Refine
Has Fulltext
- yes (81)
Is part of the Bibliography
- yes (81)
Year of publication
- 2018 (81) (remove)
Document Type
- Doctoral Thesis (80)
- Journal article (1)
Keywords
- Maus (6)
- Thrombozyt (5)
- Staphylococcus aureus (4)
- Stress (4)
- Biene (3)
- Biofilm (3)
- G-Protein gekoppelte Rezeptoren (3)
- Microscopy (3)
- Myc (3)
- Thrombose (3)
- Angst (2)
- Arteriosklerose (2)
- Aspergillus fumigatus (2)
- Cadherine (2)
- Cancer immunotherapy (2)
- Candida albicans (2)
- Entwicklung (2)
- Epigenetik (2)
- Hippocampus (2)
- Hämostase (2)
- Metabolismus (2)
- Mikroskopie (2)
- Neurogenese (2)
- Phänologie (2)
- Schlaganfall (2)
- Signaltransduktion (2)
- T-Lymphozyt (2)
- Tagesrhythmus (2)
- Taufliege (2)
- Tissue Engineering (2)
- Zelldifferenzierung (2)
- gp130 (2)
- miRNS (2)
- 3D-Kultur (1)
- 5-FU (1)
- ACL (1)
- ADHS (1)
- Acid adaptation (1)
- Acyrthosiphon pisum (1)
- Adenomatous-polyposis-coli-Protein (1)
- Aliphatics (1)
- Ameise (1)
- Ameisen (1)
- Angeborene Immunität (1)
- Animal behavior IntelliCage system (1)
- Animal model (1)
- Ant (1)
- Antigen CD28 (1)
- Antigen CD8 (1)
- Antikörper-Antwort (1)
- Anxiety (1)
- Aorta (1)
- Apis mellifera (1)
- Arbeitsgedächtnis (1)
- ArsZ (1)
- Arzneimitteldesign (1)
- Aspergillose (1)
- AstA (1)
- Atherosclerosis (1)
- Atta vollenweideri (1)
- Aurora-A (1)
- Autoantikörper (1)
- B-Lymphozyt (1)
- Bacillus subtilis (1)
- Bacteria (1)
- Bacterial infection (1)
- Bakterien (1)
- Bakteriophagen (1)
- Bildverarbeitung (1)
- BioVaSc (1)
- Biofilm formation (1)
- Biofilms (1)
- Bioinformatics (1)
- Bioinformatik (1)
- Biologie (1)
- Bipolar Disorder (1)
- Bisphosphonate (1)
- Blut-Hirn-Schranke (1)
- Blutstammzelle (1)
- Bone (1)
- Brain-derived neurotrophic factor (1)
- Bäckerhefe (1)
- Bärtierchen (1)
- C. albicans (1)
- CD28 (1)
- CD4+ (1)
- CD8 Effektorfunktionen (1)
- CD8 Gedächtnisreaktionen (1)
- CDH13 (1)
- CIDP (1)
- CLEC-2 (1)
- CRHR1 (1)
- CRISPR/Cas9 (1)
- CXCL13 (1)
- Cadherin 13 (1)
- Cadherin-13 (1)
- Calcium (1)
- Camponotus (1)
- Camponotus floridanus (1)
- Cancer (1)
- Cancer models (1)
- Cancer therapeutic resistance (1)
- Cartilage Regeneration (1)
- Caspr-1 (1)
- Catechol-0-Methyltransferase (1)
- Cell differentiation (1)
- Ceramides (1)
- Chain-length distribution (1)
- Chemoresistenz (1)
- Chemotherapeutic resistance (1)
- Chimeric antigen receptor (1)
- Chlamydia (1)
- Chlamydienkrankheit (1)
- Chromatin (1)
- Chronobiologie (1)
- Chronophin (1)
- Circadian Clock (1)
- Circadiane Uhr (1)
- Circadianer Rhythmus (1)
- Coffin-Lowry syndrome (1)
- Colonkrebs (1)
- Computational drug design (1)
- Contactin-1 (1)
- Corticoliberin (1)
- Cuticle (1)
- Cuticular water permeabilities (1)
- Cuticular waxes (1)
- Cyclics (1)
- DNA Sekundärstruktur (1)
- DNA secondary structure (1)
- DNS-Bindungsproteine (1)
- DNS-Reparatur (1)
- DRG (1)
- Dendritic cells (1)
- Dendritische Zelle (1)
- Depression (1)
- Diabetes mellitus (1)
- Differenzierungszustand (1)
- Drosophila (1)
- Drosophila Myc transcription growth PAF1 (1)
- Dualstere Liganden (1)
- Dualsteric Ligands (1)
- E. coli Nissle 1917 (1)
- EHEC (1)
- ELISA (1)
- Efflux pump (1)
- Einzelzellgelelektrophorese (1)
- Elektroencephalogramm (1)
- Emotion (1)
- Empfindlichkeit (1)
- Endogenous clock (1)
- Endozytose (1)
- Enoyl-acyl-carrier-protein-Reductase <Enoyl-[acyl-carrier-protein]-Reductase> (1)
- Enterohemorrhagic Escherichia coli (1)
- Entwicklung chimärer Antigenrezeptor T-Zellen (1)
- Entzündung (1)
- Entzündungsschmerz (1)
- Enzyminhibitor (1)
- Extracellular matrix proteins (1)
- Extrazelluläre Matrix (1)
- Fabry-Krankheit (1)
- Farbensehen (1)
- Feedforward loop (1)
- Fluconazole (1)
- Fluorescence (1)
- Fluorescence Microscopy (1)
- Fluorescence Resonance Energy Transfer (1)
- Fluoreszenzmikroskopie (1)
- Fruit (1)
- Furcht (1)
- G Protein-Coupled Receptors (1)
- GABAerge Nervenzelle (1)
- GLP-1 (1)
- GPCR (1)
- GPVI (1)
- Galactosidase <alpha-> (1)
- Gb3 accumulation (1)
- Gefühl (1)
- Gehirn (1)
- Generalisierung (1)
- Generalization (1)
- Genetics (1)
- Genexpression (1)
- Genom (1)
- Genome editing (1)
- Genomeditierung (1)
- Genotoxizität (1)
- Genregulation (1)
- Geruchssinn (1)
- Glottoplastik (1)
- Glutamin (1)
- Glycoprotein GPV (1)
- Golgi-Apparat (1)
- Guillain-Barré-Syndrom (1)
- HP1432, Hpn2 (1)
- Helicobacter pylori (1)
- Hematopoietic Stem Cell (1)
- Hemostasis (1)
- Herzinfarkt (1)
- Heubacillus (1)
- Hic-5 (1)
- Histatin 5 (1)
- Hospitalismus <Hygiene> (1)
- Host defense (1)
- Host-pathogen interaction (1)
- Hyaluronic acid (1)
- Hyaluronsäure (1)
- Hydrogel (1)
- Hämatopoetische Stammzellen (1)
- IFN-g (1)
- IL-23 (1)
- IgG-Subklasse (1)
- Image Processing (1)
- Immune Thrombocytopenia (1)
- Immunglobulin G (1)
- Immunsystem (1)
- Immuntherapie (1)
- Immunthrombozytopenie (1)
- Inhibition (1)
- Innate immunity (1)
- Innere Uhr (1)
- Insect (1)
- Insekt (1)
- Interaktion (1)
- Interferon <gamma-> (1)
- Interleukin 2 (1)
- Interleukin 6 (1)
- Interleukine (1)
- Intestinal stem cell (1)
- Intratumorale Heterogenität (1)
- Ion channel function (1)
- Ionenkanal (1)
- Ischemic stroke (1)
- Kation (1)
- Kehlkopfchirurgie (1)
- Kernspintomografie (1)
- Klima (1)
- Knochenmark (1)
- Knockout (1)
- Knockout <Molekulargenetik> (1)
- Knorpel (1)
- Kohlendioxid (1)
- Kommunikation (1)
- Komplement <Immunologie> (1)
- Komplexauge (1)
- Konditionierung (1)
- Kostimulation (1)
- Krebs <Medizin> (1)
- Krebsimmuntherapie (1)
- LAD (1)
- Lactatdehydrogenase (1)
- Lambdoide Prophagen (1)
- Leaf (1)
- Lgr5 (1)
- MDR1 (1)
- MIPs (1)
- MMN (1)
- MRI (1)
- MRR1 (1)
- MRT (1)
- MYC (1)
- Magnetic resonance imaging (1)
- Manisch-depressive Krankheit (1)
- Marine sponge-derived actinomycetes (1)
- Marine sponges (1)
- Marrow (1)
- Massenspektrometrie/Proteomics (1)
- Mediator Komplex (1)
- Megakaryocyte (1)
- Megakaryocytes (1)
- Megakaryozyt (1)
- Mek5/Erk5-Signalweg (1)
- Melanom (1)
- Melanoma (1)
- Membranlipide (1)
- Merkel cell carcinoma (1)
- Merkelzellkarzinom (1)
- Mesenchymal Stem Cell (1)
- Mesenchymal Stromal Cells (1)
- Metabolic Labeling (1)
- MicroRNAs (1)
- Mikrokerne (1)
- Mitochondria (1)
- Mitochondrium (1)
- Model (1)
- Model simulation (1)
- Mrr1 (1)
- Multicellular aggregates (1)
- Multidrug-Resistance-Related Proteine (1)
- Multizellulären Bakteriengemeinschaften (1)
- Muscarinrezeptor (1)
- Muskelzelle (1)
- Mutagenität (1)
- Mycobacterium tuberculosis (1)
- Mycobacterium tuberculosis InhA (1)
- N-MYC (1)
- NIR-Spektroskopie (1)
- NOS-I (1)
- NOS1AP (1)
- NaV1.9. oxidized phospholipids (1)
- Nahrungsaufnahme (1)
- Nestbau (1)
- Netzhaut (1)
- Neuroanatomie (1)
- Neurobiologie (1)
- Neurobiology (1)
- Neurodevelopment (1)
- Neuronale Plastizität (1)
- Neutrophils (1)
- Nodo-Paranodopathie (1)
- Non-coding RNA (1)
- Non-viral genome engineering (1)
- Nosocomial Infections (1)
- Nosokomiale Infektionen (1)
- OCT1 (1)
- OSM (1)
- OSMR (1)
- Olfaction (1)
- Oncostatin M (1)
- Onkogen (1)
- PDXP (1)
- Panic Disorder (1)
- Paniksyndrom (1)
- Partial Agonists (1)
- Partialagonismus (1)
- Pathogenität (1)
- Peptide (1)
- Permeability (1)
- Phonochirurgie (1)
- Phosphatase (1)
- Phospholipase D (1)
- Phospholipide (1)
- Plasmonic (1)
- Platelet (1)
- Poly(glycidol) (1)
- Polyneuropathie (1)
- Polysaccharide intercellular adhesin (PIA) (1)
- Post-transcriptional regulation (1)
- Pra1 (1)
- Probiotikum (1)
- Protein (1)
- Protein folding (1)
- Protein-Protein Interaktion (1)
- Proteom (1)
- Proteomics (1)
- Psychische Störung (1)
- Pyrrolidinderivate (1)
- Pyrrolidine carboxamides (1)
- RAF Kinasen (1)
- REST-Complex (1)
- RNA polymerase II (1)
- RNA sequencing (1)
- RNA-binding proteins (1)
- RNS (1)
- RSK2 (1)
- Ranvier-Schnürring (1)
- Raphe Kerne (1)
- Regulator of G protein signaling 2 (1)
- Rekonstruktion (1)
- Rezeptor (1)
- Rgs2 (1)
- Rho-Proteine (1)
- RhoGTPase (1)
- Rituximab (1)
- SIS-muc (1)
- SOCE (1)
- Saccharomyces cerevisiae (1)
- Schilddrüse (1)
- Schlaf (1)
- Schmerz (1)
- Sekundärstruktur (1)
- Sequenzanalyse <Chemie> (1)
- Serotonerge Nervenzelle (1)
- Serotonin (1)
- Sglt1 (1)
- Shigella flexneri (1)
- Simulation (1)
- Sleeping Beauty transposon (1)
- Small RNA (1)
- Soziale Insekten (1)
- Spatiotemporal analysis (1)
- Speicheldrüse (1)
- Sphingomyelinase (1)
- Spleen tyrosine kinase (1)
- Stammzelle (1)
- Staphylococci (1)
- Staphylococcus epidermidis (1)
- Stathmin (1)
- Stickstoffmonoxid-Synthase (1)
- Stimmbandchirurgie (1)
- Stimmerhöhung (1)
- Stofftransport <Biologie> (1)
- Streptomyces (1)
- Stressresistenz (1)
- Structure-based (1)
- Säugerzellen (1)
- Säugetiere (1)
- T-Zelle (1)
- T-cell engineering (1)
- T-cell therapy (1)
- TFIIIC (1)
- TRPA1 (1)
- TRPV1 (1)
- Tagesrhythmik (1)
- Targeted therapies (1)
- Thrombopoese (1)
- Thrombopoiesis (1)
- Thrombosis (1)
- Thrombozytenaggregation (1)
- Tiermodell (1)
- Timing (1)
- Transcriptome (1)
- Transkription (1)
- Transkriptionsfaktor (1)
- Transkriptom (1)
- Transpiration barrier (1)
- Transporters (1)
- Transposon (1)
- Transsexualismus (1)
- Tripartite Model (1)
- TrkB (1)
- Tropomyosin receptor kinase B (1)
- Trypanosoma brucei (1)
- Tuberkelbakterium (1)
- Tumor-Nekrose-Faktor <alpha> (1)
- Tumormetabolismus (1)
- Tumorzellen (1)
- Tumour angiogenesis (1)
- VSMC (1)
- Venusfliegenfalle (1)
- Verhalten (1)
- Virulenzfaktor (1)
- Voltage-Clamp-Methode (1)
- Wasser Fett Trennung (1)
- Xerostomie (1)
- adulte Neurogenese (1)
- airflow (1)
- antivirale Gene (1)
- anxiety (1)
- artificial diet (1)
- bSSFP (1)
- balanced steady state free precession (1)
- bees (1)
- behavioral rhythms (1)
- behaviour (1)
- bioinformatics (1)
- biomedical applications (1)
- biomedicine (1)
- blood glucose regulation (1)
- blood-brain barrier (1)
- bone marrow niche (1)
- brood rearing (1)
- building behavior (1)
- cancer (1)
- carbon dioxide (1)
- chronophin (1)
- circadian clocks (1)
- climate change (1)
- climate control (1)
- cohesin (1)
- color vision (1)
- comet assay (1)
- compound eyes (1)
- defense (1)
- dendritische Zelle (1)
- differentiation status (1)
- division of labor (1)
- early neural precursors (1)
- early-life stress (1)
- ecology (1)
- electroencephalogram (1)
- emotion processing (1)
- enoyl ACP reductase (1)
- epigenetics (1)
- evolution (1)
- fear conditioning (1)
- fitness (1)
- flytrap (1)
- foraging (1)
- frequency modulation (1)
- frühe neurale Vorläufer (1)
- genomics (1)
- genotoxic agents (1)
- genotoxische Agenzien (1)
- glucose (1)
- glycoprotein GPV (1)
- hibernation (1)
- human primary cells (1)
- icaADBC (1)
- inflammatory pain (1)
- insertion-site deep sequencing (1)
- insulin (1)
- intravenöse Immunglobuline (1)
- islets of Langerhans (1)
- jasmonate (1)
- lambdoid prophage (1)
- leaf-cutting ants (1)
- lipid rafts (1)
- mammalian cells (1)
- mass spectrometry (1)
- metabolism (1)
- miR-26 (1)
- miRNA Biogenesis (1)
- micronuclei (1)
- microrna (1)
- mouse platelets (1)
- ncRNA (1)
- near-infrared spectroscopy (1)
- nest climate (1)
- neuroblastoma (1)
- neuroepithelial progenitors (1)
- neuroepitheliale Vorläufer (1)
- neurogenesis (1)
- neuronal nitric oxide synthase (1)
- neuronale Stickstoffmonoxidsynthase (1)
- neuropsychiatric disorders (1)
- neuropsychiatrische Störungen (1)
- nicht-viraler Gentransfer (1)
- non-coding RNA (1)
- nuclesosome positioning (1)
- oxidierte Phospholipide (1)
- pancreas (1)
- pdxp (1)
- pea aphid (1)
- phosphatase (1)
- phospholipase D (1)
- platelet (1)
- platelet biogenesis (1)
- platelets (1)
- postradiogene Xerostomie (1)
- probiotica (1)
- progenitors (1)
- pyrrolidine carboxamides (1)
- rac1 inhibitors (1)
- reconstruction (1)
- research software (1)
- retinal development (1)
- rodent model (1)
- secretion (1)
- splicing (1)
- stem cells (1)
- store-operated calcium entry (1)
- stroke (1)
- stx-Phagen (1)
- stx-phages (1)
- synthetic lethal interaction (1)
- synthetisch lethale Interaktion (1)
- temperate zones (1)
- temporal organization (1)
- thyroid stimulating hormone receptor (1)
- timing (1)
- trans-Golgi network (1)
- transcriptome (1)
- tumorspezifische Therapie (1)
- varroa (1)
- vaskuläre glatte Muskelzelle (1)
- venus (1)
- waggle dance (1)
- water fat separation (1)
- wild bees (1)
- working memory (1)
- zeitgeber (1)
- zeitliche Organisation (1)
Institute
- Graduate School of Life Sciences (81) (remove)
Sonstige beteiligte Institutionen
- Expertenkreis zur Erarbeitung eines Stufenplans zur Diagnose und Therapie von Angsterkrankungen (1)
- Hans-Knöll-Institut Jena (1)
- Harvard Medical School, Boston, USA (1)
- Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Würzburg (1)
- Lehrstuhl für Tierökologie und Tropenbiologie, Universität Würzburg (1)
- Maastricht University, Department of Psychiatry and Neuropsychology (1)
- Universität Jena (1)
- Zentrum für Infektionsforschung (ZINF) Würzburg (1)
- Zentrum für Infektionsforschung ZINF Würzburg (1)
G-Quadruplex (G4)-Strukturen sind sehr stabile und polymorphe DNA und RNA Sekundärstrukturen mit einem konservierten Guanin-reichen Sequenzmotiv (G4-Motiv). Sie bestehen aus übereinander gestapelten planaren G-Quartetts, in denen je vier Guanine durch Wasserstoffbrückenbindungen zusammengehalten werden.
Da G4-Motive in Eukaryoten an bestimmten Stellen im Genom angereichert vorkommen, wird angenommen, dass die Funktion von G4-Strukturen darin besteht, biologische Prozesse positiv oder negativ zu regulieren. Aufgrund der hohen thermodynamischen Stabilität von G4 Strukturen ist davon auszugehen, dass Proteine in die Faltung, Stabilisierung und Entfaltung dieser Nukleinsäure-Strukturen regulatorisch involviert sind. Bis heute wurden viele Proteine in der Literatur beschrieben, die G4-Strukturen entwinden können. Jedoch konnten bisher nur wenige Proteine identifiziert werden, die in vivo die Faltung fördern oder G4-Strukturen stabilisieren.
Durch Yeast One-Hybrid (Y1H)-Screenings habe ich Zuo1 als neues G4 bindendes Protein identifiziert. In vitro Analysen bestätigten diese Interaktion und es stellte sich heraus, dass Zuo1 G4-Strukturen stabilisiert. Übereinstimmend mit den in vitro Daten konnte gezeigt werden, dass Zuo1 signifikant an G4-Motive im Genom von Saccharomyces ceresivisiae bindet. Genomweit überlappen G4-Motive, an die Zuo1 bindet, mit Stellen, an denen die DNA Replikation zum Stillstand kommt und vermehrt DNA Schäden vorkommen. Diese Ergebnisse legen nahe, dass Zuo1 eine Funktion während der DNA Reparatur oder in Zusammenhang mit dem Vorankommen der DNA Replikationsgabel hat, indem G4-Strukturen stabilisiert werden. Diese Hypothese wird außerdem durch genetische Experimente gestützt, wonach in Abwesenheit von Zuo1 die Genominstabilität zunimmt. Aufgrund dieser Daten war es möglich ein Model zu entwickeln, bei dem Zuo1 während der S-Phase G4-Strukturen bindet und stabilisiert wodurch die DNA Replikation blockiert wird. Diese Interaktion findet neben Stellen schadhafter DNA statt und unterstützt somit DNA Reparatur-Prozesse wie beispielsweise die Nukleotidexzisionsreparatur.
Als weiteres potentielles G4-bindendes Protein wurde Slx9 in Y1H-Screenings identifiziert. In vitro Experimente zeigten zwar, dass Slx9 mit höherer Affinität an G4-Strukturen bindet im Vergleich zu anderen getesteten DNA Konformationen, jedoch wurde in S. cerevisiae genomweit keine signifikante Bindung an G4-Motive festgestellt.
Transsexualität ist gekennzeichnet durch die dauerhafte Gewissheit, in einem Körper des falschen Geschlechts geboren worden zu sein. Bei Mann-zu-Frau-Transsexualität ist die Stimme ein oft unterschätzter Bestandteil der ganzheitlichen Therapie. Kann mit konservativer Therapie kein zufriedenstellend weiblicher Stimmklang erreicht werden, ist Phonochirurgie die Methode der Wahl. In Würzburg wird hierzu die Glottoplastik nach Wendler modifiziert durch Hagen angewendet.
An der vorliegenden Studie zur Qualitätsüberprüfung des operativen Verfahrens nahmen insgesamt 21 auf diese Art operierte Patientinnen teil, von denen 18 zu einer Nachuntersuchung in Würzburg erschienen und 3 die zugsandten Fragebögen ausfüllten. Erwartet wurden eine Anhebung der mittleren Sprechstimmlage sowie eine Veränderung weiterer objektiver Stimmparameter. Mit einer angehobenen Sprechstimmlage wurde auch eine höhere Zufriedenheit der Patientinnen mit ihrer Stimme vermutet. Nach Erfahrungsberichten vieler Patientinnen blieben Probleme beim Telefonieren jedoch weiterhin bestehen. Diese für den subjektiven Therapieerfolg sehr wichtige Situation wurde mit einer Perzeptionsstudie gezielt untersucht.
Insgesamt zeigte sich die Operation als risikoarme und effektive Therapieoption, um die mittlere Sprechstimmlage anzuheben. So lag die mittlere Sprechstimmlage der Patientinnen postoperativ im Median bei 170 Hz und somit im ausschließlich weiblichen Stimmlagenbereich. Die Anhebung der mittleren Sprechstimmlage ging mit einem leichten Verlust des Dynamikumfangs einher, der jedoch nur von ca. einem Drittel der Patientinnen als störend empfunden wurde. Zur subjektiven Erfolgskontrolle wurden Daten aus den standardisierten Fragebögen „Voice Handicap Index“ sowie „Fragebogen zur Lebenszufriedenheit“ und dem eigenen „Würzburger Fragebogen“ erhoben und ausgewertet. Die Ergebnisse dieser Fragebögen zeigten, abweichend von den guten objektiven Messwerten, deutliche Einschränkungen gegenüber einer stimmgesunden Vergleichsgruppe aus der Literatur. Diese Defizite betrafen sowohl die stimmbezogenen Fragengebiete des Voice Handicap Index, als auch die allgemeine Lebenszufriedenheit. Dennoch gaben die Patientinnen an, durch die Stimmoperation ein selbstsichereres Auftreten gegenüber fremden Personen gewonnen zu haben. Die subjektive Stimmzufriedenheit korrelierte sowohl mit der mittleren Sprechstimmlage als auch mit der Selbsteinschätzung der Weiblichkeit der Stimme. Bei Frequenz- und Dynamikumfang zeigten sich starke Differenzen zwischen objektiven Messergebnissen und subjektiver Zufriedenheit.
Für die Perzeptionsstudie zur Telefonsituation wurden von 18 Patientinnen sowie jeweils im Alter gepaarten männlichen und weiblichen Kontrollsprechern identische Sprachaufnahmen angefertigt und in der Frequenz entsprechend der Übertragungs-bandbreite am Telefon bearbeitet. Diese Stimmproben wurden 50 männlichen und 50 weiblichen zufällig ausgewählten Laienjuroren zur Bewertung hinsichtlich des Sprechergeschlechts vorgespielt. Gemessen wurde neben dem Urteil männlich oder weiblich auch die Zeitspanne von Beginn der Wiedergabe bis zur Urteilseingabe. Ungefähr 40 % der transsexuellen Patientinnen wurden mehrheitlich, also in über 50 % der Urteile als weiblich bewertet. Die Urteilszeit lag für die Sprachproben der transsexuellen Patientinnen signifikant über der Urteilszeit für männliche und weibliche Kontrollsprecher. Bezogen auf die Juroren zeigten sich Unterschiede zwischen männlicher und weiblicher Jurorengruppe: Männer ordneten die Sprachproben häufiger einem weiblichen Sprecher zu. Weibliche Juroren fällten ihr Urteil hingegen signifikant schneller als männliche Juroren. Es zeigte sich eine positive Korrelation der Geschlechtszuordnung mit der mittleren Sprechstimmlage. Die unabhängig von der Sprechstimmlage deutlich verlängerte Urteilszeit für transsexuelle Sprecherinnen zeigte jedoch, dass neben der mittleren Sprechstimmlage auch noch andere Faktoren die Geschlechtszuordnung beeinflussen. Dementsprechend existierte keine klare Grenzfrequenz, oberhalb derer Stimmen regelhaft als weiblich wahrgenommen wurden. Auch in einem in der Literatur mehrfach als ausschließlich weiblich definierten Sprechbereich wurden die Stimmen einzelner Patientinnen mehrheitlich als männlich wahrgenommen. Es konnte kein Zusammenhang der Formantfrequenzen F1 – F3 mit der Geschlechtszuordnung gefunden werden.
Zusammenfassend zeigten diese aus objektiven Stimmdaten, Eigen- und Fremd-bewertung bestehenden Ergebnisse, dass durch alleinige Operation zwar eine höhere Stimmlage, jedoch kein vollständig weiblicher Stimmklang erreicht wurde. Deswegen muss die Phonochirurgie zukünftig stärker in ein umfassendes Behandlungskonzept aus logopädischem Stimmtraining, Übungen für eine weibliche Gestik und Mimik sowie Alltagstraining eingebunden werden. Hierzu wurde in dieser Arbeit ein neuer Behandlungsalgorithmus für die Univ.-HNO-Klinik Würzburg erstellt.
Eine ausgeprägte Mundtrockenheit, Xerostomie, entsteht häufig durch eine irreversible Funktionseinschränkung der Speicheldrüsen. Diese ist unter anderem durch die Einnahme bestimmter Medikamente, Autoimmunerkrankungen, fortgeschrittenes Alter oder die Bestrahlungstherapie von Tumoren der Kopf-Hals-Region bedingt, wobei letztere eine der häufigsten Ursachen darstellt. Konsequenzen der eingeschränkten Drüsenfunktion sind herabgesetzte Speichelflussraten, eine Reduktion des Mund-pH-Werts, eine veränderte Elektrolyt- und Immunglobulin-Zusammensetzung des Speichels und somit eine Verringerung des Infektionsschutzes. Die resultierenden Komplikationen erstrecken sich von Karies und rezidivierenden Infektionen bis hin zu Pilzbesiedelungen der Mundschleimhaut. Diese schränken die Lebensqualität der Patienten stark ein und führen häufig zu Therapieunterbrechungen. Fast die Hälfte der Patienten leidet unter Depressionen oder psychischen Belastungszuständen. Es gibt wenige Therapieansätze zur Behandlung der postradiogenen Xerostomie: Pilocarpin erhöht zwar die Speichelflussraten, hat jedoch keinen signifikanten Effekt auf die Lebensqualität. Die operative Translokation der Glandula submandibularis hat den Weg in die klinische Routine noch nicht gefunden, während die intensitätsmodulierte Bestrahlung (IMRT) nicht für jeden Patienten geeignet ist; beide zeigen jedoch einen positiven Effekt auf die Lebensqualität. Gentechnische und stammzellbasierte Ansätze zur Regeneration des Drüsengewebes befinden sich im Experimentalstadium. Somit ergibt sich ein dringender Bedarf an innovativen Optionen zur Behandlung der postradiogenen Xerostomie. Das Tissue Engineering, die Erstellung einer künstlichen Speicheldrüse aus körpereigenen Zellen, böte hier ein potentielles Behandlungskonzept.
Diese Studie soll deshalb untersuchen, ob humane Speicheldrüsenepithelzellen (hSEZ) auf einer Matrix aus dezellularisiertem, porzinem Jejunum, der sogenannten Small intestinal submucosa + mucosa (SIS-muc), kultiviert werden können. Können die Zellen innerhalb der Wachstumsperiode wichtige physiologische Differenzierungsmarker beibehalten? Kann die Produktion von α-Amylase, einem der wichtigsten Enzyme des menschlichen Speichels, erhalten werden? Welchen Einfluss hat die Kokultur mit mikrovaskulären Endothelzellen (mvEZ)? Und zuletzt: Ist dezellularisierter Schweinedarm eine potentiell geeignete Matrix für das Tissue Engineering der menschlichen Speicheldrüse?
Zunächst erfolgte die Entnahme von humanem Speicheldrüsengewebe, woraus hSEZ isoliert wurden. Diese wurden dann sowohl in Mono- als auch in Kokultur mit mvEZ auf die SIS-muc aufgebracht und auf dieser kultiviert. Die SIS-muc wurde aus kurzen Schweinedarm-Segmenten gewonnen, die in einem mehrstufigen Verfahren dezellularisiert wurden. Die besiedelte SIS-muc wurde mittels konventioneller sowie Immunfluoreszenzfärbungen, Raster- und Transmissionsektronenmikroskopie (REM/TEM) sowie quantitativer Polymerasekettenreaktion (qPCR) untersucht, darüber hinaus erfolgte die Messung der α-Amylase-Enzymaktivität.
Histologisch sowie in der REM zeigte sich sowohl in der Mono- als auch in der Kokultur eine konfluente Besiedelung der SIS-muc mit hSEZ. In der Kokultur formten mvEZ einen Monolayer auf der serosalen Matrixseite. Bei der Charakterisierung der hSEZ zeigte sich in den Immunfluoreszenzaufnahmen eine starke Ausprägung von Zytokeratin, α-Amylase und Aquaporin-5 und eine moderate Ausprägung von Claudin-1. Bei der Untersuchung der Funktion der α-Amylase konnte in der Kokultur von hSEZ mit mvEZ eine im Gegensatz zur Mono- und 2D-Kultur signifikant erhöhte Enzymaktivität der α-Amylase nachgewiesen werden. In der qPCR-Analyse der α-Amylase-Genexpression war die 3D-Kultur der 2D-Kultur überlegen.
Die vorliegende Arbeit zeigt, dass die Kultur von hSEZ auf der SIS-muc möglich ist. Es konnte nachgewiesen werden, dass die Zellen in 3D-Kultur spezifische Differenzierungsmerkmale beibehalten, die in der 2D-Kultur teils verloren gehen und dass hSEZ in Kokultur mit mvEZ eine gegenüber der Monokultur signifikant erhöhte Produktion von α-Amylase aufweisen. Diese Arbeit liefert die Datengrundlage für zukünftige Studien im dynamischen Bioreaktor-Modell (BioVaSc), die auf dem Weg zur klinischen Translation notwendig sind. Somit stellt sie einen wichtigen Schritt in Richtung einer auf Tissue Engineering basierten Therapie der belastenden Xerostomie dar.
Solitary bees in seasonal environments have to align their life-cycles with favorable environmental conditions and resources. Therefore, a proper timing of their seasonal activity is highly fitness relevant. Most species in temperate environments use temperature as a trigger for the timing of their seasonal activity. Hence, global warming can disrupt mutualistic interactions between solitary bees and plants if increasing temperatures differently change the timing of interaction partners. The objective of this dissertation was to investigate the mechanisms of timing in spring-emerging solitary bees as well as the resulting fitness consequences if temporal mismatches with their host plants should occur. In my experiments, I focused on spring-emerging solitary bees of the genus Osmia and thereby mainly on O. cornuta and O. bicornis (in one study which is presented in Chapter IV, I additionally investigated a third species: O. brevicornis).
Chapter II presents a study in which I investigated different triggers solitary bees are using to time their emergence in spring. In a climate chamber experiment I investigated the relationship between overwintering temperature, body size, body weight and emergence date. In addition, I developed a simple mechanistic model that allowed me to unite my different observations in a consistent framework. In combination with the empirical data, the model strongly suggests that solitary bees follow a strategic approach and emerge at a date that is most profitable for their individual fitness expectations. I have shown that this date is on the one hand temperature dependent as warmer overwintering temperatures increase the weight loss of bees during hibernation, which then advances their optimal emergence date to an earlier time point (due to an earlier benefit from the emergence event). On the other hand I have also shown that the optimal emergence date depends on the individual body size (or body weight) as bees adjust their emergence date accordingly. My data show that it is not enough to solely investigate temperature effects on the timing of bee emergence, but that we should also consider individual body conditions of solitary bees to understand the timing of bee emergence.
In Chapter III, I present a study in which I investigated how exactly temperature determines the emergence date of solitary bees. Therefore, I tested several variants degree-day models to relate temperature time series to emergence data. The basic functioning of such degree-day models is that bees are said to finally emerge when a critical amount of degree-days is accumulated. I showed that bees accumulate degree-days only above a critical temperature value (~4°C in O. cornuta and ~7°C in O. bicornis) and only after the exceedance of a critical calendar date (~10th of March in O. cornuta and ~28th of March in O. bicornis). Such a critical calendar date, before which degree-days are not accumulated irrespective of the actual temperature, is in general less commonly used and, so far, it has only been included twice in a phenology model predicting bee emergence. Furthermore, I used this model to retrospectively predict the emergence dates of bees by applying the model to long-term temperature data which have been recorded by the regional climate station in Würzburg. By doing so, the model estimated that over the last 63 years, bees emerged approximately 4 days earlier.
In Chapter IV, I present a study in which I investigated how temporal mismatches in bee-plant interactions affect the fitness of solitary bees. Therefore, I performed an experiment with large flight cages serving as mesocosms. Inside these mesocosms, I manipulated the supply of blossoms to synchronize or desynchronize bee-plant interactions. In sum, I showed that even short temporal mismatches of three and six days in bee-plant interactions (with solitary bee emergence before flower occurrence) can cause severe fitness losses in solitary bees. Nonetheless, I detected different strategies by solitary bees to counteract impacts on their fitness after temporal mismatches. However, since these strategies may result in secondary fitness costs by a changed sex ratio or increased parasitism, I concluded that compensation strategies do not fully mitigate fitness losses of bees after short temporal mismatches with their food plants. In the event of further climate warming, fitness losses after temporal mismatches may not only exacerbate bee declines but may also reduce pollination services for later-flowering species and affect populations of animal-pollinated plants.
In conclusion, I showed that spring-emerging solitary bees are susceptible to climate change as in response to warmer temperatures bees advance their phenology and show a decreased fitness state. As spring-emerging solitary bees not only consider overwintering temperature but also their individual body condition for adjusting emergence dates, this may explain differing responses to climate warming within and among bee populations which may also have consequences for bee-plant interactions and the persistence of bee populations under further climate warming. If in response to climate warming plants do not shift their phenologies according to the bees, bees may experience temporal mismatches with their host plants. As bees failed to show a single compensation strategy that was entirely successful in mitigating fitness consequences after temporal mismatches with their food plants, the resulting fitness consequences for spring-emerging solitary bees would be severe. Furthermore, I showed that spring-emerging solitary bees use a critical calendar date before which they generally do not commence the summation of degree-days irrespective of the actual temperature. I therefore suggest that further studies should also include the parameter of a critical calendar date into degree-day model predictions to increase the accuracy of model predictions for emergence dates in solitary bees. Although our retrospective prediction about the advance in bee emergence corresponds to the results of several studies on phenological trends of different plant species, we suggest that more research has to be done to assess the impacts of climate warming on the synchronization in bee-plant interactions more accurately.
I. Timing is a crucial feature in organisms that live within a variable and changing environment. Complex mechanisms to measure time are wide-spread and were shown to exist in many taxa. These mechanisms are expected to provide fitness benefits by enabling organisms to anticipate environmental changes and adapt accordingly. However, very few studies have addressed the adaptive value of proper timing. The objective of this PhD-project was to investigate mechanisms and fitness consequences of timing decisions concerning colony phenology and foraging activity in the honey bee (Apis mellifera), a social insect species with a high degree of social organization and one of the most important pollinators of wild plants and crops. In chapter II, a study is presented that aimed to identify the consequences of disrupted synchrony between colony phenology and the local environment by manipulating the timing of brood onset after hibernation. In a follow-up experiment, the importance of environmental factors for the timing of brood onset was investigated to assess the potential of climate change to disrupt synchronization of colony phenology (Chapter III). Chapter IV aimed to prove for the first time that honey bees can use interval time-place learning to improve foraging activity in a variable environment. Chapter V investigates the fitness benefits of information exchange between nest mates via waggle dance communication about a resource environment that is heterogeneous in space and time.
II. In the study presented in chapter II, the importance of the timing of brood onset after hibernation as critical point in honey bee colony phenology in temperate zones was investigated. Honey bee colonies were overwintered at two climatically different sites. By translocating colonies from each site to the other in late winter, timing of brood onset was manipulated and consequently colony phenology was desynchronized with the local environment. Delaying colony phenology in respect to the local environment decreased the capability of colonies to exploit the abundant spring bloom. Early brood onset, on the other hand, increased the loads of the brood parasite Varroa destructor later in the season with negative impact on colony worker population size. This indicates a timing related trade-off and illustrates the importance of investigating effects of climate change on complex multi-trophic systems. It can be concluded that timing of brood onset in honey bees is an important fitness relevant step for colony phenology that is highly sensitive to climatic conditions in late winter. Further, phenology shifts and mismatches driven by climate change can have severe fitness consequences.
III. In chapter III, I assess the importance of the environmental factors ambient temperature and photoperiod as well as elapsed time on the timing of brood onset. Twenty-four hibernating honey bee colonies were placed into environmental chambers and allocated to different combinations of two temperature regimes and three different light regimes. Brood onset was identified non-invasively by tracking comb temperature within the winter cluster. The experiment revealed that ambient temperature plays a major role in the timing of brood onset, but the response of honey bee colonies to temperature increases is modified by photoperiod. Further, the data indicate the involvement of an internal clock. I conclude that the timing of brood onset is complex but probably highly susceptible to climate change and especially spells of warm weather in winter.
IV. In chapter IV, it was examined if honey bees are capable of interval time-place learning and if this ability improves foraging efficiency in a dynamic resource environment. In a field experiment with artificial feeders, foragers were able to learn time intervals and use this ability to anticipate time periods during which feeders were active. Further, interval time-place learning enabled foragers to increase nectar uptake rates. It was concluded that interval time-place learning can help honey bee foragers to adapt to the complex and variable temporal patterns of floral resource environments.
V. The study presented in chapter V identified the importance of the honey bee waggle dance communication for the spatiotemporal coordination of honey bee foraging activity in resource environments that can vary from day to day. Consequences of disrupting the instructional component of honey bee dance communication were investigated in eight temperate zone landscapes with different levels of spatiotemporal complexity. While nectar uptake of colonies was not affected, waggle dance communication significantly benefitted pollen harvest irrespective of landscape complexity. I suggest that this is explained by the fact that honey bees prefer to forage pollen in semi-natural habitats, which provide diverse resource species but are sparse and presumably hard to find in intensively managed agricultural landscapes. I conclude that waggle dance communication helps to ensure a sufficient and diverse pollen diet which is crucial for honey bee colony health.
VI. In my PhD-project, I could show that honey bee colonies are able to adapt their activities to a seasonally and daily changing environment, which affects resource uptake, colony development, colony health and ultimately colony fitness. Ongoing global change, however, puts timing in honey bee colonies at risk. Climate change has the potential to cause mismatches with the local resource environment. Intensivation of agricultural management with decreased resource diversity and short resource peaks in spring followed by distinctive gaps increases the probability of mismatches. Even the highly efficient foraging system of honey bees might not ensure a sufficiently diverse and healthy diet in such an environment. The global introduction of the parasitic mite V. destructor and the increased exposure to pesticides in intensively managed landscapes further degrades honey bee colony health. This might lead to reduced cognitive capabilities in workers and impact the communication and social organization in colonies, thereby undermining the ability of honey bee colonies to adapt to their environment.
Fabry disease (FD) is an X-linked lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3) due to α-galactosidase A deficiency. We studied α-galactosidase A knockout mice (GLA KO) as a model for sensory disturbance and pain in FD.
Pain associated behavior of young (3 months) and old (≥18 months) GLA KO mice and wildtype (WT) littermates in an inflammatory and a neuropathic pain model was investigated. Furthermore, affective and cognitive behavior was assessed in the naïve state and in an inflammatory pain model. Gene and protein expression of pain associated ion channels and Gb3 accumulation in dorsal root ganglion (DRG) neurons was determined. We also performed patch clamp analysis on cultivated DRG neurons and human embryonic kidney 293 (HEK) cells expressing voltage-gated-sodium channel 1.7 (Nav1.7) as an in vitro model of FD. Intracellular Gb3 deposits were modulated using shRNA silencing of α-galactosidase A.
After intraplantar injection of complete Freund`s adjuvant (CFA) and chronic constriction injury (CCI) of the right sciatic nerve, old GLA KO mice did not develop heat and mechanical hypersensitivity in contrast to young GLA KO and old WT mice. Additionally, we found no relevant differences between genotypes and age-groups in affective and cognitive behavior in the naïve state and after CFA injection. Gene and protein expression analysis provided no explanation for the observed sensory impairment. However, cultured DRG neurons of old GLA KO mice revealed a marked decrease of sodium and Ih-currents compared to young GLA KO and old WT mice. DRG neurons of old GLA KO mice displayed substantial intracellular accumulation of Gb3 compared to young GLA KO and old WT mice. Similar to cultured neurons, sodium currents were also decreased in HEK cells treated with shRNA and consecutively increased intracellular Gb3 deposits compared to the control condition, but could be rescued by treatment with agalsidase-alpha.
Our study unveils that, similar to patients with FD, GLA KO mice display age-dependent sensory deficits. However, contrary to patients, GLA KO mice are also protected from hypersensitivity induced by inflammation and nerve lesion due to Gb3-dependent and reversible reduction of neuronal sodium- and Ih-currents. Our data provide evidence for direct Gb3-dependent ion channel impairment in sensory DRG neurons as a potential contributor to sensory dysfunction and pain in FD.
For the differentiation of a embryonic stem cells (ESCs) to neuronal cells (NCs) a complex and coordinated gene regulation program is needed. One important control element for neuronal differentiation is the repressor element 1 silencing transcription factor (REST) complex, which represses neuronal gene expression in non-neuronal cells. Crucial effector proteins of the REST complex are small phosphatases such as the CTDSPs (C-terminal domain small phosphatases) that regulate polymerase II activity by dephosphorylating the C-terminal domain of the polymerase, thereby repressing target genes. The stepwise inactivation of REST, including the CTDSPs, leads to the induction of a neuron-specific gene program, which ultimately induces the formation of neurons. The spatio-temporal control of REST and its effector components is therefore a crucial step for neurogenesis.
In zebrafish it was shown that the REST-associated CTDSP2 is negatively regulated by the micro RNA (miR) -26b. Interestingly, the miR-26b is encoded in an intron of the primary transcript of CTDSP2. This gives the fundament of an intrinsic regulatory negative feedback loop, which is essential for the proceeding of neurogenesis. This feedback loop is active during neurogenesis, but inactive in non-neuronal cells. The reason for this is that the maturation of the precursor miR (pre-miR) to the mature miR-26 is arrested in non neuronal cells, but not in neurons. As only mature miRs are actively repressing genes, the regulation of miR-26 processing is an essential step in neurogenesis.
In this study, the molecular basis of miR-26 processing regulation in the context of neurogenesis was addressed. The mature miR is processed from two larger precursors: First the primary transcript is cleaved by the enzyme DROSHA in the nucleus to form the pre-miR. The pre-miR is exported from the nucleus and processed further through the enzyme DICER to yield the mature miR. The mature miR can regulate gene expression in association with the RNA-induced silencing complex (RISC).
Multiple different scenarios in which miR processing was regulated were proposed and experimentally tested. Microinjection studies using Xenopus leavis oocytes showed that slowdown or blockage of the nucleo-cytoplasmic transport are not the reason for delayed pre-miR-26 processing. Moreover, in vitro and in vivo miR-processing assays showed that maturation is most likely regulated through a in trans acting factor, which blocks processing in non neuronal cells.
Through RNA affinity chromatographic assays using zebrafish and murine lysates I was able to isolate and identify proteins that interact specifically with pre-miR-26 and could by this influence its biogenesis. Potential candidates are FMRP/FXR1/2, ZNF346 and Eral1, whose functional characterisation in the context of miR-biogenesis could now be addressed.
The second part of my thesis was executed in close colaboration with the laboratory of Prof. Albrecht Müller. The principal question was addressed how miR-26 influences neuronal gene expression and which genes are primarily affected. This research question could be addressed by using a cell culture model system, which mimics ex vivo the differentiation of ESCs to NCs via neuronal progenitor.
For the functional analysis of miR-26 knock out cell lines were generated by the CRISPR/Cas9 technology. miR-26 deficient ESC keep their pluripotent state and are able to develop NPC, but show major impairment in differentiating to NCs. Through RNA deep sequencing the miR-26 induced transcriptome differences could be analysed.
On the level of mRNAs it could be shown, that the expression of neuronal gene is downregulated in miR-26 deficient NCs. Interestingly, the deletion of miR-26 leads to selectively decreased levels of miRs, which on one hand regulate the REST complex and on the other hand are under transcriptional control by REST themself. This data and the discovery that induction of miR-26 leads to enrichment of other REST regulating miRs indicates that miR-26 initiates neurogenesis through stepwise inactivation of the REST complex.
Anxiety and depressive disorders result from a complex interplay of genetic and environmental factors and are common mutual comorbidities. On the level of cellular signaling, regulator of G protein signaling 2 (Rgs2) has been implicated in human and rodent anxiety as well as rodent depression. Rgs2 negatively regulates G protein-coupled receptor (GPCR) signaling by acting as a GTPase accelerating protein towards the Gα subunit.
The present study investigates, whether mice with a homozygous Rgs2 deletion (Rgs2-/-) show behavioral alterations as well as an increased susceptibility to stressful life events related to human anxiety and depressive disorders and tries to elucidate molecular underlying’s of these changes.
To this end, Rgs2-/- mice were characterized in an aversive-associative learning paradigm to evaluate learned fear as a model for the etiology of human anxiety disorders. Spatial learning and reward motivated spatial learning were evaluated to control for learning in non-aversive paradigms. Rgs2 deletion enhanced learning in all three paradigms, rendering increased learning upon deletion of Rgs2 not specific for aversive learning. These data support reports indicating increased long-term potentiation in Rgs2-/- mice and may predict treatment response to conditioning based behavior therapy in patients with polymorphisms associated with reduced RGS2 expression. Previous reports of increased innate anxiety were corroborated in three tests based on the approach-avoidance conflict. Interestingly, Rgs2-/- mice showed novelty-induced hypo-locomotion suggesting neophobia, which may translate to the clinical picture of agoraphobia in humans and reduced RGS2 expression in humans was associated with a higher incidence of panic disorder with agoraphobia. Depression-like behavior was more distinctive in female Rgs2-/- mice. Stress resilience, tested in an acute and a chronic stress paradigm, was also more distinctive in female Rgs2-/- mice, suggesting Rgs2 to contribute to sex specific effects of anxiety disorders and depression.
Rgs2 deletion was associated with GPCR expression changes of the adrenergic, serotonergic, dopaminergic and neuropeptide Y systems in the brain and heart as well as reduced monoaminergic neurotransmitter levels. Furthermore, the expression of two stress-related microRNAs was increased upon Rgs2 deletion. The aversive-associative learning paradigm induced a dynamic Rgs2 expression change. The observed molecular changes may contribute to the anxious and depressed phenotype as well as promote altered stress reactivity, while reflecting an alter basal stress level and a disrupted sympathetic tone. Dynamic Rgs2 expression may mediate changes in GPCR signaling duration during memory formation.
Taken together, Rgs2 deletion promotes increased anxiety-like and depression-like behavior, altered stress reactivity as well as increased cognitive function.
The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of
trachoma related blindness and the sexually transmitted pelvic inflammatory disease.
Being an obligate intracellular pathogen, C. trachomatis has an intricate dependency
on the survival of the host cell. This relationship is indispensible owing to the fact that
the pathogen spends a considerable fraction of its biphasic lifecycle within a
cytoplasmic vacuole inside the host cell, the so-called chlamydial inclusion. The
cellular apoptotic-signalling network is governed by several finely tuned regulatory
cascades composed of pro- and anti-apoptotic proteins that respond to changes in
the cellular homeostasis. In order to facilitate its intracellular survival, Chlamydia has
been known to inhibit the premature apoptosis of the host cell via the stabilization of
several host anti-apoptotic proteins such as cIAP2 and Mcl-1. While the pro- and
anti-apoptotic proteins are the major regulators of the host apoptotic signalling
network, a class of the small non-coding RNAs called microRNAs (miRNAs) has
increasingly gained focus as a new level of regulatory control over apoptosis.
This work investigates the changes in the host miRNA expression profile post
Chlamydia infection using a high throughput miRNA deep sequencing approach.
Several miRNAs previously associated with the modulation for apoptotic signalling
were differentially expressed upon Chlamydia infection in human endothelial cells. Of
the differentially regulated miRNAs, miR-30c-5p was of particular interest since it had
been previously shown to target the tumor suppressor protein p53. Our lab and
others have previously demonstrated that Chlamydia can downregulate the levels of
p53 by promoting its proteasomal degradation. This work demonstrates that
Chlamydia infection promotes p53 downregulation by increasing the abundance of
miR-30c-5p and a successful infection cycle is hindered by a loss of miR-30c-5p.
Over the last decade, dedicated research aimed towards a better understanding of
apoptotic stimuli has greatly improved our grasp on the subject. While extrinsic
stress, deprivation of survival signals and DNA damage are regarded as major
proponents of apoptotic induction, a significant responsibility lies with the
mitochondrial network of the cell. Mitochondrial function and dynamics are crucial to
cell fate determination and dysregulation of either is decisive for cell survival and
pathogenesis of several diseases. The ability of the mitochondrial network to perform
its essential tasks that include ATP synthesis, anti-oxidant defense, and calcium
homeostasis amongst numerous other processes critical to cellular equilibrium is tied
closely to the fission and fusion of individual mitochondrial fragments. It is, thus,
8
unsurprising that mitochondrial dynamics is closely linked to apoptosis. In fact, many
of the proteins involved regulation of mitochondrial dynamics are also involved in
apoptotic signalling. The mitochondrial fission regulator, Drp1 has previously been
shown to be transcriptionally regulated by p53 and is negatively affected by a miR-
30c mediated inhibition of p53. Our investigation reveals a significant alteration in the
mitochondrial dynamics of Chlamydia infected cells affected by the loss of Drp1. We
show that loss of Drp1 upon chlamydial infection is mediated by the miR-30c-5p
induced depletion of p53 and results in a hyper-fused architecture of the
mitochondrial network.
While it is widely accepted that Chlamydia depends on the host cell metabolism for
its intracellular growth and development, the role of mitochondria in an infected cell,
particularly with respect to its dynamic nature, has not been thoroughly investigated.
This work attempts to illustrate the dependence of Chlamydia on miR-30c-5p induced
changes in the mitochondrial architecture and highlight the importance of these
modulations for chlamydial growth and development.
The human-bacterial pathogen interaction is a complex process that results from
a prolonged evolutionary arms race in the struggle for survival. The pathogen employs
virulence strategies to achieve host colonization, and the latter counteracts using defense
programs. The encounter of both organisms results in drastic physiological changes
leading to stress, which is an ancient response accompanying infection. Recent evidence
suggests that the stress response in the host converges with the innate immune pathways
and influences the outcome of infection. However, the contribution of stress and the exact
mechanism(s) of its involvement in host defense remain to be elucidated. Using the model
bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen
Salmonella Typhimurium, this study investigated the role of host stress in the outcome of
infection.
Shigella infection is characterized by a pronounced pro-inflammatory response
that causes intense stress in host tissues, particularly the intestinal epithelium, which
constitutes the first barrier against Shigella colonization. In this study, inflammatory
stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine
stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly
inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts
in the plasma membrane by the stress-activated sphingomyelinases. Interestingly,
Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the
gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication
of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent
cells inhibiting extracellular bacterial invasion.
Additionally, this study shows that Shigella infection interferes with the host stress
granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear
depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1
and their accumulation in the cytoplasm.
Overall, this work investigated different aspects of the host stress-response in the
defense against bacterial infection. The findings shed light on the importance of the host
stress-pathways during infection, and improve the understanding of different strategies
in host-pathogen interaction.
Previous work of our group has established a role of sphingomyelinases in the regulation
of T cell responses to TCR or pathogen stimulation, and this became particularly
evident at the level of actin cytoskeletal dynamics. The formation of lipid membrane
microdomains is crucial for receptor clustering and signal induction, and therefore,
ceramide accumulation by membrane sphingomyelin breakdown is needed for signalling-
complex-assembly. Pathogen-induced overshooting of SMase activation substantially
impacted the formation of membrane protrusions, with T cell spreading as well as
a front/rear polarisation upon CD3/CD28 co-stimulation [103]. On the other hand, NSM
activation is part of the physiological TCR signal [67], indicating that a spatiotemporally
balanced NSM activation is crucial for its physiological function. It involves actin cytoskeletal
reorganisation and T cell polarisation. These two functions are also of central
importance in directional T cell migration and motility in tissues.
This thesis aims on defining the role of NSM in compartmentalisation of the T cell
membrane in polarisation and migration. Therefore, functional studies on the impact of
NSM activity in these processes had to be complemented by the development of tools
to study ceramide compartmentalisation in living T cells.
Melanoma and Merkel cell carcinoma (MCC) are highly aggressive cancers of the skin that frequently escape immune recognition and acquire resistance to chemotherapeutic agents, which poses a major obstacle to successful cancer treatment. Recently, a new class of therapeutics targeting the programmed cell death-1 (PD-1) immune checkpoint receptor has shown remarkable efficacy in the treatment of both cancers. Blockade of PD-1 on T cells activates cancer-specific immune responses that can mediate tumor regression. The data presented in this Ph.D. thesis demonstrates that PD-1 is also expressed by subsets of cancer cells in melanoma and MCC. Moreover, this work identifies PD-1 as a novel tumor cell-intrinsic growth receptor, even in the absence of T cell immunity. PD-1 is expressed by tumorigenic cell subsets in melanoma patient samples and established human and murine cell lines that also co-express ABCB5, a marker of immunoregulatory tumor- initiating cells in melanoma. Consistently, melanoma-expressed PD-1 downmodulates T effector cell functions and increases the intratumoral frequency of tolerogenic myeloid- derived suppressor cells. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, including in mice lacking adaptive immunity. Engagement of melanoma- PD-1 by its ligand PD-L1 promotes tumor growth, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuates growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor activates mTOR signaling mediators, including ribosomal protein S6. In a proof-of-concept study, tumoral expression of phospho-S6 in pretreatment tumor biopsies correlated with clinical responses to anti-PD-1 therapy in melanoma patients. In MCC, PD-1 is similarly co-expressed by ABCB5+ cancer cell subsets in clinical tumor specimens and established human cell lines. ABCB5 renders MCC cells resistant to the standard-of-care chemotherapeutic agents, carboplatin and etoposide. Antibody-mediated ABCB5 blockade reverses chemotherapy resistance and inhibits tumor xenograft growth by enhancing chemotherapy-induced tumor cell killing. Furthermore, engagement of MCC-expressed PD-1 by its ligands, PD-L1 and PD-L2, promotes proliferation and activates MCC-intrinsic mTOR signaling. Consistently, antibody- mediated PD-1 blockade inhibits MCC tumor xenograft growth and phosphorylation of mTOR effectors in immunocompromised mice. In summary, these findings identify cancer cell-intrinsic functions of the PD-1 pathway in tumorigenesis and suggest that blocking melanoma- and MCC-expressed PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. Additionally, these results establish ABCB5 as a previously unrecognized chemoresistance mechanism in MCC.
Abstract
Background: Attention-deficit/ hyperactivity disorder (ADHD) ranges among the most common neurodevelopmental disorders worldwide with a prevalence of 3-12% in childhood and 1-5% for adults. Over the last decade extensive genetic research has been conducted in order to determine its causative genetic factors. None of the so far identified susceptibility genes, however, could explain the estimated ADHD heritability of 76%. In this thesis one of the most promising candidates -Cadherin 13 (Cdh13) - was examined in terms of its influence on the central serotonergic (5-HT) system. In addition to that, the Cdh13 protein distribution pattern was analysed over time.
Methods: The developing serotonergic system was compared over three embryonic and postnatal stages (E13.5, E17.5 and P7) in different Cdh13 genotypes (WT, HZ and KO) using immunohistochemistry and various double staining protocols.
Results: The raphe nuclei of the 5-HT system develop in spite of Cdh13 absence and show a comparable mature constellation. The cells in the KO, however, are slightly more scattered than in the WT. Furthermore the dynamics of their formation is altered, with a transient delay in migration at E13.5. In early developmental stages the total amount of serotonergic cells is reduced in KO and HZ, though their proportional distribution to the raphe nuclei stays constant. Strikingly, at P7 the absolute numbers are comparable again.
Concerning the Cdh13 protein, it shows high concentrations on fibres running through hindbrain and midbrain areas at E13.5. This, however, changes over time, and it becomes more evenly spread until P7. Furthermore, its presence in serotonergic cells could be visualised using confocal microscopy. Since the described pattern is only in parts congruent to the localisation of serotonergic neurons, it is most likely that Cdh13 is present in other developing neurotransmitter systems, such as the dopaminergic one, as well.
Conclusion: It could be proven that Cdh13 is expressed in serotonergic cells and that its knockout does affect the developing serotonergic system to some degree. Its absence, however, only slightly and transiently affects the measured parameters of serotonergic system development, indicating a possible compensation of CDH13 function by other molecules in the case of Cdh13 deficiency. In addition further indicators could be found for an influence of Cdh13 on outgrowth and path finding of neuronal processes.
Cardiovascular diseases represent the leading cause of death worldwide, with myocardial infarction and strokes being the most common complications. In both cases, the appearance of an enlarged artery wall as a consequence of a growing plaque is responsible for the disturbance of the blood flow. The formation of plaques is driven by a chronic inflammatory condition known as atherosclerosis, characterized by an initial step of endothelial cell (EC) dysfunction followed by the recruitment of circulating immune cells into the tunica intima of the vessel. Accumulation of lipids and cells lead to the formation of atheromatous plaques that will define the cardiovascular outcome of an individual.
The role of the immune system in the progression of atherosclerosis has been widely recognized. By far, macrophages constitute the most abundant cell type in lesions and are known to be the major source of the lipid-laden foam cell pool during the course of the disease. However, other immune cells types, including T cells, dendritic cells (DCs) or mast cells, among others, have been described to be present in human and mouse plaques. How these populations can modulate the atherogenic process is dependent on their specialized function.
DCs constitute a unique population with the ability to bridge innate and adaptive immune responses, mainly by their strong capacity to present antigens bound to a major histocompatibility complex (MHC) molecule. Given their ability to polarize T cells and secrete cytokines, their role in atherosclerosis has gained attention for the development of new therapeutic approaches that could impact lesion growth. Hence, knowing the effect of a specific subset is an initial key step to evaluate its potential for clinical purposes. For example, the basic leucine zipper ATF-like 3 transcription factor (Batf3) controls the development of conventional dendritic cells type 1 (cDCs1), characterized by the expression of the surface markers CD8 and CD103. Initially, they were described to promote both T-helper 1 (Th1) and regulatory T cell (Treg) responses, known to accelerate and to protect against atherosclerosis, respectively. The first part of this thesis aimed to elucidate the potential role of Batf3-dependent DCs in atherosclerosis and concluded that even though systemic immune responses were mildly altered they do not modify the course of the disease and may not represent an attractive candidate for clinical studies.
DCs also have the ability to impact lesion growth through the release of a broad range of cytokines, which can either directly impact atherosclerotic plaques by modulating resident cells, or by further polarizing T cell responses. Among others, interleukin (IL) 23, a member of the IL-12 family of cytokines, has received much attention during the past year due to its connection to autoimmunity.
IL-23 is known to induce pathogenicity of Th17 cells and is responsible for the development of several autoimmune diseases including multiple sclerosis, psoriasis or rheumatoid arthritis. Interestingly, these patients often present with an accelerated course of atherosclerosis and thus, are at higher risk of developing cardiovascular events. Several epidemiological studies have pointed toward a possible connection between IL-23 and its receptor IL-23R in atherosclerosis, although their exact contribution remains to be elucidated. The second part of this thesis showed that resident antigen-presenting cells (APCs) in the aorta produced IL-23 during the steady state but this secretion was greatly enhanced after incubation with oxidized low-density lipoprotein (oxLDL). Furthermore, disruption of the IL-23R signaling led to decreased relative necrotic plaque area in lesions of Ldlr-/-Il23r-/- mice fed a high-fat diet (HFD) for 6 and 12 weeks compared to Ldlr-/- controls. A proposed mechanism involves that increased IL-23 production in the context of atherosclerosis may promote the pathogenicity of IL-23-responding T cells, especially IL-23R+ γδ T cells in the aortic root. Response to IL-23 might increase the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17 and alter the pro- and anti-inflammatory balance of cytokines in the aortic root. Altogether, these data showed that the IL-23 / IL-23R axis play a role in plaque stability.
Panic Disorder (PD) is characterized by unexpected, recurrent panic attacks, which are not restricted to certain situations, medication or stimuli. Like other anxiety disorders, PD is a multifactorial disorder and develops through the interaction of genetic and environmental risk factors. Despite an estimated heritability of up to 48%, no distinct genetic mechanism could be revealed yet. A dysregulation of the stress response has been shown in patients with PD and several studies could find an association of components of the corticotropin-releasing factor (CRF) system with PD. The corticotropin releasing hormone receptor 1 (CRHR1) is the main receptor of CRF in the brain and thus a crucial regulator of cerebral CRF signaling. Recent genetic studies found an association of certain CRHR1 single nucleotide polymorphisms (SNPs) with PD and other anxiety disorders. Among the associated CRHR1 SNPs, rs17689918 showed further evidence in a multilevel study regulating CRHR1 gene expression in panic-relevant brain regions and affecting brain activation in fMRI experiments, as well as flight behavior in a behavioral avoidance task (Weber et al, 2015). Here, we aimed to investigate the underlying neurogenetic and neurobiological mechanisms, by which the rs17689918 risk allele affects CRHR1 gene expression and receptor function, and its putative function in the pathophysiology of PD.
Due to its intronic position and the predicted change of splicing regulatory elements by the risk allele of rs17689918, the expression of alternative spliced CRHR1 isoforms was investigated using quantitative real-time PCR (qPCR) in a human post-mortem brain tissue sample. Of eight known CRHR1 isoforms, expression of three CRHR1 isoforms and the CRHR1-IT1-CRHR1 readthrough transcript variant 5 – all expressing the seven transmembrane domains needed for functional receptors – was analyzed. Subsequently, electrophysiological assays were developed to measure the receptor activity of differentially expressed CRHR1 isoforms via co-expressed Kir2.3 potassium channels in vitro. In a second approach, possible epigenetic regulation of CRHR1 expression by rs17689918 was investigated by analyses of DNA methylation patterns of a CpG Island within the CRHR1 promoter region, firstly in a case-control sample for PD and secondly in a healthy control sample, separated in high and low anxious individuals. To investigate a possible gene × epigene × environment interaction, the impact of early life stress by means of childhood trauma was evaluated via the childhood trauma questionnaire (CTQ). Finally, consequences of differential DNA methylation of the CRHR1 promoter region on gene expression were investigated by luciferase-based reporter gene assays in vitro.
The expression of CRHR1β was significantly decreased in amygdalae and midbrains of risk allele carriers. The expression of CRHR1-IT1-CRHR1 readthrough transcript variant 5 was significantly increased in forebrains and midbrains of risk allele carriers. All other analyzed isoforms showed no differences in expression between non-risk and risk allele carriers of rs17689918. The electrophysiological recordings of membrane potential showed an activation of Kir2.3 channels by CRHR1β in contrast to an inconsistent mix of activation and inhibition of Kir2.3 by the main isoform CRHR1α. DNA methylation of the CRHR1 promoter region was significantly reduced in panic disorder patients, as well as in high anxious individuals of an independent healthy control sample, but no direct relation to the rs17689918 risk allele could be discerned. However, the combination of carrying the risk allele, low DNA methylation and high CTQ scores lead to increased sum scores in the Beck Anxiety Inventory (BAI) in healthy individuals. Functional analyses revealed an activation of gene expression by decreased DNA methylation of the promoter region in vitro.
Our results revealed that rs17689918 regulates CRHR1 function by increasing the expression of alternative transcript variants with altered function. Our analyses of DNA methylation revealed decreased methylation as a new risk factor for panic disorder and high anxious behavior, which in combination with other risk factors like childhood trauma and the rs17689918 risk allele might further increase cognitive and somatic anxiety symptoms. This supports the role of CRHR1 as a plasticity gene of anxiety behavior, i.e. a gene that is highly regulated by epigenetic or post-transcriptional mechanisms in response to environmental stressors. By its role in CRF signaling, the dysregulation of CRHR1 might extensively affect the stress response and contribute to the pathophysiology of stress-related disorders like PD. The understanding of the underlying mechanisms, especially the genetic and epigenetic regulation, would however enhance CRHR1 as a target of improved future therapeutics for PD and other anxiety disorders.
For cellular viability, transcription is a fundamental process. Hereby, the DNA plays the most elemental and highly versatile role. It has long been known that promoters contain conserved and often well-defined motifs, which dictate the site of transcription initiation by providing binding sites for regulatory proteins. However, research within the last decade revealed that it is promoters lacking conserved promoter motifs and transcribing constitutively expressed genes that constitute the majority of promoters in eukaryotes. While the process of transcription initiation is well studied, whether defined DNA sequence motifs are required for the transcription of constitutively expressed genes in eukaryotes remains unknown. In the highly divergent protozoan parasite Trypanosoma brucei, most of the proteincoding genes are organized in large polycistronic transcription units. The genes within one polycistronic transcription unit are generally unrelated and transcribed by a common transcription start site for which no RNA polymerase II promoter motifs have been identified so far. Thus, it is assumed that transcription initiation is not regulated but how transcription is initiated in T. brucei is not known. This study aimed to investigate the requirement of DNA sequence motifs and chromatin structures for transcription initiation in an organism lacking transcriptional regulation. To this end, I performed a systematic analysis to investigate the dependence of transcription initiation on the DNA sequence. I was able to identify GT-rich promoter elements required for directional transcription initiation and targeted deposition of the histone variant H2A.Z, a conserved component during transcription initiation. Furthermore, nucleosome positioning data in this work provide evidence that sites of transcription initiation are rather characterized by broad regions of open and more accessible chromatin than narrow nucleosome depleted regions as it is the case in other eukaryotes. These findings highlight the importance of chromatin during transcription initiation. Polycistronic RNA in T. brucei is separated by adding an independently transcribed miniexon during trans-splicing. The data in this work suggest that nucleosome occupancy plays an important role during RNA maturation by slowing down the progressing polymerase and thereby facilitating the choice of the proper splice site during trans-splicing. Overall, this work investigated the role of the DNA sequence during transcription initiation and nucleosome positioning in a highly divergent eukaryote. Furthermore, the findings shed light on the conservation of the requirement of DNA motifs during transcription initiation and the regulatory potential of chromatin during RNA maturation. The findings improve the understanding of gene expression regulation in T. brucei, a eukaryotic parasite lacking transcriptional Regulation.
The Dual Olfactory Pathway in the Honeybee Brain: Sensory Supply and Electrophysiological Properties
(2018)
The olfactory sense is of utmost importance for honeybees, Apis mellifera. Honeybees use olfaction for communication within the hive, for the identification of nest mates and non-nest mates, the localization of food sources, and in case of drones (males), for the detection of the queen and mating. Honeybees, therefore, can serve as excellent model systems for an integrative analysis of an elaborated olfactory system.
To efficiently filter odorants out of the air with their antennae, honeybees possess a multitude of sensilla that contain the olfactory sensory neurons (OSN). Three types of olfactory sensilla are known from honeybee worker antennae: Sensilla trichoidea, Sensilla basiconica and Sensilla placodea. In the sensilla, odorant receptors that are located in the dendritic arborizations of the OSNs transduce the odorant information into electrical information. Approximately 60.000 OSN axons project in two parallel bundles along the antenna into the brain. Before they enter the primary olfactory brain center, the antennal lobe (AL), they diverge into four distinct tracts (T1-T4). OSNs relay onto ~3.000-4.000 local interneurons (LN) and ~900 projection neurons (PN), the output neurons of the AL. The axons of the OSNs together with neurites from LNs and PNs form spheroidal neuropil units, the so-called glomeruli. OSN axons from the four AL input tracts (T1-T4) project into four glomerular clusters. LNs interconnect the AL glomeruli, whereas PNs relay the information to the next brain centers, the mushroom body (MB) - associated with sensory integration, learning and memory - and the lateral horn (LH). In honeybees, PNs project to the MBs and the LH via two separate tracts, the medial and the lateral antennal-lobe tract (m/lALT) which run in parallel in opposing directions. The mALT runs first to the MB and then to the LH, the lALT runs first to the LH and then to the MB. This dual olfactory pathway represents a feature unique to Hymenoptera. Interestingly, both tracts were shown to process information about similar sets of odorants by extracting different features. Individual mALT PNs are more odor specific than lALT PNs. On the other hand, lALT PNs have higher spontaneous and higher odor response action potential (AP) frequencies than mALT PNs. In the MBs, PNs form synapses with ~184.000 Kenyon cells (KC), which are the MB intrinsic neurons. KCs, in contrast to PNs, show almost no spontaneous activity and employ a spatially and temporally sparse code for odor coding.
In manuscript I of my thesis, I investigated whether the differences in specificity of odor responses between m- and lALT are due to differences in the synaptic input. Therefore, I investigated the axonal projection patterns of OSNs housed in S. basiconica in honeybee workers and compared them with S. trichoidea and S. placodea using selective anterograde labeling with fluorescent tracers and confocal- microscopy analyses of axonal projections in AL glomeruli. Axons of S. basiconica-associated OSNs preferentially projected into the T3 input-tract cluster in the AL, whereas the two other types of sensilla did not show a preference for a specific glomerular cluster. T3- associated glomeruli had previously been shown to be innervated by mALT PNs. Interestingly, S. basiconica as well as a number of T3 glomeruli lack in drones. Therefore I set out to determine whether this was associated with the reduction of glomeruli innervated by mALT PNs. Retrograde tracing of mALT PNs in drones and counting of innervated glomeruli showed that the number of mALT-associated glomeruli was strongly reduced in drones compared to workers. The preferential projections of S. basiconica-associated OSNs into T3 glomeruli in female workers together with the reduction of mALT-associated glomeruli in drones support the presence of a female-specific olfactory subsystem that is partly innervated by OSNs from S. basiconica and is associated with mALT projection neurons. As mALT PNs were shown to be more odor specific, I suppose that already the OSNs in this subsystem are more odor specific than lALT associated OSNs. I conclude that this female-specific subsystem allows the worker honeybees to respond adequately to the enormous variety of odorants they experience during their lifetime.
In manuscript II, I investigated the ion channel composition of mALT and lALT PNs and KCs in situ. This approach represents the first study dealing with the honeybee PN and KC ion channel composition under standard conditions in an intact brain preparation. With these recordings I set out to investigate the potential impact of intrinsic neuronal properties on the differences between m- and lALT PNs and on the sparse odor coding properties of KCs. In PNs, I identified a set of Na+ currents and diverse K+ currents depending on voltage and Na+ or Ca2+ that support relatively high spontaneous and odor response AP frequencies. This set of currents did not significantly differ between mALT and lALT PNs, but targets for potential modulation of currents leading to differences in AP frequencies were found between both types of PNs. In contrast to PNs, KCs have very prominent K+ currents, which are likely to contribute to the sparse response fashion observed in KCs. Furthermore, Ca2+ dependent K+ currents were found, which may be of importance for coincidence detection, learning and memory formation.
Finally, I conclude that the differences in odor specificity between m- and lALT PNs are due to their synaptic input from different sets of OSNs and potential processing by LNs. The differences in spontaneous activity between the two tracts may be caused by different neuronal modulation or, in addition, also by interaction with LNs. The temporally sparse representation of odors in KCs is very likely based on the intrinsic KC properties, whereas general excitability and spatial sparseness are likely to be regulated through GABAergic feedback neurons.
Due to the earth´s rotation around itself and the sun, rhythmic daily and seasonal changes in illumination, temperature and many other environmental factors occur. Adaptation to these environmental rhythms presents a considerable advantage to survival. Thus, almost all living beings have developed a mechanism to time their behavior in accordance. This mechanism is the endogenous clock. If it fulfills the criteria of (1) entraining to zeitgebers (2) free-running behavior with a period of ~ 24 hours (3) temperature compensation, it is also referred to as “circadian clock”. Well-timed behavior is crucial for eusocial insects, which divide their tasks among different behavioral castes and need to respond to changes in the environment quickly and in an orchestrated fashion. Circadian rhythms have thus been studied and observed in many eusocial species, from ants to bees. The underlying mechanism of this clock is a molecular feedback loop that generates rhythmic changes in gene expression and protein levels with a phase length of approximately 24 hours. The properties of this feedback loop are well characterized in many insects, from the fruit fly Drosophila melanogaster, to the honeybee Apis mellifera. Though the basic principles and components of this loop are seem similar at first glance, there are important differences between the Drosophila feedback loop and that of hymenopteran insects, whose loop resembles the mammalian clock loop. The protein PERIOD (PER) is thought to be a part of the negative limb of the hymenopteran clock, partnering with CRYPTOCHROME (CRY). The anatomical location of the clock-related neurons and the PDF-network (a putative in- and output mediator of the clock) is also well characterized in Drosophila, the eusocial honeybee as well as the nocturnal cockroach Leucophea maderae. The circadian behavior, anatomy of the clock and its molecular underpinnings were studied in the carpenter ant Camponotus floridanus, a eusocial insect Locomotor activity recordings in social isolation proved that the majority of ants could entrain to different LD cycles, free-ran in constant darkness and had a temperature-compensated clock with a period slightly shorter than 24 hours. Most individuals proved to be nocturnal, but different types of activity like diurnality, crepuscularity, rhythmic activity during both phases of the LD, or arrhythmicity were also observed. The LD cycle had a slight influence on the distribution of these activities among individuals, with more diurnal ants at shorter light phases. The PDF-network of C. floridanus was revealed with the anti-PDH antibody, and partly resembled that of other eusocial or nocturnal insects. A comparison of minor and major worker brains, only revealed slight differences in the number of somata and fibers crossing the posterior midline. All in all, most PDF-structures that are conserved in other insects where found, with numerous fibers in the optic lobes, a putative accessory medulla, somata located near the proximal medulla and many fibers in the protocerebrum. A putative connection between the mushroom bodies, the optic lobes and the antennal lobes was found, indicating an influence of the clock on olfactory learning. Lastly, the location and intensity of PER-positive cell bodies at different times of a 24 hour day was established with an antibody raised against Apis mellifera PER. Four distinct clusters, which resemble those found in A. mellifera, were detected. The clusters could be grouped in dorsal and lateral neurons, and the PER-levels cycled in all examined clusters with peaks around lights on and lowest levels after lights off.
In summary, first data on circadian behavior and the anatomy and workings of the clock of C. floridanus was obtained. Firstly, it´s behavior fulfills all criteria for the presence of a circadian clock. Secondly, the PDF-network is very similar to those of other insects. Lastly, the location of the PER cell bodies seems conserved among hymenoptera. Cycling of PER levels within 24 hours confirms the suspicion of its role in the circadian feedback loop.
The rotation of the earth around its axis causes recurring and predictable changes in the environment. To anticipate those changes and adapt their physiology and behavior accordingly, most organisms possess an endogenous clock. The presence of such a clock has been demonstrated for several ant species including Camponotus ants, but its involvement in the scheduling of daily activities within and outside the ant nest is fairly unknown. Timing of individual behaviors and synchronization among individuals is needed to generate a coordinated collective response and to maintain colony function. The aim of this thesis was to investigate the presence of a circadian clock in different worker castes, and to determine the daily timing of their behavioral tasks within the colonies of two nectar-collecting Camponotus species.
In chapter I, I describe the general temporal organization of work throughout the worker life in the species Camponotus rufipes. Continuous tracking of behavioral activity of individually- marked workers for up to 11 weeks in subcolonies revealed an age-dependent division of labor between interior and exterior workers. After eclosion, the fairly immobile young ants were frequently nurtured by older nurses, yet they started nursing the brood themselves within the first 48 hours of their life. Only 60% of workers switched to foraging at an age range of one to two weeks, likely because of the reduced needs within the small scale of the subcolonies. Not only the transition rates varied between subcolonies, but also the time courses of the task sequences between workers did, emphasizing the timed allocation of workers to different tasks in response to colony needs.
Most of the observed foragers were present outside the nest only during the night, indicating a distinct timing of this behavioral activity on a daily level as well. As food availability, humidity and temperature levels were kept constant throughout the day, the preference for nocturnal activity seems to be endogenous and characteristic for C. rufipes. The subsequent monitoring of locomotor activity of workers taken from the subcolonies revealed the presence of a functional endogenous clock already in one-day old ants. As some nurses displayed activity rhythms in phase with the foraging rhythm, a synchronization of these in-nest workers by social interactions with exterior workers can be hypothesized.
Do both castes use their endogenous clock to schedule their daily activities within the colony? In chapter II, I analyzed behavioral activity of C. rufipes foragers and nurses within the social context continuously for 24 hours. As time-restricted access to food sources may be one factor affecting daily activities of ants under natural conditions, I confronted subcolonies with either daily pulses of food availability or ad libitum feeding. Under nighttime and ad libitum feeding, behavioral activity of foragers outside the nest was predominantly nocturnal, confirming the results from the simple counting of exterior workers done in chapter I. Foragers switched to diurnality during daytime feeding, demonstrating the flexible and adaptive timing of a daily behavior. Because they synchronized their activity with the short times of food availability, these workers showed high levels of inactivity. Nurses, in contrast, were active all around the clock independent of the feeding regime, spending their active time largely with feeding and licking the brood. After the feeding pulses, however, a short bout of activity was observed in nurses. During this time period, both castes increasingly interacted via trophallaxis within the nest. With this form of social zeitgeber, exterior workers were able to entrain in-nest workers, a phenomenon observed already in chapter I. Under the subsequent monitoring of locomotor activity under LD conditions the rhythmic workers of both castes were uniformly nocturnal independent of the feeding regime. This endogenous activity pattern displayed by both worker castes in isolation was modified in the social context in adaption to task demands.
Chapter III focuses on the potential factors causing the observed plasticity of daily rhythms in the social context in the ant C. rufipes. As presence of brood and conspecifics are likely indicators of the social context, I tested the effect of these factors on the endogenous rhythms of otherwise isolated individuals. Even in foragers, the contact to brood triggered an arrhythmic activity pattern resembling the arrhythmic behavioral activity pattern seen in nurses within the social context. As indicated in chapter I and II, social interaction could be one crucial factor for the synchronization of in nest activities. When separate groups were entrained to phase-shifted light-dark-cycles and monitored afterwards under constant conditions in pairwise contact through a mesh partitioning, both individuals shifted parts of their activity towards the activity period of the conspecific. Both social cues modulated the endogenous rhythms of workers and contribute to the context dependent plasticity in ant colonies.
Although most nursing activities are executed arrhythmically throughout the day (chapter II), previous studies reported rhythmic translocation events of the brood in Camponotus nurses. As this behavior favors brood development, the timing of the translocations within the dark nest seems to be crucial. In chapter IV, I tracked translocation activity of all nurses within subcolonies of C. mus. Under the confirmed synchronized conditions of a LD-cycle, the daily pattern of brood relocation was based on the rhythmic, alternating activity of subpopulations with preferred translocation direction either to the warm or to the cold part of the temperature gradient at certain times of the day. Although the social interaction after pulse feeding had noticeable effects on the in-nest activity in C. rufipes (chapter I and II), it was not sufficient to synchronize the brood translocation rhythm of C. mus under constant darkness (e.g. when other zeitgebers were absent). The free-running translocation activity in some nurses demonstrated nevertheless the involvement of an endogenous clock in this behavior, which could be entrained under natural conditions by other potential non-photic zeitgebers like temperature and humidity cycles.
Daily cycling of temperature and humidity could not only be relevant for in-nest activities, but also for the foraging activity outside the nest. Chapter V focuses on the monitoring of field foraging rhythms in the sympatric species C. mus and C. rufipes in relation to abiotic factors. Although both species had comparable critical thermal limits in the laboratory, foragers in C. mus were strictly diurnal and therefore foraged under higher temperatures than the predominant nocturnal foragers in C. rufipes. Marking experiments in C. rufipes colonies with higher levels of diurnal activity revealed the presence of temporally specialized forager subpopulations. These results suggest the presence of temporal niches not only between the two Camponotus species, but as well between workers within colonies of the same species.
In conclusion, the temporal organization in colonies of Camponotus ants involves not only the scheduling of tasks performed throughout the worker life, but also the precise timing of daily activities. The necessary endogenous clock is already functioning in all workers after eclosion. Whereas the light-dark cycle and food availability seem to be the prominent zeitgebers for foragers, nurses may rely more on non-photic zeitgeber like social interaction, temperature and humidity cycles.
In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis.
Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.