@phdthesis{SchellergebBirkholz2020,
  author    = {Scheller [geb. Birkholz], Inga},
  title     = {Studies on the role of actin-binding proteins in platelet production and function in mice},
  doi       = {10.25972/OPUS-16858},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168582},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {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.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Volz2020,
  author    = {Volz, Julia},
  title     = {Studies on the influence of platelets on vascular integrity in primary tumors and the role of BIN2 in platelet calcium signaling},
  doi       = {10.25972/OPUS-21742},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217427},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {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.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Lu2020,
  author    = {Lu, Yunzhi},
  title     = {Kinetics of mouse and human muscle type nicotinic receptor channels},
  doi       = {10.25972/OPUS-19268},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192688},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated. Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents ({\^i}) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of {\^i} and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits.},
  subject      = {Nicotinischer Acetylcholinrezeptor},
  language  = {en}
}
@phdthesis{Surrey2020,
  author    = {Surrey, Verena},
  title     = {Identification of affected cellular targets, mechanisms and signaling pathways in a mouse model for spinal muscular atrophy with respiratory distress type 1 (SMARD1)},
  doi       = {10.25972/OPUS-17638},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176386},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {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.},
  subject      = {Spinale Muskelatrophie},
  language  = {en}
}