@phdthesis{Schreiber2018,
  author    = {Schreiber, Benjamin},
  title     = {Selective and enhanced fluorescence by biocompatible nanocoatings to monitor G-protein-coupled receptor dynamics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173923},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane. Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand. Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{vanEeuwijk2018,
  author    = {van Eeuwijk, Judith Martina Maria},
  title     = {Studies on thrombopoiesis and spleen tyrosine kinase-mediated signaling in platelets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142933},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis. Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor  chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.},
  subject      = {Thrombose},
  language  = {en}
}
@phdthesis{Chowdhury2018,
  author    = {Chowdhury, Suvagata Roy},
  title     = {The Role of MicroRNAs in \(Chlamydia\) Infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The obligate intracellular pathogen Chlamydia trachomatis is the causative agent of trachoma related blindness and the sexually transmitted pelvic inflammatory disease. Being an obligate intracellular pathogen, C. trachomatis has an intricate dependency on the survival of the host cell. This relationship is indispensible owing to the fact that the pathogen spends a considerable fraction of its biphasic lifecycle within a cytoplasmic vacuole inside the host cell, the so-called chlamydial inclusion. The cellular apoptotic-signalling network is governed by several finely tuned regulatory cascades composed of pro- and anti-apoptotic proteins that respond to changes in the cellular homeostasis. In order to facilitate its intracellular survival, Chlamydia has been known to inhibit the premature apoptosis of the host cell via the stabilization of several host anti-apoptotic proteins such as cIAP2 and Mcl-1. While the pro- and anti-apoptotic proteins are the major regulators of the host apoptotic signalling network, a class of the small non-coding RNAs called microRNAs (miRNAs) has increasingly gained focus as a new level of regulatory control over apoptosis. This work investigates the changes in the host miRNA expression profile post Chlamydia infection using a high throughput miRNA deep sequencing approach. Several miRNAs previously associated with the modulation for apoptotic signalling were differentially expressed upon Chlamydia infection in human endothelial cells. Of the differentially regulated miRNAs, miR-30c-5p was of particular interest since it had been previously shown to target the tumor suppressor protein p53. Our lab and others have previously demonstrated that Chlamydia can downregulate the levels of p53 by promoting its proteasomal degradation. This work demonstrates that Chlamydia infection promotes p53 downregulation by increasing the abundance of miR-30c-5p and a successful infection cycle is hindered by a loss of miR-30c-5p. Over the last decade, dedicated research aimed towards a better understanding of apoptotic stimuli has greatly improved our grasp on the subject. While extrinsic stress, deprivation of survival signals and DNA damage are regarded as major proponents of apoptotic induction, a significant responsibility lies with the mitochondrial network of the cell. Mitochondrial function and dynamics are crucial to cell fate determination and dysregulation of either is decisive for cell survival and pathogenesis of several diseases. The ability of the mitochondrial network to perform its essential tasks that include ATP synthesis, anti-oxidant defense, and calcium homeostasis amongst numerous other processes critical to cellular equilibrium is tied closely to the fission and fusion of individual mitochondrial fragments. It is, thus, 8 unsurprising that mitochondrial dynamics is closely linked to apoptosis. In fact, many of the proteins involved regulation of mitochondrial dynamics are also involved in apoptotic signalling. The mitochondrial fission regulator, Drp1 has previously been shown to be transcriptionally regulated by p53 and is negatively affected by a miR- 30c mediated inhibition of p53. Our investigation reveals a significant alteration in the mitochondrial dynamics of Chlamydia infected cells affected by the loss of Drp1. We show that loss of Drp1 upon chlamydial infection is mediated by the miR-30c-5p induced depletion of p53 and results in a hyper-fused architecture of the mitochondrial network. While it is widely accepted that Chlamydia depends on the host cell metabolism for its intracellular growth and development, the role of mitochondria in an infected cell, particularly with respect to its dynamic nature, has not been thoroughly investigated. This work attempts to illustrate the dependence of Chlamydia on miR-30c-5p induced changes in the mitochondrial architecture and highlight the importance of these modulations for chlamydial growth and development.},
  subject      = {Chlamydienkrankheit},
  language  = {en}
}