, over neuronal survival, synaptic connectivity and axonal growth, down to molecular changes and epigenetic modifications are suspected to play a key roles in these diseases, resulting in life-long behavioural changes.
Genome-wide association as well as copy-number variation studies have linked cadherin-13 (CDH13) as a novel genetic risk factor to neuropsychiatric and neurodevelopmental disorders. CDH13 is highly expressed during embryonic brain development, as well as in the adult brain, where it is present in regions including the hippocampus, striatum and thalamus (among others) and is upregulated in response to chronic stress exposure. It is however unclear how CDH13 interacts with environmentally relevant cues, including stressful triggers, in the formation of long-lasting behavioural and molecular changes. It is currently unknown how the environment influences CDH13 and which long term changes in behaviour and gene expression are caused by their interaction. This work therefore investigates the interaction between CDH13 deficiency and neonatal maternal separation (MS) in mice with the aim to elucidate the function of CDH13 and its role in the response to early-life stress (ELS).
For this purpose, mixed litters of wild-type (Cdh13+/+), heterozygous (Cdh13+/-) and homozygous knockout (Cdh13-/-) mice were maternally separated from postnatal day 1 (PN1) to postnatal day 14 (PN14) for 3 hours each day (180MS; PN1-PN14). In a first series of experiments, these mice were subjected to a battery of behavioural tests starting at 8 weeks of age in order to assess motor activity, memory functions as well as measures of anxiety. Subsequently, expression of RNA in various brain regions was measured using quantitativ real-time polymerase chain reaction (qRT-PCR). A second cohort of mice was exposed to the same MS procedure, but was not behaviourally tested, to assess molecular changes in hippocampus using RNA sequencing.
Behavioural analysis revealed that MS had an overall anxiolytic-like effect, with mice after MS spending more time in the open arms of the elevated-plus-maze (EPM) and the light compartment in the light-dark box (LDB). As a notable exception, Cdh13-/- mice did not show an increase of time spent in the light compartment after MS compared to Cdh13+/+ and Cdh13+/- MS mice. During the Barnes-maze learning task, mice of most groups showed a similar ability in learning the location of the escape hole, both in terms of primary latency and primary errors. Cdh13-/- control (CTRL) mice however committed more primary errors than Cdh13-/- MS mice. In the contextual fear conditioning (cFC) test, Cdh13-/- mice showed more freezing responses during the extinction recall, indicating a reduced extinction of fear memory. In the step-down test, an impulsivity task, Cdh13-/- mice had a tendency to wait longer before stepping down from the platform, indicative of more hesitant behaviour. In the same animals, qRT-PCR of several brain areas revealed changes in the GABAergic and glutamatergic systems, while also highlighting changes in the gatekeeper enzyme Glykogensynthase-Kinase 3 (Gsk3a), both in relation to Cdh13 deficiency and MS. Results from the RNA sequencing study and subsequent gene-set enrichment analysis revealed changes in adhesion and developmental genes due to Cdh13 deficiency, while also highlighting a strong link between CDH13 and endoplasmatic reticulum function. In addition, some results suggest that MS increased pro-survival pathways, while a gene x environment analysis showed alterations in apoptotic pathways and migration, as well as immune factors and membrane metabolism. An analysis of the overlap between gene and environment, as well as their interaction, highlighted an effect on cell adhesion factors, underscoring their importance for adaptation to the environment.
Overall, the stress model resulted in increased stress resilience in Cdh13+/+ and Cdh13+/- mice, a change absent in Cdh13-/- mice, suggesting a role of CDH13 during programming and adaptation to early-life experiences, that can results in long-lasting consequences on brain functions and associated behaviours. These changes were also visible in the RNA sequencing, where key pathways for cell-cell adhesion, neuronal survival and cell-stress adaptation were altered. In conclusion, these findings further highlight the role of CDH13 during brain development, while also shedding light on its function in the adaptation and response during (early life) environmental challenges.
In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.
miR-221 is regarded as an oncogene in many malignancies, and miR-221-mediated resistance towards TRAIL was one of the first oncogenic roles shown for this small noncoding RNA. In contrast, miR-221 is downregulated in prostate cancer (PCa), thereby implying a tumour suppressive function. By using proliferation and apoptosis assays, we show a novel feature of miR-221 in PCa cells: instead of inducing TRAIL resistance, miR-221 sensitized cells towards TRAIL-induced proliferation inhibition and apoptosis induction. Partially responsible for this effect was the interferon-mediated gene signature, which among other things contained an endogenous overexpression of the TRAIL encoding gene TNFSF10. This TRAIL-friendly environment was provoked by downregulation of the established miR-221 target gene SOCS3. Moreover, we introduced PIK3R1 as a target gene of miR-221 in PCa cells. Proliferation assays showed that siRNA-mediated downregulation of SOCS3 and PIK3R1 mimicked the effect of miR-221 on TRAIL sensitivity. Finally, Western blotting experiments confirmed lower amounts of phospho-Akt after siRNA-mediated downregulation of PIK3R1 in PC3 cells. Our results further support the tumour suppressing role of miR-221 in PCa, since it sensitises PCa cells towards TRAIL by regulating the expression of the oncogenes SOCS3 and PIK3R1. Given the TRAIL-inhibiting effect of miR-221 in various cancer entities, our results suggest that the influence of miR-221 on TRAIL-mediated apoptosis is highly context- and entity-dependent.
The knee joint is a complex composite joint containing the C-shaped wedge-like menisci composed of fibrocartilage. Due to their complex composition and structure, they provide mechanical resilience to the knee joint protecting the articular cartilage. Because of the limited repair potential, meniscal injuries do not only affect the meniscus itself but also lead to altered joint homeostasis and inevitably to secondary osteoarthritis.
The meniscus was characterized focusing on its anatomy, structure and meniscal markers such as aggrecan, collagen type I (Col I) and Col II. The components relevant for meniscus tissue engineering, namely cells, Col I scaffolds, biochemical and biomechanical stimuli were studied. Meniscal cells (MCs) were isolated from meniscus, mesenchymal stem cells (MSCs) from bone marrow and dermal microvascular endothelial cells (d-mvECs) from foreskin biopsies. For the human (h) meniscus model, wedge-shape compression of a hMSC-laden Col I gel was successfully established. During three weeks of static culture, the biochemical stimulus transforming growth factor beta-3 (TGF beta-3) led to a compact collagen structure. On day 21, this meniscus model showed high metabolic activity and matrix remodeling as confirmed by matrix metalloproteinases detection. The fibrochondrogenic properties were illustrated by immunohistochemical detection of meniscal markers, significant GAG/DNA increase and increased compressive properties. For further improvement, biomechanical stimulation systems by compression and hydrostatic pressure were designed. As one vascularization approach, direct stimulation with ciclopirox olamine (CPX) significantly increased sprouting of hd-mvEC spheroids even in absence of auxiliary cells such as MSCs. Second, a cell sheet composed of hMSCs and hd-mvECs was fabricated by temperature triggered cell sheet engineering and transferred onto the wedge-shaped meniscus model. Third, a biological vascularized scaffold (BioVaSc-TERM) was re-endothelialized with hd-mvECs providing a viable vascularized network. The vascularized BioVaSc-TERM was suggested as wrapping scaffold of the meniscus model by using two suture techniques, the all-inside-repair (AIR) for the posterior horn, and the outside-in-refixation (OIR) for the anterior horn and the middle part.
This meniscus model for replacing torn menisci is a promising approach to be further optimized regarding vascularization, biochemical and biomechanical stimuli.
Development and proof of concept of a biological vascularized cell‐based drug delivery system
(2019)
A major therapeutic challenge is the increasing incidence of chronic disorders.
The persistent impairment or loss of tissue function requires constitutive on‐demand
drug availability optimally achieved by a drug delivery system ideally directly connected
to the blood circulation of the patient. However, despite the efforts and achievements in
cell‐based therapies and the generation of complex and customized cell‐specific
microenvironments, the generation of functional tissue is still unaccomplished.
This study demonstrates the capability to generate a vascularized platform technology to
potentially overcome the supply restraints for graft development and clinical application
with immediate anastomosis to the blood circulation.
The ability to decellularize segments of the rat intestine while preserving the ECM for
subsequent reendothelialization was proven. The reestablishment of a functional
arteriovenous perfusion circuit enabled the supply of co‐cultured cells capable to replace
the function of damaged tissue or to serve as a drug delivery system. During in vitro
studies, the applicability of the developed miniaturized biological vascularized scaffold
(mBioVaSc‐TERM®) was demonstrated. While indicating promising results in short term
in vivo studies, long term implantations revealed current limitations for the translation
into clinical application. The gained insights will impact further improvements of quality
and performance of this promising platform technology for future regenerative therapies.
Articular cartilage lesions that occur upon intensive sport, trauma or degenerative disease represent a severe therapeutic problem. At present, osteoarthritis is the most common joint disease worldwide, affecting around 10% of men and 18% of women over 60 years of age (302). The poor self-regeneration capacity of cartilage and the lack of efficient therapeutic treatment options to regenerate durable articular cartilage tissue, provide the rationale for the development of new treatment options based on cartilage tissue engineering approaches (281). The integrated use of cells, biomaterials and growth factors to guide tissue development has the potential to provide functional substitutes of lost or damaged tissues (2,3). For the regeneration of cartilage, the availability of mesenchymal stromal cells (MSCs) or their recruitment into the defect site is fundamental (281). Due to their high proliferation capacity, the possibility to differentiate into chondrocytes and their potential to attract other progenitor cells into the defect site, bone marrow-derived mesenchymal stromal cells (BMSCs) are still regarded as an attractive cell source for cartilage tissue engineering (80). However, in order to successfully engineer cartilage tissue, a better understanding of basic principles of developmental processes and microenvironmental cues that guide chondrogenesis is required.
Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis, enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.
Das humane Schädeldach besteht aus fünf Schädelplatten, die durch intramembranöse Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Schädelnähte aus, die eine Fusion der Schädelplatten verhindern und damit ein Schädelwachstum parallel zu Gehirnentwicklung ermöglichen. Für diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung nötig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gewährleistet wird. Störungen in diesem regulatorischen System können zu einer vorzeitigen Fusion der Schädelplatten, Kraniosynostose genannt, führen. Die Kraniosynostose ist eine der häufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Schädeldeformationen, die sekundär einen erhöhten intrakranialen Druck zur Folge haben können. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auffälligkeiten vorliegen. Bisher sind über 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil überlappen, ist eine klare klinische Diagnose häufig erschwert. Sowohl Umwelteinflüsse als auch genetische Veränderungen können die Ursache für Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Veränderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Darüber hinaus wurden chromosomale Veränderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in über 50% der Fälle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollständig geklärt.
Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufklärung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30% der Patienten konnte eine potentiell pathogene Veränderung identifiziert werden. Davon waren 23% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass möglicherweise eine MSX2 Überexpression vorliegt. Für ein normales Schädelwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert für einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgeführt. Eine simulierte Überexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium führte zu schweren Gehirn-, Herz- und Augendefekten.
Mittels NGS wurden 77% der potentiell pathogenen genetischen Veränderungen identifiziert. Hierfür wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von fünf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zusätzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. Für diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine Änderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenität dieser spezifischen Varianten zu bekräftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zukünftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenzänderungen bei Kraniosynostose Patienten sinnvoll ist.
The alarming increase in the magnitude and spatiotemporal patterns of changes in composition, structure and function of forest ecosystems during recent years calls for enhanced cross-border mitigation and adaption measures, which strongly entail intensified research to understand the underlying processes in the ecosystems as well as their dynamics. Remote sensing data and methods are nowadays the main complementary sources of synoptic, up-to-date and objective information to support field observations in forest ecology. In particular, analysis of three-dimensional (3D) remote sensing data is regarded as an appropriate complement, since they are hypothesized to resemble the 3D character of most forest attributes. Following their use in various small-scale forest structural analyses over the past two decades, these sources of data are now on their way to be integrated in novel applications in fields like citizen science, environmental impact assessment, forest fire analysis, and biodiversity assessment in remote areas. These and a number of other novel applications provide valuable material for the Forests special issue “3D Remote Sensing Applications in Forest Ecology: Composition, Structure and Function”, which shows the promising future of these technologies and improves our understanding of the potentials and challenges of 3D remote sensing in practical forest ecology worldwide.
The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules.
An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed.
Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates.
In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.
Studying how cambial age and axial height affects wood anatomical traits may improve our understanding of xylem hydraulics, heartwood formation and axial growth. Radial strips were collected from six different heights (0–11.3 m) along the main trunk of three Manchurian catalpa (Catalpa bungei) trees, yielding 88 samples. In total, thirteen wood anatomical vessel and fiber traits were observed usinglight microscopy (LM) and scanning electron microscopy (SEM), and linear models were used to analyse the combined effect of axial height, cambial age and their interaction. Vessel diameter differed by about one order of magnitude between early- and latewood, and increased significantly with both cambial age and axial height in latewood, while it was positively affected by cambial age and independent of height in earlywood. Vertical position further had a positive effect on earlywood vessel density, and negative effects on fibre wall thickness, wall thickness to diameter ratio and length. Cambial age had positive effects on the pit membrane diameter and vessel element length, while the annual diameter growth decreased with both cambial age and axial position. In contrast, early- and latewood fiber diameter were unaffected by both cambial age and axial height. We further observed an increasing amount of tyloses from sapwood to heartwood, accompanied by an increase of warty layers and amorphous deposits on cell walls, bordered pit membranes and pit apertures. This study highlights the significant effects of cambial age and vertical position on xylem anatomical traits, and confirms earlier work that cautions to take into account xylem spatial position when interpreting wood anatomical structures, and thus, xylem hydraulic functioning.
Fin development and regeneration are complex biological processes that are highly relevant in teleost fish. They share genetic factors, signaling pathways and cellular properties to coordinate formation of regularly shaped extremities. Especially correct tissue structure defined by extracellular matrix (ECM) formation is essential. Gene expression and protein localization studies demonstrated expression of fndc3a (fibronectin domain containing protein 3a) in both developing and regenerating caudal fins of zebrafish (Danio rerio). We established a hypomorphic fndc3a mutant line (fndc3a\(^{wue1/wue1}\)) via CRISPR/Cas9, exhibiting phenotypic malformations and changed gene expression patterns during early stages of median fin fold development. These developmental effects are mostly temporary, but result in a fraction of adults with permanent tail fin deformations. In addition, caudal fin regeneration in adult fndc3a\(^{wue1/wue1}\) mutants is hampered by interference with actinotrichia formation and epidermal cell organization. Investigation of the ECM implies that loss of epidermal tissue structure is a common cause for both of the observed defects. Our results thereby provide a molecular link between these developmental processes and foreshadow Fndc3a as a novel temporal regulator of epidermal cell properties during extremity development and regeneration in zebrafish.
In mammals the melanocortin 4 receptor (Mc4r) signaling system has been mainly associated with the regulation of appetite and energy homeostasis. In fish of the genus Xiphophorus (platyfish and swordtails) puberty onset is genetically determined by a single locus, which encodes the mc4r. Wild populations of Xiphophorus are polymorphic for early and late-maturing individuals. Copy number variation of different mc4r alleles is responsible for the difference in puberty onset. To answer whether this is a special adaptation of the Mc4r signaling system in the lineage of Xiphophorus or a more widely conserved mechanism in teleosts, we studied the role of Mc4r in reproductive biology of medaka (Oryzias latipes), a close relative to Xiphophorus and a well-established model to study gonadal development. To understand the potential role of Mc4r in medaka, we characterized the major features of the Mc4r signaling system (mc4r, mrap2, pomc, agrp1). In medaka, all these genes are expressed before hatching. In adults, they are mainly expressed in the brain. The transcript of the receptor accessory protein mrap2 co-localizes with mc4r in the hypothalamus in adult brains indicating a conserved function of modulating Mc4r signaling. Comparing growth and puberty between wild-type and mc4r knockout medaka revealed that absence of Mc4r does not change puberty timing but significantly delays hatching. Embryonic development of knockout animals is retarded compared to wild-types. In conclusion, the Mc4r system in medaka is involved in regulation of growth rather than puberty.
In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.