Refine
Has Fulltext
- yes (62) (remove)
Is part of the Bibliography
- yes (62)
Year of publication
- 2014 (62) (remove)
Document Type
- Doctoral Thesis (62)
Keywords
- T-Lymphozyt (8)
- Maus (6)
- Thrombozyt (5)
- Escherichia coli (3)
- Structural Biology (3)
- Thrombose (3)
- Transkriptionsfaktor (3)
- Arteriosklerose (2)
- Aufmerksamkeits-Defizit-Syndrom (2)
- Blutstillung (2)
- Calcium (2)
- Cancer (2)
- Chronophin (2)
- Dendritische Zelle (2)
- Entzündung (2)
- Genexpression (2)
- Herzinfarkt (2)
- Herzmuskelzelle (2)
- Hämostase (2)
- Immunologie (2)
- Inflammation (2)
- Inhibitor (2)
- Kernspintomographie (2)
- Monoklonaler Antikörper (2)
- Myc (2)
- PET (2)
- Platelet (2)
- Positronen-Emissions-Tomographie (2)
- Ratte (2)
- Staphylococcus aureus (2)
- Taufliege (2)
- dendritic cells (2)
- miRNS (2)
- virulence (2)
- ADHD (1)
- Adaptation (1)
- Adapterprotein (1)
- Alpaca (1)
- Analgesie (1)
- Aneuploidy (1)
- Angiogenese (1)
- Angiogenesis (1)
- Angst (1)
- Animal model (1)
- Anpassung (1)
- Antiangiogenese (1)
- Antibiotikum (1)
- Antigen CD4 (1)
- Antigen CD40 (1)
- Antigen CD8 (1)
- Assoziation (1)
- Atherosclerosis (1)
- Atomic-force-microscopy (1)
- Attention deficit / hyperactivity disorder (1)
- Aufmerksamkeitsdefizit-Syndrom (1)
- Aurora B (1)
- Autoaggressionskrankheit (1)
- Autoimmunität (1)
- Autotransporterprotein (1)
- B cells (1)
- B-Lymphozyt (1)
- B-Zell-Lymphom (1)
- BTN3A1 and Phospho-antigen presentation (1)
- Bach (1)
- Bacteria (1)
- Bakterien (1)
- Bestimmung des Relaxations-Parameters in der Magnetresonanztomografie (1)
- Bevacizumab (1)
- Bilderzeugung (1)
- Bildgebendes Verfahren (1)
- Biodiversität (1)
- Biologische Uhr (1)
- Biomaterial (1)
- Biomedicine (1)
- Bioreaktor (1)
- Borneo (1)
- Brustdrüsenentzündung (1)
- Burkitt Lymphom (1)
- Burkitt Lymphoma (1)
- CD150 (1)
- CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine (1)
- CD40-targeted bifunctional scFv-TRAIL fusion proteins (1)
- CD8+ T cells (1)
- CD8+ T-Zellen (1)
- COMT polymorphism (1)
- Calciumhomöostase (1)
- Calciumkanal (1)
- Calciumtransport (1)
- Candida albicans (1)
- Cardiac hypertrophy (1)
- Cardiomyocytes (1)
- Catecholmethyltransferase <Catechol-0-Methyltransferase> (1)
- Cell Cycle (1)
- Cellular invasion (1)
- Cerebrospinal fluid (CSF) (1)
- ChIP-Seq (1)
- Chemokin CXCL10 (1)
- Cholesterinstoffwechsel (1)
- Chromosom 6 (1)
- Chromosome 6 (1)
- Chronic Mild Stres (1)
- Chronobiologie (1)
- Circadian Rhythms (1)
- Collybistin (1)
- Colonkrebs (1)
- Compressed Sensing (1)
- Craving (1)
- Crosstalk (1)
- Cytokine (1)
- DNA Repair (1)
- DNA lesion (1)
- DNS-Bindung (1)
- DNS-Reparatur (1)
- DNS-Schaden (1)
- DNS-Schädigung (1)
- Deep sequencing (1)
- Dendritische Zellen (1)
- Desmoglein 2 (1)
- Desmoglein 3 (1)
- Desmogleine (1)
- Dexamethason (1)
- Differentielle Genexpression (1)
- Dimerisierung (1)
- Diversity (1)
- Dopamin (1)
- Dopamin-Transporter (1)
- Dopamine transporter (1)
- Drosophila (1)
- Dual RNA-seq (1)
- Dynein Light Chain (1)
- E. coli Nissle (1)
- EAE (1)
- EHEC (1)
- Eap (1)
- Echinococcus multilocularis (1)
- Einzel-Molekül (1)
- Elektroakupunktur (1)
- Elektrospinning (1)
- Encephalomyelitis (1)
- Endocytose (1)
- Entwicklung (1)
- Erwachsener (1)
- Essgewohnheit (1)
- Essverhalten (1)
- Evolution (1)
- Exon Array (1)
- Experimental Autoimmune Encephalomyelitis (EAE) (1)
- Experimentelle Autoimmune Enzephalomyelitis (EAE) (1)
- FBXO6 (1)
- Fanconi Anemia (1)
- Fanconi-Anämie (1)
- Fear conditioning (1)
- Filter (1)
- Foxp3+CD4+ regulatorische T-Zelle (1)
- Foxp3+CD4+ regulatory T cell (1)
- Froschlurche (1)
- Fuchsbandwurm (1)
- Function (1)
- Funktionelle Annotation (1)
- Funktionelle Kernspintomographie (1)
- Gen notch (1)
- Gene regulation (1)
- Gene therapy (1)
- General amplifier model (1)
- Genetics (1)
- Genregulation (1)
- Gephyrin (1)
- Gerinnungsfaktor XII (1)
- Gewebekultur (1)
- Glioblastom (1)
- Glioblastoma (1)
- Gliom (1)
- Glioma (1)
- Glucosetransporter (1)
- Glucosetransportproteine (1)
- Glutamat-Decarboxylase (1)
- Glykosylierung (1)
- Glykosyltransferase (1)
- HAD phosphatase (1)
- HIV (1)
- HIV-associated neurocognitive disorder (HAND) (1)
- HIV-assoziierte neurokognitive Störung (HAND) (1)
- Haloacid dehalogenase (1)
- Harnwegsinfektion (1)
- Heilung (1)
- Heißhunger (1)
- Helferzelle (1)
- Herzinsuffizienz (1)
- Herzruptur (1)
- Hey Proteine (1)
- Hey proteins (1)
- High throughput screening (1)
- Hochdurchsatzsequenzierung (1)
- Huwe1 (1)
- Hypertrophie (1)
- Hypertrophy (1)
- Hämsotase (1)
- ITAM (1)
- ITAM-signalling (1)
- Immunaktivierung (1)
- Immune activation (1)
- Immunsystem (1)
- Implantatentwicklung (1)
- In vivo Imaging (1)
- Infection imaging (1)
- Infektion (1)
- Infektionsbildgebung (1)
- Infektionsbiologie (1)
- Innere Uhr (1)
- Interferon <Gamma-> (1)
- Invasion (1)
- Keratinozyt (1)
- Kinasen (1)
- Klassische Konditionierung (1)
- Knochenmarktransplantation (1)
- Knockout-Maus (1)
- Kolonkarzinom (1)
- Komprimierte Abtastung (1)
- Kontingenz (1)
- Krebs <Medizin> (1)
- Kuh (1)
- LAT2 (1)
- Larve (1)
- Latrophilin receptor (1)
- Learning (1)
- Leishmaniose (1)
- Lernen (1)
- Liquor (1)
- Macrophage (1)
- Magnetic Resonance Relaxometry (1)
- Magnetresonanz-Relaxometrie (1)
- Makrophage (1)
- Makrophagen (1)
- Malaria tropica (1)
- Marine sponge (1)
- Masern (1)
- Masernvirus (1)
- Medizintechnik (1)
- Meeresschwämme (1)
- Meniskustransplantation (1)
- Metalloproteinasen (1)
- Metatranscriptomics (1)
- Methylphenidat (1)
- MicroRNA (1)
- Microbial community (1)
- Migration (1)
- Miz1 (1)
- Model Based Reconstruction Algorithms in Magnetic Resonance Imaging (1)
- Modellbasierte-Rekonstruktionsalgorithmen in der Magnetresonanztomografie (1)
- Molekularbiologie (1)
- Molekulare Bildgebung (1)
- Morris Water Maze (1)
- Multidrug-Resistenz (1)
- Multiple Sclerosis (1)
- Multiple Sklerose (1)
- Murine models of thrombosis and haemostasis (1)
- Myokardinfarkt (1)
- NF-kB (1)
- NFAT (1)
- NFATc1 (1)
- Nahrung (1)
- Nectin4 (1)
- Nervenzelle (1)
- Neuroligin 2 (1)
- Neuronale Plastizität (1)
- Neuropeptide (1)
- Neuroplasticity (1)
- Neurowissenschaften (1)
- Newman strain sae (1)
- Non-invasive imaging (1)
- Notch Signalweg (1)
- Notch signalling (1)
- Nuclear imaging (1)
- Oncolytic Virotherapy (1)
- Onkolyse (1)
- Opioidpeptide (1)
- Orai1 (1)
- PDXP (1)
- PLP phosphatase (1)
- Pemphigus (1)
- Period2 (1)
- Periphere Analgesie (1)
- Phosphatasen (1)
- Phosphatidylinositolkinase <Phosphatidylinositol-3-Kinase> (1)
- Phospholamban (1)
- Phospholipase D (1)
- Phosphorylierung (1)
- Plasmodium falciparum (1)
- Platelet count (1)
- Polymorphismus (1)
- Positron emission tomography (1)
- Posttranskriptionelle Regulation (1)
- Probiotikum (1)
- Protein TRPC6 (1)
- Protein-Tyrosin-Kinasen (1)
- Proteintyrosinphosphatase (1)
- Psoriasis (1)
- Pyridoxal 5'-phosphate phosphatase (1)
- Pyridoxalphosphat (1)
- RNS-Spleißen (1)
- Radiologische Diagnostik (1)
- Rasterionenmikroskop (1)
- Rasterkraft-Mikroskopie (1)
- Red Sea (1)
- Regulation (1)
- Relaxation Parameter Mapping in Magnetic Resonance Imaging (1)
- Reperfusion (1)
- Rezeptor (1)
- Rodung (1)
- Rotes Meer (1)
- Rupturen (1)
- Röntgenkristallographie (1)
- SERCA2a (1)
- SLFN5 (1)
- SNAP-Rezeptor (1)
- SR proteins (1)
- Salmonella enterica (1)
- Schlaganfall (1)
- Schreckreaktion (1)
- Schuppenflechte (1)
- Selbstregulation (1)
- Serotonin (1)
- Serotonin Transporter (1)
- Signaltransduktion (1)
- Single-molecule (1)
- Small-angle X-ray Scattering (1)
- Snap25 (1)
- Splicing (1)
- Stammzelle (1)
- Staupevirus (1)
- Stiff person syndrome (1)
- Stress (1)
- Stressreaktion (1)
- Symbiosis (1)
- Synapse (1)
- Synapsine (1)
- Südostasien (1)
- T cell (1)
- T cell cytotoxicity (1)
- T cells (1)
- T-Zelle (1)
- TRAIL mutants (1)
- TRPM7 kinase (1)
- TWEAK (1)
- Tagesrhythmus (1)
- Thiolase (1)
- Tiermodell (1)
- Tissue Engineering (1)
- Transcription factor (1)
- Transforming Growth Factor beta (1)
- Transgenic mice (1)
- Transkriptomanalyse (1)
- Tuberkulose (1)
- Tumor microenvironment (1)
- Tumor-Nekrose-Faktor (1)
- Tumorzelle (1)
- Tyrosinkinaseinhibitor (1)
- UPEC (1)
- Ubiquitin (1)
- Vaccinia-Virus (1)
- Verhalten (1)
- Virtuelle Realität (1)
- Virulenz (1)
- Vulnerable plaque (1)
- Vγ9Vδ2 T cells (1)
- Wirtszelle (1)
- Wundheilung (1)
- Zellkontakt (1)
- Zellmigration (1)
- Zellzyklus (1)
- Zentralnervensystem (1)
- adult attention deficit hyperactivity disorder (aADHD) (1)
- amphibia (1)
- amphiphysin (1)
- atherosclerosis (1)
- autoimmunity (1)
- bone marrow transplantation (1)
- calcium (1)
- calcium homeostasis (1)
- calcium imaging (1)
- canine (1)
- clock genes (1)
- coagulation factor XII (1)
- contextual conditioning (1)
- contingency awareness (1)
- cytokines (1)
- dendritic cells (1)
- development (1)
- eating behavior (1)
- electroacupuncture (1)
- fMRI (1)
- fear potentiated startle response (1)
- filtering (1)
- food craving (1)
- frogs (1)
- glutamate decarboxylase 65 (1)
- human (1)
- imunology (1)
- infection (1)
- interaction stress and circadian system (1)
- leishmaniasis (1)
- logging (1)
- macrophage (1)
- mastitis (1)
- methylphenidate (1)
- miR-23a cluster (1)
- murines Modell der aufsteigenden Harnwegsinfektion (1)
- myocardial infarction (1)
- neuron (1)
- neuroscience (1)
- oilpalm plantation (1)
- opioid peptide (1)
- p38MAPK (1)
- peripheral analgesia (1)
- phosphatase (1)
- psychosocial stress (1)
- receptor signaling (1)
- self-regulation (1)
- stream (1)
- th1/th2 polarization (1)
- transcriptome analysis (1)
- virtual reality (1)
- working memory (1)
- wound healing (1)
- Ölpalmenanbau (1)
- γδ T cells (1)
Institute
- Graduate School of Life Sciences (62) (remove)
Sonstige beteiligte Institutionen
The Staphylococcus aureus two component system (TCS) sae governs expression of numerous virulence factors, including Eap (extracellular adherence protein), which in turn among other functions also mediates invasion of host cells. The sae TCS is encoded by the saePQRS operon, with saeS coding for the sensor histidine kinase (SaeS) and saeR encoding the response regulator (SaeR). The saeRS system is preceded by two additional open reading frames (ORFs), saeP and saeQ, which are predicted to encode a lipoprotein (SaeP) and a membrane protein (SaeQ), respectively. Earlier, we have shown that SDS-containing subinhibitory concentrations of biocides (Perform®) and SDS alone activate sae transcription and increase cellular invasiveness in S. aureus strain Newman. The effect is associated with an amino acid exchange in the N-terminus of SaeS (L18P), specific to strain Newman.
In this work, the role of whether the two additional genes, saePQ coding for the accessory proteins SaeP and SaeQ, respectively, are involved in SDS-mediated saeRS was investigated. It could demonstrated that the lack of the SaeP protein resulted in an increased saeRS transcription without SDS stress in both SaeSL/P variants, while the SDS effect was less pronounced on sae and eap expression compared to the Newman wildtype, suggesting that the SaeP protein represses the sae system. Also, SDS-mediated inductions of sae and eap transcription along with enhanced invasion were found to be dependent on presence of the SaeSP variant in Newman wildtype. On the other hand, the study also shows that the saePQ region of the sae operon is required for fully functional two-component system saeRS under normal growth conditions, but it is not involved in SDS-mediated activation of the saeS signaling and sae-target class I gene, eap.
In the second approach, the study investigates whether SDS-induced sae expression and host cell invasion is common among S. aureus strains not carrying the (L18P) point mutation. To demonstrate this strain Newman, its isogenic saeS mutants, and various S. aureus isolates were analysed for sae, eap expression and cellular invasiveness. Among the strains tested, SDS exposure resulted only in an increase of sae transcription, Eap production and cellular invasiveness in strain Newman wild type and MRSA strain ST239-635/93R, the latter without an increase in Eap. Interestingly, the epidemic community-associated MRSA strain, USA300 LAC showed a biphasic response in sae transcription at different growth stages, which, however, was not accompanied by increased invasiveness. All other clinical isolates investigated displayed a decrease of the parameters tested. While in strain Newman the SDS effect was due to the saeSP allele, this was not the case in strain ST239-635/93R and the biphasic USA300 strains. Also, increased invasiveness of ST239-635/93R was found to be independent of Eap production. Furthermore, to investigate the global effect of SDS on sae target gene expression, strain Newman wild-type and Newman ∆sae were treated with SDS and analyzed for their transcription profiles of sae target genes using microarray assays. We could show that subinhibitory concentrations of SDS upregulate and downregulate gene expression of several signaling pathways involved in biosynthetic, metabolic pathways as well as virulence, host cell adherence, stress reponse and many hypothetical proteins.
In summary, the study sheds light on the role of the upstream region saePQ in SDS-mediated saeRS and eap expression during S. aureus SDS stress. Most importantly, the study also shows that subinhibitory SDS concentrations have pronounced strain-dependent effects on sae transcription and subsequent host cell invasion in S. aureus, with the latter likely to be mediated in some strains by other factors than the known invasin Eap and FnBP proteins. Moreover, there seems to exist more than the saeSP-mediated mechanism for SDS-induced sae transcription in clinical S. aureus isolates. These results help to further understand and clarify virulence and pathogenesis mechanisms and their regulation in S. aureus.
Atherosclerosis is considered a chronic inflammatory disease of the arterial vessel wall which is not only modulated by innate and adaptive immune responses but also by factors of the blood coagulation system.
In general hypercoagulability seems to increase the development and progression of experimental atherosclerosis in mice on an atherogenic background. In addition, the great majority of coagulation proteins including coagulation factor XII (FXII) have been detected in early and advanced human atherosclerotic lesions supporting the cross-link between the coagulation system and atherosclerosis. Moreover, FXII has been detected in close proximity to macrophages, foam cells and smooth muscle cells in these lesions and has been demonstrated to be functionally active in human plaques. Although these data indicate that factor XII may play a role in atherogenesis a direct contribution of FXII to atherogenesis has not been addressed experimentally to date. Furthermore, clinical studies examining the function of FXII in vascular disease have yielded conflicting results.
Hence, in order to investigate the function of coagulation factor XII in atherosclerosis apolipoprotein E and FXII-deficient (F12\(^{-/-}\) apoE\(^{-/-}\)) mice were employed. Compared to F12\(^{+/+}\)apoE\(^{-/-}\) controls, atherosclerotic lesion formation was reduced in F12\(^{-/-}\)apoE\(^{-/-}\) mice, associated with diminished systemic T-cell activation and Th1-cell polarization after 12 weeks of high fat diet. Moreover, a significant decrease in plasma levels of complement factor C5a was evidenced in F12\(^{-/-}\)apoE\(^{-/-}\) mice. Interestingly, C5a increased the production of interleukin-12 (IL-12) in dendritic cells (DCs) and enhanced their capacity to trigger antigen-specific interferon-gamma (IFNγ) production in OTII CD4\(^+\) T cells in vitro. Importantly, a reduction in frequencies of IL-12 expressing splenic DCs from atherosclerotic F12\(^{-/-}\)apoE\(^{-/-}\) versus F12\(^{+/+}\)apoE\(^{-/-}\) mice was observed in vivo, accompanied by a diminished splenic Il12 transcript expression and significantly reduced IL-12 serum levels.
Consequently, these data reveal FXII to play an important role in atherosclerotic lesion formation and to promote DC-induced and systemic IL 12 expression as well as pro-inflammatory T-cell responses likely at least in part via the activation of the complement system.
Der Myokardinfarkt (MI) gehört nach wie vor zu den führenden Todesursachen weltweit. Eine Minimierung der Infarktgröße, die durch die Dauer der Ischämie bestimmt wird, ist wesentlich für das Überleben und die Lebensqualität des Myokardinfarkt-Patienten. Die Reperfusion stellt aktuell eine zentrale klinische Intervention dar, um den myokardialen Schaden einzugrenzen. Dennoch führt die Reperfusion per se zu zusätzlichem Schaden am Herzen. Somit ist die Erforschung neuer Strategien zur Minimierung des myokardialen Reperfusionsschadens international von Interesse. Die Pathophysiologie des myokardialen Reperfusionsschadens ist vielschichtig und einige Komponenten sind auch heute in ihrer Wirkweise noch nicht vollständig mechanistisch verstanden. Die vorliegende Arbeit untersucht die Rolle von CD4+ T-Zellen und insbesondere deren Subpopulation der regulatorischen T-Zellen im myokardialen Reperfusionsschaden und stellt neue, auf T-Zellen abzielende, Therapien in Ergänzung zur myokardialen Reperfusion vor.
Zunächst wurde eine Infiltration von T-Zellen in das Myokard nach Ischämie-Reperfusion (I/ R) untersucht. Nach der Ischämie-Reperfusion wurden infiltrierende CD4+ T-Zellen als quantitativ führend und aktiviert identifiziert und erwiesen sich in der Infarktgrößenbestimmung als relevante Mediatoren des Reperfusionsschadens. CD25+Foxp3+ regulatorische T-Zellen (Treg) stellen eine Subpopulation von CD4+ T-Zellen mit immunsuppressiven Eigenschaften dar, die schnell und niederschwellig aktiviert werden können und kommen somit als zum Reperfusionsschaden beitragend in Frage. Mit Hilfe des DEREG (DEpletion of REGulatory T cells) -Mausmodells wurde gezeigt, dass regulatorische T-Zellen zum myokardialen Reperfusionsschaden beitragen; Treg-depletierte DEREG-Mäuse waren vor dem Reperfusionsschaden geschützt und zeigten kleinere Infarktgrößen als die Kontrolltiere. Zudem wurde mittels Transferexperimenten gezeigt, dass für den Treg-vermittelten Reperfusionsschaden die Anwesenheit von CD25- konventionellen T-Zellen (Tconv) erforderlich ist. Regulatorische T-Zellen stellen also einen in der vorliegenden Arbeit identifizierten potentiellen Angriffspunkt zur Reduktion des myokardialen Reperfusionsschadens dar.
Anhand von T-Zell-Rezeptor transgenen OT-II Mäusen und MHC (Major Histocompatibility Complex) Klasse II Knockout (KO) Tieren wurde gezeigt, dass Autoantigenerkennung im myokardialen Reperfusionsschaden eine Rolle spielt. Zur vollen T-Zell-Aktivierung notwendig ist neben dem MHC Klasse II-Signalweg und Kostimulatoren auch das Moleküle CD154 (CD40L). Die Gabe eines inhibitorischen anti-CD154-Antikörpers reduzierte die Infarktgröße in Wildtyp-Tieren sigifikant. Der myokardiale Reperfusionsschaden kann neben Zellen der adaptiven Immunität auch durch Neutrophile Granulozyten, Plättchen oder Inflammation des Endothels verstärkt werden. Knockout Mäuse mit einer Defizienz an CD4+ T-Zellen verfügten über eine verbesserte Mikroperfusion. Mechanistisch war nach 24h Reperfusion die absolute Zellzahl an Neutrophilen Granulozyten im CD4 KO im Vergleich zu Wildtyp-Mäusen unverändert; in Endothelzellen war die Regulation bestimmter Gene (VEGFα, TIMP-1 und Eng) nach I/ R im CD4 KO jedoch verändert.
Zusammengefasst zeigt die vorliegende Arbeit eine zentrale Rolle der Antigen-Erkennung durch den T-Zell-Rezeptor zur Aktivierung von CD4+ T-Zellen im myokardialen Reperfusionsschaden. In Anwesenheit von CD4+Foxp3+ T-Zellen ist der Reperfusionsschaden erhöht. Somit können CD4+Foxp3+ T-Zellen potentiell als Ziel für neuartige Therapien des Myokardinfarkts genutzt werden.
The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses.
This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type
phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors.
The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase.
The role of chronophin dimerization was studied by generating a constitutively monomeric variant,
which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization.
The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were
investigated by performing domain swapping experiments with chronophin and its paralog AUM and
subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure
determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context.
Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and
characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.
The number of fungal infections is rising in Germany and worldwide. These infections are mainly caused by the opportunistic fungal pathogen C. albicans, which especially harms immunocompromised people. With increasing numbers of fungal infections, more frequent and longer lasting treatments are necessary and lead to an increase of drug resistances, for example against the clinically applied therapeutic fluconazole. Drug resistance in C. albicans can be mediated by the Multidrug resistance pump 1 (Mdr1), a membrane transporter belonging to the major facilitator family. However, Mdr1-mediated fluconazole drug resistance is caused by the pump’s regulator, the transcription factor Mrr1 (Multidrug resistance regulator 1). It was shown that Mrr1 is hyperactive without stimulation or further activation in resistant strains which is due to so called gain of function mutations in the MRR1 gene.
To understand the mechanism that lays behind this constitutive activity of Mrr1, the transcription factor should be structurally and functionally (in vitro) characterized which could provide a basis for successful drug development to target Mdr1-mediated drug resistance caused by Mrr1. Therefore, the entire 1108 amino acid protein was successfully expressed in Escherichia coli. However, further purification was compromised as the protein tended to form aggregates, unsuitable for crystallization trials or further characterization experiments. Expression trials in the eukaryote Pichia pastoris neither yielded full length nor truncated Mrr1 protein. In order to overcome the aggregation problem, a shortened variant, missing the N-terminal 249 amino acids named Mrr1 ‘250’, was successfully expressed in E. coli and could be purified without aggregation. Similar to the wild type Mrr1 ‘250’, selected gain of function variants were successfully cloned, expressed and purified with varying yields and with varying purity. The Mrr1 `250’ construct contains most of the described regulatory domains of Mrr1. It was used for crystallization and an initial comparative analysis between the wild type protein and the variants. The proposed dimeric form of the transcription factor, necessary for DNA binding, could be verified for both, the wild type and the mutant proteins. Secondary structure analysis by circular dichroism measurements revealed no significant differences in the overall fold of the wild type and variant proteins. In vitro, the gain of function variants seem to be less stable compared to the wild type protein, as they were more prone to degradation. Whether this observation holds true for the full length protein’s stability in vitro and in vivo remains to be determined. The crystallization experiments, performed with the Mrr1 ‘250’ constructs, led to few small needle shaped or cubic crystals, which did not diffract very well and were hardly reproducible. Therefore no structural information of the transcription factor could be gained so far.
Infections with M. tuberculosis, the causative agent of tuberculosis, are the leading cause of mortality among bacterial diseases. Especially long treatment times, an increasing number of resistant strains and the prevalence of for decades persisting bacteria create the necessity for new drugs against this disease. The cholesterol import and metabolism pathways were discovered as promising new targets and interestingly they seem to play an important role for the chronic stage of the tuberculosis infection and for persisting bacteria.
In this thesis, the 3-ketoacyl-CoA thiolase FadA5 from M. tuberculosis was characterized and the potential for specifically targeting this enzyme was investigated. FadA5 catalyzes the last step of the β-oxidation reaction in the side-chain degradation pathway of cholesterol. We solved the three dimensional structure of this enzyme by X-ray crystallography and obtained two different apo structures and three structures in complex with acetyl-CoA, CoA and a hydrolyzed steroid-CoA, which is the natural product of FadA5. Analysis of the FadA5 apo structures revealed a typical thiolase fold as it is common for biosynthetic and degradative enzymes of this class for one of the structures. The second apo structure showed deviations from the typical thiolase fold. All obtained structures show the enzyme as a dimer, which is consistent with the observed dimer formation in solution. Thus the dimer is likely to be the catalytically active form of the enzyme. Besides the characteristic structural fold, the catalytic triad, comprising two cysteines and one histidine, as well as the typical coenzyme A binding site of enzymes belonging to the thiolase class could be identified. The two obtained apo structures differed significantly from each other. One apo structure is in agreement with the characteristic thiolase fold and the well-known dimer interface could be identified in our structure. The same characteristics were observed in all complex structures. In contrast, the second apo structure followed the thiolase fold only partially. One subdomain, spanning 30 amino acids, was in a different orientation. This reorientation was caused by the formation of two disulfide bonds, including the active site cysteines, which rendered the enzyme inactive. The disulfide bonds together with the resulting domain swap still permitted dimer formation, yet with a significantly shifted dimer interface. The comparison of the apo structures together with the preliminary activity analysis performed by our collaborator suggest, that FadA5 can be inactivated by oxidation and reactivated by reduction. If this redox switch is of biological importance requires further evaluation, however, this would be the first reported example of a bacterial thiolase employing redox regulation.
Our obtained complex structures represent different stages of the thiolase reaction cycle. In some complex structures, FadA5 was found to be acetylated at the catalytic cysteine and it was in complex with acetyl-CoA or CoA. These structures, together with the FadA5 structure in complex with a hydrolyzed steroid-CoA, revealed important insights into enzyme dynamics upon ligand binding and release. The steroid-bound structure is as yet a unique example of a thiolase enzyme interacting with a complex ligand. The characterized enzyme was used as platform for modeling studies and for comparison with human thiolases. These studies permitted initial conclusions regarding the specific targetability of FadA5 as a drug target against M. tuberculosis infection, taking the closely related human enzymes into account. Additional analyses led to the proposal of a specific lead compound based on the steroid and ligand interactions within the active site of FadA5.
HIV verursacht eine progressive Zerstörung des Immunsystems und führt zusätzlich durch Veränderungen im ZNS zu neurokognitiven Störungen (HIV-associated neurocognitive disorders, HAND). Die HIV-Infektion geht mit einer Dysfunktion von dopaminergen Signalwegen einher, die sich unter anderem in einer erhöhten Dopamin-Verfügbarkeit im Liquor von Therapie-naiven HIV-Patienten äußert. Der Grund für die Dysregulation der dopaminergen Signalwege in HIV-Patienten ist nicht geklärt. Aufgrund dessen war das Hauptziel dieser Arbeit die Identifizierung des pathogenetischen Mechanismus, der zu einer erhöhten Dopamin-Konzentration im Liquor von HIV-Patienten führt. Die primäre Hypothese war, dass die erhöhte Dopamin-Verfügbarkeit nicht durch das Virus selbst, sondern vielmehr durch die genetische Konstitution der HIV-Patienten hervorgerufen wird. Deshalb wurden Polymorphismen untersucht, die die dopaminerge Neurotransmission beeinflussen. Es wurde vermutet, dass a) verschiedene Genotypen dieser Polymorphismen in nicht-infizierten und HIV-infizierten Personen mit anderen Häufigkeiten auftreten, b) verschiedene Genotypen mit veränderten Dopamin-Verfügbarkeiten assoziiert sind, c) unterschiedliche Genotypen Auswirkungen auf Marker der Progression der HIV-Infektion haben und d) verschiedene Genotypen die Immunaktivierung beeinflussen. Dazu wurden in 190 HIV-infizierten und nicht-infizierten Teilnehmern unterschiedlicher Ethnien die Polymorphismen BDNF Val66Met, COMT Val108/158Met, DAT 3‘-UTR VNTR, DRD2 TaqIα, DRD3 Ser9Gly und DRD4 VNRT mit PCR, ggf. Restriktionsverdau und Agarose-Gelelektrophorese analysiert und die Expression des Dopamin-Transporters mit real time PCR bestimmt. Darüber hinaus wurden zur weiteren klinischen Charakterisierung die Immunmarker MCP-1, sCD14, suPAR und RANTES mit ELISA analysiert, da eine Erhöhung dieser Parameter mit einer beschleunigten HIV-Progression assoziiert ist. Die Bestimmung der T-Zell-Aktivierung (CD3/CD8/CD38/HLA-DR) wurde mit einer durchflusszytometrischen Analyse durchgeführt. In dieser Arbeit haben wir gezeigt, dass HIV-Patienten hochsignifikant häufiger homozygot für das 10-repeat Allel des Dopamin-Transporter-Polymorphismus sind als nicht-infizierte Personen (57,1 % bzw. 26,8 %, p = 0,001, OR = 3,93, 95 % CI 1,72 – 8,96, direkte logistische Regression). HIV-Patienten und nicht-infizierte Personen mit diesem Genotyp weisen eine signifikant höhere Dopamin-Verfügbarkeit im Liquor auf als Personen mit dem 9/10-Genotyp (p = 0,03) und eine signifikant geringere Expression des Dopamin-Transporters auf PBMCs (p = 0,05). Der DAT 10/10-Genotyp ist im Gegensatz zu anderen Genotypen in HIV-Patienten jedoch weder mit unterschiedlichen CD4+-Zellzahlen und Viruslasten noch mit einer veränderten Häufigkeit von HAND verbunden.
Zusätzlich weisen deutsche und südafrikanische nicht-infizierte und HIV-infizierte Personen mit dem DAT 10/10-Genotyp eine signifikant höhere MCP-1-Konzentration im Plasma auf als Personen mit anderen DAT-Genotypen (p = 0,0076). Keiner der Immunmarker ist mit der Dopamin-Verfügbarkeit assoziiert. Dennoch ist die Immunaktivierung in südafrikanischen HIV-Patienten im Vergleich zu nicht-infizierten Südafrikanern signifikant erhöht: HIV-Patienten zeigen im Vergleich zu nicht-infizierten Personen eine stärkere T-Zell-Aktivierung (p = 0,0001), eine erhöhte Plasma-Konzentration von MCP-1 (p = 0,0014), eine gesteigerte sCD14-Konzentration (p = 0,0004) und eine vermehrte suPAR-Konzentration im Plasma (p = 0,006). In der vorliegenden Arbeit konnte kein Nachweis erbracht werden, dass die erhöhte Immunaktivierung in den südafrikanischen HIV-Patienten durch die Koinfektion mit Echinoccocus oder durch genetische Polymorphismen bei Chemokinen hervorgerufen wird. Eine chronisch erhöhte Immunaktivierung stellt eine treibende Kraft für die Virusreplikation dar und kann letztendlich zu einer Erschöpfung des Immunsystems führen.
Der 10/10-Genotyp des DAT VNTR könnte einen Risiko-Faktor für die HIV-Infektion darstellen, da dieser eine erhöhte Dopamin-Verfügbarkeit nach sich zieht. Dopamin aktiviert HIV in chronisch infizierten T-Lymphoblasten und führt zudem zu einer erhöhten Expression und Sezernierung von TNF-α, das wiederum die Expression von HIV induziert. Diese Ergebnisse untermauern den Zusammenhang von Dopamin und HIV. Es ist jedoch nicht völlig geklärt, ob die erhöhte Dopamin-Konzentration ausschließlich durch den Genotyp hervorgerufen oder auch durch die HIV-Infektion begünstigt wird.
Due to the rotation of the earth in the solar system all inhabitants of our planet are exposed to regular environmental changes since more than 3.5 billion years. In order to anticipate these predictable changes in the environment, evolutionarily conserved biological rhythms have evolved in most organisms – ranging from ancient cyanobacteria up to human beings – and also at different levels of organization – from single cells up to behavior. These rhythms are endogenously generated by so called circadian clocks in our body and entrained to the 24 h cycle by external timing cues. In multi-cellular organisms the majority of the cells in the body is equipped with such an oscillator. In mammals, the circadian system is structured in a hierarchical fashion: A central pacemaker resides in the bilateral suprachiasmatic nucleus (SCN) of the hypothalamus, while subsidiary peripheral clocks exist in nearly every tissue and organ.
In contrast to the aforementioned recurrent environmental changes most organisms are also exposed to unpredictable changes in the environment. In order to adapt to these sudden alterations the acute activation of the stress response system, involving the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, displays a fundamental survival mechanism. However, if activation of the stress system becomes chronic, devastating somatic and affective disorders might be the consequence.
At first glance, the circadian and the stress system seem to represent two separate bodily control systems that are involved in adaptation to predictable and unpredictable stimuli, respectively. However, both systems are fundamental for survival, and thus, communicate with each other at various levels. Early studies already demonstrated that stressor exposure at different times of the diurnal cycle generates different stress effects, whereupon the type of stressor plays a pivotal role. Moreover, alterations in the SCN and peripheral circadian clocks could be shown following stressor exposure.
In cooperation with various co-workers, I investigated whether the stress responsiveness is modulated by the endogenous clock in a diurnal fashion and whether repeated psychosocial stress impacts the circadian clock depending on the time of day of stressor exposure. Therefore, male C57BL/6 mice were repeatedly exposed to a psychosocial stressor, either at the beginning of the inactive/light phase (SDL mice) or active/dark phase (SDD mice).
Subsequently, different behavioral, physiological/endocrine and immunological/ inflammatory consequences were assessed. It could be shown that the effects of repeated psychosocial stressor exposure strongly depend on the time of day of stressor exposure. The present results demonstrate that repeated daily stressor exposure has a more negative outcome when applied during the active/dark phase compared to the inactive/light phase. Stressor exposure during the active phase resulted in a loss of general activity, decreased interest in an unfamiliar conspecific, a shift towards a more pro-inflammatory body milieu, and rhythm disturbances in plasma hormones, all representing well-accepted hallmarks of depression. In contrast, C57BL/6 mice exposed to the stressor in their inactive phase exhibited minor physiological alterations that might prevent the formation of the maladaptive consequences mentioned above, thus representing beneficial adaptations.
The second focus of this thesis was put on the investigation of the effects of repeated psychosocial stressor exposure at different times of the light-dark cycle on various levels of the circadian system. An increased expression of the PERIOD2 (PER2) protein, which represents an essential core clock component, could be found in the SCN of mice repeatedly exposed to the stressor during their active phase. In consistence with the alterations in the central circadian pacemaker, the daily rhythm of different hormones and the activity rhythm were considerably affected by SDD. Mice exposed to the psychosocial stressor in their active phase showed a shifted, or absent, rhythm of the hormones corticosterone and leptin. Moreover, their activity was found to be phase-delayed, which seems to be attributable to the Period (Per) gene since Per1/Per2 double-mutants still exhibited their normal activity rhythm following 19 days of stressor exposure during the active phase. In contrast, a phase-advance in the peripheral adrenal gland clock could be seen in C57BL/6 mice subjected to the stressor during their inactive phase. This phase-shift might be required for maintaining the normal rhythmicity in hormonal release and activity.
It has previously been suggested that activation of the HPA axis upon stressor exposure at different times of the light-dark cycle is depending on whether the stressor is of physical or psychological nature. Data from the HPA axis analysis now refine previous findings, indicating that psychosocial stressors also modulate HPA axis responses based on the time of day of stressor presentation. The present results demonstrate that HPA axis activity was reduced following repeated stressor exposure during the active phase. It is reasonable to speculate that this reduced basal activity of the stress system represents a failure in HPA axis adjustment, which could contribute to the negative consequences of repeated psychosocial stressor exposure during the dark phase.
Taken together, it can be concluded that the endogenous clock in mice modulates the stress responsiveness in a circadian fashion and that repeated psychosocial stressor exposure affects the biological clock depending on the time of day of stressor presentation. Thereby, stressor exposure during the active phase results in a more negative outcome as compared to stressor experience during the inactive phase. It is assumed that the interaction between the circadian clock and the stress system is a complex issue that might ensure that the endogenous clock does not get out of synchrony in any order.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of enzymes with important functions in physiology and disease. HAD phosphatases can target diverse metabolites, lipids, DNA, and serine/threonine or tyrosine phosphorylated proteins with often high specificity (Seifried et al., 2013). These enzymes thus markedly enlarge the repertoire and substrate spectrum of mammalian phosphatases. However, the basis of HAD phosphatase substrate specificity is still elusive and a number of mammalian HAD phosphatases remain uncharacterized to date. This study characterizes the biochemical and structural properties of AUM (aspartate-based, ubiquitous, Mg2+-dependent phosphatase), a previously unexplored mammalian HAD phosphatase.
In vitro phosphatase assays of purified, recombinant AUM showed phosphatase activity towards para-nitrophenyl phosphate and adenine and guanine nucleotide di- and triphosphates. Inhibitor studies indicated that similar to other HAD superfamily members, the AUM-catalyzed dephosphorylation reaction proceeds via a pentacovalent phosphoaspartate intermediate. In line with an aspartate-based catalytic mechanism, AUM was insensitive to inhibitors of serine/threonine phosphatases. The characterization of the purified recombinant murine enzyme also revealed that AUM exists in equilibrium between dimers and tetramers.
AUM was identified as the closest, yet functionally distinct relative of chronophin, a pyridoxal 5’-phosphate and serine/threonine-directed phosphatase. Phylogenetic analyses showed that AUM and chronophin evolved via duplication of an ancestral gene at the origin of the vertebrates. In contrast to chronophin, AUM acts as a tyrosine-specific HAD-type phosphatase in vitro and in cells. To elucidate how AUM and chronophin achieve these distinct substrate preferences, comparative evolutionary analyses, biochemical approaches and structural analyses were combined. Swapping experiments of less homologous regions between AUM and chronophin were performed. The mutational analysis revealed residues important for AUM catalysis and specificity. A single differently conserved residue in the cap domain of AUM or chronophin is crucial for phosphatase specificity (AUML204, chronophinH182). The X-ray crystal structure of the AUM cap fused to the catalytic core of chronophin (CAC, PDB: 4BKM) was solved to 2.65 Å resolution. It presents the first crystal structure of the murine AUM capping domain. The detailed view of the catalytic clefts of AUM and chronophin reveals the structural basis of the divergent substrate specificities. These presented findings provide insights into the design principles of capped HAD phosphatases and show that their substrate specificity can be encoded by a small number of predictable residues. In addition, the catalytic properties of AUM were investigated, identifying a mechanism of reversible oxidation regulating the activity of AUM in vitro. AUM phosphatase activity is inhibited by oxidation and can be recovered by reduction. The underlying molecular mechanism was revealed by mutational analyses. The cysteines C35, C104 and C243, located in the AUM core domain, are responsible for the inhibition of AUM by oxidation. C293 mediates the redox-dependent tetramerization of AUM in vitro. Based on the chronophin and CAC structure, a direct impact of the oxidation of C35 on the nucleophile D34 is proposed. In addition, a redox-dependent disulfide bridge (C104, C243), connecting the core and cap domain of AUM may be important for an open/close-mechanism. This hypothesis is supported by CD spectroscopy experiments that demonstrate a structural change in AUM upon reduction. These data present the first evidence for the regulation of AUM catalysis by reversible oxidation. This finding is so far unique in the field of HAD phosphatases.
In this context, the first cell-based AUM activity assay was developed. For this, the artificial substrate pNPP was combined with the reducing agent DTT to create a specific AUM activity readout. This fractionation-based assay is the first tool to differentiate between cell lines or tissues with different AUM concentrations or activities.
Taken together, the presented biochemical characterization reveals the specificity determinants and catalytic properties of AUM. General insights into structural determinants of mammalian HAD phosphatase substrate recognition are provided and reversible oxidation as possible regulatory mechanism for AUM is proposed. These findings constitute a framework for further functional analyses to elucidate the biomedical importance of AUM.
Structural and biochemical characterization of gephyrin and various gephyrin-ligand complexes
(2014)
Efficient synaptic neurotransmission requires the exact apposition of presynaptic terminals and matching neurotransmitter receptor clusters on the postsynaptic side. The receptors are embedded in the postsynaptic density, which also contains scaffolding and regulatory proteins that ensure high local receptor concentrations. At inhibitory synapses the cytosolic scaffolding protein gephyrin assumes an essential organizing role within the postsynaptic density by the formation of self-oligomers which provide a high density of binding sites for certain -amino butyric acid type A (GABAA) and the large majority of glycine receptors (GlyR). Gephyrin contains two oligomerization domains: In isolation, the 20 kDa N-terminal G domain (GephG) and the 46 kDa E domain (GephE) trimerize and dimerize, respectively. In the full-length protein the domains are interconnected by a central ~150 amino acid linker, and only GephG trimerization is utilized, whereas GephE dimerization is prevented, thus suggesting the need for a trigger to release GephE autoinhibition, which would pave the way for the formation of higher oligomers and for efficient receptor clustering. The structural basis for this GephE autoinhibition has remained elusive so far, but the linker was reported to be sufficient for autoinhibition. This work dealt with the biochemical and structural characterization of apo-gephyrin and gephyrin in complexes with ligands which are known to promote the formation of synaptic gephyrin clusters (collybistin and neuroligin 2) and reorganize them (dynein light chain 1).
For full-length gephyrin no structural information has been available so far. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analyses described in this thesis disclosed that the gephyrin trimer forms a highly flexible assembly, which, due to the long linker, can switch between compact and extended conformational states in solution, with a preference for compact states. This partial compaction and potentially GephE autoinhibition are achieved by interactions of parts of the linker with the G and E domains, as suggested by circular dichroism spectroscopy. However, the linker on its own cannot account for GephE blockage, as size exclusion chromatography experiments coupled with multi angle light scattering detection (SEC-MALS) and SAXS analyses revealed that a gephyrin variant only encompassing the linker and GephE (GephLE) forms dimers and not monomers as suggested by an earlier study. The oligomeric state of GephLE and the observation that several gephyrin variants, in which linker segments of varying length were deleted, predominantly formed trimers, suggested the presence of a linker independent mechanism of GephE dimerization blockade. Taken together, the data indicated that linker-dependent and linker-independent mechanisms mediate gephyrin autoinhibition.
In the second project gephyrin’s interaction with DYNLL1 (Dynein LC8 Light Chain 1) was characterized. DYNLL1 is a 25 kDa dimer incorporated into the dynein motor and provides two binding sites, each of which can accommodate an octapeptide derived from gephyrin’s linker region (referred to as GephDB). Originally, DYNLL1 was regarded as a cargo adaptor, linking gephyrin-GlyR complexes to the dynein motor, thus driving their retrograde transport and leading to a decrease of synaptic gephyrin-GlyR complexes.
Building on these studies, this thesis assessed the cargo hypothesis as well as the so far unclear stoichiometry of the gephyrin-DYNLL1 complex. The cargo scenario would require ternary complex formation between gephyrin, DYNLL1 and the dynein intermediate chain (DIC) of the dynein motor. However, such a complex could not be detected by analytical size exclusion chromatography (aSEC) experiments – presumably because gephyrin and DIC competed for a common binding site in DYNLL1. This finding was consistent with a single DYNLL1 dimer capturing two linker segments of a single gephyrin trimer as suggested by a 26 kDa mass increase of the gephyrin species in the presence of DYNLL1 in SEC-MALS experiments. aSEC experiments at even higher concentrations (~20 µM gephyrin and ~80 µM DYNLL1) indicated that the affinity of GephDB was significantly impaired in the context of full-length gephyrin but also in a variant that bears only GephG and the first 39 residues of the linker (GephGL220). Presumably due to avidity effects two linkers stably associated with a single DYNLL1 dimer, whereas the third DYNLL1 binding motif remained predominantly unoccupied unless high concentrations of GephGL220 (50 µM) and DYNLL1 (200 µM) were used. These findings indicate that an interplay between GephG and the N-terminal linker segment mediates the attenuation of GephDB affinity towards DYNLL1 and that preventing DYNLL1 from the induction of higher gephyrin oligomers is either advantageous for DYNLL1-mediated reorganization of gephyrin-GlyR clusters or that DYNLL1 exerts possibly two (concentration-dependent) actions on gephyrin.
The gephyrin-collybistin-neuroligin 2 complex was the subject of the third project. Previously, collybistin and gephyrin were observed to mutually trigger their translocation to the postsynaptic membrane, where the disordered cytoplasmic tail of the postsynaptic cell adhesion molecule NL2 (NL2cyt) causes the anchoring of collybistin 2 (CB2) by binding to its SH3 domain, thereby releasing SH3 domain mediated autoinhibiton of CB2 binding to the membrane phospholipid phosphatidylinositol-3-phosphate. Critical for this event is the binding of gephyrin to both CB2 and NL2, presumably via GephE.
Following up on these previous studies biochemical data presented in this thesis confirm the formation of the ternary complex. Unexpectedly, analyses by means of native polyacrylamide gel electrophoresis pointed to: (1) The existence of a complex containing NL2cyt and CB2 lacking the SH3 domain and consequently an additional NL2 binding site in CB2. (2) Attenuated gephyrin-collybistin complex formation in the presence of the SH3 domain. (3) A requirement for high NL2cyt concentrations (> 30 µM) during the formation of the ternary complex. This might allow for the regulation by other factors such as additional binding partners or posttranslational modifications. Although of preliminary character, these results provide a starting point for future studies, which will hopefully elucidate the interplay between gephyrin, collybistin, NL2 and certain GABAA receptors.
The Fanconi anemia (FA) pathway is a replication-dependent DNA repair mechanism which is essential for the removal of interstrand crosslink (ICL) DNA damages in higher eukaryotes (Moldovan and D’Andrea, 2009). Malfunctions in this highly regulated repair network lead to genome instability (Deans and West, 2011). Pathological phenotypes of the disease FA which is caused by mutations in the eponymous pathway are very heterogeneous, involving congenital abnormalities, bone-marrow failure, cancer predisposition and infertility (Auerbach, 2009). The FA pathway comprises a complex interaction network and to date 16 FA complementation groups and associated factors have been identified (Kottemann and Smogorzewska, 2013). Additionally, components of nucleotide excision repair (NER), homologous recombination repair (HRR), and translesion synthesis (TLS) are involved and coordinated by the FA proteins (Niedzwiedz et al., 2004; Knipscheer et al., 2009). One of the FA proteins is the DEAH helicase FANCM. In complex with its binding partners FAAP24 and MHF1/2 it binds the stalled replication fork and activates the FA damage response (Wang et al., 2013). However, the exact steps towards removal of the ICL damage still remain elusive.
To decipher the underlying process of FA initiation by FANCM, this thesis mainly focuses on the archaeal FANCM homolog helicase-associated endonuclease for fork-structured DNA (Hef). Hef from the archaeal organism Thermoplasma acidophilum (taHef) differs from other archaeal Hef proteins and exclusively comprises an N-terminal helicase entity with two RecA and a thumb-like domain while others additionally contain a nuclease portion at the C-terminus. I solved the crystal structure of full-length taHef at a resolution of 2.43 Å. In contrast to the crystal structure of the helicase domain of Hef from Pyrococcus furiosus (pfHef), taHef exhibits an extremely open conformation (Nishino et al., 2005b) which implies that a domain movement of the RecA-like helicase motor domains of 61° is possible thus highlighting the flexibility of helicases which is required to translocate along the DNA. However, small-angle x-ray scattering (SAXS) measurements confirm an intermediate conformation of taHef in solution indicating that both crystal structures represent rather edge states. Most
importantly, proliferating cell nuclear antigen (PCNA) was identified as an interaction partner of Hef. This interaction is mediated by a highly conserved canonical PCNA interacting peptide (PIP) motif. Intriguingly, the presence of PCNA does not alter the ATPase nor the helicase activity of taHef, thus suggesting that the interaction is entirely dedicated to recruit taHef to the replication fork to fulfill its function. Due to a high level of flexibility the taHef-taPCNA complex could not be crystallized and therefore SAXS was utilized to determine a low-resolution model of this quaternary structure.
This newly discovered PCNA interaction could also be validated for the eukaryotic FANCM homolog Mph1 from the thermophilic fungus Chaetomium thermophilum (ctMph1). As the first step towards the characterization of this interaction I solved the crystal structure of PCNA from Chaetomium thermophilum (ctPCNA).
Furthermore, it was possible to achieve preliminary results on the putative interaction between the human proteins FANCM and PCNA (hsFANCM, hsPCNA). In collaboration with Detlev Schindler (Human Genetics, Würzburg) and Weidong Wang (National Institute on Aging, Baltimore, USA) co-immunoprecipitation (CoIP) experiments were performed using hsFANCM and hsPCNA expressed in HEK293 cells. Although an interaction was reproducibly observed in hydroxyurea stimulated cells
further experiments and optimization procedures are required and ongoing.
Studies on receptor signaling and regulation in platelets and T cells from genetically modified mice
(2014)
Receptors with tyrosine-based signaling motifs control essential functions of hematopoietic cells, including lymphocytes and platelets. Downstream of the platelet receptor glycoprotein (GP) VI and the T cell receptor (TCR) the immunoreceptor tyrosine-based activation motif (ITAM) initiates a signaling cascade that involves kinases, adapter and effector proteins and finally leads to cellular activation. This thesis summarizes the results of three studies investigating different aspects of receptor signaling and regulation in platelets and T cells.
In the first part, the impact of constitutive Ca2+ influx on TCR signaling and T cell physiology was investigated using a transgenic mouse line with a mutation in the Ca2+ sensor stromal interaction molecule 1 (STIM1). The elevated cytoplasmic Ca2+ level resulted in an altered phosphorylation pattern of the key enzyme phospholipase (PL) Cγ1 in response to TCR stimulation, but without affecting its enzymatic activity. Withdrawal of extracellular Ca2+ or inhibition of the phosphatase calcineurin restored the normal phosphorylation pattern. In addition, there was a decrease in the release of Th2-type cytokines interleukin 4, 5 and 13 upon stimulation in vitro.
The second part of the thesis deals with the role of the adapter protein growth factor receptor-bound protein 2 (Grb2) in platelets using a megakaryocyte/platelet-specific knockout mouse line. Loss of Grb2 severely impaired signaling of GPVI and C-type lectin-like receptor 2 (CLEC-2), a related hemITAM receptor. This was attributed to defective stabilization of the linker for activation of T cells (LAT) signalosome and resulted in reduced adhesion, aggregation, Ca2+ mobilization and procoagulant activity downstream of (hem)ITAM-coupled receptors in vitro. In contrast, the signaling pathways of G protein-coupled receptors (GPCRs) and the integrin αIIbβ3, which do not utilize the LAT signalosome, were unaffected. In vivo, the defective (hem)ITAM signaling caused prolonged bleeding times, however, thrombus formation was only affected under conditions where GPCR signaling was impaired (upon acetylsalicylic acid treatment). These results establish Grb2 as an important adapter protein in the propagation of GPVI- and CLEC-2-induced signals.
Finally, the proteolytic regulation of the immunoreceptor tyrosine-based switch motif (ITSM)-bearing receptor CD84 in platelets was investigated. This study demonstrated that in mice CD84 is cleaved by two distinct and independent proteolytic mechanisms upon platelet activation: shedding of the extracellular part, which is exclusively mediated by a disintegrin and metalloproteinase (ADAM) 10 and cleavage of the intracellular C-terminus by the protease calpain. Finally, the analysis of soluble CD84 levels in the plasma of transgenic mice revealed that shedding of CD84 by ADAM10 occurs constitutively in vivo.
The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell’s physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.
The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed “Dual RNA-seq”. Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host’s immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.
Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis.
Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock.
In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats.
We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD.
It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity.
Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication.
Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.
The integrity of our genome is continuously endangered by DNA damaging factors. Several cellular mechanisms have evolved to recognize and remove different types of DNA lesions. Despite the wealth of information on the three-dimensional structure and the catalytic mechanism of DNA repair enzymes, the essential process of target site search and identification remains more elusive. How can a small number of repair proteins find and detect the rare sites of damage rapidly and efficiently over an excess of millions of undamaged bases?
To address this pivotal question in DNA repair, I focused on the central players from the two DNA damage excision repair pathways in my studies: nucleotide excision repair (NER) and base excision repair (BER). As examples for completely different approaches of damage search, recognition and verification, I compared the NER protein Xeroderma pigmentosum group D (XPD) with the BER proteins human thymine DNA glycosylase (hTDG) and human 8-oxoguanine glycosylase (hOgg1).
In particular, the single molecule approach of atomic force microscopy (AFM) imaging and complementary biochemical and biophysical techniques were applied. I established a simple, optimized preparation approach, which yields homogeneous and pure samples of long (several hundreds to thousands of base pairs) DNA substrates suitable for the AFM studies with DNA repair proteins. Via this sample preparation, a single target site of interest can be introduced into DNA at a known position, which allows separate analysis of specific protein-DNA complexes bound to the lesion site and nonspecific complexes bound to non-damaged DNA.
The first part of the thesis investigates the XPD protein involved in eukaryotic NER. In general, the NER mechanism removes helix-distorting lesions – carcinogenic UV light induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs) as well as bulky DNA adducts. The 5’-3’ helicase XPD has been proposed to be one of the key players in DNA damage verification in eukaryotic NER, which is still a matter of hot debate. In the studies, I focused on XPD from the archaeal species Thermoplasma acidophilum (taXPD), which shares a relatively high sequence homology with the sequence of the human protein and may serve as a good model for its eukaryotic counterpart. Based on AFM experiments and accompanying DNA binding affinity measurements with the biosensor technology Biolayer Interferometry (BLI), a clear role of XPD in damage verification was deciphered. Specifically, the data suggested that the ATP-dependent 5’-3’ helicase activity of XPD was blocked by the presence of damage leading to stalled XPD-DNA damage verification complexes at the lesion sites.
Successful damage verification led to ATP-dependent conformational changes visible by a significant transition in DNA bend angles from ~ 50° to ~ 65° at the site of the bound protein. Remarkably, this DNA bend angle shift was observed both in the presence of ATP and ATPγs (non-hydrolyzable ATP analog) indicating that ATP-binding instead of ATP hydrolysis was sufficient to induce repair competent conformational changes of XPD. Most importantly, detailed protein binding position and DNA bend angle analyses revealed for the first time that XPD preferably recognizes a bulky fluorescein lesion on the translocated strand, whereas a CPD lesion is preferentially detected on the opposite, non-translocated strand. Despite the different recognition strategies for both types of damages, they share a common verification complex conformation, which may serve as a signal for the recruitment of further NER factors.
In the second part of the thesis, AFM imaging and a 2-Aminopurine fluorescence-based base-flipping assay were combined to investigate damage search and recognition by DNA glycosylases in BER. Exemplarily, I chose to study hTDG as a representative of the vast glycosylase family. hTDG excises thymine and uracil from mutagenic G:T and G:U mispairs contributing to cancer and genetic disease. The AFM data suggested that hTDG uses the intrinsic flexibility of G:T and G:U wobble pairs for initial damage sensing, while scanning DNA as a search complex (SC, slightly bent DNA). Remarkably, hTDG has been indicated to continuously switch between the search and interrogation conformation (IC, stronger bent DNA) during damage search. In the IC, target bases are interrogated by extrahelical base flipping, which is facilitated by protein-induced DNA bending and enhanced DNA flexibility at mismatches. AFM and fluorescence analyses revealed that the flipped base is stabilized via hTDG’s arginine finger. Correct target bases are perfectly stabilized within the enzyme’s catalytic pocket resulting in prolonged residence time and enhanced excision probability. To test for the generalizability of the proposed hTDG damage search model to BER glycosylases, identical studies were performed with a second glycosylase, hOgg1. The data on hOgg1, which removes structurally more stable 8-oxoguanine lesions, supported the hypothesis developed for lesion recognition by hTDG as a common strategy employed by BER glycosylases
Abstract
Glioblastomas, primary brain tumors, represent a tumor entity with a dismal prognosis and a median survival of only about one year. Invasion into the healthy brain parenchyma contributes substantially to the malignancy of this type of brain tumor. Therefore, a better understanding of the mechanisms promoting the invasive behavior of these brain tumors is needed to identify new therapeutic targets.
Cofilin, an actin regulatory protein, has been shown to be an important regulator of the invasive behavior of tumor cells in other types of cancer and the actin cytoskeleton is involved in the formation of a variety of cellular structures important for cell migration and invasion. Cofilin is regulated by phosphorylation on a single residue, serine 3. The aim of this thesis was to examine the role of the cofilin regulatory phosphatase chronophin for glioma cell migration and invasion.
First, it was established that chronophin depletion in the cell line GBM6840 leads to an increase in the ratio of phosphorylated cofilin to total cofilin. Higher chronophin levels were correlated with a decrease in F-actin in the cell lines GBM6840 and U87 as measured in an actin spin down assay and in a flow cytometry based assay.
Furthermore, it was shown that knockdown of chronophin in two different cell lines, GBM6840 and DBTRG-05-MG, strongly increased their invasiveness in vitro. Expression of human chronophin in the cell line U87 decreased its invasiveness substantially. There was no difference in cell proliferation between GBM6840 and DBTRG-05-MG cells expressing a chronophin targeting shRNA or a control shRNA and U87 cells transfected with an empty vector or a human chronophin encoding plasmid. The increase in invasiveness after chronophin depletion could be correlated with an increase in directionality in cell migration under 2D culture conditions in the cell lines U87 and GBM6840. Moreover, treatment with the ROCK inhibitor Y-27632 decreased directionality in GBM6840 cells under 2D culture conditions and reduced the invasiveness of GBM6840 chronophin shRNA cells back to control levels.
Expression of a non-phosphorylatable cofilin mutant, the S3A mutant, was able to reduce invasiveness and to reduce directionality under 2D culture conditions back to control levels in GBM6840 chronophin shRNA cells.
This provides important evidence for the involvement of cofilin phosphoregulation in the phenotypes described above.
In vivo, when injected into NOD-SCID mice, chronophin depleted cells showed a dramatic growth reduction as compared to control and rescue cells.
Transciptomic characterization of GBM6840 cells by microarray analysis and subsequent comparison of the data with microarray profiles of normal brain tissues and different glioma entities identified two specifically chronophin regulated transcripts potentially involved in tumor progression and invasion, MXI1 and EDIL3. Moreover, c-myc was identified as a significantly altered transcription factor after chronophin deregulation based on the number of c-myc target molecules in the microarray dataset.
MXI1 is a potential negative regulator of c-myc dependent transcription, and was strongly downregulated after chronophin knockdown in GBM6840. In line with this, the activity of a c-myc reporter plasmid was increased after chronophin depletion in GBM6840 and reduced after chronophin expression in U87 cells.
However, the protein level of the c-myc protein was reduced after chronophin depletion in GBM6840.
Finally, anaylsis of the expression of proteases known to be important for glioblastoma pathogenesis revealed no major changes in protease expression between chronophin depleted and control cells.
Therefore, a comprehensive analysis of chronophin in the context of glioma pathogenesis has been performed in this thesis. It has been shown that chronophin depletion strongly enhanced invasiveness of glioma cells and that it induced transcriptomic changes potentially involved in tumor progression. The proteins regulating cofilin phosphorylation are therefore valuable therapeutic targets for anti-invasive therapy in glioblastomas. Inhibitors for kinases upstream of cofilin, e.g. LIMKs and ROCKs, are available, and might be promising agents for anti-invasive therapy.
In this work, a model-based acceleration of parameter mapping (MAP) for the determination of the tissue parameter T1 using magnetic resonance imaging (MRI) is introduced. The iterative reconstruction uses prior knowledge about the relaxation behavior of the longitudinal magnetization after a suitable magnetization preparation to generate a series of fully sampled k-spaces from a strongly undersampled acquisition. A Fourier transform results in a spatially resolved time course of the longitudinal relaxation process, or equivalently, a spatially resolved map of the longitudinal relaxation time T1.
In its fastest implementation, the MAP algorithm enables the reconstruction of a T1 map from a radial gradient echo dataset acquired within only a few seconds after magnetization preparation, while the acquisition time of conventional T1 mapping techniques typically lies in the range of a few minutes. After validation of the MAP algorithm for two different types of magnetization preparation (saturation recovery & inversion recovery), the developed algorithm was applied in different areas of preclinical and clinical MRI and possible advantages and disadvantages were evaluated.
SNAP25 (Synaptosomal-Associated Protein of 25 kDa; part of the SNARE complex) is involved in the docking and fusion of synaptic vesicles in presynaptic neurons necessary for the regulation of neurotransmitter release, as well as in axonal growth and synaptic plasticity. In humans, different single nucleotide polymorphisms of SNAP25 have repeatedly been associated with attention deficit/hyperactivity disorder (ADHD). Thus, in this study heterozygous Snap25 knockout mice were investigated as a model of ADHD.
Heterozygous (+/-) Snap25 knockout mice as well as their wild-type (+/+) littermates were reared under control conditions or underwent a Maternal Separation (MS) procedure. Starting at the age of 2 months, mice were tested for locomotor activity in a repeated long-term Open Field (OF) task, for attention deficits and impulsive behavior in the 5 Choice Serial Reaction Time Task (5CSRTT), for anxiety-like behavior in the Light-Dark Box (LDB) and for depression-like behavior in the Porsolt Forced Swim Test (FST). The brains of these mice were subsequently tested for the expression of several ADHD related genes in a quantitative Real-Time PCR (qRT-PCR) study. Another group of female mice (+/+; +/-) underwent a one hour OF test after oral administration of 45 mg/kg Methylphenidate (MPH) or placebo.
To find an optimized dosage for this MPH challenge, a pilot study was performed. Wild-type C57BL/6 mice were tested in a long-term OF with several dosages of MPH both intraperitoneally (i.p.) and orally. The brains of these animals were afterwards investigated for neurotransmitter concentrations. In this pilot study the dosages of MPH that were similarly behaviorally effective without causing symptoms of overdosing were 7.5-15 mg/kg intraperitoneally and 30-60 mg/kg orally. However, even though it was possible to find intraperitoneal and oral doses that correlate behaviorally, the neurochemistry was mostly different.
In the study on Snap25-deficient mice, unstressed controls showed a hyperactive phenotype in the second of two long-term OF sessions (60 min) spaced three weeks apart. Considering all groups, there was a significant interaction of stress and genotype in the second session, with animals subjected to MS being overall hyperactive with no genotype differences. In the training phase of the 5CSRTT only effects of stress were found, with MS animals finding and consuming fewer rewards. In the single test trial, several genotype effects became apparent, with tendencies for the number of correct nose pokes and the number of rewards eaten, and a significant effect for the number of rewards eaten directly after the correct response. In all of these variables +/- mice performed worse than their wild-type littermates. In the LDB +/- mice entered the lit compartment of the arena earlier than the controls, thus showing attenuated anxiety-like behavior. Regarding depressive-like behavior in the FST, male +/- mice spent significantly less time struggling than male +/+ mice. In the gene expression study, +/- mice had lower expression levels of Maoa and Comt, and higher expression levels of Nos1 than wild-types. Finally, the locomotor activity response to MPH was exaggerated in +/- mice as compared to controls.
Heterozygous Snap25 knockout mice show some of the behavioral characteristics of ADHD, as for example a mild hyperactivity in a familiar environment, difficulties in the correct execution of a given task and even some behavior that can be interpreted as delay aversion. Additionally, expression levels of three ADHD related genes were changed in these animals. Although the exaggerated locomotor activity response to MPH is not to be expected of an ADHD model, the difference in the response between +/+ and +/- mice nonetheless implicates a potential dysfunction of the brain dopaminergic system.
Burkitt lymphoma (BL) is a highly aggressive B cell malignancy. Rituximab, a humanized antibody against CD20, in a combination with chemotherapy is a current treatment of choice for B-cell lymphomas including BL. However, certain group of BL patients are resistant to Rituximab therapy. Therefore, alternative treatments targeting survival pathways of BL are needed.
In BL deregulation of MYC expression, together with additional mutations, inhibits differentiation of germinal centre (GC) B cells and drives proliferation of tumor cells. Pro-apoptotic properties of MYC are counteracted through the B-cell receptor (BCR) and phosphoinositide-3-kinase (PI3K) pathway to ensure survival of BL cells. In normal B-cells BCR triggering activates both NF-κB and NFAT-dependent survival signals. Since BL cells do not exhibit constitutive NF-κB activity, we hypothesized that anti-apoptotic NFATc1A isoform might provide a major survival signal for BL cells.
We show that NFATc1 is constitutively expressed in nuclei of BL, in BL cell lines and in Eµ-Myc–induced B cell lymphoma (BCL) cells. Nuclear residence of NFATc1 in these entities depends on intracellular Ca2+ levels but is largely insensitive to cyclosporine A (CsA) treatment and therefore independent from calcineurine (CN) activity. The protein/protein interaction between the regulatory domain of NFATc1 and DNA binding domain of BCL6 likely contributes to sustained nuclear residence of NFATc1 and to the regulation of proposed NFATc1-MYC-BCL6-PRDM1 network in B-cell lymphomas.
Our data revealed lack of strict correlation between the expression of six NFATc1 isoforms in different BL-related entities suggesting that both NFATc1/alphaA and -betaA isoforms provide survival functions and that NFATc1alpha/betaB and -alpha/betaC isoforms either do not possess pro-apoptotic properties in BL cells or these properties are counterbalanced. In addition, we show that in BL entities expression of NFATc1 protein is largely regulated at post-transcriptional level, including MYC dependent increase of protein stability.
Functionally we show that conditional inactivation of Nfatc1 gene in Eµ-Myc mice prevents development of BCL tumors with mature B cell immunophenotype (IgD+). Loss of NFATc1 expression in BCL cells ex vivo results in apoptosis of tumor cells.
Together our results identify NFATc1 as an important survival factor in BL cells and, hence, as a promising target for alternative therapeutic strategies for BL.
Molecular and developmental characterization of the Echinococcus multilocularis stem cell system
(2014)
The metacestode larva of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most dangerous zoonotic diseases in the Northern Hemisphere. Unlike “typical” metacestode larvae from other tapeworms, it grows as a mass of interconnected vesicles which infiltrates the liver of the intermediate host, continuously forming new vesicles in the periphery. From these vesicles, protoscoleces (the infective form for the definitive host) are generated by asexual budding. It is thought that in E. multilocularis, as in other flatworms, undifferentiated stem cells (so-called germinative cells in cestodes and neoblasts in free-living flatworms) are the sole source of new cells for growth and development. Therefore, this cell population should be of central importance for the progression of AE.
In this work, I characterized the germinative cells of E. multilocularis, and demonstrate that they are indeed the only proliferating cells in metacestode vesicles. The germinative cells are a population of undifferentiated cells with similar morphology, and express high levels of transcripts of a novel non-autonomous retrotransposon family (ta-TRIMs). Experiments of recovery after hydroxyurea treatment suggest that individual germinative cells have extensive self-renewal capabilities. However, germinative cells also display heterogeneity at the molecular level, since only some of them express conserved homologs of fgfr, nanos and argonaute genes, suggesting the existence of several distinct sub-populations. Unlike free-living flatworms, cestode germinative cells lack chromatoid bodies. Furthermore, piwi and vasa orthologs are absent from the genomes of cestodes, and there is widespread expression of some conserved neoblast markers in E. multilocularis metacestode vesicles. All of these results suggest important differences between the stem cell systems of free-living flatworms and cestodes.
Furthermore, I describe molecular markers for differentiated cell types, including the nervous system, which allow for the tracing of germinative cell differentiation. Using these molecular markers, a previously undescribed nerve net was discovered in metacestode vesicles. Because the metacestode vesicles are non-motile, and the nerve net of the vesicle is independent of the nervous system of the protoscolex, we propose that it could serve as a neuroendocrine system. By means of bioinformatic analyses, 22 neuropeptide genes were discovered in the E. multilocularis genome. Many of these genes are expressed in metacestode vesicles, as well as in primary cell preparations undergoing complete metacestode regeneration. This suggests a possible role for these genes in metacestode development. In line with this hypothesis, one putative neuropeptide (RGFI-amide) was able to stimulate the proliferation of primary cells at a concentration of 10-7 M, and the corresponding gene was upregulated during metacestode regeneration.
Platelet activation and aggregation are essential processes for the sealing of injured vessel walls and preventing blood loss. Under pathological conditions, however, platelet aggregation can lead to uncontrolled thrombus formation, resulting in irreversible vessel occlusion. Therefore, precise regulation of platelet activation is required to ensure efficient platelet plug formation and wound sealing but also to prevent uncontrolled thrombus formation. Rapid elevations in the intracellular levels of cations are a core signaling event during platelet activation. In this thesis, the roles of Ca2+ and Mg2+ channels in the regulation of platelet function were investigated.
Orai1, the major store-operated calcium (SOC) channel in platelets, is not only vital for diverse signaling pathways, but may also regulate receptor-operated calcium entry (ROCE). The coupling between the Orai1 signalosome and canonical transient receptor potential channel (TRPC) isoforms has been suggested as an essential step in the activation of store-operated calcium entry (SOCE) and ROCE in human platelets. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms still remains to be answered. In the first part of this thesis, the functional crosstalk between Orai1 and TRPC6 was addressed. Orai1-mediated SOCE was found to enhance the activity of phospholipases (PL) C and D, to increase diacylglycerol (DAG) production and finally to regulate TRPC6-mediated ROCE via DAG, indicating that the regulation of TRPC6 channel activity seems to be independent of the physical interaction with Orai1. Furthermore, Orai1 and TRPC6 double deficiency led to a reduced Ca2+ store content and basal cytoplasmic Ca2+ concentrations, but surprisingly also enhanced ATP secretion, which may enhance Ca2+ influx via P2X1 and compensate for the severe Ca2+ deficits seen in double mutant platelets. In addition, Orai1 and TRPC6 were not essential for G protein-coupled receptor (GPCR)-mediated platelet activation, aggregation and thrombus formation.
Transient receptor potential melastatin-like 7 (TRPM7) contains a cytosolic serine/threonine protein kinase. To date, a few in vitro substrates of the TRPM7 kinase have been identified, however, the physiological role of the kinase remains unknown. In the second part of this thesis, mice with a point mutation which blocks the catalytic activity of the TRPM7 kinase (Trpm7KI) were used to study the role of the TRPM7 kinase in platelet function. In Trpm7KI platelets phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism and Ca2+ mobilization were severely impaired upon glycoprotein (GP) VI activation, indicating that the TRPM7 kinase regulates PLC function. This signaling defect in Trpm7KI platelets resulted in impaired aggregate formation under flow and protected animals from arterial thrombosis and ischemic brain infarction. Altogether, these results highlight the kinase domain of TRPM7 as a pivotal signaling moiety implicated in the pathogenesis of thrombosis and cerebrovascular events.