Refine
Is part of the Bibliography
- yes (85)
Year of publication
- 2020 (85) (remove)
Document Type
- Doctoral Thesis (84)
- Journal article (1)
Keywords
- Aufmerksamkeit (3)
- Maus (3)
- T-Lymphozyt (3)
- Thrombozyt (3)
- Angststörung (2)
- Candida albicans (2)
- Einzelmolekülmikroskopie (2)
- Evolution (2)
- FKBP52 (2)
- Graft-versus-host-disease (2)
- Herzinsuffizienz (2)
- Immunologie (2)
- Neisseria meningitidis (2)
- Sozialangst (2)
- Spinale Muskelatrophie (2)
- Tissue Engineering (2)
- cytoskeleton (2)
- fMRI (2)
- platelet (2)
- social anxiety (2)
- tDCS (2)
- 2-point Dixon (1)
- 3D spheroid culture (1)
- A2B adenosine receptor (1)
- A2BAR (1)
- AMPK (1)
- ASM (1)
- ASM-Inhibition (1)
- Adenosinrezeptor (1)
- Adhesion and Invasion (1)
- Adhesion and degranulation promoting adapter protein (1)
- Adipose Tissue Engineering (1)
- Affekt (1)
- Aktive Implantate (1)
- Analgesie (1)
- Angst (1)
- Antigen presentation (1)
- Antigen recognition (1)
- Antigenpräsentation (1)
- Arrestine (1)
- Arteriosklerose (1)
- Arzneimitteldesign (1)
- Atmungsinsuffizienz (1)
- Augenbewegung (1)
- Autoimmunity (1)
- Autoimmunität (1)
- Autophagie (1)
- Autophagy (1)
- Aversive Konditionierung (1)
- BCG (1)
- BIN2 (1)
- BOLD signal (1)
- Bacteria (1)
- Bacteria-Host Cell Interaction (1)
- Bacteroides thetaiotaomicron (1)
- Bestrahlung (1)
- Biene (1)
- Bienen <Familie> (1)
- Bildartefakte (1)
- Biologische Uhr (1)
- Bioprozessmethode (1)
- Blinatumomab (1)
- Blumeria graminis (1)
- Blut-Nerven-Barriere (1)
- Bone Marrow Dosimetry (1)
- Bone Quantification (1)
- Bone morphogenetic protein 2 (1)
- Brain derived neurotorphic factor (1)
- Brustkrebs (1)
- Butyrophilin (1)
- CAMP production (1)
- CAR T Zellen (1)
- CAR T-cells (1)
- CD1d (1)
- CLEC16A (1)
- CRISPR/Cas-Methode (1)
- Calcium signalling (1)
- Calciumkanal (1)
- Carotis Intima Media Dicke (1)
- Cas9 (1)
- Cation Homeostasis (1)
- Caveolin-1 (1)
- Ceramid (1)
- Chemokine receptors (1)
- Citalopram (1)
- Clonal competition assay (1)
- Co-Analgetika (1)
- Coactosin-like (1)
- Commensalism (1)
- Complexome (1)
- Computational Fluid Dynamics (1)
- Computerunterstütztes Verfahren (1)
- Contrast Agent Bolus Based Perfusion Magnetic Resonance Imaging (1)
- Cul4b (1)
- Cuticle (1)
- DEQCT (1)
- DNA-Reparatur (1)
- DNS (1)
- DNS-Reparatur (1)
- Darm (1)
- Dasatinib (1)
- Dendritic cells (1)
- Dendritische Zelle (1)
- Dendritische Zellen (1)
- Depression (1)
- Diabetes mellitus Typ 1 (1)
- Diphenylether (1)
- Dispersal (1)
- Dolutegravir (1)
- Droseraceae (1)
- Drosophila (1)
- Drosophila melanogaster (1)
- E. coli Nissle (1)
- E1 inhibitoren (1)
- E1 inhibitors (1)
- EEG (1)
- Electroencephalographie (1)
- Elektroencephalographie (1)
- Emotionale Informationsverarbeitung (1)
- Endorganschaden (1)
- Endothel (1)
- Entscheidungsverhalten (1)
- Enzym (1)
- Epigenetik (1)
- Epithelzelle (1)
- Escherichia coli (1)
- Extinktion (1)
- Extracellular Matrix (1)
- FRET (1)
- FRET sensors (1)
- Fat Quantification (1)
- FemX (1)
- Fettgewebe (1)
- Fettsucht (1)
- Fettsäurestoffwechsel (1)
- Fettzelle (1)
- Fitness (1)
- Fluoreszenz (1)
- Fluoreszenz-Resonanz-Energie-Transfer (1)
- Fluoreszenzmikroskopie (1)
- Fluoreszenzspektroskopie (1)
- Frizzled 5 (1)
- Funktionelle Kernspintomografie (1)
- Funktionsausfälle (1)
- Furchtextinktion (1)
- Förster Resonance Energy Transfer (1)
- G protein-coupled receptor kinase (1)
- G protein-coupled receptors (1)
- G-Protein gekoppelte Rezeptoren (1)
- GPCR dimerisation (1)
- GPCR nanodomains (1)
- GPCR signaling (1)
- GPVI (1)
- GWAS (1)
- Gap Junction (1)
- Gefäßalter (1)
- Genetics (1)
- Genexpression (1)
- Genom (1)
- Genome Instability (1)
- Genomic Selection (1)
- Germination and differentiation (1)
- Glioblastomtherapie (1)
- Glycolipid (1)
- Grad-seq (1)
- Gram-positive bacteria (1)
- HIV (1)
- HIV Drug resistance (1)
- HIV South Africa (1)
- Heart failure (1)
- Heat shock protein 90 (1)
- Helikasen (1)
- Hemmung der Proliferation schnell wachsender Krebszellen (1)
- Herzhypertrophie (1)
- Herzschrittmacher (1)
- Hfq (1)
- Hi-C (1)
- High throughput screening (1)
- Hippokampus (1)
- Hirnstimulation (1)
- Histone (1)
- Hitzeschock-Proteine (1)
- Hitzeschockprotein 90 (1)
- Hochauflösende Mikroskopie (1)
- Homöostase (1)
- Honigbienen (1)
- Host Colonization (1)
- Hypertrophie (1)
- Hypothalamus (1)
- IL-12p70 (1)
- IMP1/ZBP1 (1)
- Ighmbp2 (1)
- Immunmodulation (1)
- Immunology (1)
- Immunophilin (1)
- Immuntherapie (1)
- Individual based model (IBM) (1)
- Informationsverarbeitung (1)
- Inhibition (1)
- Integrase inhibitor (1)
- Internal Dosimetry (1)
- Ionisierende Strahlung (1)
- Irradiation (1)
- K5 capsule (1)
- Kardiologie (1)
- Kationen-Homöostase (1)
- Kinase (1)
- Kinetik (1)
- Konditionierung (1)
- Kontextkonditionierung (1)
- Kontrastmittel-gestützte MRT-Perfusionsmessung (1)
- Kontrolle (1)
- Koronare Herzkrankheit (1)
- Krebs <Medizin> (1)
- LPS (1)
- Lactobacillus reuteri (1)
- Luftfeuchtigkeit (1)
- MDSCs (1)
- MLN7243 (1)
- MM (1)
- MRI (1)
- Magnetische Resonanz (1)
- Magnetresonanztomographie (1)
- Megakaryocyte (1)
- Methyltransferase (1)
- Microbiota (1)
- Microscopy (1)
- Mikroskopie (1)
- Molecular Radiotherapy (1)
- Molekularbiologie (1)
- Moosfaserterminalen (1)
- Motoneuronenerkrankung (1)
- Multiproteinkomplex (1)
- Myeloma (1)
- N-MYC (1)
- NNMT (1)
- NSM2 (1)
- Nanofabrication (1)
- Nanofabrikation (1)
- Naturschutz (1)
- Neisseria gonorrhoeae (1)
- Nerven (1)
- Nicht-kartesische Bildgebung (1)
- Nicotiana tabacum (1)
- Non-Hodgkin-Lymphom (1)
- Non-coding RNA (1)
- Non-conventional T cell (1)
- Nuclar Medicine (1)
- Numerische Fluidmechanik (1)
- Oligomerisation (1)
- Opiatrezeptor (1)
- Opioide (1)
- Panik (1)
- Panikstörung (1)
- Paniksyndrom (1)
- Parkinson-Krankheit (1)
- Pathogenicity (1)
- Peptides (1)
- Permeabilität (1)
- Peyer's patch (1)
- Pflanzen-Bienen-Netzwerke (1)
- PhD thesis pharmacology (1)
- Pharmakodynamik (1)
- Phosphatase (1)
- Phosphoantigen (1)
- Platelets (1)
- Powdery mildew fungus (1)
- Primärtumor (1)
- ProQ (1)
- Probiotic (1)
- Profilin (1)
- Prognose (1)
- Prädiktoren und Korrelate (1)
- Psychologie (1)
- Pulswelle (1)
- Quantitative Genetics (1)
- RNA (1)
- RNA helicase (1)
- RNA-binding protein (1)
- RNS (1)
- RNS-Bindungsproteine (1)
- Raf kinase inhibitor protein (1)
- RecQ helicase (1)
- Regulation of Gene Expression (1)
- Regulatorische T-Zellen (1)
- Regulatorischer T-Lymphozyt (1)
- Remission (1)
- Renaturierung <Ökologie> (1)
- Repeats (1)
- Rezeptorpharmakologie (1)
- Roscovitine (1)
- Rothmund-Thomson-Syndrome (1)
- SMARD1 (1)
- STD patients (1)
- Sandminen (1)
- Saure Sphingomyelinase (1)
- Schmerz (1)
- Secretion (1)
- Sequenzanalyse (1)
- Serotonin (1)
- Serotonin-Reuptake-Hemmer (1)
- Serotonin-Wiederaufnahmehemmer (1)
- Sertralin (1)
- Signalkette (1)
- Signaltransduktion (1)
- Small non-messenger RNS (1)
- Soziale Wahrnehmung (1)
- Spatial heterogeneity (1)
- Sphingolipide (1)
- Sphingolipidstoffwechsel (1)
- Sphingomyelin (1)
- Sphingomyelinphosphodiesterase (1)
- Spinal muscular atrophy (DLC) (1)
- Staphylococcus (1)
- Staphylococcus aureus (1)
- Stk (1)
- Stp (1)
- Streptococcus pneumoniae (1)
- Super-resolution microscopy (1)
- Super-resolution microsopy (1)
- Superhochauflösende Mikroskopie (1)
- T Zell Selektion (1)
- T cell activation (1)
- T cell selection (1)
- T zellen (1)
- TFIIH (1)
- TFIIIC (1)
- TP53 (1)
- TP53 lesions (1)
- TRPC (1)
- TRPC-Ionenkanäle (1)
- TRPC3 (1)
- TT-Fields (1)
- TTF (1)
- Tageslänge (1)
- Taufliege (1)
- Temporal heterogeneity (1)
- Theoretical Ecology (1)
- Therapie (1)
- Therapieoutcome (1)
- Thymosin b4 (1)
- Thymus (1)
- Toleranz (1)
- Transcription Factor (1)
- Transkriptionsfaktor (1)
- Transkriptomanalyse (1)
- Trypanosoma brucei brucei (1)
- Tumor Treating Fields (1)
- Tumortherapiefeld (1)
- Type II-C CRISPR/Cas (1)
- Type VIIb secretion system (1)
- USP (1)
- Ubiquitin (1)
- Ubiquitin activating eznyme 1 (1)
- Ubiquitin-Protein-Ligase (1)
- Ubiquitin-aktivierende Enzym 1 (1)
- Ubiquitinierung (1)
- VSG (1)
- Vaccine (1)
- Vagusnervstimulation (1)
- Vakzinen (1)
- Vaskuläre Integrität (1)
- Vermeidungsreaktion (1)
- Vermeidungsverhalten (1)
- Verweildauer (1)
- Vgamma9Vdelta2 T cell (1)
- Virtuelle Realität (1)
- Visuelle Aufmerksamkeit (1)
- WNT (1)
- Wachstumsfaktor (1)
- Wildbienen (1)
- Wirbelströme (1)
- Wnt-Proteine (1)
- X-ray crystallography (1)
- Xiphophorus Melanom (1)
- Zebrabärbling (1)
- Zellkern (1)
- Zellkernarchitektur (1)
- Zellmigration (1)
- Zellskelett (1)
- Zellwand (1)
- adipocytes (1)
- adipogenic differentiation (1)
- adipose (1)
- adipose-derived (1)
- antigenic variation (1)
- anxiety conditioning (1)
- artifizielle Hautmodelle (1)
- asialoGM1 (1)
- auditory cortex (1)
- autophagy (1)
- bacterial fatty-acid biosynthesis (1)
- bee microbiota (1)
- bee-associated bacteria (1)
- bestäuberfreundliche Pflanzen (1)
- biased agonism (1)
- biomedical applications (1)
- bioprocessing (1)
- bone regeneration (1)
- cadherin-13 (1)
- carnivorous plants (1)
- chlamydia trachomatis (1)
- chromosome conformation capture (1)
- circadian clock (1)
- circuitopathies (1)
- continuous wavelet analysis (1)
- control (1)
- current source density (1)
- decision-making (1)
- deubiquitinase (1)
- ectopic bone formation (1)
- ectopic release (1)
- emojis (1)
- emotional feedback (1)
- emotional information processing (1)
- emotionale Anspannung (1)
- enoyl-ACP reductase (1)
- exotische Pflanzen (1)
- extracellular signal–regulated kinases 1/2 (1)
- eye-tracking (1)
- fMRI time series (1)
- fear extinction (1)
- fear generalization (1)
- fgf (1)
- freezing of gait (1)
- funktionelle Kernspintomographie (1)
- gastrointestinal infection (1)
- gastronintestinal microbiota (1)
- genomics (1)
- hippocampus (1)
- histone variants (1)
- honey bees (1)
- honeybees (1)
- human induced pluripotent stem cells (1)
- humanen induzierte pluripotente Stammzellen (1)
- iNKT cell (1)
- in vivo study (1)
- induzierte Phosphatasen MKP-1 und MKP-2 (1)
- inferiore frontale Gyrus (1)
- inflammatorische Gewebeschäden (1)
- inhibitor residence time (1)
- insects (1)
- intestinal mucus (1)
- intracellular calcium release (1)
- ketamine anaesthesia (1)
- lambdoid phage resistance (1)
- laminar recording (1)
- learning and behaviour (1)
- lytic phage resistance (1)
- mTOR (1)
- megachilid bees (1)
- mesenchymal stem cells (1)
- mesoscopic (1)
- metabolic modelling (1)
- metabolism (1)
- metatranscriptome (1)
- microbial transmission (1)
- microbiota (1)
- microcircuitry (1)
- mitochondria (1)
- mossy fiber terminal (1)
- motoneuron (1)
- multi-modal stimuli (1)
- mutually exclusive expression (1)
- neurobiology (1)
- neuroblastoma (1)
- neurodevelopment (1)
- next generation sequencing (1)
- nitric oxide (1)
- nuclear architecture (1)
- oligomerization (1)
- opioid receptor (1)
- parasitology (1)
- phage resistance (1)
- photoinduced electron transfer (1)
- photoinduzierter Elektronentransfer (1)
- plant-bee visitation networks (1)
- pollen foraging (1)
- pollen metabarcoding (1)
- pollinator friendly plants (1)
- population dynamics (1)
- predictors and correlates (1)
- rIFG (1)
- reduction of ERK1/2 phosphorylation (1)
- reduction of cells proliferation (1)
- resting tremor (1)
- rtPA (1)
- sand mine (1)
- scale-up (1)
- secretory properties (1)
- serotonin (1)
- signalling (1)
- single molecule microscopy (1)
- single-molecule imaging (1)
- single-molecule microscopy (1)
- site directed immobilization (1)
- social attention (1)
- solitary bee nests (1)
- spatial organization (1)
- structure-based drug design (1)
- suspension culture (1)
- sustained fear (1)
- t cell (1)
- therapy of glioblastoma (1)
- therapy outcome (1)
- transcranial direct current stimulation (1)
- transcutaneous vagus nerve stimulation (1)
- transkranielle Gleichstromstimulation (1)
- translation (1)
- ubiquitin chain formation (1)
- ubiquitin linkage specificity (1)
- ubiquitin recognition (1)
- ultimatum game (1)
- variant surface glycoprotein (1)
- virtual reality (1)
- visual attention (1)
- vmPFC (1)
- wild bees (1)
Institute
- Graduate School of Life Sciences (85) (remove)
Sonstige beteiligte Institutionen
G protein coupled receptor kinases (GRK) phosphorylate and thereby desensitize G protein coupled receptors (GPCR) including β-adrenergic receptors (βAR), which are critical regulators of cardiac function. We identified the Raf kinase inhibitor protein (RKIP) as an endogenous inhibitor of GRK2 that leads to increased cardiac contractility via βAR activation. RKIP binds to the N-terminus (aa1-185) of GRK2, which is important for the GRK2/receptor interaction. Thereby it interferes with the GRK2/receptor interaction without interference with cytosolic GRK2 target activation. In this project, the RKIP/GRK interface was investigated to develop strategies that simulate the effects of RKIP on βAR.
RKIP binding to different isoforms of GRK expressed in the heart was analyzed by protein interaction assays using full-length and N-termini of GRK2, GRK3 and GRK5: 1-53, 54-185 and 1-185. Co-immunoprecipitation (Co-IPs) and pull-down assays revealed that RKIP binds to the peptides of GRK2 and GRK3 but not to the ones of GRK5, which suggests the existence of several binding sites of RKIP within the N-termini of GRK2 and GRK3. To analyze whether the peptides of GRK2 and GRK3 are able to simulate the RKIP mediated interference of the GRK2/receptor interaction, we analyzed the β2-AR phosphorylation in the absence and presence of the peptides. Interestingly, N-termini (aa1-185) of GRK2 and GRK3 reduced β2AR phosphorylation to a comparable extent as RKIP. In line with reduced receptor phosphorylation, the peptides also reduced isoproterenol-stimulated receptor internalization as shown by [3H] CGP-12177 radioligand binding assay and fluorescence microscopy compared to control cells. Subsequently, these peptides increased downstream signaling of β2AR, i.e. the phosphorylation of the PKA substrate phosducin. In an attempt to elucidate the mechanism behind the observed effects, Co-IPs were performed in order to investigate whether the peptides bind directly to the β2-AR and block its phosphorylation by GRK2. Indeed, GRK2 1-185 and GRK3 1-185 could bind the receptor, suggesting that this way GRK2 is prevented from inhibiting the receptor. To investigate the physiological effect of GRK2 1-185, GRK3 1-185 and GRK5 1-185, their effect on neonatal mouse cardiomyocyte contractility and hypertrophy was analyzed. After long-term isoproterenol stimulation, in the presence of GRK2 1 185 and GRK3 1-185 the cross-sectional area of the cardiomyocytes showed no significant increase in comparison to the unstimulated control cells. In addition, upon isoproterenol stimulation, GRK2 1-185 and GRK3 1-185 increased the beat rate in cardiomyocytes, mimicking RKIP while the base impedance, an indicator of viability, remained stable.
The N-termini (1-185) of GRK2 and GRK3 simulated RKIP’s function and had a significant influence on β2AR phosphorylation, on its downstream signaling and internalization, could bind β2-AR, increased beat rate and did not significantly induce hypertrophy, suggesting that they may serve as a model for the generation of new and more specific targeting strategies for GRK mediated receptor regulation.
Neisseria meningitidis, a commensal β-proteobacterium residing exclusively in the human nasopharynx, is a leading cause of sepsis and epidemic meningitis worldwide. While comparative genome analysis was able to define hyperinvasive lineages that are responsible for most of the cases of invasive meningococcal disease (IMD), the genetic basis of their virulence remains unclear. Recent studies demonstrate that the type II C CRISPR/Cas system of meningococci is associated with carriage and less invasive lineages. CRISPR/Cas, an adaptive defence system against foreign DNA, was shown to be involved in gene regulation in Francisella novicida. This study shows that knockout strains of N. meningitidis lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells in a strain-dependant manner, which constitutes a central step in the pathogenesis of IMD. Consequently, this study indicates that the meningococcal CRISPR/Cas system fulfils functions beyond the defence of foreign DNA and is involved in the regulation of meningococcal virulence.
Emotionale Kontrolle ist für unsere Zusammenleben unerlässlich. Zum neuronalen Netzwerk der Emotionsverarbeitung und Emotionskontrolle gehört auch der rechte inferiore präfrontale Kortex, wobei seine Funktion häufig mit der einer Bremse verglichen wird. Die Antizipationsangst, die bei manchen Angststörungen eine Rolle spielt und das daraus resultierende Vermeidungsverhalten, bieten einen relevanten Zusammenhang, den man in der Therapie von Angsterkrankungen beeinflussen könnte. Hierbei bieten nichtinvasive Hirnstimulationsverfahren einen möglichen Ansatzpunkt und der rechte IFG ein mögliches Ziel. In dieser Studie stimulierten wir den rechten inferioren frontalen Gyrus (rIFG) mittels anodaler transkranieller Gleichstromstimulation (tDCS) um zu prüfen, ob dadurch die emotionale Anspannung moduliert werden kann. Zu diesem Zwecke wurde der rIFG bei gesunden Probanden (N = 80), aufgeteilt in eine tDCS Gruppe und eine Sham Gruppe, über einen Zeitraum von 20 Minuten mit einer Stromstärke von 2 mA und einer Elektrodengröße von 35 cm² elektrisch stimuliert. Währenddessen wurde die Hautleitfähigkeiten (SCL) als psychophysiologischer Parameter in Antizipation eines akustischen neutralen bzw. aversiven Reizes gemessen. Die Art des akustischen Reizes war dabei für die Probanden durch einen visuellen Hinweisstimulus vorhersehbar, jedoch war der Zeitpunkt der Präsentation des akustischen Reizes nicht vorhersehbar. Dadurch konnte emotionale Anspannung in Antizipation des aversiven Stimulus induziert werden, was wir durch ein insgesamt höheres SCL während der aversiven Bedingung nachweisen konnten. Wir konnten einen signifikanten Effekt der tDCS des rIFG auf die psychophysiologischen Parameter der Antizipationsangst nachweisen. Der Effekt beruhte dabei auf einem geringeren Anstieg des Hautleitfähigkeitslevels der tDCS Gruppe von neutraler zu aversiver Bedingung im Vergleich zu Sham Gruppe. Wir können daher bestätigen, dass es möglich ist die physiologische Reaktion bei emotionaler Anspannung durch tDCS des rIFG zu regulieren. Darüber hinaus können wir dadurch die angenommene Rolle des rIFG in der Emotionsregulation bestätigen. Dieser scheint daher ein vielversprechender Stimulationsort für tDCS zur Verstärkung der emotionalen Kontrolle zu sein. Auf Basis unserer Ergebnisse, könnte in zukünftigen Studien tDCS des rIFG in Kombination mit Verhaltenstherapie bei Angsterkrankungen oder zur Modulation von Vermeidungsverhalten eingesetzt werden. Durch unseren Versuch konnte damit ein grundlegender Beitrag für zukünftige Therapiestudien im Zusammenhang mit tDCS geleistet werden.
Diese Arbeit beschäftigt sich mit der Kompatibilität in der medizinischen Bildgebung unter zwei verschiedenen Aspekten: (A) Beeinflussung von Gradientenfeldern durch das Magnetsystem eines Magnetresonanztomographen. (B) Beeinflussung elektronischer Bauteile durch ionisierende Strahlung.
Imperfektionen in der Gradientenhardware (7–13) führen dazu, dass nicht die ideale zeitliche Gradientenform ausgespielt wird, sondern eine verzerrte Version der Gradienten (6,14). In der nicht-kartesischen Bildgebung führen diese resultierenden Abweichungen in den k-Raum Trajektorien zu Bildartefakten, die sich negativ auf die Diagnosestellung auswirken können. Die linearen und zeitinvarianten Eigenschaften des Gradientensystems ermöglichen die Bestimmung der Übertragungsfunktion (GSTF) (20). Diese Übertragungsfunktion kann innerhalb der Bildrekonstruktion zur Trajektorienkorrektur verwendet werden (14,15,70). In dieser Arbeit wurden mit der Feldkamera (Skope Magnetic Resonance Technologies, Zürich, Schweiz) (22,23) und der schichtselektiven Phantommethode (5,6) zwei etablierte GSTF-Messverfahren verglichen. Dabei wurde die Notwendigkeit einer Abtastzeitkompensation festgestellt, um die GSTF-Informationen entsprechend der gewählten Abtastzeit zu korrigieren (s. Abbildung 16) und die Trajektorien hinreichend zu korrigieren und damit Bildartefakte zu reduzieren. Die Langzeit- und Temperaturanalyse der GSTF zeigte für zwei verschiedene Siemens-Tomographen (Siemens Healthcare, Erlangen, Germany) eine Langzeit und Temperaturstabilität, auch bei extensiven Duty-Cyclen. Damit lässt sich auch einfach eine Pre-emphasis-Korrektur der Gradienten realisieren, was exemplarisch mit einer Zig-Zag- und einer Spiral-Sequenz gezeigt werden konnte. Die GSTF-Pre-emphasis-Korrektur lieferte dabei ähnliche Ergebnisse wie die GSTF-Post-Processing-Technik (s. Abbildung 44 und 47).
In Bezug auf die Kompatibilität in der medizinischen Bildgebung wurde in dieser Arbeit auch die Beeinflussung von medizinischen Implantaten durch ionisierende Strahlung untersucht. Herzschrittmacher, Kardioverter-Defibrillatoren oder andere aktive medizini- sche Implantate können in ihrer Funktion durch ionisierende Strahlung, die bei verschiedenen diagnostischen und therapeutischen Anwendungen appliziert wird, beeinträchtigt werden (28,97,111). In dieser Studie wurden verschiedene elektronische Bauteile, wie Kondensatoren, Transistoren, Batterien und Speicherkarten in einer gewebeäquivalenten Messumgebung bestrahlt und dabei auf ihre Funktionalität überprüft. Die Messumgebung simuliert dabei die Wechselwirkungseigenschaften von menschlichem Gewebe mit ionisierender Strahlung in einem Energiebereich von 10 keV – 6 MeV. Zudem ermöglicht sie mit der Einschubeinheit die Integration von Implantaten/elektronischen Bauteilen, sowie eine realistische Bestrahlungsplanung und Dosisverifikation (35,77). Bei den Kondensatoren zeigten sich während der Bestrahlung ein verändertes Funktionsverhalten, mit signifikant abweichenden Spannungen und Zeitkonstanten gegenüber dem unbestrahlten Zustand. Auch die Batterien haben sich während der Bestrahlung signifikant schneller entladen, als ohne Strahlungsapplikation. Nach der Bestrahlung konnten bei den untersuchten SD-Speicherkarten auch Veränderungen in den Speicherzellen festgestellt werden. Bei den Transistoren war aufgrund von Fehlern im Messsetup und dem Schaltungsdesign keine genauere teststatistische Auswertung möglich. Zusammenfassend lässt sich sagen, dass sich charakteristische Kenngrößen der untersuchten Bauteile bei Strahlungsapplikation signifikant veränderten.
Modulation CD4+ humaner Treg- und Tconv-Zellen durch Inhibition der sauren Sphingomyelinase in vitro
(2020)
Die saure Sphingomyelinase (ASM) stellt durch die Umwandlung von Sphingomyelin in Ceramid und Phosphorylcholin ein zentrales, fein reguliertes Enzym im Sphingolipidmetabolismus dar. Dadurch nimmt es Einfluss auf verschiedene zelluläre Mechanismen wie Signalvermittlung, Endo- und Exozytose und Zellaktivierung. Dementsprechend weitreichend ist auch die Bedeutung der ASM bei verschiedenen Krankheiten wie Arteriosklerose, Depression oder Neoplasien. Auch auf das Immunsystem, insbesondere auf die Signalvermittlung durch T-Zellen innerhalb des adaptiven Immunsystems, nimmt die saure Sphingomyelinase Einfluss. Aufbauend auf früheren Forschungsarbeiten zur pharmakologischen und genetischen Hemmung der ASM im Mausmodell untersuchten wir, welche Auswirkungen die Hemmung dieses Enzyms in humanen Zellkulturen auf die Population regulatorischer und konventioneller T-Zellen haben. Hierzu verwendeten wir die beiden selektiven Serotonin-Wiederaufnahmehemmer Sertralin und Citalopram; zwei antidepressiv wirksame Medikamente, die durch eine Verdrängung der ASM von der lysosomalen Membran eine hemmende Wirkung ausüben. Wir konnten zeigen, dass diese beiden Substanzen sowohl in Maus-T-Zellen, als auch in humanen T-Zellen, in der Lage sind, die Aktivität der sauren Sphingomyelinase zu inhibieren. Durch Kultivierung von Immunzellen der Maus zusammen mit den Inhibitoren konnte darüber hinaus eine Erhöhung der Treg-Zellfrequenz erreicht werden. Verschiedene Zellkulturexperimente mit humanen PBMCs zeigten weiterhin, dass unter gewissen Umständen so auch eine Vermehrung regulatorischer T-Zellen im Menschen möglich ist, und dass dies mutmaßlich durch Einbindung der ASM im CD3/CD28-Signalweg bedingt ist. In mit AntiCD3-Antikörper stimulierten experimentellen Ansätzen kam es jedoch nur bei einzelnen Individuen, die als Responder identifiziert werden konnten, zu einer Treg-Zellvermehrung. Umgekehrt kam es durch externe Zugabe von C6-Ceramid zu einer Verringerung des Anteils an regulatorischen T-Zellen. Des Weiteren wurden verschiedene Veränderungen im Expressionsverhalten von Treg- und Tconv-Zellen bezüglich CD25, CD69 und CTLA-4 in Anwesenheit der ASMInhibitoren beobachtet. Weiterhin bestätigte sich, dass die pharmakologische Hemmung der sauren Sphingomyelinase auch Auswirkungen auf die Effektorfunktion von T-Zellen hat. Während die Proliferation der Zellen weitgehend unbeeinträchtigt blieb, kam es zu einer verringerten Sekretion der Zytokine IFN-gamma, TNF, IL-5 und IL-10. In ihrer Gesamtheit sprechen diese Ergebnisse dafür, dass Inhibitoren der sauren Sphingomyelinase begünstigend auf Krankheitsgeschehen mit überschießender oder dysregulierter Aktivität des Immunsystems einwirken könnten. Immunmodulatorischen Wirkungen durch Inhibition der ASM erklären möglicherweise auch Einflüsse auf das Immunsystem, die für verschiedene Antidepressiva beschrieben wurden. Insgesamt ist die Bedeutung der sauren Sphingomyelinase innerhalb der Regulation des adaptiven Immunsystems jedoch noch ein weitgehend ungeklärtes Thema mit vielen offenen Fragen. Daher ist auch in Zukunft weitere klinische und experimentelle Forschung erforderlich, um zu klären, welchen Einfluss dieses Enzyms auf Immunzellen hat und wie sich dieser auch klinisch anwenden lässt.
Analyse der Genexpression verschiedener Kandidatengene und der Methylierung im Xiphophorus Melanom
(2020)
Das Melanom ist eine der aggressivsten Formen von malignen Tumoren beim Menschen. Bei Fischen der Gattung Xiphophorus kommt es zur spontanen Tumorformation, welche auch durch zwischenartliche Kreuzung herbeiführbar ist. Hybride mit angeborenem Melanom stellen ein nützliches Tiermodell zur Untersuchung der genetischen Grundlage der Tumorentwicklung dar. Ihre Tumorigenese hängt mit der pigmentzellspezifischen Überexpression der durch eine Mutation aktivierten Rezeptortyrosinkinase Xmrk zusammen. In reinrassigen Fischen wird die onkogene Funktion des xmrk durch den Genlocus R, welcher molekular noch nicht identifiziert wurde, unterdrückt. Zusammen mit der Überexpression von xmrk konnten mittels einer RNA-Seq Analyse weitere Gene gefunden werden, welche differenziell in den Proben von malignen und benignen Geweben des Xiphophorus exprimiert werden. Des Weiteren ist bekannt, dass die Methylierung des xmrk Promotors Einfluss auf die Expression des Genes hat.
Um die Daten der durch RNA-Seq gefundenen Kandidatengene zu validieren, wurde deren Expression in malignen und benignen Geweben der Flossen und des Rumpfes mittels qPCR quantifiziert. Zusätzlich dazu wurde die Expression einiger humaner Orthologe dieser Gene in Proben aus humanen Melanomzelllinien gemessen. Mir war es möglich zu zeigen, dass mit Ausnahme von cdkn2ab, mitfb und xirp2b alle Kandidatengene signifikant unterschiedlich in mindestens einem Vergleich von benignem und malignem Gewebe exprimiert waren. Das mit xmrk verglichen gegensätzliche Expressionsmuster von pdcd4a macht es zu einem vielversprechenden Kandidaten als vom R-Locus codierten Tumorsuppressorgen. In den humanen Melanomzelllinien konnte ausschließlich von PDGFRB keine erhöhte Expression in irgendeiner Probe nachgewiesen werden. Während die Expression von PDCD4, C-MYC und MITF in mindestens drei der vier Zelllinien mittelstark erhöht war, ließ sich bei KIT eine enorm gesteigerte Überexpression in Zellen der Linie Hermes3a nachweisen. Da drei der fünf analysierten Gene und ihre Orthologen ähnliche Expressionsmuster in Proben des Xiphophorus und der humanen Melanomzelllinien zeigen, deuten diese Ergebnisse auf die Nützlichkeit des Tiermodells zur Identifizierung entscheidender Gene und Signalwege im malignen Melanom hin. Ein zweites Ziel der Arbeit war das Erlangen tieferer Einblicke in die Methylierung des Xiphophorus Melanoms auf einer globalen und promotor- spezifischen Ebene. Um die Hypothese einer Reduzierung der globalen Methylierung zu testen, führte ich eine kolorimetrische Quantifizierung der 5-mC DNA in Kontroll- und Tumorgeweben aus. Diese Vorgehensweise zeigte zum ersten Mal eine signifikante Verminderung der methylierten globalen DNA in den benignen Läsionen und malignen Melanomen der Flossen verglichen mit dem Kontrollgewebe. Um herauszufinden, on diese Demethylierung direkt mit der Überexpression des xmrk verbunden ist, analysierte ich als nächstes die Methylierung eines CpG Dinukleotids des xmrk Promotors mithilfe von methylierungssensitiven Restriktionsendonukleasen. Obwohl nur in den Proben des exophytischen Tumorwachstums als Krebsgewebe eine verringerte Methylierung des CpG Dinukleotids verglichen mit den Kontrollen nachgewiesen werden konnte, zeigte sich die Stelle in Zellen der Xiphophorus Melanomzelllinie PSM komplett unmethyliert. Diese Ergebnisse deuten stark daraufhin, dass eine differenzierte Methylierung das onkogene Potential dieser Zellen bewirkt. Um die Effekte veränderter globaler und promotor-spezifischer Methylierung auf die Tumorigenese besser zu verstehen, sind weitere Untersuchungen nötig.
Zur Durchführung peripherer Nervenblockaden werden im klinischen Alltag nichtselektive Lokalanästhetika verwendet, die neben sensorischen auch motorische Nervenfasern blockieren. Diese Arbeit untersucht und beschreibt Grundlagen für die Verwendung selektiv wirksamer Co-Analgetika. Ziel dieser Arbeit war in diesem Kontext die Analyse der intrazellulären Signalwege, welche nach Applikation von rtPA am peripheren Nerven zur Öffnung der perineuralen Barriere und so zu einer opiat- vermittelten Analgesie führen. Gemäß unserer Hypothese bindet rtPA an den LRP-1- Rezeptor und löst eine intrazelluläre Signalkaskade aus: Erk wird phosphoryliert und inhibiert über bislang unklare Mechanismen die Claudin-1-Transkription. Claudin-1 wird weniger in die Zellmembran eingebaut und/oder verlässt durch Endozytose/ Internalisierung die Zellmembran, was zur Öffnung der perineuralen Barriere führt und den Durchtritt selektiv wirksamer Analgetika erlaubt. In der späteren Phase steht die Analyse der Wiederherstellung der Barrierefunktion der Zellmembran im Vordergrund. Die ist von zentraler Bedeutung um eine Schädigung des Nervens durch das Umgebungsmilieu zu verhindern. Vermutlich wird die Wiederherstellung der Barrierefunktion über den Wnt-Signalweg gesteuert. Die Akkumulation von b-Catenin und Cdx2 führt zu einem erneuten Anstieg der Claudin-1-Transkription. Der Claudin-1- Gehalt steigt in Western Blot-Untersuchungen jedoch bereits zu einem früheren Zeitpunkt in der Zellmembran wieder an. Dies legt nahe, dass weitere von der Transkription unabhängige Mechanismen zur Wiederherstellung der Barrierefunktion beitragen. Eine mögliche Alternative zu rtPA stellt katalytisch inaktives rtPAi dar, welches in Untersuchungen ähnliche Ergebnisse wie rtPA zeigte. Dabei könnte die Verwendung von rtPAi anstatt rtPA pathophysiologisch denkbare Komplikationen wie beispielsweise Blutungen verhindern.
In Versuchen anderer Mitglieder der Arbeitsgruppe wurde die Öffnung der perineuralen Barriere mittels immunhistochemischer und funktioneller Untersuchungen bestätigt. Auch konnten keine akute Neurotoxizität oder Blutungsgefahr beobachtet werden. Somit stellt rtPA in Kombination mit Opioiden eine mögliche Alternative zur Verbesserung der postoperativen Analgesie dar, die jedoch weiterer Untersuchungen hinsichtlich von Nutzen, Risiken und Nebenwirkungen bedarf.
Myeloid-derived suppressor cells (MDSCs) constitute of monocytic (M-MDSCs) and granulocytic cell subsets (G-MDSCs)and were initially described as suppressors of T-cell function in tumor microenvironments. Recent studies have shown the involvement of MDSCs in a number of infectious diseases including Mycobacterium tuberculosis (Mtb) infection. MDSCs are tremendously accumulated in patients with Mtb infection and exert a suppressive effect on T cell responses against mycobacteria. Mycobacterium bovis BCG, the only available vaccine against Mtb fails to protect against the adult pulmonary tuberculosis (TB). Understanding the mechanisms of MDSC suppression for immunity against mycobacterial infection will provide a rational basis to improve anti- TB vaccination and host-directed therapies against TB. In this study, we investigated the role of three lipid-rich components of the plasma membrane, Caveolin-1(Cav-1), Acid Sphingomyelinase (ASM) and asialo-GM1 on BCG-activated MDSCs.
Cav-1 is one of the vital components of caveolae (plasma membrane invaginations) which regulates apoptosis and lipid metabolism. In this work, we found that MDSCs upregulated Cav-1, TLR4 and TLR2 expression after BCG infection on the cell surface. However, Cav-1 deficiency resulted in a selective defect in the intracellular TLR2 accumulation in the M-MDSC, but not G-MDSC subset. Further analysis indicated no difference in the phagocytosis of BCG by M-MDSCs from WT and Cav1-/- mice but a reduced capacity to up-regulate surface markers, to secrete various cytokines, induce iNOS and NO production. These defects correlated with deficits of Cav1-/- MDSCs in the suppression of T cell proliferation. Among the signaling pathways that were affected by Cav-1 deficiency, we found lower phosphorylation of NF-kB and p38 mitogen-activated protein kinase (MAPK) in BCG - activated MDSCs.
ASM is an enzyme present in lysosomes and is translocated to the cell surface where it hydrolyzes sphingomyelin into ceramide. Flow cytometric studies revealed that MDSCs phagocytosed BCG independent of inhibiting ASMase using pharmacological inhibitors (amitryptiline or desipramine) or MDSCs from WT and ASM-/-. Suppression of ASMase or using ASM-/- MDSCs resulted in reduced NO production and decreased cytokine secretion by MDSCs in response to BCG. Furthermore, MDSCs inhibited by amitryptiline had impaired AKT phosphorylation upon BCG infection.
Asialo-GM1 is a ganglioside expressed on the cell surface of MDSCs reported to cooperate with TLR2 for activating ERK signaling. Here, in this study, we found that asialo-GM1 expression was upregulated specifically upon mycobacterial infection and not upon any other stimulus. We noted that the soluble form of asialo-GM1 bound to BCG. Flow cytometric studies revealed that blocking
81
asialo-GM1 did not affect the phagocytosis of BCG into MDSCs. Furthermore, blocking of asialo- GM1 had no effect on the cytokine and NO secretion or AKT signaling.
Collectively, the data presented in this work implicated that Cav-1, ASM, asialo-GM1 are dispensable for the internalization of BCG. Rather, Cav-1 and ASM are required for the functional activation of MDSCs. Although asialo-GM1 binds to BCG, we did not find any difference in the functional activation of MDSCs after blocking asialo-GM1. This study provides insights into the role of lipid raft components of the MDSC cell membrane during mycobacterial infection.
In dynamic CE MR perfusion imaging the passage of an intravenously injected CA bolus through tissue is monitored to assess the myocardial pefusion state.
To enable this, knowledge of the shape of CA wash-in through upstream epicardial vessels is required, the so-called AIF.
For technical reasons this cannot be quantified directly in the supplying vessels and is thus measured in the left ventricle, which introduces the risk of systematic errors in quantification of MBF due to bolus dispersion in coronary vessels.
This means occuring CA dispersion must be accounted in the quantification process in order to produce reliable and reproducible results.
In order to do this, CFD simulations are performed to analyze and approximate these errors and deepen insights and knowledge gained from previous CFD analyses on both idealized as well as realistic and pathologically altered 3D geometries.
In a first step, several different procedures and approaches are undertaken in order to accelerate the performed workflow, however, maintaining a sufficient degree of numerical accuracy.
In the end, the implementation of these steps makes the analysis of the cardiovascular 3D model of unprecedented detail including vessels at pre-arteriolar level feasible at all.
The findings of the Navier-Stokes simulations are thus validated with regard to different aspects of cardiac blood flow.
These include the distribution of VBF into the different myocardial regions, the areals, which can be associated to the large coronary arteries as well as the fragmentation of VBF into vessels of different diameters.
The subsequently performed CA transport simulations yield results on the one hand confirming previous studies.
On the other hand, interesting additional knowledge about the behavior of CA dispersion in coronary arteries is obtained both regarding travelled distance as well as vessel diameters.
The relative dispersion of the so-called vascular transport function, a characterizing feature of vascular networks, shows a linear decrease with vessel diameter.
This results in asymptotically decreased additional dispersion of the CA time curve towards smaller and more distal vessels.
Nonetheless, perfusion quantification errors are subject to strong regional variability and reach an average value of $(-28\pm16)$ \% at rest across the whole myocardium.
Depending on the distance from the inlet and the considered coronary tree, MBF errors up to 62 \% are observed.
The ability to differentiate into mesenchymal lineages, as well as immunomodulatory, anti-inflammatory, anti-apoptotic, and angiogenic properties give ASCs great therapeutic potential. Through their culture as multicellular, three-dimensional spheroids this potential can even be enhanced. Accordingly, 3D spheroids are not only promising candidates for the application in regenerative medicine and inflammatory disease therapy, but also for the use as building blocks in tissue engineering approaches. Due to the resemblance to physiological cell-cell and cell-matrix interactions, 3D spheroids gain higher similarity to real tissues, what makes them a valuable tool in the development of bioactive constructs equivalent to native tissues in terms of its cellular and extracellular structure. Especially, to overcome the still tremendous clinical need for adequate implants to repair soft tissue defects, 3D spheroids consisting of ASCs are a promising approach in adipose tissue engineering. Nevertheless, studies on the use of ASC-based spheroids as building blocks for fat tissue reconstruction have so far been very rare. In order to optimally exploit their therapeutic potential to further their use in regenerative medicine, including adipose tissue engineering approaches, a 3D spheroid model consisting of ASCs was characterized extensively in this work. This included not only the elucidation of the structural features, but also the differentiation capacity, gene expression, and secretory properties. In addition, the elucidation of underlying mechanisms contributing to the improved therapeutic efficiency was addressed.
Dem Endothel, welches die luminale Oberfläche aller Blutgefäße auskleidet, kommt eine wichtige Barrierefunktion zwischen Blut und Gewebe zu. Nur durch eine bedarfsgerechte Justierung dieser Barriere, die den Durchtritt von Molekülen und Zellen reguliert, kann die Gewebehomöostase aufrechterhalten werden. Dabei ist das Endothel nicht nur passive Barriere, sondern auch an dieser dynamischen Regulation aktiv beteiligt. Störungen oder Fehlregulationen dieser Prozesse führen zu Pathologien, z.B. Arteriosklerose.
Es ist seit längerem bekannt, dass Carcinoembryonic antigen–related cell adhesion molecule-1 (CEACAM1), ein Mitglied der Immunglobulin-Superfamilie, die Bildung und Morphogenese neuer Blutgefäße beeinflusst. Die spontane Entwicklung kleiner Arteriosklerose-ähnlicher Läsionen in CEACAM1 knockout (Cc1-/-) Mäusen zeigt, dass CEACAM1 auch für die Homöostase ausgereifter Blutgefäße von Bedeutung ist. Ziel dieser Dissertationsarbeit war daher, den Einfluss von CEACAM1 auf wesentliche Aspekte der Endothelfunktion in Aorten in situ bzw. in Endothelzellkulturen in vitro zu analysieren.
Es konnte zunächst gezeigt werden, dass CEACAM1-defiziente Endothelzellen im Vergleich zu Wildtyp (WT) Endothelzellen eine rundlichere Zellmorphologie mit meanderförmigen Zellgrenzen und interzellulären Lücken aufweisen. Diese morphologischen Unterschiede stimmen mit Befunden in situ an Aorten von WT und Cc1-/- Mäusen überein.
Weiterhin wurde eine Translokation der endothelialen NO-Synthase (eNOS) von der Zellmembran in den peri-nukleären Bereich bei CEACAM1-Defizienz festgestellt. Die erhobenen Daten bieten zwei mögliche Erklärungen dafür. Einerseits könnte CEACAM1 durch Interaktion mit eNOS als Membrananker fungieren. Daneben wiesen CEACAM1-defiziente Endothelzellen eine erhöhte Expression des Enzyms APT1 auf, welches eNOS depalmitoyliert. Die daraus resultierende, ebenfalls nachgewiesene geringere Palmitoylierung könnte auch zur verminderten Membran-lokalisation von eNOS beitragen.
Zur endothelialen Funktion gehört, die Adhäsion von Blutzellen an die Gefäßwand weitestgehend zu beschränken. CEACAM1-defiziente Endothelzellen zeigten im Vergleich zu WT Endothelzellen eine verstärkte Adhäsivität gegenüber murinen und humanen Monozyten. Ähnliche Unterschiede wurden für Aortenexplantate aus WT und Cc1-/- Mäusen festgestellt. Dies ist einerseits mit einer verstärkten Expression des Zelladhäsionsmoleküls ICAM-1 bei CEACAM1-Defizienz erklärbar. Darüber hinaus vermittelt die Glykokalyx anti-adhäsive Eigenschaften. Aus Vorbefunden war bekannt, dass die endotheliale Glykokalyx in der Aorta von Cc1-/- Mäuse reduziert ist. Im Rahmen dieser Arbeit konnte dies auf eine verstärkte Expression der Glykokalyx-degradierenden Enzyme MMP9, Chondroitinase sowie Hyaluronidase-2 in Cc1-/- Endothelzellen zurückgeführt werden.
Eine erhöhte Permeabilität stellt einen Indikator für ein dysfunktionales Endothel, eines der initialen Schritte in der Pathogenese der Arteriosklerose, dar. Zur Analyse der aortalen Permeabilität wurde ein modifizierter Miles-Assay etabliert. Unter Verwendung etablierter muriner Arteriosklerosemodelle konnte gezeigt werden, dass dieser Assay eine Störung der vaskulären Permeabilität bereits vor Auftreten makroskopischer Veränderungen zuverlässig detektiert.
Im Rahmen der folgenden Analysen an WT und Cc1-/- Mäusen zeigte sich ein altersabhängiger Effekt von CEACAM1 auf die Gefäßpermeabilität: Aorten von 3 Monate alten Cc1-/- Mäuse wiesen eine im Vergleich zum WT erhöhte Gefäßpermeabilität auf, welche wahrscheinlich Folge einer verzögerten Gefäßreifung ist. Im Alter von 9 Monaten zeigte sich dagegen ein entgegengesetztes Bild. Dies wurde auf eine verstärkte Expression des die Barriere schädigenden Inflammationsmediators TNF-α in 9 Monate alten WT Mäusen zurückgeführt.
Außerdem modulierte CEACAM1 die TNF-α-vermittelte Lockerung der endothelialen Barriere, indem es die Phosphorylierung von Adherens Junction Proteinen beeinflusste. Basal stabilisierte CEACAM1 die endotheliale Barriere durch Hemmung der Phosphorylierung von Caveolin-1, welches Adherens Junctions destabilisiert. Unter Einfluss von TNF-α war CEACAM1 verstärkt im Bereich von Adherens Junctions lokalisiert und rekrutierte dort Src-Kinase. Src-Kinase wiederum destabilisierte Adherens Junctions durch Phosphorylierung von β-Catenin, was in verstärkter Gefäßpermeabilität resultierte. Dagegen führte TNF-α in CEACAM1-defizienten Endothelzellen zu einer Dephosphorylierung von Caveolin-1 und β-Catenin, wodurch Adherens Junctions und damit die endotheliale Barriere stabilisiert wurden. Diese CEACAM1-abhängige differenzielle Regulation der Stabilität von Adherens Junctions unter TNF-α trägt wahrscheinlich maßgeblich zu den Unterschieden der vaskulären Permeabilität in 3 bzw. 9 Monate alten WT und Cc1-/- Mäusen bei.
Zusammenfassend konnte im Rahmen dieser Arbeit nachgewiesen werden, dass CEACAM1 zentrale Funktionen des Endothels und hierüber die Homöostase reifer Gefäße beeinflusst. Da eine Expression von CEACAM1 auch in arteriosklerotischen Plaques nachgewiesen werden konnte, soll in weiteren Untersuchungen auch der Beitrag von CEACAM1 zur arteriosklerotischen Plaquebildung analysiert werden.
The role of the adhesion and degranulation promoting adapter protein (ADAP) in platelet production
(2020)
Bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Although this process is fundamental to maintain normal platelet counts in circulation only little is known about the regulation of directed proplatelet formation.
As revealed in this thesis, ADAP (adhesion and degranulation promoting adapter protein) deficiency (constitutive as well as MK and platelet-specific) resulted in a microthrombocytopenia in mice, recapitulating the clinical hallmark of patients with mutations in the ADAP gene. The thrombocytopenia was caused by a combination of an enhanced removal of platelets from the circulation by macrophages and a platelet production defect. This defect led to an ectopic release of (pro)platelet-like particles into the bone marrow compartment, with a massive accumulation of such fragments around sinusoids. In vitro studies of cultured BM cell-derived MKs revealed a polarization defect of the demarcation membrane system, which is dependent on F-actin dynamics. ADAP-deficient MKs spread on collagen and fibronectin displayed a reduced F-actin content and podosome density in the lowest confocal plane. In addition, ADAP-deficient MKs exhibited a reduced capacity to adhere on Horm collagen and in line with that the activation of beta1-integrins in the lowest confocal plane of spread MKs was diminished. These results point to ADAP as a novel regulator of terminal platelet formation.
Beside ADAP-deficient mice, three other knockout mouse models (deficiency for profilin1 (PFN1), Wiskott-Aldrich-syndrome protein (WASP) and Actin-related protein 2/3 complex subunit 2 (ARPC2)) exist, which display ectopic release of (pro)platelet-like particles. As shown in the final part of the thesis, the pattern of the ectopic release of (pro)platelet-like particles in these genetically modified mice (PFN1 and WASP) was comparable to ADAP-deficient mice. Furthermore, all tested mutant MKs displayed an adhesion defect as well as a reduced podosome density on Horm collagen. These results indicate that similar mechanisms might apply for ectopic release.
Genome-wide association studies revealed CLEC16A as a candidate gene for Type 1 Diabetes and multiple other autoimmune disorders. The function of CLEC16A remains unknown. However, previous work showed that the CLEC16A ortholog ema and the murine Clec16a were both implicated in autophagy, a process partially required for MHC class II loading and antigen presentation. Furthermore, studies could show that autophagy was required in thymic epithelial cells for antigen presentation during T cell selection, suggesting a possible role of CLEC16A in T cell selection in the thymus. Additionally, it was postulated that CLEC16A may function as an expression quantitative trait locus for its neighboring genes and that Clec16a KD was involved in pancreatic islet function and impaired insulin secretion and glucose homeostasis. Prior to this work, Schuster et al. had created a Clec16a KD NOD mouse, which was protected from spontaneous autoimmune diabetes.
For this work it was hypothesized that CLEC16A variation serves as a Type 1 Diabetes risk gene by affecting autophagy in thymic epithelial cells, which modulates antigen presentation and shapes the T cell repertoire. To expand and complement previous findings by Schuster et al., this thesis aimed to investigate how CLEC16A modifies the function of thymic epithelial cells. For this purpose, CLEC16A KD was induced in human cells via RNA interference and autophagy was studied through immunoblotting. Additionally, inflammation of pancreatic tissue in Clec16a KD NOD mice was scored using H.E. stained pancreatic sections. Thymic transplantation experiments were conducted to test whether the effects of Clec16a KD were T cell intrinsic. Also, intraperitoneal glucose tolerance tests were performed to study glucose homeostasis in Clec16a KD NOD animals. Finally, using qPCR, gene expression levels of neighboring genes such as Dexi and Socs1 were measured to study Clec16a as an expression quantitative trait locus.
In combination with the findings of Schuster et al., this thesis demonstrates that Clec16a KD reduces the severity of insulitis and protects from onset of spontaneous diabetes in the NOD mouse. Disease protection is conveyed by impaired autophagy in TEC, which leads to altered T cell selection and hyporeactive CD4+ T cells. The effects of Clec16a KD in the NOD mouse are thymus intrinsic. Glucose homeostasis remains unchanged in the Clec16a KD NOD mouse and plays no role in disease protection. Clec16a and Dexi presented similar expression levels, but further studies are required to investigate a clear link between these two genes. Finally, impaired autophagy could be replicated in human CLEC16A KD cells, which demonstrates a conserved function of CLEC16A and suggests a possible link between CLEC16A variation and risk of autoimmune disease in human.
The human body is laden with trillions of microorganisms that belong to all three domains of life. Some species of this microbiota subsist as harmless commensals in healthy adults, but under certain circumstances, they can cause mucosal disease or even systemic, life-threatening infections. While the bacterial members of our microbiota are heavily studied today, much less attention is afforded to eukaryotic species that colonize different mucocutaneous surfaces of the human body. This dissertation focuses on identifying regulatory circuits that enable a prominent member of these eukaryotes, C. albicans, to, on the one hand, live on a specific mammalian mucosal surface as a harmless commensal and, on the other hand, proliferate as a pathogen. Since the ultimate source of many fatal Candida infections is the gastrointestinal (GI) tract of the infected individual, this organism is particularly suited to distinguishing traits essential for the gut colonization of commensal fungi and their ability to cause disease. Sequence-specific DNA-binding proteins that regulate transcription are important to most biological processes; I thus used these proteins as starting points to gain insights into 1) how a specific transcription regulator promotes virulence in C. albicans; 2) which traits C. albicans requires to inhabit the GI tract of a specific, well-defined mouse model as a harmless commensal; and 3) how three previously undescribed transcriptional regulators contribute to the commensal colonization of the digestive tract of this mouse model. Altogether, this work advances the knowledge concerning the biology of commensal fungi in the mammalian gut and genetic determinants of fungal commensalism, as well as pathogenicity.
Das Non-Hodgkin-Lymphom (NHL) steht an siebter Stelle der Inzidenzen aller Krebserkrankungen, mit jährlich steigender Tendenz. Wie kann einer so gefährlichen und heterogenen Krankheitsentität in der heutigen Medizin angemessen begegnet werden? Neben etablierten Therapien, die geraden bei rezidivierten oder refraktären NHL an ihre Grenzen stoßen, bieten experimentelle Therapieansätze neue Hoffnung: Blinatumomab ist ein bispezifischer Antikörper, der durch seine beiden Domänen als Adapter für die T-Zelle und die Tumor-Zelle fungiert und eine Zytolyse der malignen B-Zelle induziert. Bei der ALL fand Blinatumomab schon Anwendung in mehreren klinischen Studien und wurde im Dezember 2014 von der FDA in den USA zur Behandlung von Philadelphia-Chromosom-negativer rezidivierten/ refraktären B-Zell Vorläufer-ALL zugelassen. Als erste klinische Studie an NHL-Patienten wurde von 2004-2011 die MT103/104-Studie veranlasst. Im Zuge dieser unverblindeten, multizentrischen Phase I/II Studie wurden 76 Patienten mit refraktärem und rezidiviertem NHL vier bis acht Wochen mit Blinatumomab als Dauerinfusion behandelt und hierbei Informationen zu Toxizität und Tolerabilität gesammelt. Mit der Langzeitbeobachtung der Würzburger Kohorte aus dieser Studie befasst sich die vorliegende Arbeit. Ziel ist es zunächst, festzustellen, wie lange die Patienten nach Blinatumomab-Therapie im Zuge der MT103/104 Studie gesamt, rezidiv- oder therapiefrei überlebten und ob bei einem bestimmten Patientensubkollektiv ein besonders vorteilhaftes Langzeitüberleben gezeigt werden kann. Die Frage nach der Sicherheit von Blinatumomab beantwortet die Erfassung des Langzeitnebenwirkungsspektrums: Somit werden als zweiter Endpunkt die häufigsten Gründe für Krankenhausaufenthalte nach Blinatumomabtherapie, eventuelle Häufungen einer spezifischen Nebenwirkungsentität und die Reversibilität der unter der Therapie aufgetretenen Nebenwirkungen mit einem selbst entwickelten Fragebogen erfasst. Der MoCA-Test soll neurokognitive Langzeittoxizitäten ausschließen. Die Arbeit konnte nicht nur zeigen, dass Patienten, die auf Blinatumomab ansprachen gegenüber den Patienten ohne Ansprechen ein deutlich längeres Überleben zeigten, sie bestätigte die Wichtigkeit des Erhalts der effektiven Dosis von 60 µg/m²/24h für das Erreichen und den Erhalt der Progressionsfreiheit. Sechs Patienten waren bei Beobachtungsende noch in Remission. Die unterschiedlichen Eindosierungsmodi hatten keinen Effekt auf das Langzeitüberleben, können aber nebenwirkungsbedingte Therapieabbrüche während der Therapie minimieren. Alle während der Therapie aufgetretenen Nebenwirkungen waren in der Langzeitnachbeobachtung vollständig reversibel. Am häufigsten mussten Patienten auf Grund von Infektionen im Verlauf hospitalisiert werden, bei zwei Patienten traten zusätzliche Tumorerkrankungen auf, die allerdings nicht mit der Blinatumomab-Therapie assoziiert waren. Die Rate der Transformationen von indolenten in aggressive NHL war nicht erhöht. Im MoCA-Test lassen sich keine Häufungen von neurokognitiven Defiziten finden. Blinatumomab zeigt sich auch in der Langzeitbeobachtung als ein für die Behandlung von rezidivierten und refraktären NHLs effektives und sicheres Medikament.
A mouse model for genetic deletion of presynaptic BDNF from adult hippocampal mossy fiber terminals
(2020)
Brain-derived neurotrophic factor (BDNF) is a modulator and mediator of structural and functional plasticity at synapses in the central nervous system. Despite our profound knowledge about the synaptic function of BDNF at synapses, it is still controversially discussed whether synaptic BDNF acts primarily from pre- or postsynaptic sites. In the central nervous system, several studies show that mossy fiber (MF) projections formed by hippocampal granule neurons store the highest amount of BDNF. However, immunofluorescence and RNA labelling studies suggest that MF BDNF is primarily produced by granule neurons. Multiple other studies prefer the view that BDNF is primarily produced by postsynaptic neurons such as CA3 pyramidal neurons. Here, we question whether the BDNF, which is stored in the mossy fiber synapse, is primarily produced by granule neurons or whether by other cells in the MF-CA3 microcircuit. After standardization of immunolabelling of BDNF, confocal imaging confirmed the localization of BDNF in presynaptic MF terminals. This anterograde location of synaptic BDNF was also found in distinct regions of the fear and anxiety circuit, namely in the oval nucleus of the bed nucleus stria terminals (ovBNST) and in the central amygdala. To find out whether the presynaptic BDNF location is due to protein translation in the corresponding presynaptic dentate gyrus (DG) granule neuron, we developed and characterized a mouse model that exhibits BDNF deletion specifically from adult DG granule neurons. In this mouse model, loss of presynaptic BDNF immunoreactivity correlated with the specific Creactivity in granule neurons, thus confirming that MF BDNF is principally released by granule neurons. After BDNF deletion from granule neurons, we observed more immature neurons with widely arborized dendritic trees. This indicated that local BDNF deletion also affects the local adult neurogenesis, albeit Cre-mediated BDNF deletion only occur in adult granule neurons. Since BDNF is a master regulator of structural synaptic plasticity, it was questioned whether it is possible to visualize presynaptic, synapse-specific, structural plasticity in mossy fiber synapses. It was established that a combination of Cre-techniques together with targeting of GFP to membranes with the help of palmitoylation / myristoylation anchors was able to distinctly outline the synaptic structure of the BDNF-containing MF synapse. In summary, the mouse model characterized in here is suited to investigate the synaptic signalling function of presynaptic BDNF at the mossy fiber terminal, a model synapse to investigate microcircuit information processing from molecule to behaviour.
My dissertation comprises three studies: (1) an assessment of honey bee colony losses in the USA between 2014 and 2015, (2) an exploration of the potential of reclaimed sand mines as bee habitat, and (3) an evaluation of native and non-native pollinator friendly plants in regard to their attraction to bees. While the first study focuses on honey bees, the latter two studies primarily take wild bees or entire bee communities in focus.
The study on honey bee colony losses was conducted within the framework of the Bee Informed Partnership (BIP, beeinformed.org) and aligns with the annual colony loss surveys which have been conducted in the USA since the winter of 2006/2007. It was the fourth year for which summer and annual losses were calculated in addition to winter losses. Among participants, backyard beekeepers were the largest group (n = 5690), although sideline (n = 169) and commercial (n = 78) beekeepers managed the majority (91.7 %) of the 414 267 surveyed colonies. Overall, 15.1 % of the estimated 2.74 million managed colonies in the USA were included in the study. Total honey bee colony losses (based on the entirety of included colonies) were higher in summer (25.3 %) than in winter (22.3 %) and amounted to 40.6 % for the entire 2014/2015 beekeeping year. Average colony losses per beekeeper or operation were higher in winter (43.7 %) than in summer (14.7 %) and amounted to 49 % for the entire 2014/2015 beekeeping year. Due to the dominance of backyard beekeepers among participants, average losses per operation (or unweighted loss) stronger reflected this smaller type of beekeeper. Backyard beekeepers mainly named colony management issues (e.g., starvation, weak colony in the fall) as causes for mortality, while sideline and commercial beekeepers stronger emphasized parasites or factors outside their control (e.g., varroa, nosema, queen failure).
The second study took place at reclaimed sand mines. Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.
The third study was an experimental field study on pollinator friendly plants. Bees rely on the pollen and nectar of plants as their food source. Therefore, pollinator friendly plantings are often used for habitat enhancements in bee conservation. Non-native pollinator friendly plants may aid in bee conservation efforts, but have not been tested and compared with native pollinator friendly plants in a common garden experiment. In this study, we seeded mixes of 20 native and 20 non-native pollinator friendly plants in two separate plots at three sites in Maryland, USA. For two years, we recorded flower visitors to the plants throughout the blooming period and additionally sampled bees with pan traps. A total of 3744 bees (120 species) were sampled in the study. Of these, 1708 bees (72 species) were hand netted directly from flowers for comparisons between native and non-native plants. Depending on the season, bee abundance and species richness was either similar or lower (early season and for richness also late season) at native plots compared to non-native plots. Additionally, the overall bee community composition differed significantly between native and non-native plots. Furthermore, native plants were associated with more specialized plant-bee visitation networks compared to non-native plants. In general, visitation networks were more specialized in the early season than the later seasons. Four species (Bombus impatiens, Halictus poeyi/ligatus, Lasioglossum pilosum, and Xylocopa virginica) out of the five most abundant bee species (also including Apis mellifera) foraged more specialized on native than non-native plants. Our study showed that non-native plants were well accepted by a diverse bee community and had a similar to higher attraction for bees compared to native plants. However, we also demonstrated alterations in foraging behavior, bee community assemblage, and visitation networks. As long as used with caution, non-native plants can be a useful addition to native pollinator friendly plantings. This study gives a first example of a direct comparison between native and non-native pollinator friendly plants.
In this thesis, three species were investigated for the conservation of two non-conventional T cell systems, the CD1d/ iNKT cell system and the BTN3/ Vγ9Vδ2 T cell system. Non-conventional T cells are αβ or γδ T cells that do not fit into the classical mode of antigen recognition and adaptive responses. These T cells recognize antigens different from classical peptide antigens and are not restricted to the polymorphic MHC molecules but rather to non-polymorphic antigen-presenting molecules. The iNKT cell subset is restricted by the lipid antigen-presenting molecule CD1d and carries out immunomodulatory functions by rapid cytokine secretion. The molecular basis of this system, the semi-invariant iNKT TCR chains and CD1d were proven to be expressed and compared to homologs in human and rodents. Cotton rats possess multiple members of the AV14 and BV8 family and only one isoform of CD1d which is comparable to findings in the rat.
Moreover, the reactivity of primary cells to glycolipid antigens could be shown, and an iNKT
cell-like population was detected in primary cells using newly developed cotton rat CD1d oligomers. These were also applied to test the capacity of CD1d to present typical glycolipid
antigens to iNKT TCR transductants. In addition, expression of cotton rat iNKT TCR α and β chains in TCR-negative cell lines was used to show successful pairing and detection of glycolipids in the context of CD1d. In summary, the conservation of a functional CD1d/iNKT cell system in the cotton rat could be shown, and tools were developed to study this cell subset in the course of infectious diseases. The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis that indicate cell stress, transformation or infection. Up to this date, phosphoantigen-reactive γδ T cells have only been shown in primate species. However, evidence for the existence and functional conservation of the genes implied in the BTN3/Vγ9Vδ2 T cell system was found in several placental mammal species,
and two candidate species were chosen for further investigation. The nine-banded armadillo, a valuable model for leprosy research, was shown to possess homologous genes to TRGV9, TRDV2 and BTN3. In this study, the expression of productive rearrangements of TRDV2 gene segments could be shown in peripheral blood samples, but no evidence was found for the expression of a functional TRGV9 rearrangement or BTN3 molecules. Moreover, determinants of phosphoantigen-reactive Vγ9Vδ2 T cells and functional BTN3 molecules were found to still be prevalent in armadillo genes. This makes the armadillo an interesting model to study the structural determinants that allow phosphoantigen recognition by a functional Vγ9Vδ2 T cell subset although this species is merely a witness for a functional system in a placental mammal ancestor. In contrast, alpacas were shown to express functional Vγ9Vδ2 T cells which conserved many features of the human counterpart. Expression of Vγ9Vδ2 pairings could be shown by single-cell PCR and functional phosphoantigenreactive pairings were observed. This phosphoantigen reactivity was also shown in PBMC cultures with a newly developed antibody specific for alpaca Vδ2Jδ4 chains. Moreover, a more detailed study of the alpaca TCR repertoire showed similarities to “γδ high” species like
camelids and cattle which possess an extended family of TRDV genes. The γ and δ loci of alpaca
TCR genes were drafted based on genomic information and cDNA studies and provide an overview for more detailed studies. Conservation of phosphoantigen recognition by the single BTN3 molecule of alpacas was shown in 293T knock out cell lines, and BTN3 detection on PBMCs was investigated with a newly developed alpaca BTN3-specific antibody. These findings prove the existence of a functional BTN3-dependent phosphoantigen-reactive Vγ9Vδ2 T cell subset and provide a basis for the future study of this cell system in a non-primate species. Moreover, as the first non-primate candidate species with the BTN3/Vγ9Vδ2 T cell system the alpaca is an important outgroup for research in this field. The use of a single BTN3 variant in contrast to three human isoforms that work together renders the alpaca a unique and to this date indispensable model for Vγ9Vδ2 T cells.
In conclusion, this study provides an overview of the applicability of new animal models in the
study of the non-conventional T cell subsets iNKT cells and Vγ9Vδ2 T cells and leads the way for a better understanding of structural and functional relationships.
Angsterkrankungen sowie die posttraumatische Belastungsstörung sind weit verbreitete psychische Erkrankungen. Trotz gut evaluierter Therapiemethoden gibt es immer noch therapierefraktäre oder rezidivierend erkrankende Patienten, für die nicht-invasive Hirnstimulationsverfahren wie die transkranielle Gleichstromstimulation (tDCS) eine zusätzliche Option darstellen können. Diese Studie untersuchte daher die förderliche Wirkung der tDCS auf das Extinktionslernen, dem neuronalen Hintergrundmechanismus der Expositionstherapie. Für die Untersuchung der Extinktionsprozesse wurde ein Ein-Tages-Furchtkonditionierungsparadigma mit weiblichen Gesichtern als konditionierte Stimuli (CS) und einem 95 dB lauten weiblichen Schrei als unkonditionierten Stimulus verwendet. Die tDCS zielte darauf ab den ventromedialen präfrontalen Kortex (vmPFC), ein wichtiges Kontrollareal der Extinktion, zu aktivieren, wohingegen furchtgenerierende dorsomediale Hirnareale von der Stimulation ausgespart bleiben sollten. Hierfür wurden zwei ca. 4 x 4 cm große Elektroden in bitemporaler Anordnung etwas unterhalb der EEG 10-20-Positionen F7 und F8 appliziert und ein Gleichstrom mit einer Stärke von 1.5 mA verwendet. Die 20- minütige Stimulation startete während einer 10-minütigen Pause zwischen Akquisition und Extinktion und lief bis zum Ende der Extinktion durch. Die gesunden Probanden wurden randomisiert und doppelt verblindet zwei sham- und zwei real-Stimulationsgruppen mit jeweils entgegengesetzten Stromflussrichtungen zugeordnet. Zur Messung der Furchtreaktion dienten die elektrodermale Reaktion sowie subjektive Arousal- und Valenzbewertungen. Zusätzlich wurde die Kontingenzerwartung sowie verschiedene Fragebögen zu Depressivität, Affekt, State- und Trait-Angst, Angstsensitivität und Händigkeit erhoben. Die Untersuchung der Effekte von tDCS und Stromflussrichtung erfolgte bei allen erfolgreich konditionierten Probanden (N = 84) mittels generalisierten Schätzgleichungen. Erwartet wurde insbesondere eine Verbesserung des frühen Extinktionslernens in den real-Stimulationsgruppen, wobei vermutetet wurde, dass rechts und links anodaler Stromfluss nicht zu identischen Resultaten führen würde. Die Ergebnisse wiesen auf eine Verbesserung der frühen Extinktion unter tDCS hin. Der Effekt spiegelte sich in den Maßen der elektrodermalen Aktivität in einer stärkeren Reduktion der CS+/CS- Diskrimination und einem beschleunigten Reaktionsverlust auf CS+ wider. Der vermittelnde Mechanismus kann im intendierten Aktivitätsanstieg des vmPFC liegen, eine Steigerung der dopaminergen Neurotransmission ist jedoch ebenso denkbar. Zusätzlich ist auch die Verbesserung der Prozessierung von prediction errors durch die Veränderung der Dopaminsekretion bzw. Aktivitätssteigerung im vmPFC, Orbitofrontalkortex und mittleren temporalen Gyrus möglich. Die subjektiven Valenz- und Arousalbewertungen zeigten sich während des gesamten Experiments unbeeinflusst von der tDCS. Neben diesem Haupteffekt kam es zu weiteren nicht erwarteten Effekten. Einer dieser bedeutsamen Nebeneffekte war ein kurzer initialer Reaktionsanstieg auf den CS- zu Beginn des ersten und zweiten Extinktionsblocks in beiden real-Stimulationsgruppen, der u. a. mitverantwortlich für deren stärkeren Verlust der CS+/CS- Diskrimination war. Auch negative Auswirkungen auf die stimulierten Personen – insbesondere in Kombination mit Angsterkrankungen – können eine denkbare Folge hiervon sein. Daher stellt dieser Nebeneffekt eine wichtige Limitation des Hauptergebnisses dar, dessen Ursachen dringend in weiteren Studien evaluiert werden sollten. Als mögliche Gründe werden ein Verlust der Sicherheitsinformation des CS-, Angstgeneralisierungseffekte sowie ein erhöhtes Maß an sustained fear vermutet. Darüber hinaus wurden unerwarteterweise auch keinerlei Unterschiede der Stromflussrichtung während der frühen Extinktion manifest, in der späten bzw. gesamten Extinktion zeigten sich jedoch verschiedene Vor- und Nachteile. Vorteilhaft an der rechts anodalen im Vergleich zur links anodalen Stimulation war ein geringerer gemittelter Reaktionsanstieg auf CS+ und CS- zu Beginn des zweiten Extinktionsblocks. Dieser Effekt beruhte vermutlich auf einer Steigerung der Emotionsregulation durch Stimulation des rechten inferioren frontalen Gyrus. Als nachteilig erwies sich jedoch, dass die Reduktion der State-Angst während der Extinktion unter rechts anodaler tDCS geringer ausfiel. Bei Angstpatienten gibt es Hinweise auf eine Unteraktivierung des linken Frontalkortex, sodass angstreduzierende Effekte durch linksfrontale Aktivierung denkbar sind. Die Wahl der Stromflussrichtung sollte demnach je nach gewünschten Effekten und Angstausmaß der stimulierten Probanden abgewogen werden. Aufgrund der experimentellen Anordnung ergeben sich einige Limitationen dieser Studie. Der gesamte Extinktionsvorgang war in allen Gruppen nur von sehr kurzer Dauer, dadurch hielten auch die positiven Effekte in den real-Stimulationsgruppen nicht lange an. Zudem fand keine Testung des Extinktionsrecalls statt, sodass keine Aussage über die langfristige Wirkung der tDCS gemacht werden kann. Da die Stimulation direkt nach der Akquisition gestartet wurde, kann es neben bzw. anstelle einer Verbesserung des Extinktionslernens auch zu einer Störung der Furchtkonsolidierung und dadurch zu einer geringeren Furchtexpression gekommen sein. Zudem ist der vmPFC, das Hauptstimulationsziel dieser Studie, ebenso an der Suppression von Furchtreaktionen beteiligt, somit könnte auch dieser Mechanismus für die gefundenen Effekte verantwortlich sein. Eine Replikation der Studienergebnisse in einem mehrtägigem Konditionierungsparadigma wäre damit sinnvoll, um die Dauer und Hintergründe der gefundenen Effekte besser zu verstehen. Insgesamt bilden die Ergebnisse dieser Studie eine gute Basis zur Anwendung der tDCS des vmPFC zur Verbesserung des Extinktionslernens. Die Schwächen des hier getesteten Stimulationsprotokolls sollten jedoch in künftigen Studien weiter evaluiert und reduziert werden. Falls Testungen an Angstpatienten schließlich zu Erfolgen führen, könnte die tDCS des vmPFC als günstige und leicht anwendbare Ergänzung zu Expositionstherapien bei Patienten mit bisher therapieresistenten oder rezidivierenden Angsterkrankungen eingesetzt werden.
Transient Receptor Potential (TRP; C-classical; TRPC) Kanäle sind Ionenkanäle in der Plasmamembran und erlauben einen nicht selektiven Ca2+-Einstrom in die Zelle. Durch die Stimulation von Gq-Protein-gekoppelten Rezeptoren (GqPCRs) wird dieser Ca2+-Einstrom erhöht, wodurch über Calmodulin, die Phosphatase Calcineurin aktiviert wird. Der Transkriptionsfaktor nuclear factor of activated T-cells (NFAT) wird durch Calcineurin dephosphoryliert und wandert in den Nucleus, wo er mit anderen Transkriptionsfaktoren interagiert und Hypertrophie-induzierende Gene aktiviert. TRPC3 ist hierbei eine der relevantesten Isoformen für die Entwicklung einer Myokardhypertrophie, wie sie im Rahmen zahlreicher kardiovaskulärer Erkrankungen zu finden ist. Eine kardiale Hypertrophie ist an der Pathogenese der Herzinsuffizienz beteiligt und stellt somit einen wichtigen Risikofaktor für den plötzlichen Herztod dar. Aus diesem Grund ist es von besonderer Bedeutung die Regulation von TRPC3 Kanälen und deren Einfluss auf hypertrophe Prozesse genauer zu untersuchen.
In Vorversuchen wurde FK506 bindendes Protein 52 (FKBP52) als neuer Interaktionspartner von TRPC3 im Herzen gefunden. Die dabei gefundene FKBP52-Bindestelle von TRPC3 lag erstaunlicherweise außerhalb der zu erwartenden Bindestelle mit den vermeintlichen FKBP Bindemotiven.
FKBP52 ist ein Immunophilin, das als cis/trans Isomerase fungiert und dadurch an der Regulation von verschiedenen Ionenkanälen beteiligt ist, darunter auch TRPC-Kanäle. Es zeigte sich, dass alle Domänen von FKBP52, bis auf die TPR3-Domäne und der C Terminus, in der Lage waren, mit TRPC3 zu interagieren. Aufgrund der Funktion der FKBPs und der Tatsache, dass TRPC3 eine Rolle in der Entwicklung einer kardialen Hypertrophie spielt, sollte in dieser Arbeit untersucht werden, ob FKBP52 die Aktivität und die nachgeschalteten hypertrophen Signalwege von TRPC3 beeinflusst.
Die Downregulation von FKBP52 führte zu einer verstärkten TRPC3-abhängigen hypertrophen Antwort in neonatalen Rattenkardiomyozyten (engl. neonatal rat cardiomyocytes, NRCs). Der gleiche Effekt war sowohl in NRCs und in adulten Rattenkardiomyozyten (engl. adult rat cardiomyocytes, ARCs) zu sehen, wenn Peptidyl-Prolyl-cis-trans-Isomerase (PPIase) defiziente Mutanten von FKBP52 überexprimiert wurde. Verkürzte FKBP52 Mutanten erhöhten ebenfalls die TRPC3-abhängige Aktivität von Calcineurin, was durch eine verstärkte Translokation von NFAT in den Nucleus von NRCs zu sehen war. Außerdem konnte in NRCs und in menschlichen embryonalen Nierenzellen (engl. human embryonic kidney cells, HEK 293 Zellen), die die PPIase defizienten Mutanten exprimierten, ein erhöhter Ca2+-Einstrom in die Zelle beobachtet werden. Das gleiche war nach Downregulation von FKBP52 in HEK 293 Zellen, die TRPC3 überexprimieren (T3.9 Zellen), zu sehen. Eine funktionelle Interaktion von FKBP52 und TRPC3 konnte auch in elektrophysiologischen Messungen bestätigt werden. Nach der Interaktion von TRPC3 mit den FKBP52 Mutanten zeigte sich eine erhöhte TRPC3-Aktivität.
Die Daten zeigen somit, dass TRPC3-Kanäle durch FKBP52 reguliert werden und diese Regulation abhängig von der PPIase Funktion ist. Eine Interaktion von TRPC3 mit vollfunktionsfähigem FKBP52 könnte vor einer Ca2+-Überlastung und einer damit einhergehenden pathologischen Hypertrophie des Herzens schützen.
A continuous arms race between the development of novel antibiotics and the evolution of corresponding resistance mechanisms in bacteria has been observed, since antibiotic agents like arsphenamines (e.g. Salvarsan, developed by Paul Ehrlich [1]), sulphonamides (e.g. Prontosil, Gerhard Domagk [2]) and penicillin (Alexander Fleming [3]) were first applied to effectively cure bacterial infections in the early 20th century. The rapid emergence of resistances in contrast to the currently lagging discovery of antibiotics displays a severe threat to human health. Some serious infectious diseases, such as tuberculosis or melioidosis, which were either thought to be an issue only in Third-World countries in case of tuberculosis, or regionally restricted with respect to melioidosis, are now on the rise to expand to other areas. In contrast, methicillin-resistant Staphylococcus aureus (MRSA) is already present in clinical setups all over the world and causes severe infections in immunocompromised patients. Thus, there is an urgent need for new and effective antimicrobial agents, which impair vital functions of the pathogen’s metabolism.
One central metabolic pathway is represented by the bacterial fatty-acid synthesis pathway (FAS II), which is essential for the synthesis of long and branched-chain fatty acids, as well as mycolic acids. These substances play a major role as modulating components of the properties of the most important protective barrier – the cell envelope. The integrity of the bacterial cell wall and the associated membrane(s) is crucial for cell growth and for protection against physical strain, intrusion of antibiotic agents and regulation of uptake of ions and other small molecules. Thus, this central pathway represents a promising target for antibiotic action against pathogens to combat infectious diseases. The last and rate-limiting step is catalysed by the trans-2-enoyl-ACP reductase (ENR) FabI or InhA (in mycobacteria), which has been demonstrated to be a valuable target for drug design and can be addressed, amongst others, by diphenyl ether (DPE) compounds, derived from triclosan (TCL) – the first one of this class which was discovered to bind to ENR enzymes [4, 5].
Based on this scaffold, inhibitors containing different combinations of substituents at crucial positions, as well as a novel type of substituent at position five were investigated regarding their binding behaviour towards the Burkholderia pseudomallei and Mycobacterium tuberculosis ENR enzymes bpFabI and InhA, respectively, by structural, kinetic and in-vivo experiments. Generally, substitution patterns modulate the association and dissociation velocities of the different ENR inhibitors in the context of the two-step slow-onset binding mechanism, which is observed for both enzymes. These alterations in the rapidity of complex formation and decomposition have a crucial impact on the residence time of a compound and hence, on the pharmacokinetic properties of potential drug candidates. For example, the substituents at the 2’-position of the DPE scaffold influence the ground- and transition state stability during the binding process to bpFabI, whereas 4’-substituents primarily alter the transition state [6]. The novel triazole group attached to the 5-position of the scaffold, targeting the hydrophobic part of the substrate-binding pocket in InhA, significantly enhances the energy barrier of the transition state of inhibitor binding [7] and decelerates the association- as well as the dissociation processes. Combinations with different substituents at the 2’-position can enhance or diminish this effect, e.g. by ground-state stabilisation, which will result in an increased residence time of the respective inhibitor on InhA.
Further structural investigations carried out in this work, confirm the proposed binding mode of a customised saFabI inhibitor [8], carrying a pyridone moiety on the DPE scaffold to expand interactions with the protein environment. Structural and preliminary kinetic data confirm the binding of the same inhibitor to InhA in a related fashion. Comparisons with structures of the ENR inhibitor AFN-1252 [9] bound to ENR enzymes from other organisms, addressing a similar region as the pyridone-moiety of the DPE inhibitor, suggest that also the DPE inhibitor bears the potential to display binding to homologues of saFabI and InhA and may be optimised accordingly.
Both of the newly investigated substituents, the pyridone moiety at the 4’-position as well as the 5-triazole substituent, provide a good starting point to modify the DPE scaffold also towards improved kinetic properties against ENR enzymes other than the herein studied and combining both groups on the DPE scaffold may have beneficial effects. The understanding of the underlying binding mechanism is a crucial factor to promote the dedicated design of inhibitors with superior pharmacokinetic characteristics.
A second target for a structure-based drug-design approach is the interaction surface between ENR enzymes and the acyl-carrier protein (ACP), which delivers the growing acyl chain to each distinct enzyme of the dissociated FAS-II system and presumably recognises its respective interaction partner via electrostatic contacts. The interface between saACP and saFabI was investigated using different approaches including crosslinking experiments and the design of fusion constructs connecting the ACP and the FabI subunits via a flexible linker region of varying lengths and compositions. The crosslinking studies confirmed a set of residues to be part of the contact interface of a previously proposed complex model [10] and displayed high crosslinking efficiency of saACP to saFabI when mutated to cysteine residues. However, crystals of the complex obtained from either the single components, or of the fusion constructs usually displayed weak diffraction, which supports the assumption that complex formation is highly transient. To obtain ordered crystals for structural characterisation of the complex it is necessary to trap the complex in a fixed state, e.g. by a high-affinity substrate attached to ACP [11], which abolishes rapid complex dissociation. For this purpose, acyl-coupled long-residence time inhibitors might be a valuable tool to elucidate the detailed architecture of the ACP-FabI interface. This may provide a novel basis for the development of inhibitors that specifically target the FAS-II biosynthesis pathway.
The molecular chaperone Hsp90 facilitates the folding and activation of a wide array of structurally and functionally diverse client proteins. Hsp90 presents a central node of protein homeostasis and is frequently involved in the development of many human diseases. Although Hsp90 is a promising target for disease treatment, the mechanism by which Hsp90 facilitates client recognition and maturation is poorly understood.
The shape of the homodimeric protein resembles a molecular clamp that opens and closes in response to binding and hydrolysis of ATP. Structural studies reveal a network of distinct local conformational rearrangements that coordinate the slow transition into the hydrolysis-active, closed state configuration (time order of minutes). However, the kinetics of local conformational changes remain elusive because spectroscopic tools that can detect them have been missing so far.
Fluorescence quenching of extrinsic fluorophores by the natural amino acid Tryptophan is based on a photoinduced electron transfer (PET) reaction, which requires sub-nanometer contact between fluorophore and Tryptophan. This quenching mechanism has been developed into a 1-nm spectroscopic tool for the detection of rapid protein folding dynamics. Within the scope of this doctoral thesis, PET-reporter systems were designed to investigate the kinetics of local conformational motions that are part of the mechanistic core of the Hsp90 chaperone cycle. ATP-triggered kinetics of closure of the ATP-lid as well as swapping of the N-terminal ß-strand across subunits and association of the N-terminal and middle-domain were estimated in solution. Bulk experiments revealed that local motions occur on similar timescales and are in good agreement with the ATP-hydrolysis rate. Functional mutations demonstrated that local motions act cooperatively. Furthermore, the lid was shown to close via a two-step process consisting of a rapid lid-reconfiguration in direct response to ATP-binding, followed by slow closure of the lid. The co-chaperone Aha1 seems to act early in the chaperone cycle by remodelling of the lid and by stabilization of apo Hsp90 in a NM-domain pre-associated conformation.
A two-colour single-molecule PET microscopy method was developed to observe local motions at remote positions simultaneously and in real-time. Thus, directionality within the network of local conformational changes could be revealed. In a first attempt, the feasibility of detecting PET-complexes on the single-molecule surface was tested on Hsp90 constructs that report on only one motion (one-colour single-molecule PET microscopy). PET-quenched complexes could be distinguished from photobleached fluorophores through oxidation by molecular oxygen, resulting in fluorescence recovery. In two-colour experiments, a dimmed state was identified for PET-quenched complexes, but not for all of the used PET-reporter systems. Results suggest that local motions occur simultaneously within the time-resolution of the experiment (0.3 sec). Furthermore, bi-exponential kinetics of transition into the closed clamp configuration indicate a more complex mechanism of clamp-closure than of clamp-opening, which could be well described by a mono-exponential function.
Humans tend to believe in what they can see with their own eyes. Hence, visualization methods like microscopy have always been extremely popular since their invention in the 17th century. With the advent of super-resolution microscopy, the diffraction limit of ~200 - 250 nm could be overcome to enable more detailed insights into biological samples. Especially the single molecule localization microscopy method dSTORM offers the possibility of quantitative bioimaging. Hereby, the repetitive photoswitching of organic dyes in the presence of thiols is exploited to enable a lateral resolution of 20 nm. Another, recently introduced super-resolution method is expansion microscopy (ExM) which physically expands the sample to increase the resolution by the expansion factor from four to even twenty. To enable this, the sample is embedded into a hydrogel, homogenized using an unspecific proteinase and expanded in distilled water. Within this thesis, both methods were used to shed light on plasma membrane receptor distributions and different bacterial and fungal pathogens. In the first part of this thesis dSTORM was used to elucidate the “Receptome”, the entirety of all membrane receptors, of the cell line Jurkat T-cells and primary T-cells. Within this project we could successfully visualize and quantify the distribution of the plasma membrane receptors CD2, CD3, CD4, CD5, CD7, CD11a, CD20, CD28, CD45, CD69 and CD105 with receptor densities ranging from 0.8 cluster/µm² in case of CD20 and 81.4 cluster/µm² for the highly abundant CD45 in activated primary T-cells at the basal membrane. Hereby, we could also demonstrate a homogeneous distribution of most receptors, while only few were clustered. In the case of CD3-clusters were detected in Jurkat T-cells and in primary activated T-cells, but not in naïve ones, demonstrating the activation of this receptor. This was followed by the application of dSTORM to three different clinical projects involving the receptors CD38, BCMA and CD20 which are immunotherapeutic targets by monoclonal antibodies and CAR T-cells. In the first two projects dSTORM was applied to determine the receptor upregulation upon exposure of various drugs to MM1.S cells or primary multiple myeloma patient cells. This increase in membrane receptor expression can subsequently enhance the efficacy of therapies directed against these receptors. Within the CD20-project, the superior sensitivity of dSTORM compared to flow cytometry could be demonstrated. Hereby, a substantially higher fraction of CD20-positive patient cells was detected by dSTORM than by flow cytometry. In addition, we could show that by dSTORM CD20-positive evaluated cells were eradicated by immunotherapeutic CAR T-cell treatment. These studies were followed by whole cell super-resolution imaging using both LLS-3D dSTORM and 10x ExM to exclude any artifacts caused by interactions with the glass surface. In 10x ExM signal amplification via biotinylated primary antibodies and streptavidin ATTO 643 was essential to detect even single antibodies directed against the heterodimer CD11a with standard confocal microscopes. Albeit probably not quantitative due to the process of gelation, digestion and expansion during the ExM protocol, even some putative dimers of the receptor CD2 could be visualized using 10x ExM-SIM, similar to dSTORM experiments. Within the second part of this thesis, expansion microscopy was established in bacterial and fungal pathogens. ExM enabled not only an isotropic fourfold expansion of Chlamydia trachomatis, but also allowed the discrimination between the two developmental forms by the chlamydial size after expansion into reticulate and elementary bodies. Hereafter, a new α-NH2-ω-N3-C6-ceramide was introduced enabling an efficient fixation and for the first time the use of lipids in both, 4x and 10x ExM, termed sphingolipid ExM. This compound was used to investigate the ceramide uptake and incorporation into the cell membrane of Chlamydia trachomatis and Simkania negevensis. For Chlamydia trachomatis the combined resolution power of 10x ExM and SIM even allowed the visualization of both bacterial membranes within a distance of ~30 nm. Finally, ExM was applied to the three different fungi Ustilago maydis, Fusarium oxysporum and Aspergillus fumigatus after enzymatic removal of the fungal cell wall. In case of Ustilago maydis sporidia this digestion could be applied to both, living cells resulting in protoplasts and to fixed cells, preserving the fungal morphology. This new protocol could be demonstrated for immunostainings and fluorescent proteins of the three different fungi.
The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors.
In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.
Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication.
Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform.
Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis.
Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks.
Deubiquitinases are regulators of the ubiquitin proteasome system that counteract the ubiquitination cascade by removing ubiquitin from substrates and cleaving ubiquitin chains. Due to their involvment in various important pathways, they are associated with several diseases and may thus present promising drug targets. The two related ubiquitin specific proteases USP25 and USP28 share a highly conserved amino acid sequence but perform distinct biological functions. USP28 plays roles in cell cycle regulation and was also linked to several types of cancer. It adopts oncogenic functions by rescuing the oncoproteins MYC and JUN from proteasomal degradation, which is induced by the E3-ligase SCF (FBW7). Opposingly, USP28 also regulates the stability of the tumor suppressor FBW7 itself. USP25 contributes to a balanced innate immune system by stabilizing TRAF3 and TRAF6 and lately was found to promote Wnt-signaling by deubiquitinating TNKS.
Due to the high level of identity of both proteases, a recent attempt to inhibit USP28 led to cross reactivity against USP25. In our study, we characterized both USP25 and USP28 structurally and functionally using x-ray crystallography, biochemical as well as biophysical approaches to determine similarities and differences that can be exploited for the development of specific inhibitors.
The crystal structure of the USP28 catalytic domain revealed a cherry-couple like dimer that mediates self-association by an inserted helical subdomain, the USP25/28 catalytic domain inserted domain (UCID). In USP25, the UCID leads to formation of a tetramer composed of two interlinked USP28-like dimers. Structural and functional analysis revealed that the dimeric USP28 is active, whereas the tetrameric USP25 is auto inhibited. Disruption of the tetramer by a cancer-associated mutation or a deletion-variant activates USP25 through dimer formation in in vitro assays and leads to an increased stability of TNKS in cell studies. Furthermore, in vitro data showed that neither ubiquitin nor substrate binding led to the activation of the USP25 tetramer construct. With the structure of the C-terminal domain of USP25, we determined the last unknown region in the enzyme as a separately folded domain that mediates substrate interactions.
Combined the structures of the USP25 and USP28 catalytic domains and the functional characterization of both enzymes provide novel insights into the regulation of USPs by oligomerization. Furthermore, we identified individual features of each protease that might be explored for the development of specific small molecule inhibitors.
Amplification of N-MYC is a poor prognostic and survival marker of neuroblastoma. To broaden the scope of knowledge in N-MYC cancer biology, interactors of N-MYC should be investigated. TFIIIC complex was identified as a new protein interacting partner of N-MYC. TFIIIC is a core component of RNAPIII transcription machinery which is important for the synthesis of tRNA genes. TFIIIC recognizes and binds to B-box located internal of tRNA genes which subsequently initiate the RNAPIII transcription process. Apart from the role in RNAPIII transcription machinery, TFIIIC is an architectural protein. TFIIIC binds to thousands of sites across the genome without RNAPIII and TFIIIB. These binding loci are known as Extra TFIIIC (ETC) sites at which TFIIIC perform its role in genome organization. However, knowledge of TFIIIC is mostly restricted to studies conducted in yeasts, the exact function of TFIIIC and how it regulates N-MYC remains to be elucidated. To obtain a better overview about TFIIIC functions, two TFIIIC subunits (TFIIIC5 and TFIIIC2) which represent sub-complexes A and B were chosen for investigation. ChIP-seq experiment of RNAPIII transcription machinery was performed. It showed that both TFIIIC subunits functioned together as a complex. Next, joint binding sites of two TFIIIC subunits and N-MYC were identified. The data revealed that co-occupancies between N-MYC and TFIIIC subunits had different preference on genomic distribution. Furthermore, TFIIIC5 exhibited strong binding association with architectural proteins RAD21 and CTCF whereas TFIIIC2 was only modestly enriched with these two proteins. Both TFIIIC subunits showed equal but weak enrichment with accessory protein CAPH2. Despite the weak association with other architectural proteins, TFIIIC2 binds preferentially to repetitive elements SINE. In order to understand how TFIIIC5 affects other architectural proteins in chromatin binding, cells were depleted of TFIIIC protein upon doxycycline induction of shRNA. N-MYC binding was not affected. Yet, 50% reduction of RAD21 binding to joint N-MYC/TFIIIC sites was noticed. CAPH2 binding was increased at some joint sites while some did not respond. Lastly, CTCF did not show changes in binding under the effect of TFIIIC5 knockdown. In summary, the data indicated TFIIIC subunits from different sub-complexes diverge in functions other than tRNA synthesis. The association of TFIIIC5 with architectural proteins and TFIIIC2 with SINE elements were suggested to be distinct mechanisms to regulate N-Myc directly or indirectly.
In reconstructive and plastic surgery, there exists a growing demand of adequate tissue implants, since currently available strategies for autologous transplantation are limited by complications including transplant failure and donor site morbidity. By developing in vitro and in vivo autologous substitutes for defective tissue sites, adipose tissue engineering can address these challenges, although there are several obstacles to overcome. One of the major limitations is the sufficient vascularization of in vitro engineered large constructs that remains crucial and demanding for functional tissues. Decellularized jejunal segments may represent a suitable scaffolding system with preexisting capillary structures that can be repopulated with human microvascular endothelial cells (hMVECs), and a luminal matrix applicable for the adipogenic differentiation of human adipose-derived stem cells (hASCs). Hence, co-culture of these cells in jejunal segments, utilizing a custom-made bioreactor system, was characterized in terms of vascularization and adipose tissue development. Substantial adipogenesis of hASCs was demonstrated within the jejunal lumen in contrast to non-induced controls, and the increase of key adipogenic markers was verified over time upon induction. The development of major extracellular matrix components of mature adipose tissue, such as laminin and collagen IV, was shown within the scaffold in induced samples. Successful reseeding of the vascular network with hMVECs was demonstrated in long-term culture and co-localization of vascular structures and adipogenically differentiated hASCs was observed. Therefore, these results represent a novel approach for in vitro engineering of vascularized adipose tissue constructs that warrants further investigations in preclinical studies.
Another still existing obstacle in adipose tissue engineering is the insufficient knowledge about the applied cells, for instance the understanding of how cells can be optimally expanded and differentiated for successful engineering of tissue transplants. Even though hASCs can be easily isolated from liposuction of abdominal fat depots, yielding low donor site morbidity, huge numbers of cells are required to entirely seed complex and large 3D matrices or scaffolds. Thus, cells need to be large-scale expanded in vitro on the premise of not losing their differentiation capacity caused by replicative aging. Accordingly, an improved differentiation of hASCs in adipose tissue engineering approaches remains still desirable since most engineered constructs exhibit an inhomogeneous differentiation pattern. For mesenchymal stem cells (MSCs), it has been shown that growth factor application can lead to a significant improvement of both proliferation and differentiation capacity. Especially basic fibroblast growth factor (bFGF) represents a potent mitogen for MSCs, while maintaining or even promoting their osteogenic, chondrogenic and adipogenic differentiation potential. As there are currently different contradictory information present in literature about the applied bFGF concentration and the explicit effect of bFGF on ASC differentiation, here, the effect of bFGF on hASC proliferation and differentiation capacity was investigated at different concentrations and time points in 2D culture. Preculture of hASCs with bFGF prior to adipogenic induction showed a remarkable effect, whereas administration of bFGF during culture did not improve adipogenic differentiation capacity. Furthermore, the observations indicated as mode of action an impact of this preculture on cell proliferation capacity, resulting in increased cellular density at the time of adipogenic induction. The difference in cell density at this time point appeared to be pivotal for increased adipogenic capacity of the cells, which was confirmed in a further experiment employing different seeding densities. Interestingly, furthermore, the obtained results suggested a cell-cell contact-mediated mechanism positively influencing adipogenic differentiation. As a consequence, subsequently, studies were conducted focusing on intercellular communication of these cells, which has hardly been investigated to date.
Despite the multitude of literature on the differentiation capacity of ASCs, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in hASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in hASCs was demonstrated histologically and on the gene and protein expression level and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of hASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, these results demonstrate the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of hASCs and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.
Antigenic variation of surface proteins is a commonly used strategy among pathogens to evade the host immune response [63]. The mechanism underlying antigenic variation relies on monoallelic exclusion of a single gene from a hypervariable multigene family combined with repeated, systematic changes in antigen expression. In many systems, these gene families are arranged in subtelomeric contingency loci that are subject to both transcriptional repression and enhanced mutagenesis and recombination [16].
Eviction of a selected gene from a repressed antigen repertoire can be achieved e.g. by recombination into a dedicated, transcriptionally permissive site or by local epigenetic alterations in chromatin composition of the selected gene.
Both processes are ultimately affected by genome architecture. Architectural proteins controlling antigenic variation have, however, remained elusive in any pathogen.
The unicellular protozoan parasite Trypanosoma brucei evades the host immune response by periodically changing expression of a single variant surface glycoprotein (VSG) from a repertoire of ~3000 VSG genes – the largest mutually exclusively expressed gene family described today. To activate a selected VSG gene, it needs to be located in a dedicated expression site that becomes subject to relocation into a distinct, transcriptionally active subnuclear compartment, the expression site body (ESB). Whereas this emphasizes the importance of nuclear architecture in regulating antigen expression in T. brucei, the mechanisms underlying spatial positioning of DNA in T. brucei are not well understood.
In this study I applied genome-wide chromosome conformation capture (Hi-C) to obtain a comprehensive picture of the T. brucei genome in three dimensions, both in procyclic and bloodstream form parasites. Hi-C revealed a highly structured nucleus with megabase chromosomes occupying distinct chromosome territories. Further, specific trans interactions between chromosomes, among which are clusters of centromeres, rRNA genes and procyclins became apparent. With respect to antigenic variation, Hi-C revealed a striking compaction of the subtelomeric VSG gene repertoire and a strong clustering of transcriptionally repressed VSG-containing expression sites. Further, Hi-C analyses confirmed the spatial separation of the actively transcribed from the silenced expression sites in three dimensions.
I further sought to characterize architectural proteins mediating nuclear architecture in T. brucei. Whereas CTCF is absent in non-metazoans, we found cohesin to be expressed throughout the cell cycle, emphasizing a function beyond sister chromatid cohesion in S-phase.
By Chromatin-Immunoprecipitation with sequencing (ChIPseq), I found cohesin enrichment to coincide with the presence of histone H3 vari- ant (H3.V) and H4 variant (H4.V). Most importantly, cohesin and the histone variants were enriched towards the VSG gene at silent and active expression sites.
While the deletion of H3.V led to increased clustering of expression sites in three dimensions and increased chromatin accessibility at expression site promoters, the additional deletion of H4.V increased chromatin accessibility at expression sits even further.
RNAseq showed that mutually exclusive VSG expression was lost in H3.V and H4.V single and double deletion mutants. Immunofluorescence imaging of surface VSGs, flow cytometry and single-cell RNAseq revealed a progressive loss of VSG-2 expression, indicative of an increase in VSG switching rate in the H3.V/H4.V double deletion mutants. Using long-read sequencing technology, we found that VSG switching occurred via recombination and concluded, that the concomitant increase in spatial proximity and accessibility among expression sites facilitated the recombination event.
I therefore identified the histone variants H3.V and H4.V to act at the interface of global nuclear architecture and chromatin accessibility and to represent a link between genome architecture and antigenic variation.
Plants have evolved many mechanisms to defend against herbivores and pathogens. In many cases, these mechanisms took other duties. One example of such a neofunction- alisation would be carnivory. Carnivory evolved from the defence against herbivores. Instead of repelling the predator with a bitter taste, the plant kills it and absorbs its nutrients. A second example can be found in the pollination process. Many of the genes involved here were originally part of defence mechanisms against pathogens. In this thesis, I study these two examples on a genomic and transcriptomic level. The first project, Genomics of carnivorous Droseraceae, aims at obtaining annotated genome sequences of three carnivorous plants. I assembled the genome of Aldrovanda vesiculosa, annotated those of A. vesiculosa, Drosera spatulata and Dionaea muscipula and com- pared their genomic contents. Because of the high repetitiveness of the D. muscipula genome, I also developed reper, an assembly free method for detection, classification and quantification of repeats. With that method, we were able to study the repeats without the need of incorporating them into a genome assembly. The second large project investigates the role of DEFL (defensin-like) genes in pollen tube guidance in tobacco flowers. We sequenced the transcriptome of the SR1 strain in different stages of the pollination process. I assembled and annotated the transcriptome and searched for differentially expressed genes. We also used a method based on Hidden- Markov-Models (HMM) to find DEFLs, which I then analysed regarding their expression during the different stages of fertilisation. In total, this thesis results in annotated genome assemblies of three carnivorous Droser- aceae, which are used as a foundation for various analyses investigating the roots of car- nivory, insights into the role of DEFLs on a transcriptomic level in tobacco pollination and a new method for repeat identification in complex genomes.
Die spinale Muskelatrophie (SMA) ist eine monogenetische Erkrankung, bei der es durch den Verlust des SMN Proteins zur Degeneration der α-Motoneurone im Rückenmark kommt. Abhängig vom Schweregrad zeigen die Patienten bereits innerhalb der ersten Lebensmonate ausgeprägte Lähmungen der Skelettmuskulatur und eine Zwerchfellparese einhergehend mit einer reduzierten Lebenserwartung. Mithilfe von Mausmodellen für die SMA konnte gezeigt werden, dass der Motoneuronenverlust bei Smn-defizienten Mäusen mit Störungen der Neurotransmission an der motorischen Endplatte und mit Differenzierungsstörungen der Motoneurone einhergeht. Die Differenzierungs-störungen primärer Smn-defizienter Motoneurone sind eng gekoppelt mit einer verminderten Clusterbildung spannungsabhängiger Kalziumkanäle im distalen axonalen Bereich. Dies wiederum führt zu einer verminderten Frequenz spontaner Kalziumeinströme am Axonterminus und hat eine veränderte axonale Elongation zur Folge.
Es wurden folgende Aspekte in Bezug auf die Verstärkung und die Induktion spontaner Kalziumeinströme in Mausmodellen für spinale Muskelatrophien in dieser Arbeit adressiert:
1) Lassen sich spontane Kalziumeinströme in Smn-defizienten Motoneuronen durch die externe Applikation von Kalziumkanalagonisten verstärken?
2) Sind spontane Kalziumeinströme in primären Motoneuronen durch den Brain-derived-neurotrophic-factor (BDNF) induzierbar?
3) Zeigen primäre Motoneurone eines Mausmodells für spinale Muskelatrophie mit Ateminsuffizienz Typ 1 (SMARD1) ebenfalls veränderte Kalziumtransienten?
Die Ergebnisse meiner Arbeit zeigen, dass durch den Kalziumkanalagonisten R-Roscovitine die Frequenz der spontanen Kalziumeinströme im distalen Axon von Smn-defizienten Motoneuronen signifikant erhöht wird. Dies hat wiederum einen regulierenden Effekt auf die Differenzierung der SMA Motoneurone zur Folge. Smn-defiziente Motoneurone zeigen somit keine Unterschiede mehr in Bezug auf Axonlängen und Wachstumskegelflächen im Vergleich zu Kontrollzellen. Für R-
10
Roscovitine ist neben der agonistischen Wirkung am Kalziumkanal auch ein inhibitorischer Effekt auf die Cyclin-abhängige Kinase 5 beschrieben. Es konnte jedoch gezeigt werden, dass die erhöhten Kalziumtransienten unter der Behandlung mit R-Roscovitine durch eine direkte Bindung an die Cav2 Kalziumkanäle verursacht werden und nicht durch eine Cdk5 Blockade. Dafür spricht die schnelle und reversible Wirkung von R-Roscovitine, sowie die Aufhebung des R-Roscovitines Effekts bei gleichzeitiger Gabe des Cav2.2 Antagonisten ω-Conotoxin MVIIC.
Der zweite Aspekt dieser Arbeit behandelt den Einfluss der neurotrophen Faktoren BDNF, CNTF und GDNF auf die Kalziumtransienten am Wachstumskegel wildtypischer Motoneurone. Der Vergleich der neurotrophen Faktoren zeigt, dass nur BDNF eine induzierende Wirkung auf spontane Kalziumtransienten am Wachstumskegel hat.
Der letzte Abschnitt dieser Arbeit beschäftigt sich mit den Kalziumtransienten bei Motoneuronen aus dem Nmd2J (SMARD1) Mausmodell. Die SMARD1 gilt als eigenständige Form der spinalen Muskelatrophien mit unterschiedlicher Genetik und unterschiedlichen klinischen Merkmalen. Die Motoneurone weisen in Bezug auf die Kalziumtransienten keine Unterschiede zwischen Wildtyp und Nmd2J Mutante auf. Es ergibt sich somit kein Hinweis darauf, dass die Degeneration der Motoneurone bei der SMARD1 von einer Störung der Kalziumhomöostase im distalen axonalen Bereich ausgeht.
Staphylococcus aureus ist ein Kommensale, der die menschliche Haut und Schleimhaut der Nase und des Rachens besiedelt. Der Keim verursacht aufgrund zahlreicher Virulenzfaktoren leichte aber auch schwere Infektionen wie Pneumonie, Endokarditis oder Sepsis. Die Behandlung von S. aureus-Infektionen gestaltet sich heutzutage schwierig, da der Keim Resistenzen gegen verschiedenste Antibiotika ausgebildet hat. Zur Bekämpfung dieser Resistenzen werden neue Antibiotika benötigt, die u.a. mit der Zellphysiologie und der Zellwandwandsynthese der Bakterien interferieren.
Die Zellphysiologie und Zellwandsynthese wird abhängig von der Wachstumsphase und Umwelt-einflüssen in den Bakterien streng reguliert. Neben den Zweikomponentensystemen sind Serin/Threonin-Proteinkinasen und -Phosphatasen wesentliche Sensoren und Regulatoren der Bakterien. Durch Phosphorylierung und Dephosphorylierung bewirken diese beiden Systeme eine Hemmung oder Aktivierung der entsprechenden Zielproteine. Dadurch kann sich die Bakterienzelle an innere und äußere Reize anpassen. In dieser Arbeit wurde die konservierte Serin/Threonin-Proteinkinase Stk und die Serin/Threonin-Phosphatase Stp von S. aureus untersucht. Die beiden Proteine Stk und Stp haben einen großen Einfluss auf die Signalweiterleitung, den zentralen Metabolismus, die Stressantwort, die Antibiotikaresistenz und die Virulenz von S. aureus.
Im ersten Teil dieser Arbeit wird dargelegt, dass Stk und Stp in der bakteriellen Membran lokalisiert sind, dort miteinander interagieren und antagonistisch Zielproteine phosphorylieren bzw. dephospho-rylieren. Die Deletion der Phosphatase Stp bewirkt, dass zahlreiche Proteine in der Zelle permanent phosphoryliert und daher vermutlich nur noch eingeschränkt funktionstüchtig sind. Die ausbleibende Dephosphorylierung der Proteine in der stp-Mutante hat einen dramatischen Effekt auf die Zellwand-synthese und die Virulenz von S. aureus. So hat die stp-Mutante eine verdickte Zellwand und ist weniger virulent als die stk-Mutante und der Wildtypstamm. Im Rahmen dieser Arbeit wird erstmals eine Erklärung präsentiert, die die strukturellen Besonderheiten von Stk und deren Auswirkung auf die Zellwandsynthese zusammenführt: In der stp-Mutante akkumulieren Zellwandvorläufer in der Zelle, da vermutlich die entsprechenden Zellwandsyntheseproteine durch Stk-vermittelte Phosphorylierung gehemmt werden. Die Proteine FemXAB nehmen eine zentrale Rolle in der Zellwandsynthese ein, indem sie die Pentaglycin-Interpeptidbrücke des Zellwandvorläufers Pentaglycin-Lipid II syntheti-sieren. Stk wird durch die Bindung seiner extrazellulären Domänen an Pentaglycin-Lipid II aktiviert. In der vorliegenden Arbeit konnte FemX als in vitro Substrat von Stk und Stp identifiziert werden. Die permanente Phosphorylierung von FemX in der stp-Mutante führt zur verminderten Synthese der Pentaglycin-Brücken am Lipid II und infolgedessen zum Einbau von unvollständigen Muropeptiden in den neuen Peptidoglycanstrang. Diese strukturelle Veränderung führt zur Verdickung der Zellwand und folglich zur verminderten Empfindlichkeit gegenüber der Glycyl-Glycinpeptidase Lysostaphin. Neben FemX interagiert Stk mit weiteren Zellwandsyntheseproteinen wie FemAB und einigen Zellteilungsproteinen. Diese Ergebnisse verdeutlichen, dass Stk das Vorkommen seines extrazellulären Liganden Lipid II detektiert und dementsprechend die Zellwandsynthese über FemX reguliert.
Im zweiten Teil der Arbeit wurde anhand verschiedener Omics-Techniken die stk-, stp- und stk/stp-Mutante im Vergleich zum S. aureus NewmanHG Wildtyp charakterisiert. Dabei zeigten sich teilweise große Unterschiede zwischen der stp-Mutante und den anderen Stämmen. Mit diesen Unter-suchungen konnten Ergebnisse aus anderen Studien bestätigt und mit weiteren Daten untermauert werden. So lässt sich die verminderte Virulenz der stp-Mutante mit der reduzierten Expression und Sekretion von Toxinen wie Hämolysinen und Leukozidinen erklären. Dies führt zu einer verminderten Hämolyse von Erythrozyten und einer verminderten Immunantwort gegen diese Toxine im Infektions-versuch. Stk und Stp phosphorylieren bzw. dephosphorylieren Transkriptionsfaktoren und Antwort-regulatoren von Zweikomponentensystemen, was zu der veränderten Expression und Sekretion der Virulenzfaktoren führt. Die Analyse der Mutanten offenbart, dass Stk ein negativer und Stp ein positiver Regulator der Virulenz in S. aureus ist. Außerdem regulieren Stk und Stp zentrale Aspekte des Metabolismus in S. aureus. So ist die Konzentration an Nukleotidtriphosphaten in der stp-Mutante reduziert, was auf eine verminderte Expression der Gene der Pyrimidinsynthese zurückzuführen ist. Anhand dieser Ergebnisse wird deutlich, dass Stk und Stp wesentliche Aspekte der Zellphysiologie wie die Zellwandsynthese, den zentralen Metabolismus und die Virulenz von S. aureus regulieren.
Divalent cations are important second messengers triggering various signal transduction events in platelets. Whereas calcium channel blockers have an established antithrombotic effect and the regulation of Ca2+ homeostasis has been elucidated in platelets, the molecular regulation of Mg2+ and Zn2+ homeostasis has not been investigated so far.
In the first part of the thesis, the role of -type serine-threonine kinase linked to transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in platelets was investigated. Using Trpm7R/R mice with a point mutation deleting the kinase activity, we showed that the TRPM7 kinase regulates platelet activation via immunoreceptor tyrosine-based activation motif (ITAM), hem(ITAM) and protease-activated receptor (PAR) signaling routes. Furthermore, Trpm7R/R mice were protected from in vivo thrombosis and stroke, thus establishing TRPM7 kinase as a promising anti-thrombotic target.
In the second part of the thesis, the role of TRPM7 channel in a megakaryocyte (MK) and platelet-specific knockout mouse, Trpm7fl/fl-Pf4Cre, was investigated. Here, we observed that depending on the type of stimulation, Trpm7fl/fl-Pf4Cre platelets showed either enhanced or inhibited responses. Although Trpm7fl/fl-Pf4Cre mice were thrombocytopenic, no differences to wildtype mice were observed in models of in vivo thrombosis and stroke. The above two studies highlight that inhibition of TRPM7 kinase but not the channel itself (in MKs and platelets) may be a promising anti-thrombotic strategy.
Besides TRPM7, we investigated the role of magnesium transporter 1 (MAGT1) in platelet Mg2+ homeostasis and found that MAGT1 primarily regulates receptor-operated calcium entry (ROCE) in platelets specifically upon GPVI activation. This physiological crosstalk is triggered by protein kinase C (PKC) isoforms. Platelets from Magt1-/y mice hyper-reacted to GPVI and thromboxane A2 (TXA2) receptor stimulation in vitro. Consequently, Magt1-/y platelets were found to be pro-thrombotic in disease models of thrombosis and stroke.
To compare platelet ITAM-signaling to the immune system, we further investigated the role of MAGT1 in T and B cells. We described the primary role of MAGT1 in mice under pathogen-free conditions. Magt1-/y B cells showed dysregulated Mg2+ and Ca2+ homeostasis upon B-cell receptor activation, thereby altering Syk, LAT, phospholipase C (PLC)2 and PKC phosphorylation. In contrast to human MAGT1-deficient T cells, development and effector functions of mouse Magt1-/y T cells showed no alterations.
Finally, in the last part of the thesis, we described methods to measure intracellular free zinc [Zn2+]i in human and mouse platelets with storage pool disease (SPD). We propose to measure the [Zn2+]i status in SPD platelets as a relatively easy diagnostic to screen platelet granule abnormalities.
Kinetics and timing of IL-12 production by dendritic cells for Th1 polarization \(in\) \(vivo\)
(2020)
Dendritic cell (DC) based vaccines rely on the quality of DC maturation to induce antigen presentation, co-stimulation, lymph node migration and the release of heterodimeric IL-12p70 in case of T helper type-1 cell (Th1) polarization. In contrast, DCs that cannot secrete IL-12p70 (e.g. after cytokine cocktail maturation) readily induce Th1 cells when injected into mice and humans. Since it was also previously suggested that DCs are capable of activating other DCs in a bystander fashion, we tested here for the DC source of IL-12p70 for Th1 polarization in a murine DC vaccination model. Migration of the injected murine bone marrow-derived DCs (BM-DCs) was essential for antigen delivery to the lymph node. However, they contributed only partially to antigen presentation, and induced a non-polarized Th0 state of the cognate T cells producing IL-2 but no IFN-. Instead, endogenous dermal migratory XCR1+ cDC1s underwent re-programming by the injected BM-DCs to acquire bystander antigen presentation and IL-12 release for Th1 polarization in the lymph node. Genetic deficiency of migratory DCs and specifically of XCR1+ migratory DCs completely abolished Th1 priming. The kinetic of cell interactions in the draining lymph nodes appeared step-wise as i) injected DCs with cognate T cells, ii) injected DCs with bystander XCR1+ DCs, and iii) bystander XCR1+ DCs with T cells. The transcriptome of the bystander DCs showed a down-regulation of Treg and Th2/Th9 inducing genes, and up-regulation of genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory BM-DCs direct T cell priming and bystander DC activation, but not Th1 polarization which is mediated by endogenous IL-12p70+ XCR1+ migratory bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.
Platelet activation and aggregation at sites of vascular injury involves massive cytoskeletal re-organization, which is required for proper platelet function. Moreover, the cytoskeleton plays central roles in megakaryo- and thrombopoiesis. Thus, cytoskeletal protein aberrations can be the underlying reason for many pathological phenotypes. Although intensive research is carried out to identify the key players involved in cytoskeletal reorganization, the signaling cascades orchestrating these complex processes are still poorly understood. This thesis investigates the role of three actin-binding proteins, Coactosin-like (Cotl) 1, Profilin (Pfn) 1 and Thymosin (T) β4, in platelet formation and function using genetically modified mice.
ADF-H-containing proteins such as Twinfilin or Cofilin are well characterized as regulators of thrombopoesis and cytoskeletal reorganization. Although Cotl1 belongs to the ADF-H protein family, lack of Cotl1 did not affect platelet count or cytoskeletal dynamics. However, Cotl1-deficiency resulted in significant protection from arterial thrombus formation and ischemic stroke in vivo. Defective GPIb-vWF interactions and altered second wave mediator release present potential reasons for the beneficial effect of Cotl1-deficiency. These results reveal an unexpected function of Cotl1 as a regulator of thrombosis and hemostasis, establishing it as a potential target for a safe therapeutic therapy to prevent arterial thrombosis or ischemic stroke.
Recent studies showed that the organization of the circumferential actin cytoskeleton modulates calpain-mediated αIIbβ3 integrin closure, thereby also controlling αIIbβ3 integrin localization. The second part of this thesis identified the actin-sequestering protein Pfn1 as a central regulator of platelet integrin function as Pfn1-deficient platelets displayed almost abolished αIIbβ3 integrin signaling. This translated into a profound protection from arterial thrombus formation and prolonged tail bleeding times in vivo which was caused by enhanced calpain-dependent integrin closure. These findings further emphasize the importance of a functional actin cytoskeleton for intact platelet function in vitro and in vivo.
Tβ4 is a moonlighting protein, acting as one of the major actin-sequestering proteins in cells of higher eukaryotes and exerting various paracrine functions including anti-inflammatory, immunomodulatory and pro-angiogenic effects. Although excessively studied, its role for cytoskeletal dynamics, the distinction between endo- and exogenous protein function and its uptake and release mechanisms are still poorly understood. Constitutive Tβ4-deficiency resulted in thrombocytopenia accompanied by a largely diminished G-actin pool in platelets and divergent effects on platelet reactivity. Pre-incubation of platelets with recombinant Tβ4 will help to understand the function of endo- and exogenous protein, which is under current investigation.
Cellular therapies using chimeric antigen receptor (CAR) modified T cells to eradicate tumor cells have been a major breakthrough in the treatment of hematologic malignancies. However, there are no measures to control CAR T cell activity after infusion, which is mostly required in cases of CAR T cell overreaction, e.g. cytokine release syndrome, or in the case of T cell failure, e.g. caused by exhaustion.
In our study, we identified the tyrosine kinase inhibitor (TKI) dasatinib (© Sprycel) as a suitable agent to steer CAR T cells in vitro and in vivo. We show that single treatment of CD4+ and CD8+ CAR T cells with dasatinib conferred either partial or complete inhibition, depending on the applied concentration. The blockade was immediate and encompassed spe-cific lysis, cytokine secretion and proliferation following antigen encounter. The mechanism relied on reduced phosphorylation of key kinases in the CAR signaling cascade, which led to abrogation of nuclear factor of activated T-cells (NFAT) signaling. Importantly, inhibition was fully reversible by dasatinib withdrawal. In vivo, dasatinib blocked CAR T cell function without impairing the engraftment of CAR T cells or their subsequent anti tumor function once dasatinib administration was discontinued. We therefore introduce dasatinib as a new tool to efficiently block CAR T cells in vitro and in vivo, with data suggesting that dasatinib can be used in a clinical setting to mitigate toxicity after adaptive transfer of CAR modified T cells and other forms of T cell based immunotherapy.
Additionally we show that intermittent inhibition of CAR T cells by dasatinib im-proves the efficacy of CAR T cell therapy. By pausing T cells for short periods of time in vi-vo, upregulation of programmed death protein 1 (PD-1) and subsequent induction of exhaus-tion was prevented, which increased the expansion of T cells and the rate of tumor eradica-tion. Our data therefore suggest that dasatinib can additionally be used to overcome T cell exhaustion that is induced by massive tumor burden and upregulation of inhibitory receptors.
Hintergrund:
Eine Panikattacke beginnt typischerweise mit der Wahrnehmung einer physiologischen oder psychischen Veränderung, die von der Person als bedrohlich eingestuft wird. Während in klassischen neuroanatomischen Modellen der Panikstörung die Amygdala in der sich anschließenden aufschaukelnden Symptomatik in den Mittelpunkt gestellt wurde, erweitern aktuelle Studien dieses amygdalozentrische Bild und lenken die Aufmerksamkeit auf extratemporale neuronale Netzwerke. Dysfunktionen im neuronalen Aufmerksamkeitsnetzwerk, relevant für die Wahrnehmung und Regulierung exterozeptiver und interozeptiver Prozesse, könnten zur Entstehung einer Panikstörung beitragen. Weiterhin scheinen bestimmte Risikogenotypen für die Panikstörung wie z.B. im Adenosin Rezeptor 2A (ADORA2A) oder dem Neuropeptid S Rezeptor (NPSR1) Gen und die entsprechenden Neurotransmittersysteme in der Regulierung der Aufmerksamkeitsnetzwerke involviert zu sein.
Fragestellung:
Dysfunktionen im noradrenergen bottom-up Alertingnetzwerk und in der dopaminergen exekutiven top-down Aufmerksamkeitskontrolle könnten in einem neurokognitiven Entstehungsmodell der Panikstörung eine wichtige Rolle spielen. Mit Hilfe funktioneller Bildgebung soll die Funktion des neuronalen Aufmerksamkeitsnetzwerkes in einer nichtklinischen Stichprobe abhängig von genetischen Risikofaktoren und einer klinischen Stichprobe von und nach einer kognitiven Verhaltenstherapie untersucht werden.
Methoden:
Im nichtklinischen Teil der Untersuchung wurden in Studie 1 47 gesunde Versuchspersonen für die NPSR1 rs324981 Variante stratifiziert rekrutiert. Mittels fMRT wurde die Aktivität des Alertingnetzwerks und des Executive Control Netzwerks auf neuronaler Ebene mit dem Attentional Networt Test (ANT) untersucht. In Studie 2 wurde bei N=65 Versuchspersonen stratifiziert für die ADORA2A rs5751876 Variante als zusätzliches Verhaltensmaß die Fähigkeit zur interozeptiven Wahrnehmung in Bezug zur Konnektivität im insulären Ruhenetzwerk untersucht. Im klinischen Teil der Untersuchung (Studie 3) wurden 44 Patienten mit Panikstörung sowie eine entsprechend große und gematchte Kontrollgruppe rekrutiert. Es wurden fMRT Ruhemessungen vor und nach Abschluss einer kognitiven Verhaltenstherapie erhoben. Als zusätzliches Verhaltensmaß wurde die selbstberichtete Aufmerksamkeitskontrolle zwischen der Patienten- und der Kontrollgruppe verglichen.
Ergebnisse:
Träger des NPSR1 TT und des ADORA2A TT Risikogenotyps für Angst und Angsterkrankungen zeigten eine erhöhte Aktivität in Teilen des Alertingnetzwerks. Die Aktivität im Executive Control Netzwerk war arealabhängig teilweise erhöht, teilweise reduziert. Innerhalb eines interozeptiven Netzwerks zeigten Träger des ADORA2A TT Genotyps Hinweise auf eine dysfunktionale fronto-striatale-insuläre Interaktion. Im klinischen Teil der Studie zeigten Patienten mit Panikstörung eine reduzierte Konnektivität des dorsolateralen Präfrontalkortex (dlPFC) im fronto-parietalen Aufmerksamkeitsnetzwerk. Die Konnektivität innerhalb dieses Netzwerks korrelierte mit Defiziten in selbstberichteter Aufmerksamkeitskontrolle bei Patienten mit Panikstörung. Nach Abschluss der Therapie zeigte sich bei Patienten, die von der Therapie profitiert hatten, wieder eine Zunahme oder Verbesserung der Konnektivität mit dem dlPFC.
Schlussfolgerung:
Die Ergebnisse dieser Untersuchung betonen die Rolle dysfunktionaler interozeptiver und exterozeptiver Aufmerksamkeitsnetzwerke in der Entstehung von Angsterkrankungen. Bei Patienten mit Panikstörung sowie gesunden Versuchspersonen mit bestimmten prädisponierenden genetischen Variationen scheint eine Dysbalance des neuronalen Aufmerksamkeitsnetzwerks bzgl. der Abstimmung von bottom-up und top-down Netzwerken vorzuliegen.
This thesis aims for a better understanding of the mechanisms underlying anxiety as well as trauma- and stressor-related disorders and the development of new therapeutic approaches. I was first interested in the associative learning mechanisms involved in the etiology of anxiety disorders. Second, I explored the therapeutic effects of transcutaneous vagus nerve stimulation (tVNS) as a promising new method to accelerate and stabilize extinction learning in humans.
For these purposes, I applied differential anxiety conditioning protocols realized by the implementation of virtual reality (VR). Here, a formerly neutral virtual context (anxiety context, CTX+) is presented whereby the participants unpredictably receive mildly aversive electric stimuli (unconditioned stimulus, US). Another virtual context (safety context, CTX-) is never associated with the US. Moreover, extinction of conditioned anxiety can be modeled by presenting the same contexts without US delivery. When unannounced USs were administered after extinction, i.e. reinstatement, the strength of the “returned” conditioned anxiety can provide information on the stability of the extinction memory.
In Study 1, I disentangled the role of elemental and conjunctive context representations in the acquisition of conditioned anxiety. Sequential screenshots of two virtual offices were presented like a flip-book so that I elicited the impression of walking through the contexts. Some pictures of CTX+ were paired with an US (threat elements), but not some other screenshots of the same context (non-threat elements), nor the screenshots depicting CTX- (safety elements). Higher contingency ratings for threat compared to non-threat elements revealed elemental representation. Electro-cortical responses showed larger P100 and early posterior negativity amplitudes elicited by screenshots depicting CTX+ compared to CTX- and suggested conjunctive representation. These results support the dual context representation in anxiety acquisition in healthy individuals.
Study 2 addressed the effects of tVNS on the stabilization of extinction learning by using a context conditioning paradigm. Potentiated startle responses as well as higher aversive ratings in CTX+ compared to CTX- indicate successful anxiety conditioning. Complete extinction was found in startle responses and valence ratings as no differentiation between CTX+ and CTX- suggested. TVNS did not affect extinction or reinstatement of anxiety which may be related to the inappropriate transferability of successful stimulation parameters from epilepsy patients to healthy participants during anxiety extinction.
Therefore, in Study 3 I wanted to replicate the modulatory effects of tVNS on heart rate and pain perception by the previously used parameters. However, no effects of tVNS were observed on subjective pain ratings, on pain tolerance, or on heart rate. This led to the conclusion that the modification of stimulation parameters is necessary for a successful acceleration of anxiety extinction in humans.
In Study 4, I prolonged the tVNS and, considering previous tVNS studies, I applied a cue conditioning paradigm in VR. Therefore, during acquisition a cue (CS+) presented in CTX+ predicted the US, but not another cue (CS-). Both cues were presented in a second context (CTX-) and never paired with the US. Afterward, participants received either tVNS or sham stimulation and underwent extinction learning. I found context-dependent cue conditioning only in valence ratings, which was indicated by lower valence for CS+ compared to CS- in CTX+, but no differential ratings in CTX-. Successful extinction was indicated by equal responses to CS+ and CS-. Interestingly, I found reinstatement of conditioned fear in a context-dependent manner, meaning startle response was potentiated for CS+ compared to CS- only in the anxiety context. Importantly, even the prolonged tVNS had no effect, neither on extinction nor on reinstatement of context-dependent cue conditioning. However, I found first evidence for accelerated physiological contextual extinction due to less differentiation between startles in CTX+ compared to CTX- in the tVNS than in the sham stimulated group.
In sum, this thesis first confirms the dual representation of a context in an elemental and a conjunctive manner. Second, though anxiety conditioning and context-dependent cue conditioning paradigms worked well, the translation of tVNS accelerated extinction from rats to humans needs to be further developed, especially the stimulation parameters. Nevertheless, tVNS remains a very promising approach of memory enhancement, which can be particularly auspicious in clinical settings.
The Role of Attentional Control and Fear Acquisition and Generalization in Social Anxiety Disorder
(2020)
Although Social Anxiety Disorder (SAD) is one of the most prevalent mental disorders, still little is known about its development and maintenance. Cognitive models assume that deviations in attentional as well as associative learning processes play a role in the etiology of SAD. Amongst others, deficits in inhibitory attentional control as well as aberrations during fear generalization, which have already been observed in other anxiety disorders, are two candidate mechanisms that might contribute to the onset and retention of SAD. However, a review of the literature shows that there is a lack of research relating to these topics. Thus, the aim of the present thesis was to examine in which way individuals with SAD differ from healthy controls regarding attentional control and generalization of acquired fear during the processing of social stimuli.
Study 1 tested whether impairment in the inhibitory control of attention is a feature of SAD, and how it might be influenced by emotional expression and gaze direction of an interactional partner. For this purpose, individuals with SAD and healthy controls (HC) participated in an antisaccade task with faces displaying different emotional expressions (angry, neutral and happy) and gaze directions (direct and averted) serving as target stimuli. While the participants performed either pro- or antisaccades in response to the peripherally presented faces, their gaze behavior was recorded via eye-tracking, and ratings of valence and arousal were obtained. Results revealed that both groups showed prolonged latencies and increased error rates in trials with correct anti- compared to prosaccades. However, there were no differences between groups with regard to response latency or error rates, indicating that SAD patients did not exhibit impairment on inhibitory attentional control in comparison to HC during eye-tracking. Possible explanations for this finding could be that reduced inhibitory attentional control in SAD only occurs under certain circumstances, for example, when these individuals currently run the risk of being negatively evaluated by others and not in the mere presence of phobic stimuli, or when the cognitive load of a task is so high that it cannot be unwound by compensatory strategies, such as putting more effort into a task.
As not only deviations in attentional, but also associative learning processes might be pathogenic markers of SAD, these mechanisms were further addressed in the following experiments. Study 2 is the first that attempted to investigate the generalization of conditioned fear in patients with SAD. To this end, patients with SAD and HC were conditioned to two neutral female faces serving as conditioned stimuli (CS+: reinforced; CS-: non-reinforced) and a fearful face paired with a loud scream serving as unconditioned stimulus (US). Fear generalization was tested by presenting morphs of the two faces (GS: generalization stimuli), which varied in their similarity to the original faces. During the whole experiment, self-report ratings, heart rate (HR) and skin conductance responses (SCR) were recorded. Results demonstrated that SAD patients rated all stimuli as less pleasant and more arousing, and overestimated the occurrence of the US compared to HC, indicating a general hyperarousal in individuals with SAD. In addition, ratings and SCR indicated that both groups generalized their acquired fear from the CS+ to intermediate GSs as a function of their similarity to the CS+. However, except for the HR data, which indicated that only SAD patients but not HC displayed a generalization response in this measure, most of the results did not support the hypothesis that SAD is characterized by overgeneralization. A plausible reason for this finding could be that overgeneralization is just a key characteristic of some anxiety disorders and SAD is not one of them. Still, other factors, such as comorbidities in the individuals with SAD, could also have had an influence on the results, which is why overgeneralization was further examined in study 3.
The aim of study 3 was to investigate fear generalization on a neuronal level. Hence, high (HSA) and low socially anxious participants (LSA) underwent a conditioning paradigm, which was an adaption of the experimental design used study 2 for EEG. During the experiment, steady-state visually evoked potentials (ssVEPs) and ratings of valence and arousal were recorded. Analyses revealed significant generalization gradients in all ratings with highest fear responses to the CS+ and a progressive decline of these reactions with increasing similarity to the CS-. In contrast, the generalization gradient on a neuronal level showed highest amplitudes for the CS+ and a reduction in amplitude to the most proximal, but not distal GSs in the ssVEP signal, which might be interpreted as lateral inhibition in the visual cortex. The observed dissociation among explicit and implicit measures points to different functions of behavioral and sensory cortical processes during fear generalization: While the ratings might reflect an individual’s consciously increased readiness to react to threat, the lateral inhibition pattern in the occipital cortex might serve to maximize the contrast among stimuli with and without affective value and thereby improve adaptive behavior. As no group differences could be observed, the finding of study 2 that overgeneralization does not seem to be a marker of SAD is further consolidated.
In sum, the conducted experiments suggest that individuals with SAD are characterized by a general hyperarousal during the exposition to disorder-relevant stimuli as indicated by enhanced arousal and reduced valence ratings of the stimuli compared to HC. However, the hypotheses that reduced inhibitory attentional control and overgeneralization of conditioned fear are markers of SAD were mostly not confirmed. Further research is required to elucidate whether they only occur under certain circumstances, such as high cognitive load (e.g. handling two tasks simultaneously) or social stress (e.g. before giving a speech), or whether they are not characteristics of SAD at all. With the help of these findings, new interventions for the treatment of SAD can be developed, such as attentional bias modification or discrimination learning.
People who suffer Social Anxiety Disorder (SAD) are under substantial personal distress and endure impaired normal functioning in at least some parts of everyday life. Next, to the personal suffering, there are also the immense public health costs to consider, as SAD is the most common anxiety disorder and thereby one of the major psychiatric disorders in general. Over the last years, fundamental research found cognitive factors as essential components in the development and maintenance of social fears. Following leading cognitive models, avoidance behaviors are thought to be an important factor in maintaining the developed social anxieties. Therefore, this thesis aims to deepen the knowledge of avoidance behaviors exhibited in social anxiety, which allows to get a better understanding of how SAD is maintained.
To reach this goal three studies were conducted, each using a different research approach. In the first study cutting-edge Virtual Reality (VR) equipment was used to immerse participants in a virtual environment. In this virtual setting, High Socially Anxious (HSA) individuals and matched controls had to execute a social Approach-Avoidance Task (AAT). In the task, participants had to pass a virtual person displaying neutral or angry facial expressions. By using a highly immersive VR apparatus, the first described study took the initial step in establishing a new VR task for the implicit research on social approach-avoidance behaviors. By moving freely through a VR environment, participants experienced near real-life social situations. By tracking body and head movements, physical and attentional approach-avoidance processes were studied.
The second study looked at differences in attention shifts initiated by gaze-cues of neutral or emotional faces. Comparing HSA and controls, enabled a closer look at attention re-allocation with special focus on social stimuli. Further, context conditioning was used to compare task performance in a safe and in a threatening environment. Next to behavioral performance, the study also investigated neural activity using Electroencephalography (EEG) primarily looking at the N2pc component.
In the third study, eye movements of HSA and Low Socially Anxious (LSA) were analyzed using an eye-tracking apparatus while participants executed a computer task. The participants’ tasks consisted of the detection of either social or non-social stimuli in complex visual settings. The study intended to compare attention shifts towards social components between these two tasks and how high levels of social anxiety influence them. In other words, the measurements of eye movements enabled the investigation to what extent social attention is task-dependent and how it is influenced by social anxiety.
With the three described studies, three different approaches were used to get an in-depth understanding of what avoidance behaviors in SAD are and to which extent they are exhibited. Overall, the results showed that HSA individuals exhibited exaggerated physical and attentional avoidance behavior. Furthermore, the results highlighted that the task profoundly influences attention allocation. Finally, all evidence indicates that avoidance behaviors in SAD are exceedingly complex. They are not merely based on the fear of a particular stimulus, but rather involve highly compound cognitive processes, which surpass the simple avoidance of threatening stimuli. To conclude, it is essential that further research is conducted with special focus on SAD, its maintaining factors, and the influence of the chosen research task and method.
Recently, it was shown that MDA-MB-231 breast cancer cells express very high levels of the A2BAR as the sole adenosine receptor subtype, and stimulation of the A2BAR in MDA-MB-231 cells triggers an unusual inhibitory signal on ERK1/2 phosphorylation. The ERK1/2 pathway is reported to be associated with the control of growth, proliferation and differentiation of cells and as such might serve as a promising target for tumor treatment. The present study investigated signaling mechanisms involved in linking A2BAR to ERK1/2 phosphorylation in MDA-MB-231 cells. The A2BAR mediated reduction of ERK1/2 phosphorylation and of proliferation of MDA-MB-231 cell is in good agreement with previous results from (Dubey et al., 2005). These observations provide support to the hypothesis that activation of A2BAR could attenuate the growth of some types of cancer cell and argue against a stimulation of proliferation resulting from the activation of A2BAR as discussed by (Fernandez-Gallardo et al., 2016). AC activation by forskolin has recently been shown to enhance the activity of the chemotherapeutic agent doxorubicin in TNBC cells via a mechanism dependent on the PKA-mediated inhibition of ERK1/2 phosphorylation. Furthermore, forskolin also increased the sensitivity of MDA-MB-231 and MDA-MB-468 triple negative breast cancer cells to 5-fluorouracil and taxol (Illiano et al., 2018), and sustains the evidence of anticancer activity mediated by cAMP/PKA-mediated ERK1/2 inhibition. Similar to these studies, a reduced amount of pERK1/2 was also observed after stimulation of AC with FSK, application of cAMP-AM or inhibition of PDE-4. The inhibition of ERK1/2 phosphorylation was mimicked by UTP and abolished with the PLC inhibitor U73122 or by chelating intracellular Ca2+ with BAPTA-AM. These results point to an important role for both cAMP and Ca2+ signaling in the pathway leading to a decrease in ERK1/2 phosphorylation. This study encourages the idea that A2BAR could be used as target in cancer therapy. But A2BAR did not only stimulate signaling cascades associated with cell survival and proliferation reduction, but also key phases relevant in angiogenesis like Ca2+ mobilization (Kohn et al., 1995). Whereas the potency toward AC and Ca2+ are similar for the diverse agonists, the potency to promote ERK1/2 reduction is much higher. Interestingly, the proliferation of MDA-MB-231 cells is inhibited by low nanomolar agonist concentration which is inactive in Ca2+ mobilization. This means that it is certainly possible to reduce the proliferation without promoting angiogenesis. LUF6210 is particularly interesting when considering that it preferentially stimulates a reduction in ERK1/2 phosphorylation over Ca2+ and therefore may not promote angiogenesis. LUF6210 is therapeutically appealing as adjuvant in treatment of cancer. Given that stimulation of AC can activate a reduction of ERK1/2 phosphorylation and proliferation in cancer cells, agonist bias toward Gs-AC-PKA-mediated ERK1/2 inhibition represent a potential therapy of various malignancies. The fact that the reduction of ERK1/2 phosphorylation followed by reduced proliferation observed in MDA-MB-231 cells were mediated by the activation of the A2BAR illustrates the importance of this receptor subtype in cancer. A2BARs must be considered as a key factor in cancer treatment and deserve attention for the development of new therapeutic strategies.
Potential evolutionary responses to landscape heterogeneity and systematic environmental trends
(2020)
Over the course of the last century, humans have witnessed drastic levels of global environmental change that endangered both, the survival of single species as well as biodiversity itself. This includes climate change, in both environmental means and in variance and subsequently frequent extreme weather events, as well as land use change that species have to cope with.
With increasing urbanization, increasing agricultural area and increasing intensification, natural habitat is not only lost, but also changes its shape and distribution in the landscape. Both aspects can heavily influence an individual's fitness and therefore act as a selective force promoting evolutionary change.
This way climate change influences individuals' niches and dispersal. Local adaptation and dispersal are not independent of each other. Dispersal can have two opposite effects on local adaptation. It can oppose local adaptation, by promoting the immigration of maladapted indi-
viduals or favor local adaptation by introducing better adapted genotypes. Which of those effects of dispersal on local adaptation emerges in a population depends on the dispersal strategies and the spatial structure of the landscape. In principle an adaptive response can include adjustment of the niche optimum as well as habitat tolerance (niche width) or (instead) ecological tracking of adequate conditions by dispersal and range shifting. So
far, there has been no extensive modeling study of the evolution of the environmental niche optimum and tolerance along with dispersal probability in complex landscapes. Either only dispersal or (part of ) the environmental niche can evolve or the landscapes used are not realistic but rather a very abstract representation of spatial structures.
I want to try and disentangle those different effects of both local adaptation and dispersal during global change, as well as their interaction, especially considering the separation between the effects of increasing mean and increasing variance. For this, I implemented an individual based model (IBM), with escalating complexity.
I showed that both on a temporal as well as on a spatial scale, variation can be more influential then mean conditions.
Indeed, the actual spatial configuration of this heterogeneity and the relationship between spatial and temporal heterogeneity affect the evolution of the niche and of dispersal probability more than temporal or spatial mean conditions. I could show that in isolated populations, an increase of an environmental attribute's mean or variance can lead to extinction, under certain conditions. In particular, increasing variance cannot be tracked forever, since increasing tolerance has distinct limits of feasibility. Increasing mean conditions can also occur too fast to be tracked, especially from generalist individuals. When expanding the model to the metapopulation level without a temporal environmental trend, the degree of spatial vs.temporal heterogeneity influenced the evolution of random dispersal heavily. With increasing spatial heterogeneity, individuals from extreme and rare patches
evolve from being philopatric to dispersive, while individuals from average patches switch in the opposite direction.
With the last expansion to a different set of landscapes with varying degrees of edge density, I could show that edge effects are strong in pseudo-agricultural landscapes, while
in pseudo-natural habitats they were hardly found, regardless of emigration strategy. Sharp edges select against dispersal in the edge patches and could potentially further isolate populations in agricultural landscapes.
The work I present here can also be expanded further and I present several suggestions on what to do next. These expansions could help the realism of the model and eventually shed light on its bearing on ecological global change predictions. For example species distribution models or extinction risk models would be more precise, if they included both spatial and temporal variation. The current modeling practices might not be suffcient to
describe the possible outcomes of global change, because spatio-temporal heterogeneity and its influence on species' niches is too important to be ignored for longer.
Parkinson’s disease (PD) is among the most common neurodegenerative conditions, and it is characterized by the progressive loss of dopaminergic neurons and a great variability in clinical expression. Despite several effective medications, it still causes disability as all patients show treatment-resistant symptoms and complications.
A possible reason for this therapeutic-burden and great clinical variability lies in a probable misconception about its pathophysiology, one that focuses on neurodegeneration, while largely neglecting its functional consequences and the related compensatory changes. In this thesis, I expand on the hypothesis that some PD symptoms have a dysfunctional origin and reflect derangements of neural network dynamics, the means by which brain coordination supports any motor behaviour. In particular, I have investigated resting tremor and freezing of gait, two common symptoms with an enigmatic mechanism and suboptimal management.
In the case of tremor, I predicted a pathological change in response to dopamine loss, which included the activation of noradrenergic (NA) neurons of the locus coeruleus (LC) projecting to the cerebellum. This compensatory LC activation that supports dopaminergic neurons might indeed come at the expense of tremor development. To assess the role of LC-NA in tremor development, I recorded tremor occurrence in the reserpinized rat model of PD, one of very few showing tremor, after selective lesioning (with the neurotoxin DSP-4) of the LC-NA terminal axons. DSP-4 induced a severe reduction of LC-NA terminal axons in the cerebellar cortex and this was associated with a significant reduction in tremor development. Unlike its development, tremor frequency and the akinetic rigid signs did not differ between the groups, thus suggesting a dopaminergic dependency. These findings suggest that the LC-NA innervation of the cerebellum has a critical role for PD tremor, possibly by exerting a network effect, which gates the cerebello-thalamic-cortical circuit into pathological oscillations upon a dopaminergic loss in the basal ganglia.
In contrast, for the study of freezing of gait, I worked with human PD subjects and deep brain stimulation, a therapeutic neuromodulation device that in some prototypes also allows the recording of neural activity in freely-moving subjects. Gait freezing is a disabling PD symptom that suddenly impairs effective stepping, thus causing falls and disability. Also in this study, I hypothesized that the underlying pathophysiology may be represented by dysfunctional neural network dynamics that abruptly impair locomotor control by affecting the communication in the supraspinal locomotor network. To test this hypothesis, I investigated the coupling between the cortex and the subthalamic nucleus, two main nodes of the supraspinal locomotor network, in freely-moving subjects PD patients and also performed molecular brain imaging of striatal dopamine receptor density and kinematic measurements. I found that in PD patients, walking is associated with cortical-subthalamic stable coupling in a low-frequency band (i.e. θ-α rhythms). In contrast, these structures decoupled when gait freezing occurred in the brain hemisphere with less dopaminergic innervation. These findings suggest that freezing of gait is a “circuitopathy”, with dysfunctional cortical-subcortical communication.
Altogether the results of my experiments support the hypothesis that the pathophysiology of PD goes beyond neurodegenerative (loss-of-function) processes and that derangement of neural network dynamics coincides with some disabling PD symptoms, thus suggesting that PD can be interpreted as the combination of multiple circuitopathies.
Gastrointestinal infections account for high morbidity and mortality in humans every year across the globe. The increasing emergence of antibiotic resistance among the gastrointestinal pathogens and the induction of virulence factors by antibiotics makes it highly risky to only depend on antibiotic therapy for intestinal infections. Most of these infections are associated with an imbalance in the gut microbial population whereas the restoration of the balance with probiotic supplements can result in an improvement of the health condition. Probiotics are therefore considered as successful support in the treatment of gastrointestinal infections.
E. coli Nissle 1917 (EcN) is the active component of the probiotic medication Mutaflor® and has been used in the treatment of various gastrointestinal disorders for more than 100 years. Several studies have reported antagonistic effects of EcN against enterohemorrhagic E. coli (EHEC) in vitro and in vivo. However, detailed investigations on the probiotic mechanisms and safety aspects of EcN are a pre-requisite, for administering EcN to treat EHEC infected patients or to use EcN as a prophylactic for the patient’s close contacts.
In this regard, the first part of the study aimed to understand the nature and behaviour of EcN in the presence of pathogenic or non-pathogenic E. coli strains. Transcriptomic analysis was deployed to this end. We investigated the changes in EcN’s transcriptome after different time points of coculture with the EHEC strain EDL933 or the K-12 strain MG1655. The transcriptome data reported a strain-specific response in EcN at all the investigated time points (3 h, 5 h, 7 h and 8 h) of coincubation. The alterations in gene regulation of EcN were highly pronounced in initial timepoints (3 h and 5 h) of coincubation with EDL933, which gradually decreased over time. In the presence of MG1655, the alterations were strongly differentially regulated only at later time points (7 h and 8 h). The unique transcriptional response of EcN towards two different E. coli strains, that are genetically more than 98 % identical, was startling.
12
More importantly, this can be considered as a beneficial trait of EcN over a chemical-pharmaceutical preparation like an antibiotic that might act identically on all target cells.
Bacteriophages are one of the most abundant members of gut microbiota. On the one hand, the infection of a probiotic strain by a lysogenic phage could transfer genetic material coding for pathogenic factors or antibiotic resistance into an otherwise beneficial probiotic bacterium and thereby converting it into a virulent pathogenic bacterium. On the other hand, infection by a lytic phage could result in bacterial lysis and prevent the bacterium from exerting its probiotic effect. Thus, in order to successfully establish and colonise the gut, it is crucial for any probiotic to be resistant against phage infections. To address this, in the second part of the study, we investigated the phage resistance of EcN towards the lysogenic lambda and the lytic T4 phage.
EcN showed complete resistance against tested phages and was also able to inactivate these phages upon coincubation. In the case of lambda phages, the resistance was attributed to the presence of a lambdoid prophage (prophage 3) in the genome of EcN. In addition, the overexpression of one of the early genes of EcN’s prophage 3 (i.e. phage repressor gene pr) in the phage sensitive MG1655 conferred partial protection against lambda phage infection. Moreover, the inactivation was mediated by binding of lambda phages to its receptor LamB. Experiments with lytic T4 phages revealed that the EcN’s K5 polysaccharide capsule was crucial for its T4 phage resistance, while its lipopolysaccharide (LPS) inactivated the T4 phages. Apart from protecting itself, EcN displayed even a protective role for the tested K-12 strains, by interfering with the lysogeny and lysis by these phages.
In summary, this work highlights two novel positive traits of the probiotic strain EcN: i) the strain-specific response that was evident from the global transcriptome analysis of EcN when incubated with other E. coli strains, and ii) lytic and lysogenic phage resistance. Both these traits are additional safety aspects for a well-characterised probiotic strain and encourage its application in therapeutics.
Nicotinamide N-methyltransferase (NNMT) is a new regulator of energy homeostasis. Its expression is increased in models of obesity and diabetes. An enhanced NNMT level is also caused by an adipose tissue-specific knockout of glucose transporter type 4 (GLUT4) in mice, whereas the overexpression of this glucose transporter reduced the NNMT expression. Furthermore, the knockdown of the enzyme prevents mice from diet-induced obesity (DIO) and the recently developed small molecule inhibitors for NNMT reverses the DIO. These previous findings demonstrated the exclusive role of NNMT in adipose tissue and further make it to a promising target in obesity treatment. However, the regulation mechanism of this methyltransferase is not yet clarified.
The first part of the thesis focus on the investigation whether pro-inflammatory signals are responsible for the enhanced NNMT expression in obese adipose tissue because a hallmark of this tissue is a low-level chronic inflammation. Indeed, the NNMT mRNA in our study was elevated in obese patients compared with the control group, whereas the GLUT4 mRNA expression does not differ between lean and obese humans. To analyze whether pro inflammatory signals, like interleukin (IL 6) and tumor necrosis factor α (TNF-α), regulate NNMT expression 3T3-L1 adipocytes were treated with these cytokines. However, IL 6, TNF α, and leptin, which is an alternative activator of the JAK/STAT pathway, did not affect the NNMT protein or mRNA level in differentiated 3T3-L1 adipocytes. The mRNA and protein levels were measured by quantitative polymerase chain reaction (qPCR) and western blotting.
In the second part of this study, 3T3-L1 adipocytes were cultivated with varying glucose concentrations to show whether NNMT expression depends on glucose availability. Further studies with activators and inhibitors of AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) signaling pathways were used to elucidate the regulation mechanism of the enzyme.
The glucose deprivation of differentiated 3T3-L1 adipocytes led to a 2-fold increase in NNMT expression. This effect was confirmed by the inhibition of the glucose transports with phloretin as well as the inhibition of glycolysis with 2-deoxyglucose (2-DG). AMPK serves as an intracellular energy sensor and the pharmacological activation of it enhanced the NNMT expression. This increase was also caused by the inhibition of mTOR. Conversely, the activation of mTOR using MHY1485 prevented the effect of glucose deprivation on NNMT. Furthermore, the NNMT up-regulation was also blocked by the different autophagy inhibitors.
Taken together, NNMT plays a critical role in autophagy in adipocytes, because an inhibition of this process prevented the augmented NNMT expression during glucose starvation. Moreover, the effect on NNMT protein and mRNA level depends on AMPK and mTOR. However, pro-inflammatory signals did not affect the expression. Further in vivo studies have to clarify whether AMPK activation and mTOR inhibition as well as autophagy are responsible for the increased NNMT levels in obese adipose tissue. In future this methyltransferase emerges as an awesome therapeutic target for obesity.
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in early
developmental processes such as cell proliferation, migration, and differentiation.
Recent research in humans showed that the brain 5-HT system and CDH13 are
interlinked in the genetics of neurodevelopmental disorders including attention-
deficit/hyperactivity disorder and autism spectrum disorder (Lesch et al., 2008;
Neale et al., 2008; Neale, Medland, Ripke, Anney, et al., 2010; Neale, Medland,
Ripke, Asherson, et al., 2010; Sanders et al., 2011; Sanders et al., 2015; Zhou et
al., 2008). This study introduces Cadherin-13 (CDH13), a cell adhesion protein, as
a contributor to the development and function of the 5-HT system. Our
experiments show that the absence of CDH13 increases the density of 5-HT
neurons in the developing dorsal raphe (DR) and increases the 5-HT innervation
of the prefrontal cortex in mouse embryonic stages. CDH13 is also observed in
radial glial cells, an important progenitor cell type linked to neuronal migration.
A three-dimensional reconstruction carried out with super-resolution microscopy,
identifies 5-HT neurons intertwined with radial glial cells, and CDH13 clusters at
contact points between these cells. This indicates a potential contribution of
CDH13 to the migration of DR 5-HT neurons. As CDH13 is strongly expressed in
5-HT neurons, we asked whether the selective deletion of CDH13 from these cells
is sufficient to generate the alterations observed in the Cdh13 constitutive
knockout mouse line.
In 5-HT conditional Cdh13 knockout mice (Cdh13 cKO) an increase in DR 5-HT
neurons in the embryonic and adult brains is observed, as well as 5-HT
hyperinnervation of cortical regions. Therefore, illustrating that the lack of CDH13
from 5-HT neurons alone impacts DR formation and serotonergic innervation.
Behavioral testing conducted on Cdh13 cKO mice showed delayed learning in
visuospatial learning and memory processing, as well as, changes in sociability
parameters. To find out how CDH13 localizes in human 5-HT neurons, CDH13 was
visualized in neurons that derived from human induced pluripotent stem cells
(iPSC). Super-resolution microscopy confirmed CDH13 expression in a subgroup
of induced human neurons positive for typical hallmarks of 5-HT neurons, such as
expression of Tph2, the neuron-specific tryptophan hydroxylase, and synaptic
structures. In summary, the work included in this thesis presents a detailed
analysis of CDH13 expression and localization in the 5-HT system and shows that
deletion of CDH13 from 5-HT neurons affects specific higher-order functions of the
brain.
The human body is colonized by trillions of microbes from all three domains of life – eukaryotes, bacteria and archaea. The lower gastrointestinal tract is the most densely colonized part of the body, harbouring a diverse and dynamic community of microbes. While the importance of bacteria in this so-called microbiota is well acknowledged, the role of commensal fungi remains underexplored. The most prominent fungus of the human gastrointestinal microbiota is Candida albicans. This fungus occasionally causes life-threatening disseminated infections in individuals with debilitated immune defences. It is this “pathogenic” facet that has received the most attention from researchers in the past, leaving many aspects of its “commensal” lifestyle understudied. Using gnotobiotic mice as a model system to explore the biology of C. albicans in the mammalian gut, in this dissertation I establish the global response of the host to C. albicans monocolonization as well as the spatial distribution of the fungus in the intestine in the context of co-colonization with single gut bacterial species. The fungus elicited transcriptome changes in murine intestinal tissue, which included the activation of a reactive oxygen species-related defence mechanism and the induction of regulators of the circadian clock circuitry. Both responses have previously been described in the context of a complete bacterial microbiota. Imaging the intestine of animals monocolonized with the fungus or co-colonized with C. albicans and the gut bacteria Bacteroides thetaiotaomicron or Lactobacillus reuteri revealed that the fungus was embedded in a B. thetaiotaomicron-promoted outer mucus layer in the murine colon. The gel-like outer mucus constitutes a unique microhabitat, distinct in microbial composition from the adjacent intestinal lumen. This finding indicates that bacteria can shape the specific microhabitat occupied by the fungus in the intestine. Overall, the results described in this dissertation suggest that gnotobiotic mice constitute a valuable tool to dissect multiple aspects of the interactions among host, commensal fungi and cohabiting bacteria.
Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively renders tumor vessels highly permeable, causing massive intratumoral hemorrhage. While these results establish platelets as potential targets for anti-tumor therapy, depletion is not a treatment option due to the essential role of platelets for hemostasis. This thesis demonstrates for the first time that functional inhibition of glycoprotein (GP) VI on the platelet surface rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion but without inducing systemic bleeding complications. Both, the intratumoral bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, GPVI inhibition increased intra-tumoral accumulation of co-administered chemotherapeutic agents, thereby resulting in a profound anti-tumor effect. In summary, this thesis manifests platelet GPVI as a key regulator of vascular integrity specifically in growing tumors, serving as a potential basis for the development of anti-tumor strategies.
In the second part of this thesis, light is shed on the modulating role of bridging integrator 2 (BIN2) in platelet Ca2+ signaling. Stromal interaction molecule 1 (STIM1) mediated store-operated calcium entry (SOCE) is the major route of Ca2+ influx in platelets, triggered by inositol trisphosphate receptor (IP3R)-dependent Ca2+ store release. In this thesis, the BAR domain superfamily member BIN2 was identified as the first Ca2+ signaling modulator, interacting with both, STIM1 and IP3R in platelets. Deletion of BIN2 resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. These results establish BIN2 as a central regulator of platelet Ca2+ signaling.
The third part of this thesis focuses on the effect of the soluble neuronal guidance protein Sema7A on platelet function. Rosenberger et al. discovered that Sema7A cleavage from red blood cells increases the formation of platelet-neutrophil complexes, thereby reinforcing thrombo-inflammation in myocardial ischemia-reperfusion injury (MIRI). This thesis establishes soluble Sema7A as a stimulator of platelet thrombus formation via its interaction with platelet GPIbα, thereby reinforcing PNC formation. Thus, interfering with the GPIb-Sema7A interaction during MIRI represents a potential strategy to reduce cardiac damage and improve clinical outcome following MI.
Sharpening super-resolution by single molecule localization microscopy in front of a tuned mirror
(2020)
The „Resolution Revolution" in fluorescence microscopy over the last decade has given rise to a variety of techniques that allow imaging beyond the diffraction limit with a resolution power down into the nanometer range. With this, the field of so-called super-resolution microscopy was born. It allows to visualize cellular architecture at a molecular level and thereby achieve a resolution level that had been previously only accessible by electron microscopy approaches.
One of these promising techniques is single molecule localization microscopy (SMLM) in its most varied forms such as direct stochastic optical reconstruction microscopy (dSTORM) which are based on the temporal separation of the emission of individual fluorophores. Localization analysis of the subsequently taken images of single emitters eventually allows to reconstruct an image containing super-resolution information down to typically 20 nm in a cellular setting. The key point here is the localization precision, which mainly depends on the image contrast generated the by the individual fluorophore’s emission. Thus, measures to enhance the signal intensity or reduce the signal background allow to increase the image resolution achieved by dSTORM. In my thesis, this is achieved by simply adding a reflective metal-dielectric nano-coating to the microscopy coverslip that serves as a tunable nano-mirror.
I have demonstrated that such metal-dielectric coatings provide higher photon yield at lower background and thus substantially improve SMLM performance by a significantly increased localization precision, and thus ultimately higher image resolution. The strength of this approach is that ─ except for the coated cover glass ─ no specialized setup is required. The biocompatible metal-dielectric nano-coatings are fabricated directly on microscopy coverslips and have a simple three-ply design permitting straightforward implementation into a conventional fluorescence microscope. The introduced improved lateral resolution with such mirror-enhanced STORM (meSTORM) not only allows to exceed Widefield and Total Internal Reflection Fluorescence (TIRF) dSTORM performance, but also offers the possibility to measure in a simplified setup as it does not require a special TIRF objective lens.
The resolution improvement achieved with meSTORM is both spectrally and spatially tunable and thus allows for dual-color approaches on the one hand, and selectively highlighting region above the cover glass on the other hand, as demonstrated here.
Beyond lateral resolution enhancement, the clear-cut profile of the highlighted region provides additional access to the axial dimension. As shown in my thesis, this allows for example to assess the three-dimensional architecture of the intracellular microtubule network by translating the local localization uncertainty to a relative axial position. Even beyond meSTORM, a wide range of membrane or surface imaging applications may benefit from the selective highlighting and fluorescence enhancing provided by the metal-dielectric nano-coatings. This includes for example, among others, live-cell Fluorescence Correlation Spectroscopy and Fluorescence Resonance Energy Transfer studies as recently demonstrated.
Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen. It causes blinding trachoma and sexually transmitted disease such as chlamydia, pelvic inflammatory disease and lymphogranuloma venereum. Ct has a unique biphasic development cycle and replicates in an intracellular vacuole called inclusion. Normally it has two forms: the infectious form, elementary body (EB); and the non-infectious form, reticulate body (RB). Ct is not easily amenable to genetic manipulation. Hence, to understand the infection process, it is crucial to study how the metabolic activity of Ct exactly evolves in the host cell and what roles of EB and RB play differentially in Ct metabolism during infection. In addition, Ct was found regularly coinfected with other pathogens in patients who got sexually transmitted diseases (STDs). A lack of powerful methods to culture Ct outside of the host cell makes the detailed molecular mechanisms of coinfection difficult to study.
In this work, a genome-scale metabolic model with 321 metabolites and 277 reactions was first reconstructed by me to study Ct metabolic adaptation in the host cell during infection. This model was calculated to yield 84 extreme pathways, and metabolic flux strength was then modelled regarding 20hpi, 40hpi and later based on a published proteomics dataset. Activities of key enzymes involved in target pathways were further validated by RT-qPCR in both HeLa229 and HUVEC cell lines. This study suggests that Ct's major active pathways involve glycolysis, gluconeogenesis, glycerolphospholipid biosynthesis and pentose phosphate pathway, while Ct's incomplete tricarboxylic acid cycle and fatty acid biosynthesis are less active. EB is more activated in almost all these carbohydrate pathways than RB. Result suggests the survival of Ct generally requires a lot of acetyl-CoA from the host. Besides, both EB and RB can utilize folate biosynthesis to generate NAD(P)H but may use different pathways depending on the demands of ATP. When more ATP is available from both host cell and Ct itself, RB is more activated by utilizing energy providing chemicals generated by enzymes associated in the nucleic acid metabolism. The forming of folate also suggests large glutamate consumption, which is supposed to be converted from glutamine by the glutamine-fructose-6-phosphate transaminase (glmS) and CTP synthase (pyrG).
Then, RNA sequencing (RNA-seq) data analysis was performed by me in a coinfection study. Metatranscriptome from patient RNA-seq data provides a realistic overview. Thirteen patient samples were collected and sequenced by our collaborators. Six male samples were obtained by urethral swab, and seven female samples were collected by cervicovaginal lavage. All the samples were Neisseria gonorrhoeae (GC) positive, and half of them had coinfection with Ct. HISAT2 and Stringtie were used for transcriptomic mapping and assembly respectively, and differential expression analysis by DESeq2, Ballgown and Cuffdiff2 are parallelly processed for comparison. Although the measured transcripts were not sufficient to assemble Ct's transcriptome, the differential expression of genes in both the host and GC were analyzed by comparing Ct positive group (Ct+) against Ct-uninfected group. The results show that in the Ct+ group, the host MHC class II immune response was highly induced. Ct infection is associated with the regulation of DNA methylation, DNA double-strand damage and ubiquitination. The analysis also shows Ct infection enhances host fatty acid beta oxidation, thereby inducing mROS, and the host responds to reduce ceramide production and glycolysis. The coinfection upregulates GC's own ion transporters and amino acid uptake, while it downregulates GC's restriction and modification systems. Meanwhile, GC has the nitrosative and oxidative stress response and also increases the ability for ferric uptake especially in the Ct+ group compared to Ct-uninfected group.
In conclusion, methods in bioinformatics were used here in analyzing the metabolism of Ct itself, and the responses of the host and GC respectively in a coinfection study with and without Ct. These methods provide metabolic and metatranscriptomic details to study Ct metabolism during infection and Ct associated coinfection in the human microbiota.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods of data analysis, which have until now mainly been used to model neural responses of electrophysiological recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
Insect microbiota plays an essential role on the hosts’ health and fitness, regulating their development, nutrition and immunity. The natural microbiota of bees, in particular, has been given much attention, largely because of the globally reported bee population declines. However, although the worker honey bee has been associated with distinctive and specialized microbiota, the microbiota of solitary bees has not been examined in detail, despite their enormous ecological importance. The main objectives of the present thesis were a) the bacterial community description for various solitary bee species, b) the association of the solitary bee microbiota with ecological factors such as landscape type, c) the relation of the bee foraging preferences with their nest bacterial microbiota, d) the examination of the nest building material contribution to the nest microbiota, e) the isolation of bacterial strains with beneficial or harmful properties for the solitary bee larvae and f) the pathological investigation of bacteria found in deceased solitary bee larvae.
The findings of the present study revealed a high bacterial biodiversity in the solitary bee nests. At the same time, the bacterial communities were different for each bee host species. Furthermore, it was shown that the pollen bacterial communities underwent compositional shifts reflecting a reduction in floral bacteria with progressing larval development, while a clear landscape effect was absent. The examination of the nest pollen provisions showed different foraging preferences for each included bee species. Both the pollen composition and the host species identity had a strong effect on the pollen bacteria, indicating that the pollen bacterial communities are the result of a combinatory process. The introduced environmental material also contributed to the nest natural microbiome. However, although the larval microbiota was significantly influenced by the pollen microbiota, it was not much associated with that of the nest material.
Two Paenibacillus strains isolated from O. bicornis nests showed strong antifungal activities, while several isolated strains were able to metabolize various oligosaccharides which are common in pollen and nectar. Screening for potential pathogenic bacteria in the nests of O. bicornis unveiled bacterial taxa, which dominated the bacterial community in deceased larvae, while at the same time they were undetectable in the healthy individuals.
vi
Finally, larvae which were raised in vitro developed distinct bacterial microbiomes according to their diet, while their life span was affected.
The present thesis described aspects of the microbiota dynamics in the nests of seven megachilid solitary bee nests, by suggesting which transmission pathways shape the established bacterial communities and how these are altered with larval development. Furthermore, specific bacterial taxa were associated with possible services they might provide to the larvae, while others were related with possible harmful effects. Future studies should integrate microbiota examination of different bee generations and parallel investigation of the microbiota of the nests and their surrounding environment (plant community, soil) to elucidate the bacterial transmission paths which establish the nest microbiota of solitary bees. Functional assays will also allow future studies to characterize specific nest bacteria as beneficial or harmful and describe how they assist the development of healthy bees and the fitness of bee populations.
Ketamine is commonly used as an anaesthetic agent and has more recently gained attention as an antidepressant. It has been linked to increased stimulus‐locked excitability, inhibition of interneurons and modulation of intrinsic neuronal oscillations. However, the functional network mechanisms are still elusive. A better understanding of these anaesthetic network effects may improve upon previous interpretations of seminal studies conducted under anaesthesia and have widespread relevance for neuroscience with awake and anaesthetized subjects as well as in medicine. Here, we investigated the effects of anaesthetic doses of ketamine (15 mg kg\(^{-1}\) h\(^{-1}\)i.p.) on the network activity after pure‐tone stimulation within the auditory cortex of male Mongolian gerbils (Meriones unguiculatus). We used laminar current source density (CSD) analysis and subsequent layer‐specific continuous wavelet analysis to investigate spatiotemporal response dynamics on cortical columnar processing in awake and ketamine‐anaesthetized animals. We found thalamocortical input processing within granular layers III/IV to be significantly increased under ketamine. This layer‐dependent gain enhancement under ketamine was not due to changes in cross‐trial phase coherence but was rather attributed to a broadband increase in magnitude reflecting an increase in recurrent excitation. A time–frequency analysis was indicative of a prolonged period of stimulus‐induced excitation possibly due to a reduced coupling of excitation and inhibition in granular input circuits – in line with the common hypothesis of cortical disinhibition via suppression of GABAergic interneurons.
The ubiquitination of proteins controls a multitude of physiological processes. This versatility of ubiquitin as a molecular signal arises from the diverse ways by which it can be attached to target proteins. Different ubiquitination patterns are then translated into different downstream consequences. Due to the enormous complexity of possible ubiquitin modifications, the ubiquitination machinery must be highly specific and tightly controlled. Ubiquitination proceeds through an enzymatic cascade, the last step of which is catalyzed by the E3 enzyme family. E3 enzymes are the crucial regulators since they dictate the specificity of substrate selection and modification.
Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity, regulation and specificity in this crucial ligase are incompletely understood.
One aim of this study was to unravel the role of the a1’-helix N-terminal to the HECT domain that was found to be a key element mediating regulation and oligomerization in other HECT ligases. I found that most N-terminally extended HECT domain constructs were insoluble when expressed in E. coli, indicating that additional regions N-terminal to the tested fragments may be essential to protect this highly hydrophobic helix from causing aggregation.
Another question addressed in this study was how E6AP builds ubiquitin chains. Using single-turnover experiments, I showed that ubiquitin-loaded E6AP is unable to transfer an additional ubiquitin molecule onto a stably linked ubiquitin-E6AP complex. This indicates that E6AP cannot assemble chains on its active site and may instead follow a sequential addition mechanism in which one ubiquitin molecule is transferred at a time to the target protein.
Using NMR spectroscopy and extensive mutational analyses, the determinants of ubiquitin recognition by the C-lobe of E6AP were unraveled and assigned to particular steps in the catalytic cycle. A functionally critical interface was identified that is specifically required during thioester formation between the C-terminus of ubiquitin and the ligase active site. This interface resembles the one utilized by NEDD4-type enzymes, suggesting a conserved ubiquitin binding mode across HECT ligases, independent of their linkage specificities. Moreover, I identified critical surface patches on ubiquitin and in the N- and C-terminal portions of the catalytic domain of E6AP that are important for the subsequent step of isopeptide bond formation. I also uncovered key determinants of the Lys48-linkage specificity of E6AP, both in the E6AP HECT domain and ubiquitin itself. This includes the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site, Lys48. It is thus tempting to speculate that ubiquitin linkage formation by E6AP is substrate-assisted. Taken together, my results improve our mechanistic understanding of the structure-function relationship between E6AP and ubiquitin, thus providing a basis for ultimately manipulating the functions of this HECT ligase for therapeutic applications.
Gene expression and transfer of the genetic information to the next generation forms the basis of cellular life. These processes crucially rely on DNA, thus the preservation, transcription and translation of DNA is of fundamental importance for any living being. The general transcription factor TFIIH is a ten subunit protein complex, which consists of two subcomplexes: XPB, p62, p52, p44, p34, and p8 constitute the TFIIH core, CDK7, CyclinH, and MAT1 constitute the CAK. These two subcomplexes are connected via XPD. TFIIH is a crucial factor involved in both, DNA repair and transcription. The central role of TFIIH is underlined by three severe disorders linked to failure of TFIIH in these processes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Only limited structural and functional data of TFIIH are available so far. Here, the model organism Chaetomium thermophilum was utilized with the aim to structurally and functionally characterize TFIIH. By combining the expression and purification of single TFIIH subunits with the co-expression and co-purification of dual complexes, a unique and powerful modular system of the TFIIH core subunits could be established, encompassing all proteins in high quality and fully functional. This system permits the step-wise assembly of TFIIH core, thereby making it possible to assess the influence of the intricate interaction network within TFIIH core on the overall enzymatic activities of TFIIH, which has not been possible so far. Utilizing the single subunits and dual complexes, a detailed interaction network of TFIIH core was established, revealing the crucial role of the p34 subunit as a central scaffold of TFIIH by linking the two proteins p44 and p52. Our studies also suggest that p62 constitutes the central interface of TFIIH to the environment rather than acting as a scaffold. TFIIH core complexes were assembled and investigated via electron microscopy. Preliminary data indicate that TFIIH adopts different conformational states, which are important to fulfill its functions in transcription and DNA repair. Additionally, a shortened construct of p62 was used to develop an easy-to-use, low cost strategy to overcome the crystallographic phase problem via cesium derivatization.
Knowing then defeating: The Ubiquitin activating enzyme, a promising target for cancer therapy
(2020)
Ubiquitin is a 76 amino acid long polypeptide, which is present throughout eukaryotes in a highly conserved fashion. Ubiquitin can modify proteins by becoming covalently attached to them. Eukaryotic cells employ ubiquitin to maintain and regulate fundamental cellular processes like protein degradation, the immune response and transcriptional and translational regulation. Transfer of ubiquitin to the substrate is achieved by the catalysis of three classes of enzymes namely E1, E2 and E3. Together these enzymes form a pyramidal hierarchy, where E1 stands at the apex and E3 enzymes form the base of the pathway.
The ubiquitin activating enzyme 1 (UBA1) plays a major role in ubiquitylation being the ubiquitin-dedicated E1 enzyme. In addition, it is the only enzyme in this pathway to use ATP as an energy source to catalyze two important reactions. The products of these reactions, ubiquitin adenylate and ubiquitin thioester, are the essential intermediate states of ubiquitin, for being conjugated to the target protein. With the help of X-ray crystallography and biochemical approaches, snapshots of multiple catalytic states of UBA1, where it is bound to Mg-ATP, ubiquitin and the E2 Ubc13 as substrates could be captured. With the help of these high-resolution crystal structures, deeper insights into the enzymatic mechanism of UBA1 could be attained. The resulting insights into the catalytic cycle were further validated by biochemical assays. It could be shown that ATP acts as a molecular switch to induce the enzyme’s open conformation. Ubiquitin-binding to the enzyme leads to domain rotations, which facilitate the recruitment of a cognate E2 enzyme. The interdomain communication as well as the cross-talk with the substrates and the products fuel the enzymatic cycle of UBA1.
Due to the proven efficacy of proteasome inhibitors for cancer treatment, which block degradation of proteins labeled with ubiquitin, enzymes participating in the ubiquitylation cascade have been targeted by researchers for the development of novel anti-cancer therapeutics. UBA1 inhibition has been shown to preferentially induce cell death in malignant cells, and it can also be used as a strategy to overcome resistance against proteasome inhibitors. MLN7243, an adenosyl sulfamate inhibitor developed by Millenium Pharmaceutical to specifically target UBA1, is currently in Phase-I clinical trials for the treatment of solid tumors. UBA1 could be crystallized in complex with three adenosyl sulfamate inhibitors covalently linked to ubiquitin, which are promising drug candidates for cancer therapy. The inhibitors employed, MLN7243, MLN4924 and ABPA3, show distinct specificities towards different E1 enzymes. With the help of crystal structures the specificity determinants of these inhibitors could be deciphered, which were further confirmed by inhibition assays as well as molecular dynamics simulations. Together these crystal structures provide a starting point for developing E1-specific inhibitors, which, besides their potential for medicinal purposes, are important tools to better understand the function of the ubiquitin system as well as the action of ubiquitin-like proteins.
Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor.
Comparative analysis of insect circadian clocks: a behavioural, anatomical, and molecular study
(2020)
Biological clocks are endogenous oscillators that give organisms the sense of time. Insects, as the largest taxonomic group, offer fascinating models to study the evolution of clocks and their adaptation to various environments. Although the laboratory fruit fly, Drosophila melanogaster, led the role in the field of circadian biology as it provides a powerful genetic experimental tool, new model insect species need to be established to understand photoperiodic responses and to enable comparative studies. This work reports the behavioural, anatomical, and molecular characterization of the circadian clock of five insect species. The malt fly Chymomyza costata carries a D. melanogaster-like clock network, which supports circadian rhythms under rhythmic environment but cannot self-sustain when isolated from external time cues. The olive fly Bactrocera oleae is the major pest of olive plantations and the characterization of its circadian clock will improve future pest management strategies. The linden bug Pyrrhocoris apterus, a well suited model for investigating circadian and photoperiodic timing interactions, shows high degree of homology of the clock network with D. melanogaster. The scuttle flies Megaselia scalaris and Megaselia abdita represent new fascinating models to study how the clock network controls circadian behaviour. Overall, this work highlights high degree of homology between different circadian clock systems, but at the same time also dramatic differences in terms of circadian behaviour and neuro-anatomical expression of clock components. These have been mainly discussed in regards to the evolution of clocks in Diptera, and the adaptation of clocks to high latitudes.
Induction of ectopic bone formation by site directed immobilized BMP2 variants \(in\) \(vivo\)
(2020)
In contrast to common bone fractures, critical size bone defects are unable to self-regenerate and therefore external sources for bone replacement are needed. Currently, the gold standard to treat critical size bone fractures, resulting from diseases, trauma or surgical interventions, is the use of autologous bone transplantation that is associated with several drawbacks such as postoperative pain, increased loss of blood during surgery and extended operative time.
The field of bone tissue engineering focuses on the combination of biomaterials and growth factors to circumvent these adverse events and thereby to improve critical size bone defects treatment.
To this aim, a promising approach is represented by using a collagen sponge soaked with one of the most powerful osteoinductive proteins, the bone morphogenetic protein 2 (BMP2). After the approval by the Food and Drug Administration (FDA), BMP2 was used to successfully treat several severe bone defects. However, the use of BMP2 delivery systems is associated with severe side effects such as inflammation, swelling, ectopic bone formation outside of the site of implantation and breathing problems if implanted in the area of the cervical spine. The occurrence of severe side effects is related to the supraphysiological amounts of the applied protein at the implantation site. The BMP2 is typically adsorbed into the scaffold and diffuses rapidly after implantation. Therefore, intensive research has been conducted to improve the protein’s retention ability, since a prolonged entrapment of the BMP2 at the implantation site would induce superior bone formation in vivo due to a minimized protein release. By controlling the release from newly designed materials or changing the protein immobilization methods, it seems possible to improve the osteoinductive properties of the resulting BMP2-functionalized scaffolds.
The combination of biocompatible and biodegradable scaffolds functionalized with a covalently immobilized protein such as BMP2 would constitute a new alternative in bone tissue engineering by eliminating the aforementioned severe side effects. One of the most common immobilization techniques is represented by the so-called EDC/NHS chemistry. This coupling technique allows covalent biding of the growth factor but in a non-site direct manner, thus producing an implant with uncontrollable and unpredictable osteogenic activities. Therefore, the generation of BMP2 variants harboring functional groups that allow a site-directed immobilization to the scaffold, would enable the production of implants with reproducible osteogenic activity.
The new BMP2 variants harbor an artificial amino acid at a specific position of the mature polypeptide sequence. The presence of the unnatural amino acid allows to use particular covalent immobilization techniques in a highly specific and site directed manner. The two selected BMP2 variants, BMP2 E83Plk and BMP2 E83Azide, were expressed in E. coli, renatured and purified by cation exchange chromatography. The final products were intensively analyzed in terms of purity and biological activity in vitro. The two BMP2 variants enabled the application of different coupling techniques and verify the possible options for site directed immobilization to the scaffold.
Intensive analyses on the possible side effects caused by the coupling reactions and on the quantification of the coupled protein were performed. Both click chemistry reactions showed high reaction efficacies when the BMP2 variants were coupled to functionalized fluorophores. Quantification by ELISA and scintillation counting of radioactively labeled protein revealed different outcomes. Moreover, the amounts of protein detected for the BMP2 variants coupled to microspheres were similar to that of the wild type protein. Therefore, it was not possible to conclude whether the BMP2 variants were covalently coupled or just adsorbed.
BMP2 variants being immobilized to various microspheres induced osteogenic differentiation of C2C12 cells in vitro, but only in those cells that were located in close proximity to the functionalized beads. This selectivity strongly indicates that the protein is for a great portion covalently coupled and not just adsorbed. Moreover, the difference between the covalently coupled BMP2 variants and the adsorbed BMP2 WT was confirmed in vivo. Injection of the BMP2-functionalized microspheres in a rat model induced subcutaneous bone formation.
The main aim of the animal experiment was to prove whether covalently coupled BMP2 induces bone formation at significant lower doses if compared to the amount being required if the protein is simply adsorbed. To this aim, several BMP2 concentrations were tested in this animal experiment. The BMP2 variants, being covalently immobilized, were hypothesized to be retained and therefore bio-available at the site of implantation for a prolonged time. However, in the animal experiments, lower doses of either coupled or adsorbed protein were unable to induce any bone formation within the 12 weeks.
In contrast, the highest doses induced bone formation that was first detected at week 4. During the 12 weeks of the experiment, an increase in bone density and a steady state bone volume was observed. These results were obtained only for the covalently coupled BMP2 E83Azide but not for BMP2 E83Plk that did not induce bone formation in any condition. The negative outcome after application of BMP2 E83Plk suggested that the coupling reaction might have provoked changes in the protein structure that extremely influenced its osteogenic capabilities in vivo.
However, the histological examination of the different ossicles induced either by BMP2 WT or BMP2 E83Azide, revealed clear morphological differences. BMP2 WT induced a bone shell-like structure, while the covalently coupled protein induced uniform bone formation also throughout the inner part. The differences between the two newly formed bones can be clearly associated with the different protein delivery mechanisms. Thus, the developed functionalized microspheres constitute a new interesting strategy that needs further investigations in order to be able to be used as replacement of the currently used BMP2 WT loaded medical devices.
T cell infiltration into the intestine occurs after priming and activation in the mesenteric lymph nodes and Peyer’s patches and subsequent trafficking via the blood circulation. We hypothesized that additionally to the vascular trafficking route, a fraction of T cells in the Peyer’s patches directly migrate into the adjacent lamina propria of the small intestine. To test this hypothesis, we employed a mouse model of acute Graft-versus-Host Disease to study the direct T cell migration from the Peyer’s patches to the adjacent lamina propria.
First, we analyzed the border of Peyer’s patches on histological sections and found that the Peyer’s patch is not enclosed by a capsule or basement membrane. Thus, the tissue architecture allows for direct access to the surrounding tissue. With whole-mount light sheet fluorescence microscopy we quantified a three-dimensional gradient of T cells around Peyer’s patches on day 2.5 and day 3 after transplantation. This gradient evened out at day 4 and day 6 when high numbers of T cells started to evenly infiltrate the intestine from the blood circulation. We confirmed that gradient-forming T cells around Peyer’s patches resided within the tissue parenchyma of the lamina propria and not inside lymphatic vessels.
To positively prove that the recently activated donor T cells around Peyer’s patches have egressed directly from that patch, we established a protocol for intravital photoconversion of T cells inside Peyer’s patches. 12 h after photoconversion inside a single Peyer’s patch, photoconverted T cells resided only around this particular Peyer’s patch and not elsewhere in the small intestine. This indicated that the T cells did not infiltrate via the blood but migrated to the adjacent lamina propria of the small intestine. Dynamic intravital two-photon microscopy revealed that these T cells next to the Peyer’s patch migrated in a random pattern. This suggested that these cells did not follow a positive chemoattractive gradient once they had reached the lamina propria. Laser-capture microdissection combined with RNA sequencing of the mucosa near the Peyer’s patch identified a wide range of migration-promoting factors. These included chemokines, co-stimulatory receptors and migration-associated intracellular molecules, which are candidates to promote this direct migration from Peyer’s patches.
Altogether, we demonstrate for the first time that additionally to the vascular trafficking route, a fraction of T cells migrates directly from the Peyer’s patch to the surrounding mucosa. This mechanism implies so far unrecognized regional specification of Peyer’s-patch-primed T cells. Our findings may impact treatment strategies to avoid intestinal inflammation or foster immunity after oral vaccination.
To this day, opioids represent the most effective class of drugs for the treatment of severe pain. On a molecular level, all opioids in use today are agonists at the μ-opioid receptor (μ receptor). The μ receptor is a class A G protein-coupled receptor (GPCR). GPCRs are among the biological structures most frequently targeted by pharmaceuticals. They are membrane bound receptors, which confer their signals into the cell primarily by activating a variety of GTPases called G proteins. In the course of the signaling process, the μ receptor will be phosphorylated by GRKs, increasing its affinity for another entity of signaling proteins called β-arrestins (β-arrs). The binding of a β-arr to the activated μ receptor will end the G protein signal and cause the receptor to be internalized into the cell. Past research showed that the μ receptor’s G protein signal puts into effect the desired pain relieving properties of opioid drugs, whereas β-arr recruitment is more often linked to adverse effects like obstipation, tolerance, and respiratory depression. Recent work in academic and industrial research picked up on these findings and looked into the possibility of enhancing G protein signaling while suppressing β-arr recruitment. The conceptual groundwork of such approaches is the phenomenon of biased agonism. It appreciates the fact that different ligands can change the relative contribution of any given pathway to the overall downstream signaling, thus enabling not only receptor-specific but even pathway-specific signaling.
This work examined the ability of a variety of common opioid drugs to specifically activate the different signaling pathways and quantify it by means of resonance energy transfer and protein complementation experiments in living cells. Phosphorylation of the activated receptor is a central step in the canonical GPCR signaling process. Therefore, in a second step, expression levels of the phosphorylating GRKs were enhanced in search for possible effects on receptor signaling and ligand bias.
In short, detailed pharmacological profiles of 17 opioid ligands were recorded. Comparison with known clinical properties of the compounds showed robust correlation of G protein activation efficacy and analgesic potency. Ligand bias (i.e. significant preference of any path- way over another by a given agonist) was found for a number of opioids in native HEK293 cells overexpressing μ receptor and β-arrs. Furthermore, overexpression of GRK2 was shown to fundamentally
change β-arr pharmacodynamics of nearly all opioids. As a consequence, any ligand bias as detected earlier was abolished with GRK2 overexpression, with the exception of buprenorhin. In summary, the following key findings stand out: (1) Common opioid drugs exert biased agonism at the μ receptor to a small extent. (2) Ligand bias is influenced by expression levels of GRK2, which may vary between individuals, target tissues or even over time. (3) One of the opioids, buprenorhin, did not change its signaling properties with the overexpression of GRK2. This might serve as a starting point for the development of new opioids which could lack the ability of β-arr recruitment altogether and thus might help reduce adverse side effects in the treatment of severe pain.
Graft-versus-Host Disease (GvHD) stellt einen häufigen, den Gesamterfolg einer allogenen hämatopoetischen Stammzelltransplantation limitierenden Faktor dar. Bei dieser Komplikation attackieren vor allem alloreaktive T-Lymphozyten des Stammzellspenders gesunde Körperzellen des Patienten. Infolgedessen kommt es zu Gewebeschäden in den Zielorganen Haut, Leber und Darm. Die Behandlung der GvHD erfordert eine effektive Immunsuppression, was wiederum Graft-versusTumor-Effekte kompromittiert und den Rückfall der malignen Grunderkrankung bedingen kann. Viele Patienten sprechen aus bisher ungeklärten Gründen nicht auf die klassische immunsuppressive Therapie mit Steroiden oder second-line Therapien an. Neue zelluläre Therapien zur Behandlung der refraktären GvHD sind auf dem Vormarsch, bedürfen aber einer weiterführenden klinischen Testung, auch um die exakten Wirkungsmechanismen zu verstehen. Idealerweise könnten neue Testsysteme das GvHD-Potential von allogenen Stammzellpräparaten oder aber das immunsuppressive Potential von neuen GvHD-Therapien vorhersagen, bevor diese in klinischen Studien eingesetzt werden. Ziel der vorliegenden Arbeit war es, ein erstes, in multiplen Replikaten einsetzbares, humanes organotypisches Gewebemodell zur Simulation einer GvHD-Reaktion am Beispiel der Haut zu etablieren.
Zu diesem Zweck wurden artifizielle humane Hautmodelle unter statischen (KollagenHautmodelle) und dynamischen Kulturbedingungen (vaskularisierte Hautmodelle) generiert. Die Injektion unstimulierter PBMCs (engl. peripheral blood mononuclear cells) führte zu keinen histomorphologischen Veränderungen in den KollagenHautmodellen. Im Gegensatz dazu hatte die Injektion vorstimulierter allogener PBMCs eine Zerstörung der epidermalen Strukturen der Kollagen-Hautmodelle zur Folge, welche vergleichbar waren mit Gewebeschäden bei einer akuten GvHD der Haut. Dieselben Schädigungen der Epidermis wurden durch die Injektion von Mediumüberständen vorstimulierter PBMCs in die Kollagen-Hautmodelle erreicht. Im Kulturmedium der Kollagen-Hautmodelle wurden hohe Konzentrationen von Interleukin 2 und 17, Interferon gamma sowie Tumornekrosefaktor alpha gemessen, wodurch auf die Beteiligung von Zytokinen an der inflammatorischen Reaktion geschlossen werden konnte. Auch im komplexeren vaskularisierten Hautmodell verursachte die Injektion vorstimulierter PBMCs histomorphologische Veränderungen entsprechend einer akuten Haut-GvHD sowie einen zeitabhängigen Anstieg proinflammatorischer Zytokine.
Zusammenfassend zeigen die Resultate dieser Arbeit, dass die Induktion einer starken Inflammations- und Immunreaktion in artifiziellen humanen Hautmodellen, welche histomorphologisch eine GvHD imitiert, möglich ist. Dieses Modell könnte als Grundlage für die Entwicklung eines klinisch relevanten Testsystems zur Bestimmung des GvHD-Restpotentials oder zur Festlegung der immunsuppressiven Kapazität innovativer Zellpräparate dienen. Somit könnten humane artifizielle GvHDModelle in klinischen Studien eingesetzt werden und die Erfahrungen aus Tiermodellen ergänzen sowie erste in vitro Ergebnisse im humanen System liefern, welche dann mit dem tatsächlichen klinischen Resultat verglichen werden könnten.
Despite the large number of G protein-coupled receptors (GPCRs) expressed in the central nervous system (CNS), little is known about their location, organization, and dynamics in functional nanodomains at synapses. Class C GPCRs including metabotropic glutamate receptors (mGluRs) and the γ-aminobutyric acid subtype B receptor (GABABR) mediate several key functions in synaptic transmission. However, it is still insufficiently understood how these receptors function at synapses to modulate neurotransmission. One limitation is the availability of techniques to examine receptors with high spatiotemporal resolution in physiologically relevant cells. To investigate the distribution and spatiotemporal dynamics of mGluR4 and GABABR in cerebellar slices and cultured hippocampal neurons, I used advanced imaging techniques, including single-molecule imaging and superresolution microscopy with high spatial (10-20 nm) and temporal (20 ms) resolution.
The presynaptic active zone (AZ) is a highly organized structure that specializes in neurotransmitter release. mGluR4 is a prototypical presynaptic class C GPCR. mGluR4 mediates an inhibitory effect on presynaptic glutamate release mainly via the inhibition of P/Q type voltage dependent calcium channels (CaV2.1). In this study, I analyzed the organization of mGluR4 at the synapse between parallel fibers and Purkinje cells in the mouse cerebellum with near-molecular resolution using two-color direct stochastic optical reconstruction microscopy (dSTORM). Quantitative analyses revealed a four-fold mGluR4 enrichment at parallel fiber AZs. I found that an AZ contains 29 mGluR4 nanoclusters on average. Each nanocluster contains one or two mGluR4s, with few nanoclusters containing three or more receptors. To assess the spatial distribution of mGluR4 relative to functional active zone elements such as CaV2.1 and Munc 18-1 (an essential component of the synaptic secretory machinery), a distance-based colocalization analysis was used. The analysis revealed positive correlation between mGluR4 and both proteins at a distance of 40 nm. Interestingly, mGluR4 showed a higher positive correlation to Munc 18-1 in comparison to CaV2.1. These results suggest that mGluR4 might directly inhibit the exocytotic machinery to reduce glutamate release from the synaptic vesicles in addition to its role in the inhibition of presynaptic calcium influx. The revealed high degree of mGluR4 organization may provide a new ultrastructural basis to explain the depressive effect of mGluR4 on the neurotransmission.
Moreover, I directly imaged GABABR dynamic behavior with high spatiotemporal resolution in living hippocampal neurons utilizing single-molecule total internal reflection fluorescence microscopy (TIRFM). To this purpose, the GABAB1 subunit was engineered with an N-terminal SNAP-tag to enable specific labeling with bright organic fluorophores. On the plasma membrane surface, immobile and mobile GABABRs were detected at both synaptic and extrasynaptic compartments. A mean square displacement analysis (MSD) revealed characteristic dynamic patterns of GABABR depending on receptor location inside or outside of the synapses. The majority of receptors belonging to the extrasynaptic pool displayed rapid and free diffusion. In contrast, approximately 80% of receptors residing at the synaptic compartments were immobile or confined within limited regions. Receptors located at pre- and post-synaptic sites showed a similar behavior. GABABR lateral diffusion patterns inside and outside synapses might be important for the regulation of efficacy of synaptic inhibition.
Altogether, this study puts forward previously unknown GPCR nanoscopic details in functional nanodomains. GPCR spatial organization might be important for the efficiency, fidelity, and rapid signaling required for synaptic transmission.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal monogenic motoneuron disease in children with unknown etiology caused by mutations in the immunoglobulin μ-binding protein 2 (IGHMBP2) gene coding for DNA/RNA ATPase/helicase. Despite detailed knowledge of the underlying genetic changes, the cellular mechanisms leading to this disease are not well understood. In the Nmd2J ("neuromuscular disorder") mouse, the mouse model for the juvenile form of SMARD1 patients, in which similar pathological features as diaphragmatic paralysis and skeletal muscle atrophy are observed. Ex vivo studies in Nmd2J mice showed that loss of the motor axon precedes atrophy of the gastrocnemius muscle and does not correlate with neurotransmission defects in the motor endplate. The already described independent myogenic anomalies in the diaphragm and heart of the Nmd2J mouse raised the question whether spinal motoneuron degeneration develops cell autonomously. Ighmbp2 is predominantly localized in the cytoplasm and seems to bind to ribosomes and polysomes, suggesting a role in mRNA metabolism.
In this Ph.D. thesis, morphological and functional analyses of isolated Ighmbp2-deficient (Ighmbp2-def.) motoneurons were performed to answer the question whether the SMARD1 phenotype results from dysregulation of protein biosynthesis. Ighmbp2-deficient motoneurons show only negligible morphological alterations with respect to a slight increase in axonal branches. This observation is consistent with only minor changes of transcriptome based on RNA sequencing data from Ighmbp2-deficient motoneurons. Only the mRNA of fibroblast growth factor receptor 1 (Fgfr1) showed significant up-regulation in Ighmbp2-deficient motoneurons. Furthermore, no global aberrations at the translational level could be detected using pulsed SILAC (Stable Isotope Labeling by Amino acids in cell culture), AHA (L-azidohomoalanine) labeling and SUnSET (SUrface SEnsing of Translation) methods. However, a reduced β-actin protein level was observed at the growth cones of Ighmbp2-deficient motoneurons, which was accompanied with a reduced level of Imp1 protein, a known β-actin mRNA interactor. Live-cell imaging studies using fluorescence recovery after photobleaching (FRAP) showed translational down-regulation of eGFPmyr-β-actin 3'UTR mRNA in the growth cones and the cell bodies, although the amount of β-actin mRNA and the total protein amount in Ighmbp2-deficient motoneurons showed no aberrations. This compartment-specific reduction of β-actin protein occurred independently of a non-existent direct IGHMBPF2 binding to β-actin mRNA. Fgfr1, which was upregulated on the RNA level, did not show an increased protein amount in Ighmbp2-deficient motoneurons, whereas a reduced amount could be detected. Interestingly, a correlation could be found between the reduced amount of the Imp1 protein and the increased Fgfr1 mRNA, since the IMP1 protein binds the FGFR1 mRNA and thus could influence the transport and translation of FGFR1 mRNA. In summary, all data suggest that Ighmbp2 deficiency leads to a local but modest disturbance of protein biosynthesis, which might contribute to the motoneuron defects of SMARD1.
Blumeria graminis, the obligate biotrophic grass powdery mildew, is a highly pathogenic fungus capable of inflicting foliar diseases and of causing severe yield losses. There is asexual and sexual propagation in the life cycle of B. graminis. In the epidemiological processes of this pathogen, both types of spores - asexual conidia and sexual ascospores – are crucial.
Conidia of B. graminis are demonstrated to perceive cuticular very-long-chain aldehydes as molecular signal substances notably promoting germination and differentiation of the infection structure (the appressorium) – the prepenetration processes – in a concentration- and chain-length-dependent manner. Conidial germination and appressorium formation are known to be dramatically impeded by the presence of free water on the host surface. However, sexually formed ascospores are reported to easily germinate immersed in water. There are abundant assays on conidial prepenetration processes. However, with respect to the stimulating effects of very-long-chain aldehydes and to the influence of the presence of free water, ascosporic prepenetration processes are still obscure.
In order to study the effects of very-long-chain aldehydes on the ascosporic prepenetration processes of wheat powdery mildew fungus B. graminis f. sp. tritici, Formvar®-based in vitro systems were applied to exclude the secondary host effects (such as host resistance) and to reproducibly provide homogeneous hydrophobic substratum surfaces. By the presence of even-numbered very-long-chain aldehydes (C22 - C30), the appressorium formation of the ascospores was notably triggered in a chain-length dependent manner. N-octacosanal (C28) was the most inducing aldehyde tested. Unlike conidia, ascospores could easily differentiate immersed in water and showed a more variable differentiation pattern even with a single germ tube differentiating an appressorium.
To evaluate the alternative management against barley powdery mildew fungus Blumeria graminis f. sp. hordei, the suppressing effects of UV-C irradiation on the developmental processes of conidia on artificial surfaces (in vitro) and on host leaf surfaces (in vivo) were assayed. In vitro and in vivo, a single dose of 100 J m-2 UV-C was adequate to decrease conidial germination to < 20 % and to reduce appressorium formation to values < 5 %. UV-C irradiation negatively affected colony pustule size and vegetative propagation. Under photoperiodic conditions of 2h light/16h dark, 6h dark/12h light or 6h dark/18h light, UV-C-treated conidia showed photoreactivation (photo-recovery). White light-mediated photoreactivation was most effective immediately after UV-C irradiation, suggesting that a prolonged phase of darkness after UV-C application increased the efficacy of management against B. graminis. UV-C irradiation increased transcript levels of three putative photolyase genes in B. graminis, indicating those were probably involved in photoreactivation processes. However, mere white light or blue light (wavelength peak, 475 nm) could not induce the up-regulation of these genes.
To determine whether visible light directly impacted the prepenetration and penetration processes of this powdery mildew pathogen, conidia of Blumeria graminis f. sp. hordei and Blumeria graminis f. sp. tritici were inoculated onto artificial surfaces and on host leaf surfaces. Samples were analyzed after incubation periods under light conditions (white light intensity and spectral quality). Increasing white light intensities directly impaired conidial prepenetration processes in vitro but not in vivo. Applying an agar layer under the wax membrane compensated for conidial water loss as a consequence of high white light irradiation. Light stimulated in vitro and in vivo the appressorium elongation of B. graminis in a wavelength-dependent manner. Red light was more effective to trigger the elongation of appressorium than blue light or green light assayed.
Taken together, the findings of this study demonstrate that 1) a host surface recognition principle based on cuticular very-long-chain aldehydes is a common feature of B. graminis f. sp. tritici ascospores and conidia; 2) the transcriptional changes of three putative photolyase genes in B. graminis are mediated in a UV-C-dependent manner; 3) light directly affected the (pre)penetration processes of B. graminis.
Die Mitogen-aktivierten Proteinkinasen ERK1/2 (extrazellulär Signal-regulierte Kinase 1 und 2) sind die Effektorkinasen der Raf/MEK/ERK-Kaskade und verknüpfen externe Stimuli mit der intrazellulä-ren Antwort, wodurch sie wichtige Schlüsselmoleküle der zellulären Signaltransduktion darstellen. Zahlreiche Studien belegen die Beteiligung von ERK1/2 an der Entstehung pathologischer kardialer Hypertrophie. Genauso ist bekannt, dass ERK1/2 anti-apoptotische, kardioprotektive Eigenschaften besitzen.
So führte, wie in dieser Arbeit gezeigt, eine Hemmung der katalytischen ERK1/2-Aktivität durch den MEK-Inhibitor PD98059 zu einer signifikanten Reduktion der hypertrophen Antwort von Kardiomy-ozyten auf den Stimulus Phenylephrin. Dies war allerdings mit einem Anstieg der Apoptoserate in diesen Zellen verbunden, wodurch sich eine Hemmung der totalen ERK-Aktivität als nicht praktika-bel für die Behandlung pathologischer kardialer Hypertrophie herauskristallisierte. In früheren Un-tersuchungen wurde eine Autophosphorylierung von ERK an Threonin 188 (murines ERK2) entdeckt und als Trigger für ERK1/2-vermitteltes hypertrophes Wachstum identifiziert. Diese Autophospho-rylierung steuert die nukleäre Lokalisation von ERK1/2 und ermöglicht so die Aktivierung nukleärer ERK-Zielproteine sowie hypertrophes Wachstum. Eine Interferenz mit der ERKThr188-Phosphorylierung konnte schon in vitro und in vivo erfolgreich einer pathologischen Hypertrophie entgegenwirken, ohne Einfluss auf physiologisches Herzwachstum oder die zytosolischen, anti-apoptotischen Effekte von ERK1/2 zu nehmen. Einen initialen Schritt für das Zustandekommen dieser Autophosphorylierung an Threonin 188 stellt dabei die Dimerisierung von ERK dar. In der vorliegenden Arbeit wurde daher die Inhibition der ERK-Dimerisierung im Hinblick auf die Behand-lung ERKThr188-vermittelter pathologischer Hypertrophie untersucht. Dabei sollte die endogene ERK-Dimerisierung mithilfe eines selbst generierten Peptids unterbunden werden. In Übereinstimmung mit den Ergebnissen zu einer dimerisierungsdefizienten ERK2-Mutante (ERK2-Δ4) konnte das Peptid in vitro und in vivo erfolgreich pathologisch hypertrophes Herzwachstum mindern. Dabei führte es sogar zu einem Rückgang des apoptotischen Zelltodes, ausgelöst durch eine Aortenligation, füh-ren. Es zeigte sich, dass das Peptid die nukleäre Translokation von ERK2 verhindert und dadurch nukleäre ERK-Substrate geringer aktiviert werden. Da eine Dysregulation in der Raf/MEK/ERK-Kaskade auch die Entstehung von Tumoren begünstigen kann, sollte schließlich untersucht wer-den, ob das Prinzip der Hemmung nukleärer ERK-Effekte auch die Proliferation von Krebszellen beeinflussen kann. Es stellte sich heraus, dass die Peptid-vermittelte Hemmung der ERK-Dimerisierung auch die Proliferation von Kolonkarzinomzelllinien mit unterschiedlichen Mutations-stadien der Raf/MEK/ERK-Kaskade reduziert.
In der vorliegenden Arbeit konnte somit die Intervention mit der ERK-Dimerisierung als Target der ERKThr188-Autophosphorylierung als translationale Strategie zur Reduktion nukleärer ERK-Effekte herausgearbeitet werden. Dies bietet die Möglichkeit ERK-vermittelte pathologische kardialer Hy-pertrophie und ERK-vermittelte Tumor-Proliferation zu behandeln, ohne kardiotoxische Nebenwir-kungen zu verursachen.
From the simplest single-cellular organism to the most complex multicellular life forms, genetic information in form of DNA represents the universal basis for all biological processes and thus for life itself. Maintaining the structural and functional integrity of the genome is therefore of paramount importance for every single cell. DNA itself, as an active and complex macromolecular structure, is both substrate and product of many of these biochemical processes. A cornerstone of DNA maintenance is thus established by the tight regulation of the multitude of reactions in DNA metabolism, repressing adverse side reactions and ensuring the integrity of DNA in sequence and function. The family of RecQ helicases has emerged as a vital class of enzymes that facilitate genomic integrity by operating in a versatile spectrum of nucleic acid metabolism processes, such as DNA replication, repair, recombination, transcription and telomere stability. RecQ helicases are ubiquitously expressed and conserved in all kingdoms of life. Human cells express five different RecQ enzymes, RecQ1, BLM, WRN, RecQ4 and RecQ5, which all exhibit individual as well as overlapping functions in the maintenance of genomic integrity. Dysfunction of three human RecQ helicases, BLM, WRN and RecQ4, causes different heritable cancer susceptibility syndromes, supporting the theory that genomic instability is a molecular driving force for cancer development. However, based on their inherent DNA protective nature, RecQ helicases represent a double-edged sword in the maintenance of genomic integrity. While their activity in normal cells is essential to prevent cancerogenesis and cellular aging, cancer cells may exploit this DNA protective function by the overexpression of many RecQ helicases, aiding to overcome the disadvantageous results of unchecked DNA replication and simultaneously gaining resistance against chemotherapeutic drugs. Therefore, detailed knowledge how RecQ helicases warrant genomic integrity is required to understand their implication in cancerogenesis and aging, thus setting the stage to develop new strategies towards the treatment of cancer.
The current study presents and discusses the first high-resolution X-ray structure of the human RecQ4 helicase. The structure encompasses the conserved RecQ4 helicase core, including a large fraction of its unique C- terminus. Our structural analysis of the RecQ4 model highlights distinctive differences and unexpected similarities to other, structurally conserved, RecQ helicases and permits to draw conclusions about the functional implications of the unique domains within the RecQ4 C-terminus. The biochemical characterization of various RecQ4 variants provides functional insights into the RecQ4 helicase mechanism, suggesting that RecQ4 might utilize an alternative DNA strand separation technique, compared to other human RecQ family members. Finally, the RecQ4 model permits for the first time the analysis of multiple documented RecQ4 patient mutations at the atomic level and thus provides the possibility for an advanced interpretation of particular structure-function relationships in RecQ4 pathogenesis.
Depressionen gehören zu den häufigsten psychischen Erkrankungen. Neben Symptomen wie Niedergeschlagenheit, Interessenlosigkeit oder Schlafstörungen sind Depressionen auch durch Defizite in kognitiven Funktionen, wie z.B. Aufmerksamkeitsprozessen oder der Wahrnehmung, und eine negativ verzerrte Informationsverarbeitung gekennzeichnet.
Aufgrund der hohen Prävalenz, der starken psychosozialen Funktionseinschränkungen durch depressive Erkrankungen und deren rezidivierenden Charakter besteht die Notwendigkeit, die therapeutischen Interventionen zur Behandlung affektiver Störungen zu verbessern, dadurch die Krankheitsphase der Patienten zu verkürzen und letztendlich auch die Kosten für das Gesundheitssystem zu reduzieren. In diesem Zusammenhang werden in den letzten Jahren verstärkt mögliche Prädiktoren und Korrelate des Therapieerfolgs untersucht. Hierfür könnten negativ verzerrte Informationsverarbeitungsprozesse und Defizite in kognitiven Funktionen objektive Marker darstellen.
Im ersten Teil dieser Arbeit wurde der Covariation Bias, der als Überschätzung des Zusammenhangs zwischen einem krankheitsrelevanten Stimulus und einer aversiven Konsequenz definiert wird, in einem Querschnittsdesign bei schwer depressiven Patienten zu Behandlungsbeginn im Vergleich zu einer Gruppe von Patienten nach einer sechswöchigen Behandlung sowie einer gesunden Kontrollgruppe untersucht. Diese kognitive Verzerrung war bei Patienten mit schwererer Symptomatik unabhängig vom Behandlungszeitpunkt stärker ausgeprägt. Zudem prädizierte der Covariation Bias zu Behandlungsbeginn das Therapieoutcome nach sechs Behandlungswochen dahingehend, dass Patienten mit einer stärkeren kognitiven Verzerrung ein schlechteres Ansprechen auf die Therapie zeigten.
Im zweiten Teil dieser Arbeit wurde das Emotional Processing Paradigma, das aus Aufgaben zur Emotionserkennung und zur Aufmerksamkeitslenkung besteht, zum ersten Mal bei schwer depressiven Patienten im intraindividuellen Verlauf der Behandlung eingesetzt und in Zusammenhang mit dem Therapieerfolg gestellt. Neben Hinweisen darauf, dass Patienten, bei denen sich in den ersten Behandlungswochen unter anderem die Salienz negativer Emotionen verringerte, mit höherer Wahrscheinlichkeit remittierten, zeigten sich vor allem zeitlich stabile Unterschiede im Sinne einer Trait-Variablen zwischen Patienten, die auf die initiale Therapie ansprachen, und Patienten, die keine bedeutsame Verbesserung erfuhren, in den globalen kognitiven Funktionen: Patienten, bei denen es zu keiner klinisch relevanten Verbesserung durch die Therapie kam, wiesen stärkere Defizite auf.
Zusammengenommen weisen die Ergebnisse der vorliegenden Arbeit auf ein stabiles Muster von Defiziten in globalen kognitiven Funktionen bei Patienten mit Depressionen hin. Diese Abweichungen liegen jedoch nicht bei allen schwer depressiven Patienten gleichermaßen vor. Bei Patienten mit Defiziten scheint das Therapieoutcome schlechter zu sein. Somit könnten diese Prozesse der Informationsverarbeitung und kognitive Defizite auf neuropsychologischer Ebene Prädiktoren und Korrelate des Therapieoutcomes darstellen. Im Sinne der personalisierten Medizin könnte in Zukunft die Diagnostik um die Parameter der Informationsverarbeitungsprozesse ergänzt werden und so die Prognose des Therapieerfolgs verbessert und die Behandlung der Patienten individualisiert werden.
Investigation of dynamic processes of prototypical class A GPCRs by single-molecule microscopy
(2020)
In this work, two projects were pursued.
In the first project, I investigated two different subtypes of opioid receptors, which play a key role as target for analgesia. A set of subtype specific fluorescent ligands for μ opioid receptor (MOR) and δ opioid receptor (DOR) was characterised and used to gain insights into the diffusion behaviour of those receptors. It was shown that the novel ligands hold photophysical and pharmacological properties making them suitable for single-molecule microscopy. Applying them to wild-type receptors expressed in living cells revealed that both sub-types possess a heterogeneous diffusion behaviour. Further- more, the fluorescent ligands for the MOR were used to investigate homodomerisation, a highly debated topic. The results reveal that only ≈ 5 % of the receptors are present as homodimers, and thus the majority is monomeric. G-protein coupled receptors (GPCRs) play a major role as drug targets. Accordingly, understanding the activation process is very important. For a long time GPCRs have been believed to be either active or inactive. In recent years several studies have shown, that the reality is more complex, involving more substates. [1, 2, 3, 4] In this work the α 2A AR was chosen to investigate the activation process on a single-molecule level, thus being able to distinguish also rare or short-lived events that are hidden in ensemble mea- surements. With this aim, the receptor was labelled intracellular with two fluorophores using supported membranes. Thus it was possible to acquire movies showing qualita- tively smFRET events. Unfortunately, the functionality of the used construct could not be demonstrated. To recover the functionality the CLIP-tag in the third intracellular loop was replaced successfully with an amber codon. This stop codon was used to insert an unnatural amino acid. Five different mutants were created and tested and the most promising candidate could be identified. First ensemble FRET measurements indicated that the functionality might be recovered but further improvements would be needed. Overall, I could show that single-molecule microscopy is a versatile tool to investigate the behaviour of typical class A GPCRs. I was able to show that MOR are mostly monomeric under physiological expression levels. Furthermore, I could establish intra- cellular labelling with supported membranes and acquire qualitative smFRET events.
This thesis explores the development of monoaminergic systems in the central nervous system (CNS) of zebrafish. The serotonergic cells of the hypothalamus pose the main focus of the present work. Most vertebrates except for mammals possess serotonin (5-HT) synthesising cells in more than one region of the CNS. In zebrafish such regions are, e.g. the hypothalamus, the raphe nuclei and the spinal cord. Serotonin functions as a neurotransmitter and neuromodulator in the CNS. Presumably due to its neuromodulatory tasks hypothalamic serotonergic cells are in contact with the cerebrospinal fluid (CSF), which expands the field of potential serotonergic targets tremendously. This highlights that serotonergic CSF-contacting (CSF-c) cells are vital for the execution of many functions and behaviours. Further, the hypothalamic serotonergic clusters constitute the largest population of serotonergic cells in the CNS of zebrafish. Together, these facts emphasise the need to understand the development and function of serotonergic CSF-c cells in the hypothalamus. Few studies have dealt with this subject, hence, information about the development of these cells is scarce. The zinc-finger transcription factor fezf2, and Fibroblast growth factor (Fgf)-signalling via the ETS-domain transcription factor etv5b are known to regulate serotonergic cell development in the hypothalamus (Bosco et al., 2013; Rink and Guo, 2004). However, the main Fgf ligand responsible for this mediation has not been determined prior to this work. The present thesis identifies Fgf3 as a crucial Fgf ligand. To achieve this result three independent strategies to impair Fgf3 activity have been applied to zebrafish embryos: the fgf3t24152 mutant, an fgf3 morpholino-based knock-down and the CRISPR/Cas9 technique. The investigations show that Fgf3 regulates the development of monoaminergic CSF-c cells in the hypothalamus. Additionally, Fgf3 impacts on cells expressing the peptide hormone arginine vasopressin (avp). Most interestingly, the requirement for Fgf3 by these cells follows a caudo-rostral gradient with a higher dependence on Fgf3 by caudal cells. This also seems to be the case for dopaminergic CSF-c cells in the hypothalamus (Koch et al., 2014). Moreover, etv5b a downstream target of Fgf-signalling is demonstrated to be under the control of Fgf3. With regard to serotonergic CSF-c cell development, it is shown that fgf3 is expressed several hours before tph1a and 5-HT (Bellipanni et al., 2002; Bosco et al., 2013). Together with the result that the hypothalamus is already smaller before mature serotonergic CSF-c cells appear, this argues for an early impact of Fgf3 on serotonergic specification. This hypothesis is supported by several findings in this study: the universal decrease of proliferating cells in the hypothalamus and simultaneous increase of cell death after fgf3 impairment. Complementary cell fate experiments confirm that proliferating serotonergic progenitors need Fgf3 to commit serotonergic specification. Further, these results corroborate findings of an earlier study stating that hypothalamic serotonergic progenitors require Fgf-signalling via etv5b to maintain the progenitor pool (Bosco et al., 2013). Additionally, the transcriptome of the hypothalamus has been analysed and 13 previously overlooked transcripts of Fgf ligands are expressed at developmental stages. The transcriptome analysis provides evidence for a self-compensatory mechanism of fgf3 since expression of fgf3 is upregulated as a consequence of its own impairment. Moreover, the Fgf-signalling pathway appears to be mildly affected by fgf3 manipulation. Together, Fgf-signalling and especially Fgf3 are established to be of critical importance during hypothalamic development with effects on serotonergic, dopaminergic CSF-c and avp expressing cells. Furthermore, this thesis provides two strategies to impair the tph1a gene. Both strategies will facilitate investigations regarding the function of hypothalamic serotonergic CSF-c cells. Finally, the presented findings in this study provide insights into the emergence of the posterior recess region of the hypothalamus, thereby, contributing to the understanding of the evolution of the vertebrate hypothalamus.
Bone marrow dosimetry is a topic of high interest in molecular radiotherapy. Predicting the level of hematological toxicity is one of the most important goals of nuclear medicine radiation dosimetry. To achieve this, it is necessary to quantify the absorbed dose to the active bone marrow, thus aiming at administering the most efficient therapy with a minimum level of adverse effects in the patient. The anatomical complexity of trabecular bone and bone marrow leads to the need of applying non-nuclear medicine imaging methods for determining the spatial distribution of soft tissue, adipose tissue, and bone in spongiosa.
Therefore, the two objectives of this dissertation are: i) to apply magnetic resonance imaging (MRI) for quantification of the fat volume fraction, and ii) to validate a method based on dual-energy quantitative computed tomography (DEQCT) for quantification of the trabecular bone volume fraction.
In a first step, an MRI sequence (two-point Dixon) for fat-water separation was validated in a 3 Tesla system by quantifying the fat volume fraction in a phantom and the lumbar vertebrae of volunteers and comparing with magnetic resonance spectroscopy (MRS). After successful validation, the fat volume fraction was retrospectively measured in the five lumbar vertebrae of 44 patient images acquired in the clinical routine. The two-point Dixon showed a good quantification of the fat volume fraction in the phantom experiment (-9.8% maximum relative error with respect to the nominal values). In the volunteers, a non-significant difference between MRI and MRS was found for the quantification of the fat volume fraction in volumes-of-interest with similar dimensions and position in both quantification methodologies (MRI and MRS). In the study with patient data, the marrow conversion (red → yellow marrow) was found to be age-dependent, and slower in males (0.3% per year) than in females (0.5% per year). Also, considerable variability of the fat volume fraction in patients of similar ages and the same gender was observed.
These results enable the use of two-point Dixon MRI in the quantification of the fat volume fraction in the bone marrow. Additionally, the constant marrow conversion during adulthood suggests that a patient-specific approach should replace the assumption of a constant cellularity volume fraction of 0.7 (reference man) (1,2) as proposed by the International Commission on Radiological Protection (ICRP).
In a second step, a quantification method based on DEQCT was validated in two CT systems: i) a clinical CT integrated into a SPECT/CT and ii) a dual-source computed tomography (DSCT) system. The method was applied in two phantoms: the first was used to validate the DEQCT method by the quantification of the hydroxyapatite volume fraction in three vials of 50 ml each and three different hydroxyapatite concentrations (100 mg/cm3, 200 mg/cm3, 300 mg/cm3). The second phantom was the European spine phantom (ESP), an anthropomorphic spine phantom. It was used to quantify the bone mineral content (BMC) on the whole vertebra and the hydroxyapatite volume fraction (VFHA) in the spongiosa region of each vertebra of the phantom. Lastly, the BMC of lumbar vertebrae 1 (LV1) and 2 (LV2) was measured in a patient using DEQCT and dual-energy X-ray absorptiometry (DEXA). Furthermore, the hydroxyapatite volume fraction (VFHA) and the bone volume fraction (VFB) was calculated for both the whole vertebrae and the spongiosa region of LV1 and LV2.
The measured and nominal hydroxyapatite volume fraction in the vial phantom showed a good correlation (maximum relative error: 14.2%). The quantification of the BMC on the whole vertebra and the VFHA on the spongiosa region showed larger relative errors than in the validation phantom. The quantification of BMC on LV1 and LV2 showed relative errors between DEXA and DSCT equal to 7.6% (LV1) and -8.4% (LV2). Also, the values of the VFHA (mineral bone) were smaller than the VFB. This result is consistent with the bone composition (mineral bone plus organic material).
The DEQCT method enables the quantification of hydroxyapatite (mineral bone) and bone (mineral bone plus organic material) in a clinical setting. However, the method showed an overestimation of the quantified mineral bone volume fraction. This overestimation might be related to the lack of detailed information on the CT X-ray spectra and detector sensitivity. Also, the DEQCT method showed a dependency on the CT reconstruction kernel and the chemical description of the materials to be quantified.
Based on the results of this work, the feasibility for quantifying the fat volume fraction and the bone volume fraction in the spongiosa in a clinical setting has been demonstrated/proven. Furthermore, the differences in fat volume fraction in females and males, as well as the variability of the fat volume fraction in subjects of similar ages, questions the approximation of the cellularity volume fraction by only a single ICRP reference value in bone marrow dosimetry for molecular radiotherapy. Lastly, this study presents the first approach for non-invasive quantification of the bone volume fraction (mineral bone plus organic material) for improved bone marrow dosimetry.
Complex formation between macromolecules constitutes the foundation of most cellular processes. Most known complexes are made up of two or more proteins interacting in order to build a functional entity and therefore enabling activities which
the single proteins could otherwise not fulfill. With the increasing knowledge about
noncoding RNAs (ncRNAs) it has become evident that, similar to proteins, many of
them also need to form a complex to be functional. This functionalization is usually executed by specific or global RNA-binding proteins (RBPs) that are specialized
binders of a certain class of ncRNAs. For instance, the enterobacterial global RBPs
Hfq and ProQ together bind >80 % of the known small regulatory RNAs (sRNAs),
a class of ncRNAs involved in post-transcriptional regulation of gene expression.
However, identification of RNA-protein interactions so far was performed individually by employing low-throughput biochemical methods and thereby hindered the discovery of such interactions, especially in less studied organisms such
as Gram-positive bacteria. Using gradient profiling by sequencing (Grad-seq), the
present thesis aimed to establish high-throughput, global RNA/protein complexome resources for Escherichia coli and Streptococcus pneumoniae in order to provide a
new way to investigate RNA-protein as well as protein-protein interactions in these
two important model organisms.
In E. coli, Grad-seq revealed the sedimentation profiles of 4,095 (∼85 % of
total) transcripts and 2,145 (∼49 % of total) proteins and with that reproduced
its major ribonucleoprotein particles. Detailed analysis of the in-gradient distribution of the RNA and protein content uncovered two functionally unknown
molecules—the ncRNA RyeG and the small protein YggL—to be ribosomeassociated. Characterization of RyeG revealed it to encode for a 48 aa long, toxic protein that drastically increases lag times when overexpressed. YggL was shown to
be bound by the 50S subunit of the 70S ribosome, possibly indicating involvement
of YggL in ribosome biogenesis or translation of specific mRNAs.
S. pneumoniae Grad-seq detected 2,240 (∼88 % of total) transcripts and 1,301
(∼62 % of total) proteins, whose gradient migration patterns were successfully reconstructed, and thereby represents the first RNA/protein complexome resource
of a Gram-positive organism. The dataset readily verified many conserved major
complexes for the first time in S. pneumoniae and led to the discovery of a specific
interaction between the 3’!5’ exonuclease Cbf1 and the competence-regulating ciadependent sRNAs (csRNAs). Unexpectedly, trimming of the csRNAs by Cbf1 stabilized the former, thereby promoting their inhibitory function. cbf1 was further shown
to be part of the late competence genes and as such to act as a negative regulator of
competence.
Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies.
A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx.
Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis.
Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches.
The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli.
Quantitative genetics is the study of continuously distributed traits and their ge-
netic components. Recent developments in DNA sequencing technologies and
computational systems allow researchers to conduct large scale in silico studies.
However, going from raw DNA reads to genomic prediction of quantitative traits
with the help of neural networks is a long and error-prone process. In the course
of this thesis, many steps involved in this process will be assessed in depth. Chap-
ter 2 will feature a study that compares the landscape of chloroplast genome as-
sembly tools. Chapter 3 will present a software to perform genome-wide associa-
tion studies using modern tools, which allow GWAS-Flow to outperform current
state of the art software packages. Chapter 4 will give an in depth introduc-
tion to machine learning and the nature of quantitative traits and will combine
those to genomic prediction with artificial neural networks and compares the re-
sults to those of algorithms based on linear mixed models. Finally, in Chapter 5
the results from the previous chapters are summarized and used to elucidate the
complex nature of studies concerning quantitative genetics.
Einleitung:
TRPC-Kanäle spielen eine wichtige Rolle in der Pathologie der Herzinsuffizienz und kardialen Hypertrophie. Diese Effekte werden unter anderem über den Calcineurin-NFAT-Signalweg vermittelt. Ein wichtiger Interaktionspartner und Regulator von TRPC-Kanälen ist das Protein FKBP52. Mittels eines Yeast Two-Hybrid Systems wurde in einer kardialen cDNA library eine Interaktion zwischen einem C-terminalen Fragment von TRPC3 (AS 742-848), welches außerhalb der bekannten FKBP-Bindungsdomäne (AS 703-714) liegt, und FKBP52 beobachtet. Da dies eine weitere Bindungsstelle in FKBP52 vermuten ließ, erzeugten wir ein Fragment von FKBP52, welches FKBP52s genannt wurde und dem die funktionell relevante PPIase I-Domäne mit der bekannten Bindungsstelle fehlt. Eine erste Co-IP zwischen diesem Fragment und TRPC3 war erfolgreich.
Ziel:
Die Bestimmung, ob die Anwesenheit des verkürzten FKBP52 in vivo die Komplexbildung aus TRPC3 bzw. TRPC4 und dem Wildtyp-FKBP52 unterdrückt. Zusätzlich, ob FKBP52s die Interaktion zwischen TRPC3 bzw. TRPC4 und Calcineurin in vivo unterbricht und damit die Aktivierung des Calcineurin-NFAT-Signalweges hemmt.
Methoden:
Co-Immunopräzipitationen (Co-IP) wurden mit HEK-293-Zellen durchgeführt, die mit cDNA transfiziert wurden, welche Gene für TRPC3, TRPC4, Calcineurin A und FKBP52s enthielt. Zur Bestimmung der nukleären Translokation von NFATc1 mittels Fluoreszenzmikroskopie wurden HEK-293-Zellen mit TRPC3, TRPC4, GFP-NFATc1 ± FKBP52s transfiziert. Die statistische Analyse erfolgte mit einer One-Way ANOVA.
Ergebnisse:
In dieser Arbeit konnte gezeigt werden, dass FKBP52 sowohl mit TRPC3 als auch mit TRPC4 interagiert. Ebenso wurde festgestellt, dass FKBP52 auch ohne seine katalytische PPIase I-Domäne Bindungen mit TRPC3 bzw. TRPC4 eingeht. Dieses FKBP52-Konstrukt nimmt ebenso an der Komplexbildung mit TRPC3 bzw. TRPC4 und Calcineurin teil. Des Weiteren ließ sich für TRPC3 zeigen, dass unter Stimulation mit Carbachol (GPCR-Agonist) bei Anwesenheit dieses gekürzten FKBP52 eine signifikant geringere Aktivierung und Wanderung des Transkriptionsfaktors NFAT in den Nucleus erfolgte.
Schlussfolgerung:
FKBP52 spielt daher eine wichtige Rolle in dieser Signalkaskade, indem es entscheidend an der Aktivierung von Calcineurin und dessen Rekrutierung zum TRPC-Kanalkomplex beteiligt ist und damit auch an der Aktivierung des Calcineurin-NFAT-Signalweges.
We are living in a system that underlies permanent environmental changes due to the rotation of our planet. These changes are rhythmic with the most prominent one having a period of about 24 hours, but also shorter and longer rhythms characterize our environment. To cope with the ever-changing environmental conditions, it is thought to be beneficial if an organism can track and anticipate these changes. The so called endogenous clocks enable this and might provide a fitness advantage. To investigate and unravel the mechanism of endogenous clocks Chronobiologists have used different model organisms. In this thesis Drosophila melanogaster was used as model organism with its about 150 clock neurons representing the main endogenous clock of the fly in the central brain.
The molecular mechanisms and the interlocked feedback loops with the main circadian key players like period, timeless, clock or cycle are under investigation since the 1970s and are characterized quite well so far. But the impact of a functional endogenous clock in combination with diverse factors and the resulting fitness advantages were analysed in only a few studies and remains for the most part unknown. Therefore the aim of this thesis was to unravel the impact of Drosophila melanogaster`s endogenous clock on the fitness of the fly. To achieve this goal different factors – like day length, humidity and food composition – were analyzed in wild type CS and three different period mutants, namely perL, perS and per01, that carry a point mutation altering or abolishing the free-running period of the fruit fly as well as a second arrhythmic strain, clkAR.
In competition assay experiments wild type and clock mutant flies competed for up to 63 generations under a normal 24 hour rhythm with 12 hours light/day and 12 hours darkness/night (LD12:12) or T-cycles with 19 or 29 hours, according to the mutants free-running period, or constant light (LL) in case of the arrhythmic mutant as well as under natural-like outdoor conditions in two consecutive years. Overall the wild type CS strain was outcompeting the clock mutant strains independent of the environmental conditions. As the perL fly strain elongated their free-running period, the competition experiments were repeated with naturally cantonized new fly strains. With these experiments it could be shown that the genetic background of the fly strains – which are kept for decades in the lab, with backcrosses every few years – is very important and influences the fitness of flies. But also the day length impacts the fitness of the flies, enabling them to persist in higher percentage in a population under competition. Further factors that might influence the survival in a competing population were investigated, like e.g. mating preferences and locomotor activity of homo- and heterozygous females or sperm number of males transferred per mating. But these factors can still not explain the results in total and play no or only minor roles and show the complexity of the whole system with still unknown characteristics.
Furthermore populations of flies were recorded to see if the flies exhibit a common locomotor activity pattern or not and indeed a population activity pattern could be recorded for the first time and social contact as a Zeitgeber could be verified for Drosophila melanogaster.
In addition humidity and its impact on the flies´ fitness as well as a potential Zeitgeber was examined in this thesis. The flies experienced different relative humidities for eclosion and wing expansion and humidity cycle phase shifting experiments were performed to address these two different questions of fitness impact and potential Zeitgeber. The fruit fly usually ecloses in the morning hours when the relative humidity is quite high and the general assumption was that they do so to prevent desiccation. The results of this thesis were quite clear and demonstrate that the relative humidity has no great effect on the fitness of the flies according to successful eclosion or wing expansion and that temperature might be the more important factor. In the humidity cycle phase shifting experiments it could be revealed that relative humidity cannot act as a Zeitgeber for Drosophila melanogaster, but it influences and therefore masks the activity of flies by allowing or surpressing activity at specific relative humidity values.
As final experiments the lifespan of wild type and clock mutant flies was investigated under different day length and with different food qualities to unravel the impact of these factors on the fitness and therefore survival of the flies on the long run. As expected the flies with nutrient-poor minimum medium died earlier than on the nutrient-rich maximum medium, but a small effect of day length could also be seen with flies living slightly longer when they experience environmental day length conditions resembling their free-running period. The experiments also showed a fitness advantage of the wild type fly strain against the clock mutant strains for long term, but not short term (about the first 2-3 weeks).
As a conclusion it can be said that genetic variation is important to be able to adapt to changing environmental conditions and to optimize fitness and therefore survival. Having a functional endogenous clock with a free-running period of about 24 hours provides fitness advantages for the fruit fly, at least under competition. The whole system is very complex and many factors – known and unknown ones – play a role in this system by interacting on different levels, e.g. physiology, metabolism and/or behavior.
The present dissertation aims to shed light on different mechanisms of socio-emotional feedback in social decision-making situations. The objective is to evaluate emotional facial expressions as feedback stimuli, i.e., responses of interaction partners to certain social decisions. In addition to human faces, artificial emojis are also examined due to their relevance for modern digital communication. Previous research on the influence of emotional feedback suggests that a person's behavior can be effectively reinforced by rewarding stimuli. In the context of this dissertation, the differences in the feedback processing of human photographs and emojis, but also the evaluation of socially expected versus socially unexpected feedback were examined in detail in four studies. In addition to behavioral data, we used the electroencephalogram (EEG) in all studies to investigate neural correlates of social decision-making and emotional feedback.
As the central paradigm, all studies were based on a modified ultimatum game. The game is structured as follows: there is a so-called proposer who holds a specific amount of money (e.g., 10 cents) and offers the responder a certain amount (e.g., 3 cents). The responder then decides whether to accept or reject the offer. In the version of the ultimatum game presented here, different types of proposers are introduced. After the participants have accepted or rejected in the role of the responder, the different proposers react to the participant’s decision with specific emotional facial expressions. Different feedback patterns are used for the individual experiments conducted in the course of this dissertation.
In the first study, we investigated the influence of emotional feedback on decision-making in the modified version of the ultimatum game. We were able to show that a proposer who responds to the acceptance of an offer with a smiling face achieves more accepted offers overall than a control proposer who responds to both accepted and rejected offers with a neutral facial expression. Consequently, the smile served as a positive reinforcement. Similarly, a sad expression in response to a rejected offer also resulted in higher acceptance rates as compared to the control identity, which could be considered an expression of compassion for that proposer. On a neuronal level, we could show that there are differences between simply looking at negative emotional stimuli (i.e., sad and angry faces) and their appearance as feedback stimuli after rejected offers in the modified ultimatum game. The so-called feedback-related negativity was reduced (i.e., more positive) when negative emotions appeared as feedback from the proposers. We argued that these findings might show that the participants wanted to punish the proposers by rejecting an offer for its unfairness and therefore the negative feedback met their expectations. The altered processing of negative emotional facial expressions in the ultimatum game could therefore indicate that the punishment is interpreted as successful. This includes the expectation that the interaction partner will change his behavior in the future and eventually make fairer offers.
In the second study we wanted to show that smiling and sad emojis as feedback stimuli in the modified ultimatum game can also lead to increased acceptance rates. Contrary to our assumptions, this effect could not be observed. At the neural level as well, the findings did not correspond to our assumptions and differed strongly from those of the first study. One finding, however, was that the neural P3 component showed how the use of emojis as feedback stimuli particularly characterizes certain types of proposers. This is supported by the fact that the P3 is increased for the proposer who rewards an acceptance with a smile as well as for the proposer who reacts to rejection with a sad emoji compared to the neutral control proposer.
The third study examined the discrepancy between the findings of the first and second study. Accordingly, both humans and emojis representing the different proposers were presented in the ultimatum game. In addition, emojis were selected that showed a higher similarity to known emojis from common messenger services compared to the second study. We were able to replicate that the proposers in the ultimatum game, who reward an acceptance of the offer with a smile, led to an increased acceptance rate compared to the neutral control proposers. This difference is independent of whether the proposers are represented by emojis or human faces. With regard to the neural correlates, we were able to demonstrate that emojis and human faces differ strongly in their neural processing. Emojis showed stronger activation than human faces in the face-processing N170 component, the feedback-related negativity and the P3 component. We concluded that the results of the N170 and feedback-related negativity could indicate a signal for missing social information of emojis compared to faces. The increased P3 amplitude for emojis might imply that emojis appear unexpectedly as reward stimuli in a social decision task compared to human faces.
The last study of this project dealt with socially unexpected feedback. In comparison to the first three studies, new proposer identities were implemented. In particular, the focus was on a proposer who reacted to the rejection of an offer unexpectedly with a smile and to the acceptance with a neutral facial expression. According to the results, participants approach this unexpected smile through increased rejection, although it is accompanied by financial loss. In addition, as reported in studies one and three, we were able to show that proposers who respond to the acceptance of an offer with a smiling face and thus meet the expectations of the participants have higher offer acceptance rates than the control proposer. At the neuronal level, especially the feedback from the socially unexpected proposer led to an increased P3 amplitude, which indicates that smiling after rejection is attributed a special subjective importance.
The experiments provide new insights into the social influence through emotional feedback and the processing of relevant social cues. Due to the conceptual similarity of the studies, it was possible to differentiate between stable findings and potentially stimulus-dependent deviations, thus creating a well-founded contribution to the current research. Therefore, the novel paradigm presented here, and the knowledge gained from it could also play an important role in the future for clinical questions dealing with limited social competencies.
Background: Integrase strand transfer inhibitors (INSTIs) are the latest addition to the array of antiretroviral compounds used to treat an infection with Human Immunodeficiency Virus (HIV). Due to their high efficacy and increased tolerability, INSTIs have become an integral part of first-line therapy in most high-income countries over the past years. However, little is known about HIV-1’s genetic inter- and intra-subtype diversity on the Integrase (IN)-gene and its impact on the emergence of INSTI-resistance. In the absence of a functional cure, long-term efficacy of first-line compounds remains paramount for reducing virological failure and curbing on-going HIV transmissions. South Africa, harbouring more than 20% of the global HIV burden (7.7 / 37.9 million people), requires international attention in order to globally pursue UNAIDS’ (Joint United Nations Programme on HIV/AIDS) 90-90-90 goals and the road to ending the HIV/AIDS (Acquired immunodeficiency syndrome) pandemic by 2030.
Methods: In this study, the prevalence of INSTI-resistance associated mutations (RAM) was investigated in a cohort of 169 archived drug-naïve blood samples from multiple collection sites around Cape Town, South Africa. Viral RNA was isolated from plasma samples, the integrase fragment amplified by RT-PCR and subsequently sequenced by Sanger-sequencing. Additionally, all publicly available drug-naïve, South African IN sequences, isolated before the availability of the first INSTIs in 2007, were retrieved from the Los Alamos HIV sequence database (n=284). All sequences were analysed for RAMs using the Stanford HIV Drug resistance database. The identification of polymorphism in the South African subtype C IN consensus sequence allowed for comparative analyses with global subtype B, as well as subtype C sequences, from countries other than South Africa.
Results: The IN gene could be amplified and sequenced in 95/169 samples (56%). Phylogenetic inference revealed close homology between three sequence-pairs, warranting the exclusion of 3/95 sequences from further analyses. Of the 92 samples used for mutational analyses, 86/92 (93.5%) belonged to subtype C, 5/92 (5.4%) to subtype B and 1/92 (1.1%) to subtype A. The prevalence of major and accessory INSTI RAMs was 0/92 (0%) and 1/91 (1.1%), respectively, similar to the observed rates of 8/284 (2.8%) and 8/284 (2.8%) in the database sequences (p = 0.2076 and p = 0.6944, Fisher’s exact test). Compared to subtype B IN sequences, 15 polymorphisms were significantly enriched in South African subtype C sequences (corrected p<0.0015. Fisher’s exact test, Bonferroni post-hoc procedure).
Compared to subtype C IN sequences isolated outside South Africa, four polymorphisms were significantly enriched in this study cohort (corrected p<0.0014, Fisher’s exact test, Bonferroni post-hoc procedure). The highest prevalence margin was observed for the polymorphism Met50Ile being present in 60.1% of South African subtype C sequences, compared to 37% in non-South African subtype C sequences.
Conclusions: The low prevalence of major and minor RAMs in all South African Integrase sequences predicts a high susceptibility to INSTIs, however, the presence of natural polymorphisms, in particular Met50Ile, in the majority of sequences warrants further monitoring under therapeutic pressure, as their role in mutational pathways leading to INSTI- resistance is yet to be determined. Additionally, this study revealed the presence of substantial inter- and intra-subtype diversity within the HIV-1 Subtype C IN-gene. These results implicate the need for more research on a regional, potentially patient-specific level, as mutational insights from other diverse backgrounds may not accurately represent the South African context. The implementation of a national pre-treatment INSTI-resistance screening program may provide necessary insights into the development of mutational pathways leading to INSTI-resistance under therapeutic pressure for the South African context and thereby bring South Africa one step closer to achieving UNAIDS 90-90-90 goals and ending the AIDS epidemic by 2030.
Despite the advancement in the treatment from genotoxic drugs to more targeted therapies, multiple myeloma (MM) remains incurable. MM is known for its complex genetic heterogeneity as different genetic lesion accrue over the course of the disease. The current work focuses on the functional analysis of genetic lesions found at the time of diagnosis and relapse and their potential role regarding therapy response and refractory disease. Genetic lesions involving tumor suppressor gene TP53, are found at diagnosis and tend to accrue during disease progression. Different types of mono- and biallelic TP53 alterations were emulated in the AMO1 cell line model, were functionally characterized and tested for their potential role in therapy response. Both types of single hit TP53 alteration (deletion 17p and TP53 point mutations) were found to have similar adverse effects on the functionality of the p53 system and response to genotoxic drugs which were completely abolished in the case of double hit TP53 alterations (no p53 expression, or mutant overexpression in wild type TP53 deletion background). Whereas, sensitivity to proteasome inhibitors remained unaltered. Using the clonal competition assay (CCA), single TP53 hit clones were found to have a fitness advantage over wildtype cells. Proliferative cell fitness was further enhanced in double hit TP53 clones, as they dominated wildtype and single hit TP53 clones in the CCA. Presence of external selection pressure in the form of low dose melphalan expedited the intrinsic fitness advantage. Alterations found in CUL4B, a component of CRL4-CRBN protein complex, a target of immunomodulatory drugs (IMiDs), were also functionally analyzed in the current study. Hotspot mutations and mutations found in IMiDs refractory patients were modelized in L363 cells and their role in IMiDs sensitivity was studied. CUL4B mutations were found not to be involved in providing lenalidomide resistance to the cell, whereas knocking CUL4B out was observed to provide negative fitness to the cells in CCA. In the presence of external selection pressure, these clones showed fitness, which was lost in the case of lenalidomide withdrawal. This shows that some alterations may play a role in refractory patients only in the presence of therapy, and as soon as therapy is discontinued, these altered clones may disappear such as clones with alterations in CUL4B. On the other hand, some alterations provide drug-independent intrinsic positive fitness, however, be further enhanced by drug exposure, such as seen in case of TP53 altered clones. Therefore, close monitoring and functional analysis of evolving clones is desired during disease progression, as it can be helpful in therapeutic guidance to achieve a better outcome for patients.
TTFields sind eine Therapieoption des GBM, welche als alternierende elektrische Felder den Aufbau des mitotischen Spindelapparates stören. Gleichzeitig überwacht der SAC, mit seiner Schlüsselkomponente der Kinase MPS1, eine korrekte Anheftung der Spindelfasern an die Kinetochore der Chromosomen. Eine Inhibition des SAC durch den Inhibitor MPS1-IN-3 in Kombination mit Vincristin führt zu einem synergistischen Effekt auf das Tumorwachstum in vitro und in vivo. Aus diesen Erkenntnissen folgerten wir die Hypothese, dass eine SAC-Inhibition die Wirkung von TTFields verstärken könnte. Um dies zu testen, wurden Zellen der Zelllinien U87 und GaMG über 72h mit TTFields, MPS1-IN-3 oder einer Kombination aus den beiden behandelt. Anschließend wurden die Zellen gezählt, es wurde eine Analyse des Zellzyklus vorgenommen und apoptotische Zellen wurden via TUNEL-Assay detektiert. Die Kombinationsbehandlung aus TTFields und MPS1-IN-3 führte zu einer Reduktion der Zellzahl (U87: -54,3% vs. TTFields, p=0,0046; -52,9% vs. MPS1-IN-3, p=0,0026; GaMG: -74,3% vs. TTFields, p=0,0373; -84% vs. MPS1-IN-3, p<0,00001). Nur 28,1% mehr Zellen als ausgesät waren bei der Zelllinie U87 zu finden (TTFields: 179,1%; MPS1-IN-3: 168,3%), während es bei GaMG-Zellen sogar 62% weniger Zellen als ausgesät waren. Im Zellzyklus zeigte sich eine Abnahme der Zellen von der G1-Phase (U87: -59,9% vs. TTFields, p=0,0007; -42,1% vs. IN-3, p=0,0426; GaMG: -45,1% vs. TTFields, p=0,0276; -51,6% vs. IN-3, p=0,0020), während es zu einem massiven Anstieg von toten Zellen kam (U87: 2,9fach vs. TTFields, p=0,0022; 2,2fach vs. IN-3, p=0,0046; GaMG: 5,6fach vs. TTFields, p=0,0078; 7,8fach vs. IN-3, p=0,0005). Diese Zellen ließen sich im TUNEL-Assay als durch Apoptose zu Grunde gegangene Zellen weiter identifizieren (U87: 5,4fach vs. TTFields, p=0,0489; 6,2fach vs. IN-3, p=0,0278; GaMG: 8,9fach vs. IN-3, p=0,0110). Diese Ergebnisse sind erste und wichtige Hinweise für eine Verstärkung der Wirkung von TTFields durch eine Inhibition des SAC und liefern eine gute Grundlage für weitere Forschung zur Verbesserung der Therapie des GBM.
Humans in our environment are of special importance to us. Even if our minds are
fixated on tasks unrelated to their presence, our attention will likely be drawn
towards other people’s appearances and their actions.
While we might remain unaware of this attentional bias at times, various studies have demonstrated the preferred visual scanning of other humans by recording eye movements in laboratory settings. The present thesis aims to investigate the circumstances under and the mechanisms by which this so-called social attention operates.
The first study demonstrates that social features in complex naturalistic scenes are prioritized in an automatic fashion. After 200 milliseconds of stimulus presentation, which is too brief for top-down processing to intervene, participants targeted image areas depicting humans significantly more often than would be expected from a chance distribution of saccades. Additionally, saccades towards these areas occurred earlier in time than saccades towards non-social image regions. In the second study, we show that human features receive most fixations even when bottom-up information is restricted; that is, even when only the fixated region was visible and the remaining parts of the image masked, participants still fixated on social image regions longer than on regions without social cues. The third study compares the influence of real and artificial faces on gaze patterns during the observation of dynamic naturalistic videos. Here we find that artificial faces, belonging to humanlike statues or machines, significantly predicted gaze allocation but to a lesser extent than real faces. In the fourth study, we employed functional magnetic resonance imaging to investigate the neural correlates of reflexive social attention. Analyses of the evoked blood-oxygenation level dependent responses pointed to an involvement of striate and extrastriate visual cortices in the encoding of social feature space.
Collectively, these studies help to elucidate under which circumstances social
features are prioritized in a laboratory setting and how this prioritization might be achieved on a neuronal level. The final experimental chapter addresses the question whether these laboratory findings can be generalized to the real world. In this study, participants were introduced to a waiting room scenario in which they interacted with a confederate. Eye movement analyses revealed that gaze behavior heavily depended on the social context and were influenced by whether an interaction is currently desired. We further did not find any evidence for altered gaze behavior in socially anxious participants. Alleged gaze avoidance or hypervigilance in social
anxiety might thus represent a laboratory phenomenon that occurs only under very specific real-life conditions. Altogether the experiments described in the present
thesis thus refine our understanding of social attention and simultaneously
challenge the inferences we can draw from laboratory research.
Die nicht-invasive Gefäßdiagnostik stellt einen wichtigen Pfeiler in der Prävention von Herz-Kreislauferkrankungen dar. Während lange Zeit die sonographische Messung der cIMT, als morphologisches Korrelat der Gefäßalterung, als Goldstandard galt, ist in den letzten Jahren in Gestalt der Pulswellenanalyse/PWV-Messung eine Technik weiterentwickelt worden, die, als funktionelles Korrelat der Gefäßalterung, aufgrund der leichteren Durchführbarkeit und geringerer Untersucherabhängigkeit und Kosten vielversprechend ist. So erlaubt die Messung der Pulswelle mittels gewöhnlicher Blutdruckmanschetten, genau wie die cIMT, die Berechnung des individuellen Gefäßalters und die Diagnostik für das Vorliegen eines Endorganschadens der Blutgefäße.
Um die Messergebnisse der beiden Untersuchungen miteinander zu vergleichen, wurden beide in der EUROASPIRE-IV Studie an Patienten mit koronarer Herzkrankheit durchgeführt. Die Auswertung der Messergebnisse der mit dem Vascular Explorer durchgeführten Pulswellenanalyse/PWV-Messung ergab überraschenderweise, dass die Mehrheit der herzkranken Patienten weder eine vaskuläre Voralterung noch einen Endorganschaden der Blutgefäße aufweisen. Im Falle der cIMT-Messung war Gegenteiliges der Fall, was trotz der medikamentösen Therapie der Patienten so zu erwarten war. Weiterhin zeigte sich lediglich eine geringe Korrelation zwischen den Messergebnissen beider Untersuchungen. Die Determinanten der einzelnen Messwerte aus cIMT und Pulswellenanalyse/PWV-Messung waren deckungsgleich mit den in der Literatur beschriebenen Faktoren, wenn auch viele der sonst signifikanten Regressoren das Signifikanzniveau in unserer Auswertung nicht unterschritten.
Eine Limitation der funktionellen Gefäßdiagnostik liegt derzeit darin, dass die Messergebnisse stark von dem verwendeten Messgerät abhängen. Es liegen noch zu wenig Vergleichsstudien vor, um die Messergebnisse, speziell von neueren Geräten wie dem Vascular Explorer, auf andere zu übertragen. Bei der Berechnung des Gefäßalters sollten daher optimalerweise gerätespezifische Normwerte vorliegen, was beim Vascular Explorer nicht der Fall ist. Gleiches gilt für die Verwendung des PWVcf-Grenzwerts für die Diagnose eines Endorganschadens der Blutgefäße.
Analog hat auch die Messung der cIMT gewisse Einschränkungen. So wäre eine weitere Standardisierung der Messorte (A. carotis communis vs Bulbus vs A. carotis interna), zwischen denen sich die durchschnittliche cIMT erheblich unterscheidet, sowie der Messparameter (Minimal- vs Maximal- vs Mittelwert) wünschenswert. Die universelle Anwendung eines cIMT-Grenzwerts zur Diagnose eines Endorganschadens der Blutgefäße ist daher kritisch zu sehen. Dies zeigt sich auch darin, dass in den neuesten Leitlinien der bislang geltende Grenzwert angezweifelt und kein aktuell gültiger Grenzwert mehr genannt wird.
Wir interpretieren unsere Ergebnisse dahingehend, dass unsere Messung der cIMT die zu erwartende pathologische Gefäßalterung bei Patienten mit koronarer Herzkrankheit besser widerspiegelt als die Messung der Pulswelle mit dem Vascular Explorer. Welche der beiden Untersuchungen hinsichtlich der prognostischen Wertigkeit überlegen ist, muss im Rahmen von Längsschnittstudien geklärt werden.
T cells play an essential role in the immune system. Engaging the T cell receptor (TCR) initiates a cascade of signaling events that activates the T cells. Neutral sphingomyelinase (NSM) is a member of a superfamily of enzymes responsible for the hydrolysis of sphingomyelin into phosphocholine and ceramide. Sphingolipids are essential mediators in signaling cascades involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation.
Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether a deregulated metabolic activity supports the hyper-reactivity of NSM2 deficient T cells. This work demonstrates that the ablation of NSM2 activity affects the metabolism of the quiescent CD4+ T cells. These accumulate ATP in mitochondria and increase basal glycolytic activity by increasing the basal glucose uptake and GLUT1 receptor expression, which, altogether, raises intracellular ATP levels and boosts cellular respiration. The increased basal metabolic activity is associated with rapid phosphorylation of S6, a mTORC1 target, as well as enhanced elevation total ATP levels within the first hour after CD3/CD28 costimulation. Increased metabolic activity in resting NSM2 deficient T cells does, however, not support sustained stimulated responses. While elevated under steady-state conditions and elevated early after co-stimulation in NSM2 deficient CD4+ T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 hours of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4+ T cells yet fails to support sustained T cell responses upon antigenic stimulation without affecting T cell survival.
Structural and functional elucidation of the Type VIIb secretion system from Staphylococcus aureus
(2020)
The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion.
EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 Å resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely.
The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 Å. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition.