@phdthesis{RamirezPasos2019,
  author    = {Ramirez Pasos, Uri Eduardo},
  title     = {Subthalamic Nucleus Neural Synchronization and Connectivity during Limbic Processing of Emotional Pictures: Evidence from Invasive Recordings in Patients with Parkinson's Disease},
  doi       = {10.25972/OPUS-16985},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169850},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In addition to bradykinesia and tremor, patients with Parkinson's disease (PD) are known to exhibit non-motor symptoms such as apathy and hypomimia but also impulsivity in response to dopaminergic replacement therapy. Moreover, a plethora of studies observe differences in electrocortical and autonomic responses to both visual and acoustic affective stimuli in PD subjects compared to healthy controls. This suggests that the basal ganglia (BG), as well as the hyperdirect pathway and BG thalamocortical circuits, are involved in affective processing. Recent studies have shown valence and dopamine-dependent changes in synchronization in the subthalamic nucleus (STN) in PD patients during affective tasks. This thesis investigates the role of dopamine, valence, and laterality in STN electrophysiology by analyzing event-related potentials (ERP), synchronization, and inter-hemispheric STN connectivity. STN recordings were obtained from PD patients with chronically implanted electrodes for deep brain stimulation during a passive affective picture presentation task. The STN exhibited valence-dependent ERP latencies and lateralized 'high beta' (28-40 Hz) event-related desynchronization. This thesis also examines the role of dopamine, valence, and laterality on STN functional connectivity with the anterior cingulate cortex (ACC) and the amygdala. The activity of these limbic structures was reconstructed using simultaneously recorded electroencephalographic signals. While the STN was found to establish early coupling with both structures, STN-ACC coupling in the 'alpha' range (7-11 Hz) and uncoupling in the 'low beta' range (14-21 Hz) were lateralized. Lateralization was also observed at the level of synchrony in both reconstructed sources and for ACC ERP amplitude, whereas dopamine modulated ERP latency in the amygdala. These results may deepen our current understanding of the STN as a limbic node within larger emotional-motor networks in the brain.
},
  subject      = {Nucleus subthalamicus},
  language  = {en}
}
@phdthesis{Bathon2019,
  author    = {Bathon, Kerstin},
  title     = {Mutations in protein kinase A catalytic subunit as a cause of adrenal Cushing's syndrome: mechanisms and functional consequences},
  doi       = {10.25972/OPUS-16893},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168937},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Protein kinase A (PKA) is the main effector of cyclic-adenosine monophosphate (cAMP) and plays an important role in steroidogenesis and proliferation of adrenal cells. In a previous study we found two mutations (L206R, 199_200insW) in the main catalytic subunit of protein kinase A (PKA C) to be responsible for cortisol-producing adrenocortical adenomas (CPAs). These mutations interfere with the formation of a stable holoenzyme, thus causing constitutive PKA activation. More recently, we identified additional mutations affecting PKA C in CPAs associated with overt Cushing syndrome: S213R+insIILR, 200_201insV, W197R, d244 248+E249Q, E32V. This study reports a functional characterization of those PKA Cmutations linked to CPAs of Cushing's patients. All analyzed mutations except for E32V showed a reduced interaction with at least one tested regulatory (R) subunit. Interestingly the results of the activity differed among the mutants and between the assays employed. For three mutants (L206R, 199_200insW, S213R+insIILR), the results showed enhanced translocation to the nucleus. This was also observed in CRISPR/Cas9 generated PRKACA L206R mutated HEK293T cells. The enhanced nuclear translocation of this mutants could be due to the lack of R subunit binding, but also other mechanisms could be at play. Additionally, I used an algorithm, which predicted an effect of the mutation on substrate specificity for four mutants (L206R, 199_200insW, 200_201insV, d244 248+E249Q). This was proven using phosphoproteomics for three mutants (L206R, 200_201insV, d244 248+E249Q). In PRKACA L206R mutated CPAs this change in substrate specificity also caused hyperphosphorylation of H1.4 on serine 36, which has been reported to be implicated in mitosis. Due to these observations, I hypothesized, that there are several mechanisms of action of PRKACA mutations leading to increased cortisol secretion and cell proliferation in adrenal cells: interference with the formation of a stable holoenzyme, altered subcellular localization and a change in substrate specificity. My data indicate that some PKA C mutants might act via just one, others by a combination of these mechanisms. Altogether, these findings indicate that several mechanisms contribute to the development of CPAs caused by PRKACA mutations. Moreover, these findings provide a highly illustrative example of how alterations in a protein kinase can cause a human disease.},
  subject      = {Proteinkinase A},
  language  = {en}
}
@phdthesis{Segerer2019,
  author    = {Segerer, Gabriela},
  title     = {Characterization of cell biological and physiological functions of the phosphoglycolate phosphatase AUM},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123847},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer. Still, most of the mammalian HAD phosphatases remain functionally uncharacterized. This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos. To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation. The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells. These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling. Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase. To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions. Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20\% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under hypoxic (~1\% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3'-phosphate (GADP). Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism. Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.},
  subject      = {Phosphoglykolatphosphatase},
  language  = {en}
}
@phdthesis{SchmittgebWolf2019,
  author    = {Schmitt [geb. Wolf], Karen},
  title     = {Studies on the role of platelet serotonin in platelet function, hemostasis, thrombosis and stroke},
  doi       = {10.25972/OPUS-13471},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134711},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Platelet activation and aggregation are important processes in hemostasis resulting in reduction of blood loss upon vessel wall injury. However, platelet activation can lead to thrombotic events causing myocardial infarction and stroke. A more detailed understanding of the regulation of platelet activation and the subsequent formation of thrombi is essential to prevent thrombosis and ischemic stroke. Cations, platelet surface receptors, cytoskeletal rearrangements, activation of the coagulation cas-cade and intracellular signaling molecules are important in platelet activation and thrombus formation. One such important molecule is serotonin (5 hydroxytryptamin, 5 HT), an indolamine platelet agonist, biochemically derived from tryptophan. 5 HT is secreted from the enterochromaffin cells into the gastrointestinal tract (GI) and blood. Blood borne 5 HT has been proposed to regulate hemostasis by acting as a vaso-constrictor and by triggering platelet signaling through 5 HT2A receptor. Although platelets do not synthetize 5 HT, they take it up from the blood and store it in their dense granules which are secreted upon platelet activation. To identify the molecu-lar composite of the 5 HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke, 5 HT transporter knock out mice (5Htt / ) were analyzed in different in vitro and in vivo assays and in a model of is-chemic stroke. In 5Htt / platelets, 5 HT uptake from the blood was completely abol-ished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC 2) were reduced. These observed in vitro defects in 5Htt / platelets could be normalized by the addition of exogenous 5 HT. Moreover, reduced 5 HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt / mice. Surprisingly, in the transient middle cerebral artery occlusion model (tMCAO) of ischemic stroke 5Htt / mice showed near-ly normal infarct volumes and a neurological outcome comparable to control mice. Although secreted platelet 5 HT does not appear to play a crucial role in the devel-opment of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and thus plays an important role in thrombus stabilization. To further investigate the role of cations, granules and their contents and regulation of integrin activation in the process of thrombus formation, genetically modified mice were analyzed in the different in vivo thrombosis models. Whereas Tph1 / mice (lacking the enzyme responsible for the production of 5 HT in the periphery), Trpm7KI (point mu-tation in the kinase domain of Trpm7 channel, lacking kinase activity) and Unc13d / /Nbeal2 / mice (lacking α granules and the release machinery of dense granules) showed a delayed thrombus formation in vivo, MagT1y/ mice (lacking a specific Mg2+ transporter) displayed a pro thrombotic phenotype in vivo. Trpm7fl/fl Pf4Cre (lacking the non specific Mg2+ channel) and RIAM / mice (lacking a potential linker protein in integrin "inside out" signaling) showed no alterations in thrombus formation upon injury of the vessel wall.},
  subject      = {Serotonin},
  language  = {en}
}
@phdthesis{Alexander2019,
  author    = {Alexander, Stephanie},
  title     = {Collective cancer cell invasion \(in\) \(vivo\): function of β1 and β3 integrins in perivascular invasion and resistance to therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85435},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Pro-migratory signals mediated by the tumor microenvironment contribute to the cancer progression cascade, including invasion, metastasis and resistance to therapy. Derived from in vitro studies, isolated molecular steps of cancer invasion programs have been identified but their integration into the tumor microenvironment and suitability as molecular targets remain elusive. The purpose of the study was to visualize central aspects of tumor progression, including proliferation, survival and invasion by real-time intravital microscopy. The specific aims were to monitor the kinetics, mode, adhesion and chemoattraction mechanisms of tumor cell invasion, the involved guidance structures, and the response of invasion zones to anti-cancer therapy. To reach deeper tumor regions by optical imaging with subcellular resolution, near-infrared and infrared excited multiphoton microscopy was combined with a modified dorsal skinfold chamber model. Implanted HT-1080 fibrosarcoma and B16/F10 and MV3 melanoma tumors developed zones of invasive growth consisting of collective invasion strands that retained cell-cell contacts and high mitotic activity while invading at velocities of up to 200 μm per day. Collective invasion occurred predominantly along preexisting tissue structures, including blood and lymph vessels, collagen fibers and muscle strands of the deep dermis, and was thereby insensitive to RNAi based knockdown and/or antibody-based treatment against β1 and β3 integrins, chemokine (SDF-1/CXCL12) and growth factor (EGF) signaling. Therapeutic hypofractionated irradiation induced partial to complete regression of the tumor main mass, yet failed to eradicate the collective invasion strands, suggesting a microenvironmentally privileged niche. Whereas no radiosensitization was achieved by interference with EGFR or doxorubicin, the simultaneous inhibition of β1 and β3 integrins impaired cell proliferation and survival in spontaneously growing tumors and strongly enhanced the radiation response up to complete eradication of both main tumor and invasion strands. In conclusion, collective invasion in vivo is a robust process which follows preexisting tissue structures and is mainly independent of established adhesion and chemoattractant signaling. Due to its altered biological response to irradiation, collective invasion strands represent a microenvironmentally controlled and clinically relevant resistance niche to therapy. Therefore supportive regimens, such as anoikisinduction by anti-integrin therapy, may serve to enhance radio- and chemoefficacy and complement classical treatment regimens.},
  subject      = {Tumorzelle},
  language  = {en}
}
@phdthesis{Baig2019,
  author    = {Baig, Ayesha Anjum},
  title     = {Studies on platelet interactions with the coagulation system and on modulators of platelet (hem)ITAM signaling in genetically modified mice},
  doi       = {10.25972/OPUS-16488},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164888},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Activated platelets and coagulation jointly contribute to physiological hemostasis. However, pathological conditions can also trigger unwanted platelet activation and initiation of coagulation resulting in thrombosis and precipitation of ischemic damage of vital organs such as the heart or brain. The specific contribution of procoagulant platelets, positioned at the interface of the processes of platelet activation and coagulation, in ischemic stroke had remained uninvestigated. The first section of the thesis addresses this aspect through experiments conducted in novel megakaryocyte- and platelet-specific TMEM16F conditional KO mice (cKO). cKO platelets phenocopied defects in platelets from Scott Syndrome patients and had severely impaired procoagulant characteristics. This led to decelerated platelet-driven thrombin generation and delayed fibrin formation. cKO mice displayed prolonged bleeding times and impaired arterial thrombosis. However, infarct volumes in cKO mice were comparable to wildtype (WT) mice in an experimental model of ischemic stroke. Therefore, while TMEM16F-regulated platelet procoagulant activity is critical for hemostasis and thrombosis, it is dispensable for cerebral thrombo-inflammation in mice. The second section describes the generation and initial characterization of a novel knockin mouse strain that expresses human coagulation factor XII (FXII) instead of endogenous murine FXII. These knockin mice had normal occlusion times in an experimental model of arterial thrombosis demonstrating that human FXII is functional in mice. Therefore, these mice constitute a valuable tool for testing novel pharmacological agents against human FXII - an attractive potential target for antithrombotic therapy. Glycoprotein (GP)VI and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represent a major pathway for platelet activation. The last section of the thesis provides experimental evidence for redundant functions between the two members of the Grb2 family of adapter proteins - Grb2 and Gads that lie downstream of GPVI and CLEC-2 stimulation. In vitro and in vivo studies in mice deficient in both Grb2 and Gads (DKO) revealed that DKO platelets had defects in (hem)ITAM-stimulation-specific activation, aggregation and signal transduction that were more severe than the defects observed in single Grb2 KO or Gads KO mice. Furthermore, the specific role of these adapters downstream of (hem)ITAM signaling was essential for maintenance of hemostasis but dispensable for the known CLEC-2 dependent regulation of blood-lymphatic vessel separation.},
  subject      = {Blutgerinnung},
  language  = {en}
}
@phdthesis{TshitengeTshitenge2019,
  author    = {Tshitenge Tshitenge, Dieudonn{\´e}},
  title     = {Isolation and Structural Elucidation of Novel Anti-Infective Naphthylisoquinoline Alkaloids from Ancistrocladus ealaensis, and Phytochemical Analysis of Two Congolese Medicinal Plants},
  doi       = {10.25972/OPUS-15417},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154175},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Herein described are the isolation, structural elucidation, and biological evaluation of highly thrilling monomeric and dimeric new naphthylisoquinoline alkaloids from A. ealaensis. The separation, chiral resolution, and characterization of a series of stereoisomeric 2,3-dihydrobenzofuran neolignans are also reported. The analytical and phytochemical analysis on two Congolese antimalarial herbal drugs is part of the last chapter of the results. In this last case, major concerns on widely used Congolese herbal drugs are discussed.},
  subject      = {Naphthylisochinolinalkaloide},
  language  = {en}
}
@phdthesis{Fleischmann2019,
  author    = {Fleischmann, Pauline Nikola},
  title     = {Starting foraging life: Early calibration and daily use of the navigational system in \(Cataglyphis\) ants},
  doi       = {10.25972/OPUS-15995},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159951},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Cataglyphis ants are famous for their navigational abilities. They live in hostile habitats where they forage as solitary scavengers covering distances of more than hundred thousand times their body lengths. To return to their nest with a prey item - mainly other dead insects that did not survive the heat - Cataglyphis ants constantly keep track of their directions and distances travelled. The navigational strategy is called path integration, and it enables an ant to return to the nest in a straight line using its home vector. Cataglyphis ants mainly rely on celestial compass cues, like the position of the sun or the UV polarization pattern, to determine directions, and they use an idiothetic step counter and optic flow to measure distances. In addition, they acquire information about visual, olfactory and tactile landmarks, and the wind direction to increase their chances of returning to the nest safe and sound. Cataglyphis' navigational performance becomes even more impressive if one considers their life style. Most time of their lives, the ants stay underground and perform tasks within the colony. When they start their foraging careers outside the nest, they have to calibrate their compass systems and acquire all information necessary for navigation during subsequent foraging. This navigational toolkit is not instantaneously available, but has to be filled with experience. For that reason, Cataglyphis ants perform a striking behavior for up to three days before actually foraging. These so-called learning walks are crucial for the success as foragers later on. In the present thesis, both the ontogeny and the fine-structure of learning walks has been investigated. Here I show with displacement experiments that Cataglyphis ants need enough space and enough time to perform learning walks. Spatially restricted novices, i. e. na{\"i}ve ants, could not find back to the nest when tested as foragers later on. Furthermore, ants have to perform several learning walks over 1-3 days to gain landmark information for successful homing as foragers. An increasing number of feeder visits also increases the importance of landmark information, whereas in the beginning ants fully rely on their path-integration vector. Learning walks are well-structured. High-speed video analysis revealed that Cataglyphis ants include species-specific rotational elements in their learning walks. Greek Cataglyphis ants (C. noda and C. aenescens) inhabiting a cluttered pine forest perform voltes, small walked circles, and pirouettes, tight turns about the body axis with frequent stopping phases. During the longest stopping phases, the ants gaze back to their nest entrance. The Tunisian Cataglyphis fortis ants inhabiting featureless saltpans only perform voltes without directed gazes. The function of voltes has not yet been revealed. In contrast, the fine structure of pirouettes suggests that the ants take snapshots of the panorama towards their homing direction to memorize the nest's surroundings. The most likely hypothesis was that Cataglyphis ants align the gaze directions using their path integrator, which gets directional input from celestial cues during foraging. To test this hypothesis, a manipulation experiment was performed changing the celestial cues above the nest entrance (no sun, no natural polarization pattern, no UV light). The accurately directed gazes to the nest entrance offer an easily quantifiable readout suitable to ask the ants where they expect their nest entrance. Unexpectedly, all novices performing learning walks under artificial sky conditions looked back to the nest entrance. This was especially surprising, because neuronal changes in the mushroom bodies and the central complex receiving visual input could only be induced with the natural sky when comparing test animals with interior workers. The behavioral findings indicated that Cataglyphis ants use another directional reference system to align their gaze directions during the longest stopping phases of learning walk pirouettes. One possibility was the earth's magnetic field. Indeed, already disarraying the geomagnetic field at the nest entrance with an electromagnetic flat coil indicated that the ants use magnetic information to align their looks back to the nest entrance. To investigate this finding further, ants were confronted with a controlled magnetic field using a Helmholtz coil. Elimination of the horizontal field component led to undirected gaze directions like the disarray did. Rotating the magnetic field about 90°, 180° or -90° shifted the ants' gaze directions in a predictable manner. Therefore, the earth's magnetic field is a necessary and sufficient reference system for aligning nest-centered gazes during learning-walk pirouettes. Whether it is additionally used for other navigational purposes, e. g. for calibrating the solar ephemeris, remains to be tested. Maybe the voltes performed by all Cataglyphis ant species investigated so far can help to answer this question..},
  subject      = {Cataglyphis},
  language  = {en}
}
@phdthesis{Schubert2019,
  author    = {Schubert, Frank Klaus},
  title     = {The circadian clock network of \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157136},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism. The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons. To further study the functional hierarchy within the clock network and to monitor the "ticking clock" over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Tian2019,
  author    = {Tian, Yuehui},
  title     = {Characterization of novel rhodopsins with light-regulated cGMP production or cGMP degradation},
  doi       = {10.25972/OPUS-16814},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168143},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Photoreceptors are widely occurring in almost all kingdoms of life. They mediate the first step in sensing electromagnetic radiation of different wavelength. Absorption spectra are found within the strongest radiation from the sun and absorption usually triggers downstream signaling pathways. Until now, mainly 6 classes of representative photoreceptors are known: five water-soluble proteins, of these three classes of blue light-sensitive proteins including LOV (light-oxygen-voltage), BLUF (blue-light using FAD), and cryptochrome modules with flavin (vitamin B-related) nucleotides as chromophore; while two classes of yellow and red light-sensitive proteins consist of xanthopsin and phytochrome, respectively. Lastly, as uniquely integral membrane proteins, the class of rhodopsins can usually sense over a wide absorption spectrum, ranging from ultra-violet to green and even red light. Rhodopsins can be further divided into two types, i.e., microbial (type I) and animal (type II) rhodopsins. Rhodopsins consist of the protein opsin and the covalently bound chromophore retinal (vitamin A aldehyde). In this thesis, I focus on identification and characterization of novel type I opsins with guanylyl cyclase activity from green algae and a phosphodiesterase opsin from the protist Salpingoeca rosetta. Until 2014, all known type I and II rhodopsins showed a typical structure with seven transmembrane helices (7TM), an extracellular N-terminus and a cytosolic C-terminus. The proven function of the experimentally characterized type I rhodopsins was membrane transport of ions or the coupling to a transducer which enables phototaxis via a signaling chain. A completely new class of type I rhodopsins with enzymatic activity was identified in 2014. A light-activated guanylyl cyclase opsin was discovered in the fungus Blastocladiella emersonii which was named Cyclop (Cyclase opsin) by Gao et al. (2015), after heterologous expression and rigorous in-vitro characterization. BeCyclop is the first opsin for which an 8 transmembrane helices (8TM) structure was demonstrated by Gao et al. (2015). Earlier (2004), a novel class of enzymatic rhodopsins was predicted to exist in C. reinhardtii by expressed sequence tag (EST) and genome data, however, no functional data were provided up to now. The hypothetical rhodopsin included an N-terminal opsin domain, a fused two-component system with histidinekinase and response regulator domain, and a C-terminal guanylyl cyclase (GC) domain. This suggested that there could be a biochemical signaling cascade, integrating light-induction and ATP-dependent phosphate transfer, and as output the light-sensitive cGMP production. One of my projects focused on characterizing two such opsins from the green algae Chlamydomonas reinhardtii and Volvox carteri which we then named 2c-Cyclop (two-component Cyclase opsin), Cr2c-Cyclop and Vc2c-Cyclop, respectively. My results show that both 2c-Cyclops are light-inhibited GCs. Interestingly, Cr2c-Cyclop and Vc2c-Cyclop are very sensitive to light and ATP-dependent, whereby the action spectra of Cr2c-Cyclop and Vc2c-Cyclop peak at ~540 nm and ~560 nm, respectively. More importantly, guanylyl cyclase activity is dependent on continuous phosphate transfer between histidine kinase and response regulator. However, green light can dramatically block phosphoryl group transfer and inhibit cyclase activity. Accordingly, mutation of the retinal-binding lysine in the opsin domain resulted in GC activity and lacking light-inhibition. A novel rhodopsin phosphodiesterase from the protist Salpingoeca rosetta (SrRhoPDE) was discovered in 2017. However, the previous two studies of 2017 claimed a very weak or absent light-regulation. Here I give strong evidence for light-regulation by studying the activity of SrRhoPDE, expressed in Xenopus laevis oocytes, in-vitro at different cGMP concentrations. Surprisingly, hydrolysis of cGMP shows a ~100-fold higher turnover than that of cAMP. Light can enhance substrate affinity by decreasing the Km value for cGMP from 80 μM to 13 μM, but increases the maximum turnover only by ~30\%. In addition, two key single mutants, SrRhoPDE K296A or K296M, can abolish the light-activation effect by interrupting a covalent bond of Schiff base type to the chromophore retinal. I also demonstrate that SrRhoPDE shows cytosolic N- and C- termini, most likely via an 8-TM structure. In the future, SrRhoPDE can be a potentially useful optogenetic tool for light-regulation of cGMP concentration, possibly after further improvements by genetic engineering.},
  language  = {en}
}
@phdthesis{Derakhshani2019,
  author    = {Derakhshani, Shaghayegh},
  title     = {Measles virus infection enhances dendritic cell migration in a 3D environment},
  doi       = {10.25972/OPUS-18918},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189182},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system. The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs. Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions.},
  subject      = {Dendritische Zelle},
  language  = {en}
}
@phdthesis{Yu2019,
  author    = {Yu, Sung-Huan},
  title     = {Development and application of computational tools for RNA-Seq based transcriptome annotations},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176468},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In order to understand the regulation of gene expression in organisms, precise genome annotation is essential. In recent years, RNA-Seq has become a potent method for generating and improving genome annotations. However, this Approach is time consuming and often inconsistently performed when done manually. In particular, the discovery of non-coding RNAs benefits strongly from the application of RNA-Seq data but requires significant amounts of expert knowledge and is labor-intensive. As a part of my doctoral study, I developed a modular tool called ANNOgesic that can detect numerous transcribed genomic features, including non-coding RNAs, based on RNA-Seq data in a precise and automatic fashion with a focus on bacterial and achaeal species. The software performs numerous analyses and generates several visualizations. It can generate annotations of high-Resolution that are hard to produce using traditional annotation tools that are based only on genome sequences. ANNOgesic can detect numerous novel genomic Features like UTR-derived small non-coding RNAs for which no other tool has been developed before. ANNOgesic is available under an open source license (ISCL) at https://github.com/Sung-Huan/ANNOgesic. My doctoral work not only includes the development of ANNOgesic but also its application to annotate the transcriptome of Staphylococcus aureus HG003 - a strain which has been a insightful model in infection biology. Despite its potential as a model, a complete genome sequence and annotations have been lacking for HG003. In order to fill this gap, the annotations of this strain, including sRNAs and their functions, were generated using ANNOgesic by analyzing differential RNA-Seq data from 14 different samples (two media conditions with seven time points), as well as RNA-Seq data generated after transcript fragmentation. ANNOgesic was also applied to annotate several bacterial and archaeal genomes, and as part of this its high performance was demonstrated. In summary, ANNOgesic is a powerful computational tool for RNA-Seq based annotations and has been successfully applied to several species.},
  subject      = {Genom},
  language  = {en}
}
@phdthesis{Rydzek2019,
  author    = {Rydzek, Julian},
  title     = {NF-κB/NFAT Reporter Cell Platform for Chimeric Antigen Receptor (CAR)-Library Screening},
  doi       = {10.25972/OPUS-17918},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179187},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Immunotherapy with engineered T cells expressing a tumor-specific chimeric antigen receptor (CAR) is under intense preclinical and clinical investigation. This involves a rapidly increasing portfolio of novel target antigens and CAR designs that need to be tested in time- and work-intensive screening campaigns in primary T cells. Therefore, we anticipated that a standardized screening platform, similar as in pharmaceutical small molecule and antibody discovery, would facilitate the analysis of CARs by pre-selecting lead candidates from a large pool of constructs that differ in their extracellular and intracellular modules. Because CARs integrate structural elements of the T cell receptor (TCR) complex and engage TCR-associated signaling molecules upon stimulation, we reasoned that the transcription factors nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT) could serve as surrogate markers for primary T cell function. The nuclear translocation of both transcription factors in primary T cells, which we observed following CAR stimulation, supported our rationale to use NF-κB and NFAT as indicators of CAR-mediated activation in a screening platform. To enable standardized and convenient analyses, we have established a CAR-screening platform based on the human T cell lymphoma line Jurkat that has been modified to provide rapid detection of NF-κB and NFAT activation. For this purpose, Jurkat cells contained NF-κB and NFAT-inducible reporter genes that generate a duplex output of cyan fluorescent protein (CFP) and green fluorescent protein (GFP), respectively. Upon stimulation of NF-κB/NFAT reporter cells, the expression of both fluorophores could be readily quantified in high-throughput screening campaigns by flow cytometry. We modified the reporter cells with CD19-specific and ROR1-specific CARs, and we co-cultured them with antigen-positive stimulator cells to analyze NF-κB and NFAT activation. CAR-induced reporter signals could already be detected after 6 hours. The optimal readout window with high-level reporter activation was set to 24 hours, allowing the CAR-screening platform to deliver results in a rapid turnaround time. A reporter cell-screening campaign of a spacer library with CARs comprising a short, intermediate or long IgG4-Fc domain allowed distinguishing functional from non-functional constructs. Similarly, reporter cell-based analyses identified a ROR1-CAR with 4-1BB domain from a library with different intracellular signal modules due to its ability to confer high NF-κB activation, consistent with data from in vitro and in vivo studies with primary T cells. The results of both CAR screening campaigns were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary T cells (21 days). We further challenged the reporter cells in a large-scale screening campaign with a ROR1 CAR library comprising mutations in the VH CDR3 sequence of the R11 scFv. This region is crucial for binding the R11 epitope of ROR1, and we anticipated that mutations here would cause a loss of specificity and affinity for most of the CAR variants. This provided the opportunity to determine whether the CAR screening platform was able to retrieve functional constructs from a large pool of CAR variants. Indeed, using a customized pre enrichment and screening strategy, the reporter cells identified a functional CAR variant that was present with a frequency of only 6 in 1.05x10^6. As our CAR-screening platform enabled the analysis of activating signal modules, it encouraged us to also evaluate inhibitory signal modules that change the CAR mode of action. Such an inhibitory CAR (iCAR) can be used in logic gates with an activating CAR to interfere with T cell stimulation. By selecting appropriate target antigens for iCAR and CAR, this novel application aims to improve the selectivity towards tumor cells, and it could readily be studied using our screening platform. Accordingly, we tested CD19-specific iCARs with inhibitory PD-1 signal module for their suppressive effect on reporter gene activation. In logic gates with CAR or TCR stimulation, a decrease of NF-κB and NFAT signals was only observed when activating and inhibitory receptors were forced into spatial proximity. These results were further verified by experiments with primary T cells. In conclusion, our reporter cell system is attractive as a platform technology because it is independent of testing in primary T cells, exportable between laboratories, and scalable to enable small- to large-scale screening campaigns of CAR libraries. The pre-selection of appropriate lead candidates with optimal extracellular and intracellular modules can reduce the number of CAR constructs to be investigated in further in vitro and in vivo studies with primary T cells. We are therefore confident that our CAR-screening platform based on NF-κB/NFAT reporter cells will be useful to accelerate translational research by facilitating the evaluation of CARs with novel design parameters.},
  subject      = {Antigenrezeptor},
  language  = {en}
}
@phdthesis{Agnetta2019,
  author    = {Agnetta, Luca},
  title     = {Novel Photoswitchable and Dualsteric Ligands Acting on Muscarinic Acetylcholine Receptors for Receptor Function Investigation},
  doi       = {10.25972/OPUS-18717},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187170},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {G protein-coupled receptor research looks out for new technologies to elucidate the complex processes of receptor activation, function and downstream signaling with spatiotemporal resolution, preferably in living cells and organisms. A thriving approach consists in making use of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness and high degree of orthogonality regarding biological processes. This is realized by the incorporation of molecular photoswitches, which are able to effectively respond to light, such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic acetylcholine receptors belong to class A GPCRs and have received special attention in this regard due to their role as a prototypic pharmacological system and their therapeutic potential. They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and thus regulate diverse important biological processes, especially many neurological functions in our brain. In this work, the application of photopharmacological tool compounds to muscarinic receptors is presented, consisting of pharmacophores extended with azobenzene as light-responsive motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind concomitantly to the orthosteric and allosteric binding site of the receptor, which is demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective photoswitchable ligands, suitable for further ex vivo and in vivo studies. Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated extensively with regard to their photochemical behavior and pharmacological profile, outlining the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho- fluoro azobenzene. For the first time on a GPCR it was examined, which impact the different substitution pattern has on both the binding and the activity on the M1 receptor. Results show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit a considerably different biological profile that has to be investigated carefully. The achievements gained in this study can give important new insights into the binding mode and time course of activation processes, enabling precise spatial and temporal resolution of the complex signaling pathway of muscarinic receptors. Due to their role as exemplary model system, these findings may be useful for the investigation into other therapeutically relevant GPCRs.},
  subject      = {Muscarinrezeptor},
  language  = {en}
}
@phdthesis{Kaymak2019,
  author    = {Kaymak, Irem},
  title     = {Identification of metabolic liabilities in 3D models of cancer},
  doi       = {10.25972/OPUS-18154},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.},
  subject      = {Tumor},
  language  = {en}
}
@phdthesis{Gorelashvili2019,
  author    = {Gorelashvili, Maximilian Georg},
  title     = {Investigation of megakaryopoiesis and the acute phase of ischemic stroke by advanced fluorescence microscopy},
  doi       = {10.25972/OPUS-18600},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186002},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood. Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation. Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood. Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Flechsenhar2019,
  author    = {Flechsenhar, Aleya Felicia},
  title     = {The Ubiquity of Social Attention - a Detailed Investigation of the Underlying Mechanisms},
  doi       = {10.25972/OPUS-18452},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184528},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {This dissertation highlights various aspects of basic social attention by choosing versatile approaches to disentangle the precise mechanisms underlying the preference to focus on other human beings. The progressive examination of different social processes contrasted with aspects of previously adopted principles of general attention. Recent research investigating eye movements during free exploration revealed a clear and robust social bias, especially for the faces of depicted human beings in a naturalistic scene. However, free viewing implies a combination of mechanisms, namely automatic attention (bottom-up), goal-driven allocation (top-down), or contextual cues and inquires consideration of overt (open exploration using the eyes) as well as covert orienting (peripheral attention without eye movement). Within the scope of this dissertation, all of these aspects have been disentangled in three studies to provide a thorough investigation of different influences on social attention mechanisms. In the first study (section 2.1), we implemented top-down manipulations targeting non-social features in a social scene to test competing resources. Interestingly, attention towards social aspects prevailed, even though this was detrimental to completing the requirements. Furthermore, the tendency of this bias was evident for overall fixation patterns, as well as fixations occurring directly after stimulus onset, suggesting sustained as well as early preferential processing of social features. Although the introduction of tasks generally changes gaze patterns, our results imply only subtle variance when stimuli are social. Concluding, this experiment indicates that attention towards social aspects remains preferential even in light of top-down demands. The second study (section 2.2) comprised of two separate experiments, one in which we investigated reflexive covert attention and another in which we tested reflexive as well as sustained overt attention for images in which a human being was unilaterally located on either the left or right half of the scene. The first experiment consisted of a modified dot-probe paradigm, in which peripheral probes were presented either congruently on the side of the social aspect, or incongruently on the non-social side. This was based on the assumption that social features would act similar to cues in traditional spatial cueing paradigms, thereby facilitating reaction times for probes presented on the social half as opposed to the non-social half. Indeed, results reflected such congruency effect. The second experiment investigated these reflexive mechanisms by monitoring eye movements and specifying the location of saccades and fixations for short as well as long presentation times. Again, we found the majority of initial saccades to be congruently directed to the social side of the stimulus. Furthermore, we replicated findings for sustained attention processes with highest fixation densities for the head region of the displayed human being. The third study (section 2.3), tackled the other mechanism proposed in the attention dichotomy, the bottom-up influence. Specifically, we reduced the available contextual information of a scene by using a gaze-contingent display, in which only the currently fixated regions would be visible to the viewer, while the remaining image would remain masked. Thereby, participants had to voluntarily change their gaze in order to explore the stimulus. First, results revealed a replication of a social bias in free-viewing displays. Second, the preference to select social features was also evident in gaze-contingent displays. Third, we find higher recurrent gaze patterns for social images compared to non-social ones for both viewing modalities. Taken together, these findings imply a top-down driven preference for social features largely independent of contextual information. Importantly, for all experiments, we took saliency predictions of different computational algorithms into consideration to ensure that the observed social bias was not a result of high physical saliency within these areas. For our second experiment, we even reduced the stimulus set to those images, which yielded lower mean and peak saliency for the side of the stimulus containing the social information, while considering algorithms based on low-level features, as well as pre-trained high-level features incorporated in deep learning algorithms. Our experiments offer new insights into single attentional mechanisms with regard to static social naturalistic scenes and enable a further understanding of basic social processing, contrasting from that of non-social attention. The replicability and consistency of our findings across experiments speaks for a robust effect, attributing social attention an exceptional role within the general attention construct, not only behaviorally, but potentially also on a neuronal level and further allowing implications for clinical populations with impaired social functioning.},
  subject      = {Aufmerksamkeit},
  language  = {en}
}
@phdthesis{Tiwarekar2019,
  author    = {Tiwarekar, Vishakha Rakesh},
  title     = {The APOBEC3G-regulated host factors REDD1 and KDELR2 restrict measles virus replication},
  doi       = {10.25972/OPUS-17952},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179526},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Measles is an extremely contagious vaccine-preventable disease responsible for more than 90000 deaths worldwide annually. The number of deaths has declined from 8 million in the pre-vaccination era to few thousands every year due to the highly efficacious vaccine. However, this effective vaccine is still unreachable in many developing countries due to lack of infrastructure, while in developed countries too many people refuse vaccination. Specific antiviral compounds are not yet available. In the current situation, only an extensive vaccination approach along with effective antivirals could help to have a measles-free future. To develop an effective antiviral, detailed knowledge of viral-host interaction is required. This study was undertaken to understand the interaction between MV and the innate host restriction factor APOBEC3G (A3G), which is well-known for its activity against human immunodeficiency virus (HIV). Restriction of MV replication was not attributed to the cytidine deaminase function of A3G, instead, we identified a novel role of A3G in regulating cellular gene functions. Among two of the A3G regulated host factors, we found that REDD1 reduced MV replication, whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby affecting viral release. REDD1, a negative regulator of mTORC1 signalling impaired MV replication by inhibiting mTORC1. A3G regulated REDD1 expression was demonstrated to inversely correlate with MV replication. siRNA mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced REDD1 levels and simultaneously increased MV titres. Also, direct depletion of REDD1 improved MV replication in PBL, indicating its role in A3G mediated restriction of MV. Based on these finding, a new role of rapamycin, a pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing MV replication in Vero as well as in human PBL. The ER and Golgi resident receptor KDELR2 indirectly affected MV by competing with MV-H for cellular chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer assist in MV-H folding and subsequent surface expression. Taken together, the two A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for mediating its antiviral activity against MV.},
  language  = {en}
}
@phdthesis{Anany2019,
  author    = {Anany, Mohamed Ahmed Mohamed Mohamed},
  title     = {Enhancement of Toll-like receptor3 (TLR3)-induced death signaling by TNF-like weak inducer of apoptosis (TWEAK)},
  doi       = {10.25972/OPUS-18975},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily (TNFSF) and is as such initially expressed as type II class transmembrane glycoprotein from which a soluble ligand form can be released by proteolytic processing. While the expression of TWEAK has been detected at the mRNA level in various cell lines and cell types, its cell surface expression has so far only been documented for dendritic cells, monocytes and interferon-γ stimulated NK cells. The fibroblast growth factor-inducible-14 (Fn14) is a TRAF2-interacting receptor of the TNF receptor superfamily (TNFRSF) and is the only receptor for TWEAK. The expression of Fn14 is strongly induced in a variety of non-hematopoietic cell types after tissue injury. The TWEAK/Fn14 system induces pleiotropic cellular activities such as induction of proinflammatory genes, stimulation of cellular angiogenesis, proliferation, differentiation, migration and in rare cases induction of apoptosis. On the other side, Toll-like receptor3 (TLR3) is one of DNA- and RNA-sensing pattern recognition receptors (PRRs), plays a crucial role in the first line of defense against virus and invading foreign pathogens and cancer cells. Polyinosinic-polycytidylic acid poly(I:C) is a synthetic analog of dsRNA, binds to TLR3 which acts through the adapter TRIF/TICAM1, leading to cytokine secretion, NF-B activation, IRF3 nuclear translocation, inflammatory response and may also elicit the cell death. TWEAK sensitizes cells for TNFR1-induced apoptosis and necroptosis by limiting the availability of protective TRAF2-cIAP1 and TRAF2-cIAP2 complexes, which interact with the TNFR1-binding proteins TRADD and RIPK1. In accordance with the fact that poly(I:C)-induced signaling also involves these proteins, we found enhanced necroptosis-induction in HaCaT and HeLa-RIPK3 by poly(I:C) in the presence of TWEAK (Figure 24). Analysis of a panel of TRADD, FADD, RIPK1 and caspase-8 knockout cells revealed furthermore similarities and differences in the way how these molecules act in cell death signaling by poly(I:C)/TWEAK and TNF and TRAIL. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for these responses in TNF and TRAIL signaling. Lack of FADD protein abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis. Moreover, we observed that both long and short FLIP rescued HaCaT and HeLa-RIPK3 cells from poly(I:C)-induced apoptosis or necroptosis. To sum up, our results demonstrate that TWEAK, which is produced by interferon stimulated myeloid cells, controls the induction of apoptosis and necroptosis by the TLR3 ligand poly(I:C) and may thus contribute to cancer or anti-viral immunity treatment.},
  subject      = {Immunologe},
  language  = {en}
}
@phdthesis{Turakhiya2019,
  author    = {Turakhiya, Ankit},
  title     = {Functional characterization of the role of ZFAND1 in stress granule turnover},
  doi       = {10.25972/OPUS-16375},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163751},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1. In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS).},
  subject      = {ubiquitin},
  language  = {en}
}
@phdthesis{Nieberler2019,
  author    = {Nieberler, Matthias},
  title     = {The physiological role of autoproteolysis of the Adhesion GPCR Latrophilin/dCIRL},
  doi       = {10.25972/OPUS-16589},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165894},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {G protein-coupled receptors of the Adhesion family (aGPCRs) comprise the second largest group within the GPCR realm with over 30 mammalian homologs. They contain a unique structure with unusually large extracellular domains (ECDs) holding many structural folds known to mediate cell-cell and cell-matrix interactions. Furthermore, aGPCRs undergo autoproteolytic cleavage at the GPCR proteolysis site (GPS), an integral portion of the GPCR autoproteolysis inducing (GAIN) domain. Thus far, it is largely unknown if and how self-cleavage affects aGPCR activation and signaling and how these signals may shape the physiological function of cells. Latrophilin, alternatively termed the calcium-independent receptor of α-latrotoxin (CIRL) constitutes a highly conserved, prototypic aGPCR and has been assigned roles in various biological processes such as synaptic development and maturation or the regulation of neurotransmitter release. The Drosophila melanogaster homolog dCIRL is found in numerous sensory neurons including the mechanosensory larval pentascolopidial chordotonal organs (CHOs), which rely on dCIRL function in order to sense mechanical cues and to modulate the mechanogating properties of present ionotropic receptors. This study reveals further insight into the broad distribution of dCirl expression throughout the larval central nervous system, at the neuromuscular junction (NMJ), as well as subcellular localization of dCIRL in distal dendrites and cilia of chordotonal neurons. Furthermore, targeted mutagenesis which disabled GPS cleavage of dCIRL left intracellular trafficking in larval CHOs unaffected and proved autoproteolysis is not required for dCIRL function in vivo. However, substitution of a threonine residue, intrinsic to a putative tethered agonist called Stachel that has previously been documented for several other aGPCRs, abrogated receptor function. Conclusively, while this uncovered the presence of Stachel in dCIRL, it leaves the question about the biological relevance of the predetermined breaking point at the GPS unanswered. In an independent approach, the structure of the "Inter-RBL-HRM" (IRH) region, the region linking the N-terminal Rhamnose-binding lectin-like (RBL) and the hormone receptor motif (HRM) domains of dCIRL, was analyzed. Results suggest random protein folding, excessive glycosylation, and a drastic expansion of the size of IRH. Therefore, the IRH might represent a molecular spacer ensuring a certain ECD dimension, which in turn may be a prerequisite for proper receptor function. Taken together, the results of this study are consistent with dCIRL's mechanoceptive faculty and its role as a molecular sensor that translates mechanical cues into metabotropic signals through a yet undefined Stachel-dependent mechanism.},
  subject      = {Latrophilin},
  language  = {en}
}
@phdthesis{Gulve2019,
  author    = {Gulve, Nitish},
  title     = {Subversion of Host Genome Integrity by Human Herpesvirus 6 and \(Chlamydia\) \(trachomatis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162026},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Ovarian cancer is one of the most common gynecological malignancies in the world. The prevalence of a microbial signature in ovarian cancer has been reported by several studies till date. In these microorganisms, Human herpesvirus 6 (HHV-6) and Chlamydia trachomatis (C.tr) are especially important as they have significantly high prevalence rate. Moreover, these pathogens are directly involved in causing DNA damage and thereby disrupting the integrity of host genome which is the underlying cause of any cancer. This study focuses on how the two pathogens, HHV-6 and C. trachomatis can affect the genome integrity in their individual capacities and thereby may drive ovarian epithelial cells towards transformation. HHV-6 has unique tendency to integrate its genome into the host genome at subtelomeric regions and achieve a state of latency. This latent virus may get reactivated during the course of life by stress, drugs such as steroids, during transplantation, pregnancy etc. The study presented here began with an interesting observation wherein the direct repeat (DR) sequences flanking the ends of double stranded viral genome were found in unusually high numbers in human blood samples as opposed to normal ratio of two DR copies per viral genome. This study was corroborated with in vitro data where cell lines were generated to mimic the HHV-6 status in human samples. The same observation of unusually high DR copies was found in these cell lines as well. Interestingly, fluorescence in situ hybridization (FISH) and inverse polymerase chain reaction followed by southern blotting showed that DR sequences were found to be integrated in nontelomeric regions as opposed to the usual sub-telomeric integration sites in both human samples and in cell lines. Sanger sequencing confirmed the non-telomeric integration of viral DR sequences in the host genome. Several studies have shown that C. trachomatis causes DNA damage and inhibits the signaling cascade of DNA damage response. However, the effect of C. trachomatis infection on process of DNA repair itself was not addressed. In this study, the effect of C. trachomatis infection on host base excision repair (BER) has been addressed. Base excision repair is a pathway which is responsible for replacing the oxidized bases with new undamaged ones. Interestingly, it was found that C. trachomatis infection downregulated polymerase β expression and attenuated polymerase β- mediated BER in vitro. The mechanism of the polymerase β downregulation was found to be associated with the changes in the host microRNAs and downregulation of tumor suppressor, p53. MicroRNA-499 which has a binding site in the polymerase β 3'UTR was shown to be upregulated during C. trachomatis infection. Inhibition of miR-499 using synthetic miR-499 inhibitor indeed improved the repair efficiency during C. trachomatis infection in the in vitro repair assay. Moreover, p53 transcriptionally regulates polymerase β and stabilizing p53 during C. trachomatis infection enhanced the repair efficiency. Previous studies have shown that C. trachomatis can reactivate latent HHV-6. Therefore, genomic instability due to insertions of unstable 'transposon-like' HHV-6 DR followed by compromised BER during C. trachomatis infection cumulatively support the hypothesis of pathogenic infections as a probable cause of ovarian cancer},
  subject      = {Chlamydia trachomatis},
  language  = {en}
}
@phdthesis{Sauer2019,
  author    = {Sauer, Markus},
  title     = {DHX36 function in RNA G-quadruplex-mediated posttranscriptional gene regulation},
  doi       = {10.25972/OPUS-18395},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183954},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The expression of genetic information into proteins is a key aspect of life. The efficient and exact regulation of this process is essential for the cell to produce the correct amounts of these effector molecules to a given situation. For this purpose, eukaryotic cells have developed many different levels of transcriptional and posttranscriptional gene regulation. These mechanisms themselves heavily rely on interactions of proteins with associated nucleic acids. In the case of posttranscriptional gene regulation an orchestrated interplay between RNA-binding proteins, messenger RNAs (mRNA), and non-coding RNAs is compulsory to achieve this important function. A pivotal factor hereby are RNA secondary structures. One of the most stable and diverse representatives is the G-quadruplex structure (G4) implicated in many cellular mechanisms, such as mRNA processing and translation. In protein biosynthesis, G4s often act as obstacles but can also assist in this process. However, their presence has to be tightly regulated, a task which is often fulfilled by helicases. One of the best characterized G4-resolving factors is the DEAH-box protein DHX36. The in vitro function of this helicase is extensively described and individual reports aimed to address diverse cellular functions as well. Nevertheless, a comprehensive and systems-wide study on the function of this specific helicase was missing, so far. The here-presented doctoral thesis provides a detailed view on the global cellular function of DHX36. The binding sites of this helicase were defined in a transcriptome-wide manner, a consensus binding motif was deviated, and RNA targets as well as the effect this helicase exerts on them were examined. In human embryonic kidney cells, DHX36 is a mainly cytoplasmic protein preferentially binding to G-rich and G4-forming sequence motifs on more than 4,500 mRNAs. Loss of DHX36 leads to increased target mRNA levels whereas ribosome occupancy on and protein output of these transcripts are reduced. Furthermore, DHX36 knockout leads to higher RNA G4 levels and concomitant stress reactions in the cell. I hypothesize that, upon loss of this helicase, translationally-incompetent structured DHX36 target mRNAs, prone to localize in stress granules, accumulate in the cell. The cell reacts with basal stress to avoid cytotoxic effects produced by these mis-regulated and structured transcripts.},
  subject      = {RNS},
  language  = {en}
}
@phdthesis{MendesPereira2019,
  author    = {Mendes Pereira, Lenon},
  title     = {Morphological and Functional Ultrashort Echo Time (UTE) Magnetic Resonance Imaging of the Human Lung},
  doi       = {10.25972/OPUS-18317},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183176},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In this thesis, a 3D Ultrashort echo time (3D-UTE) sequence was introduced in the Self-gated Non-Contrast-Enhanced Functional Lung Imaging (SENCEFUL) framework. The sequence was developed and implemented on a 3 Tesla MR scanner. The 3D-UTE technique consisted of a nonselective RF pulse followed by a koosh ball quasi-random sampling order of the k-space. Measurements in free-breathing and without contrast agent were performed in healthy subjects and a patient with lung cancer. A gating technique, using a combination of different coils with high signal correlation, was evaluated in-vivo and compared with a manual approach of coil selection. The gating signal offered an estimation of the breathing motion during measurement and was used as a reference to segment the acquired data into different breathing phases. Gradient delays and trajectory errors were corrected during post-processing using the Gradient Impulse Response Function. Iterative SENSE was then applied to determine the fully sampled data. In order to eliminate signal changes caused by motion, a 3D image registration was employed, and the results were compared to a 2D image registration method. Ventilation was assessed in 3D and regionally quantified by monitoring the signal changes in the lung parenchyma. Finally, image quality and quantitative ventilation values were compared to the standard 2D-SENCEFUL technique. 3D-UTE, combined with an automatic gating technique and SENCEFUL MRI, offered ventilation maps with high spatial resolution and SNR. Compared to the 2D method, UTE-SENCEFUL greatly improved the clinical quality of the structural images and the visualization of the lung parenchyma. Through-plane motion, partial volume effects and ventilation artifacts were also reduced with a three-dimensional method for image registration. UTE-SENCEFUL was also able to quantify regional ventilation and presented similar results to previous studies.},
  subject      = {Kernspintomografie},
  language  = {en}
}
@phdthesis{delOlmoToledo2019,
  author    = {del Olmo Toledo, Valentina},
  title     = {Evolution of DNA binding preferences in a family of eukaryotic transcription regulators},
  doi       = {10.25972/OPUS-18789},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Regulation of gene expression by the control of transcription is essential for any cell to adapt to the environment and survive. Transcription regulators, i.e. sequence-specific DNA binding proteins that regulate gene expression, are central elements within the gene networks of most organisms. Transcription regulators are grouped into distinct families based on structural features that determine, to a large extent, the DNA sequence(s) that they can recognise and bind. Less is known, however, about how the DNA binding preferences can diversify within transcription regulator families during evolutionary timescales, and how such diversification can affect the biology of the organism. In this dissertation I study the SREBP (sterol regulatory element binding protein) family of transcriptional regulators in yeasts, and in Candida albicans in particular, as an experimental system to address these questions. The SREBPs are conserved from fungi to humans and represent a subgroup of basic helix-loop-helix DNA binding proteins. Early chromatin immunoprecipitation experiments with SREBPs from humans and yeasts showed that these proteins bound in vivo to the canonical DNA sequence, termed E-box, most basic helix-loop-helix proteins bind to. By contrast, most recent analysis carried out with less-studied fungal SREBPs revealed a non-canonical DNA motif to be the most overrepresented sequence in the bound regions. This study aims to establish the intrinsic DNA binding preferences of key branches of this family and to determine how the divergence in DNA binding affinities originated. To this end, I combined phylogenetic and ancestral reconstruction with extensive biochemical characterisation of key SREBP proteins. The results indicated that while the most-studied SREBPs (in mammals) indeed show preference for the E-box, a second branch of the family preferentially binds the non-E-box, and a third one is able to bind both sequences with similar affinity. The preference for one or the other DNA sequence is an intrinsic property of each protein because their purified DNA binding domain was sufficient to recapitulate their in vivo binding preference. The ancestor that gave rise to these two different types of SREBPs (the branch that binds E-box and the one that binds non-E-box DNA) appears to be a protein with a broader DNA binding capability that had a slight preference for the non-canonical motif. Thus, the results imply these two branches originated by either enhancing the original ancestral preference for non-E-box or tilting it towards the E-box DNA and flipping the preference for this sequence. The main function associated with members of the SREBP family in most eukaryotes is the control of lipid biosynthesis. I have further studied the function of these proteins in the lineage that encompasses the human associated yeast C. albicans. Strikingly, the three SREBPs present in the fungus' genome contribute to the colonisation of the mammalian gut by regulating cellular processes unrelated to lipid metabolism. Here I describe that two of the three C. albicans SREBPs form a regulatory cascade that regulates morphology and cell wall modifications under anaerobic conditions, whereas the third SREBP has been shown to be involved in the regulation of glycolysis genes. Therefore, I posit that the described diversification in DNA binding specificity in these proteins and the concomitant expansion of targets of regulation were key in enabling this fungal lineage to associate with animals.},
  subject      = {Candida albicans},
  language  = {en}
}
@phdthesis{Gomes2019,
  author    = {Gomes, Sara Ferreira Martins},
  title     = {Induced Pluripotent Stem Cell-derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection},
  doi       = {10.25972/OPUS-18855},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188550},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier. Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis. In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs.},
  subject      = {Neisseria meningitidis},
  language  = {en}
}
@phdthesis{SoaresMachado2019,
  author    = {Soares Machado, J{\´e}ssica},
  title     = {Dosimetry-based Assessment of Radiation-associated Cancer risk for \(^9\)\(^9\)\(^m\)Tc-MAG3 Scans in Infants and Optimization of Administered Activities for \(^6\)\(^8\)Ga-labelled Peptides in Children and Adolescents},
  doi       = {10.25972/OPUS-19264},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192640},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In 2006, 0.18 Mio pediatric nuclear medicine diagnostic exams were performed worldwide. However, for most of the radiopharmaceuticals used data on biokinetics and, as a consequence on dosimetry, are missing or have not been made publicly available. Therefore, most of the dosimetry assessments presented today for diagnostic agents in children and adolescents rely on the biokinetics data of adults. Even for one of the most common nuclear medicine exams for this patient group, renal scintigraphy with 99mTc-MAG3 for assessing renal function measured data on biokinetics is available only from a study performed on four children of different ages. In particular, renal scans are among the most frequent exams performed on infants and toddlers. Due to the young age, this patient group can be classified as a risk group with a higher probability of developing stochastic radiation effects compared to adults. As there are only limited data on biokinetics and dosimetry in this patient group, the aim of this study is to reassess the dosimetry and the associated radiation risk for a larger number of infants undergoing 99mTc-MAG3 renal scans based on a retrospective analysis of existing patient data. Data were collected retrospectively from 34 patients younger than 20 months with normal (20 patients) and abnormal renal function (14 patients) undergoing 99mTc-MAG3 scans. The patient-specific organ activity was estimated based on a retrospective calibration which was performed based on a set of two 3D-printed infant kidneys (newborns: 8.6 ml; 1-year-old: 23.4 ml) filled with known activities. Both phantoms were scanned at different positions along the anteroposterior axis inside a water phantom, providing depth- and size-dependent attenuation correction factors for planar imaging. Time-activity curves were determined by drawing kidney, bladder, and whole body regions-of-interest for each patient, and subsequently applying the calibration factor for conversion of counts to activity. Patient-specific time-integrated activity coefficients were obtained by integrating the organ-specific time-activity curves. Absorbed and effective dose coefficients for each patient were assessed with OLINDA/EXM for the provided newborn and 1-year-old phantom. Based on absorbed dose values, the radiation risk estimation was performed individually for each of the 34 patients with the National Cancer Institute's Radiation Risk Assessment Tool. The patients' organ-specific mean absorbed dose coefficients for the patients with normal renal function were 0.04±0.03 mGy/MBq for the kidneys and 0.27±0.24 mGy/MBq for the bladder. This resulted in a mean effective dose coefficient of 0.02±0.02 mSv/MBq. Based on the dosimetry results, the evaluation of the excess lifetime risk (ELR) for the development of radiation-induced cancer showed that the group of newborns has an ELR of 16.8 per 100,000 persons, which is higher in comparison with the 1-year-old group with an ELR of 14.7 per 100,000 persons. With regard to the 14 patients with abnormal renal function, the mean values for the organ absorbed dose coefficients for the patients were: 0.40±0.34 mGy/MBq for the kidneys and 0.46±0.37 mGy/MBq for the bladder. The corresponding effective dose coefficients (mSv/MBq) was: 0.05±0.02 mSv/MBq. The mean ELR (per 100,000 persons) for developing cancer from radiation exposure for patients with abnormal renal function was 29.2±18.7 per 100,000 persons. As a result, the radiation-associated stochastic risk increases with the organ doses, taking age- and gender-specific influences into account. Overall, the lifetime radiation risk associated with the 99mTc-MAG3 scans is very low in comparison to the general population risk for developing cancer. Furthermore, due to the increasing demand for PET-scans in children and adolescents with 68Ga-labelled peptides, in this work published data sets for those compounds were analyzed to derive recommendations for the administered activities in children and adolescents. The recommendation for the activities to be administered were based on the weight-independent effective dose model, proposed by the EANM Pediatric Dosage Card for application in pediatric nuclear medicine. The aim was to derive recommendations on administered activities for obtaining age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card, to determine the radiopharmaceutical class of 68Ga-labeled peptides ("multiples"), and to calculate the baseline activities based on the biokinetics of these compounds and an upper limit of the administered activity of 185 MBq for an adult. Analogous to 18F-fluoride, a minimum activity of 14 MBq is recommended. As a result, for those pediatric nuclear medicine applications involving 68Ga-labeled peptides, new values for the EANM dosage card were proposed and implemented based on the results derived in this work. Overall, despite the low additional radiation-related cancer risk, all efforts should be undertaken to optimize administered activities in children and adolescents for obtaining sufficient diagnostic information with minimal associated radiation risk.},
  subject      = {Biokinetics},
  language  = {en}
}
@phdthesis{ALHijailan2019,
  author    = {AL-Hijailan, Reem Saud},
  title     = {Establishment of endothelialized cardiac tissue using human induced pluripotent stem cells generated cardiomyocytes},
  doi       = {10.25972/OPUS-17397},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173979},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Cardiovascular diseases are considered the leading cause of death worldwide according to the World Health Organization. Heart failure is the last stage of most of these diseases, where loss of myocardium leads to architectural and functional decline. The definitive treatment option for patients with CVDs is organ or tissue transplantation, which relies on donor availability. Therefore, generating an autologous bioengineered myocardium or heart could overcome this limitation. In addition, generating cardiac patches will provide ventricular wall support and enable reparative stem cells delivery to damaged areas. Although many hurdles still exist, a good number of researches have attempted to create an engineered cardiac tissue which can induce endogenous cardiac repair by replacing damaged myocardium. The present study provided cardiac patches in two models, one by a detergent coronary perfusion decellularization protocol that was optimized, and the other that resulted in a 3D cell-free extracellular matrix with intact architecture and preserved s-glycosaminoglycan and vasculature conduits. Perfusion with 1\% Sodium dodecyle sulfate (SDS) under constant pressure resulted in cell-free porcine scaffold within two and cell-free rat scaffold in 7 days, whereas scaffold perfused with 4\% sodium deoxycholate (SDO) was not able to remove cells completely. Re-reendothelialization of tissue vasculature was obtained by injecting human microvascular endothelial cell and human fibroblast in 2:1 ratio in a dynamic culture. One-week later, CD31 positive cells and endothelium markers were observed, indicating new blood lining. Moreover, functionality test of re-endothelialized tissue revealed improvement in clotting seen in decellularized tissues. When the tissue was ready to be repopulated, porcine induced pluripotent stem cells (PiPSc) were generated by transfected reprogramming of porcine skin fibroblast and then differentiated to cardiac cells following a robust protocol, for an autologous cardiac tissue model. However, due to the limitation in the PiPSc cell number, alternatively, human induced pluripotent stem cells generated cardiac cells were used. For reseeding a coculture of human iPSc generated cardiac cells, human mesenchymal stem cells and human fibroblast in 2:1:1 ratio respectively were used in a dynamic culture for 6-8 weeks. Contractions at different areas of the tissue were recorded at an average beating rate of 67 beats/min. In addition, positive cardiac markers (Troponin T), Fibroblast (vemintin), and mesenchymal stem cells (CD90) were detected. Not only that, but by week 3, MSC started differentiating to cardiac cells progressively until few CD90 positive cells were very few by week 6 with increasing troponin t positive cells in parallel. Electrophysiological and drug studies were difficult to obtain due to tissue thickness and limited assessment sources. However, the same construct was established using small intestine submucosa (SISer) scaffold, which recorded a spontaneous beating rate between 0.88 and 1.2 Hz, a conduction velocity of 23.9 ± 0.74 cm s-1, and a maximal contraction force of 0.453 ± 0.015 mN. Moreover, electrophysiological studies demonstrated a drug-dependent response on beating rate; a higher adrenalin frequency was revealed in comparison to the untreated tissue and isoproterenol administration, whereas a decrease in beating rate was observed with propranolol and untreated tissue. The present study demonstrated the establishment of vascularized cardiac tissue, which can be used for human clinical application.},
  language  = {en}
}
@phdthesis{Nelke2019,
  author    = {Nelke, Lena},
  title     = {Establishment and optimization of 3-dimensional mamma carcinoma models for therapy simulation and drug testing},
  doi       = {10.25972/OPUS-17228},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172280},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Breast cancer is the most common cancer among women worldwide and the second most common cause of cancer death in the developed countries. As the current state of the art in first-line drug screenings is highly ineffective, there is an urgent need for novel test systems that allow for reliable predictions of drug sensitivity. In this study, a tissue engineering approach was used to successfully establish and standardize a 3-dimensional (3D) mamma carcinoma test system that was optimized for the testing of anti-tumour therapies as well as for the investigation of tumour biological issues. This 3D test system is based on the decellularised scaffold of a porcine small intestinal segment and represents the three molecular subsets of oestrogen receptor-positive, HER2/Neu-overexpressing and triple negative breast cancer (TNBC). The characterization of the test system with respect to morphology as well as the expression of markers for epithelial-mesenchymal transition (EMT) and differentiation indicate that the 3D tumour models cultured under static and dynamic conditions reflect tumour relevant features and have a good correlation with in vivo tumour tissue from the corresponding xenograft models. In this respect, the dynamic culture in a flow bioreactor resulted in the generation of tumour models that exhibited best reflection of the morphology of the xenograft material. Furthermore, the proliferation indices of 3D models were significantly reduced compared to 2-dimensional (2D) cell culture and therefore better reflect the in vivo situation. As this more physiological proliferation index prevents an overestimation of the therapeutic effect of cytostatic compounds, this is a crucial advantage of the test system compared to 2D culture. Moreover, it could be shown that the 3D models can recapitulate different tumour stages with respect to tumour cell invasion. The scaffold SISmuc with the preserved basement membrane structure allowed the investigation of invasion over this barrier which tumour cells of epithelial origin have to cross in in vivo conditions during the process of metastasis formation. Additionally, the data obtained from ultrastructural analysis and in situ zymography indicate that the invasion observed is connected to a tumour cell-associated change in the basement membrane in which matrix metalloproteinases (MMPs) are also involved. This features of the model in combination with the mentioned methods of analysis could be used in the future to mechanistically investigate invasive processes and to test anti-metastatic therapy strategies. The validation of the 3D models as a test system with respect to the predictability of therapeutic effects was achieved by the clinically relevant targeted therapy with the monoclonal antibody trastuzumab which induces therapeutic response only in patients with HER2/Neu-overexpressing mamma carcinomas due to its specificity for HER2. While neither in 2D nor in 3D models of all molecular subsets a clear reduction of cell viability or an increase in apoptosis could be observed, a distinct increase in antibody-dependent cell-mediated cytotoxicity (ADCC) was detected only in the HER2/NEU-overexpressing 3D model with the help of an ADCC reporter gene assay that had been adapted for the application in the 3D model in the here presented work. This correlates with the clinical observations and underlines the relevance of ADCC as a mechanism of action (MOA) of trastuzumab. In order to measure the effects of ADCC on the tumour cells in a direct way without the indirect measurement via a reporter gene, the introduction of an immunological component into the models was required. This was achieved by the integration of peripheral blood mononuclear cells (PBMCs), thereby allowing the measurement of the induction of tumour cell apoptosis in the HER2/Neu-overexpressing model. Hence, in this study an immunocompetent model could be established that holds the potential for further testing of therapies from the emergent field of cancer immunotherapies. Subsequently, the established test system was used for the investigation of scientific issues from different areas of application. By the comparison of the sensitivity of the 2D and 3D model of TNBC towards the water-insoluble compound curcumin that was applied in a novel nanoformulation or in a DMSO-based formulation, the 3D test system was successfully applied for the evaluation of an innovative formulation strategy for poorly soluble drugs in order to achieve cancer therapy-relevant concentrations. Moreover, due to the lack of targeted therapies for TNBC, the TNBC model was applied for testing novel treatment strategies. On the one hand, therapy with the WEE1 kinase inhibitor MK 1775 was evaluated as a single agent as well as in combination with the chemotherapeutic agent doxorubicin. This therapy approach did not reveal any distinct benefits in the 3D test system in contrast to testing in 2D culture. On the other hand, a novel therapy approach from the field of cellular immunotherapies was successfully applied in the TNBC 3D model. The treatment with T cells that express a chimeric antigen receptor (CAR) against ROR1 revealed in the static as well as in the dynamic model a migration of T cells into the tumour tissue, an enhanced proliferation of T cells as well as an efficient lysis of the tumour cells via apoptosis and therefore a specific anti-cancer effect of CAR-transduced T cells compared to control T cells. These results illustrate that the therapeutic application of CAR T cells is a promising strategy for the treatment of solid tumours like TNBC and that the here presented 3D models are suitable for the evaluation and optimization of cellular immunotherapies. In the last part of this work, the 3D models were expanded by components of the tumour stroma for future applications. By coculture with fibroblasts, the natural structures of the intestinal scaffold comprising crypts and villi were remodelled and the tumour cells formed tumour-like structures together with the fibroblasts. This tissue model displayed a strong correlation with xenograft models with respect to morphology, marker expression as well as the activation of dermal fibroblasts towards a cancer-associated fibroblast (CAF) phenotype. For the integration of adipocytes which are an essential component of the breast stroma, a coculture with human adipose-derived stromal/stem cells (hASCs) which could be successfully differentiated along the adipose lineage in 3D static as well as dynamic models was established. These models are suitable especially for the mechanistic analysis of the reciprocal interaction between tumour cells and adipocytes due to the complex differentiation process. Taken together, in this study a human 3D mamma carcinoma test system for application in the preclinical development and testing of anti-tumour therapies as well as in basic research in the field of tumour biology was successfully established. With the help of this modular test system, relevant data can be obtained concerning the efficacy of therapies in tumours of different molecular subsets and different tumour stages as well as for the optimization of novel therapy strategies like immunotherapies. In the future this can contribute to improve the preclinical screening and thereby to reduce the high attrition rates in pharmaceutical industry as well as the amount of animal experiments.},
  subject      = {Brustkrebs},
  language  = {en}
}
@phdthesis{Wagner2019,
  author    = {Wagner, Martin},
  title     = {Chronic Kidney Disease as an Important Co-morbid Condition in Coronary Heart Disease Patients},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175498},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In patients with coronary heart disease (CHD) the control of the modifiable "traditional" cardiovascular risk factors such as hypertension, dyslipidemia, diabetes, achieving/maintaining normal body weight and smoking cessation is of major importance to improve prognosis. Guideline recommendations for secondary CHD prevention include specific treatment targets for blood pressure, lipid levels, and markers of glucose metabolism for both younger and older patients. Chronic kidney disease (CKD) has been identified as a "non-traditional" risk factor for worse outcome in CHD patients, as it is associated with a markedly increased risk for subsequent CV events and mortality. The specific objectives of the current thesis-project are to investigate (a) the quality of care in a recent sample of German CHD patients and to investigate variation of risk factor control between younger and elder patients (≤70 versus >70 years), (b) to analyze the prevalence of CKD across Europe in stable CHD patients in the outpatient setting and during a hospital stay for CHD, (c) to investigate the level of awareness of CKD in German CHD patients and their treating physicians. Data from the European-wide EUROASPIRE IV study were used that include data on 7998 CHD patients in the ambulatory setting (study visit) and during a hospital stay for CHD (index). The German EUROASPIRE IV study center in W{\"u}rzburg recruited 536 patients in 2012-2013. Risk factor control was compared against the current recommendations of the European Society of Cardiology. CKD was described by stages of glomerular filtration rate (eGFR) and albuminuria. German patients were asked in an additional kidney specific module whether they have ever been told by a physician about renal impairment. The fact that CKD or acute kidney injury (AKI) was mentioned in prominent parts of the hospital discharge letter as well as correct ICD-coding of CKD or AKI served as a proxy for physician's awareness of CKD. The majority of German CHD patients was treated with the recommended drug therapies including e.g. β-blockers, anti-platelets and statins. However, treatment targets for blood pressure and LDL-cholesterol levels were not achieved in many patients (45\% and 53\%, respectively) and glycemic control in diabetic CHD patients with HbA1-levels <7\% was insufficient (61\%). A minority of patients reported on current smoking (10\%), but unhealthy life-styles e.g. overweight/obesity (85\%/37\%) were frequent. Patterns of care differed between younger and older CHD patients while older patients were less likely to receive the recommended medical CHD-therapy, were more likely to have uncontrolled blood pressure and also to be diabetic. However, a greater proportion of diabetic patients >70 years was achieving the HbA1c target, and less elder patients were current smokers or were obese. About 17\% of patients on average had CKD (eGFR< 60 ml/min/1.73m²) in the entire European sample at the study visit, and an additional 10\% had albuminuria despite preserved eGFR, with considerable variation among countries. Impaired kidney function was observed in every fifth patient admitted for CHD in the entire European dataset of the EUROASPIRE IV study. Of the German CHD patients with CKD at the study visit, only a third were aware of their renal impairment. A minority of these patients was being seen by nephrologists, however, with a higher likelihood of CKD awareness and specialist care in more advanced stages of CKD. About a third of patients admitted for CHD showed either CKD or AKI during the hospital stay, but the discharge letter mentioned chronic or acute kidney disease only in every fifth of these patients. In contrast, correct ICD coding of CKD or AKI was more complete, but still suboptimal. In summary, quality of secondary prevention in German CHD patients indicates considerably room for improvement, with life-style modifications may become an even greater factor in prevention campaigns than medical treatment into certain target ranges. Preventive therapies should also consider different needs in older individuals acknowledging physical and mental potential, other comorbidities and drug-interactions with co-medication. CKD is common in CHD patients, not only in the elderly. Since CHD and CKD affect each other and impact on worse prognosis of each other, raising the awareness of CKD among patients and physicians and considering CKD in medical therapy may improve prognosis and slow disease progression of CHD as well as CKD.},
  language  = {en}
}
@phdthesis{Mohammadi2019,
  author    = {Mohammadi, Milad},
  title     = {Role of oxidized phospholipids in inflammatory pain},
  doi       = {10.25972/OPUS-19240},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192402},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Introduction: During inflammation, reactive oxygen species (ROS) such as Hydrogen peroxide accumulate at the inflammation site and by oxidizing lipids, they produce metabolites such as 4-hydroxynonenal (4-HNE) and oxidized phospholipids (OxPLs). Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) are ligand gated ion channels that are expressed on nociceptors and their activation elicits pain. Hydrogen peroxide and 4-HNE are endogenous ligands for TRPA1 and their role in inflammatory pain conditions has been shown. OxPLs play a major pro-inflammatory role in many pathologies including atherosclerosis and multiple sclerosis. E06/T15 is a mouse IgM mAb that specifically binds oxidized phosphatidylcholine. D-4F is an apolipoprotein A-I mimetic peptide with a very high affinity for OxPLs and possess anti-inflammatory properties. E06 mAb and D-4F peptide protect against OxPLs-induced damage in atherosclerosis in vivo. Methods: To investigate the role of ROS and their metabolites in inflammatory pain, I utilized a combination of diverse and complex behavioral pain measurements and binding assays. I examined E06 mAb and D-4F as local treatment options for hypersensitivity evoked by endogenous and exogenous activators of TRPA1 and TRPV1 as well as in inflammatory and OxPL-induced pain models in vivo. 4-HNE, hydrogen peroxide as ROS source and mustard oil (AITC) were used to activate TRPA1, while capsaicin was used to activate TRPV1. Results: Intraplantar injection of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) into rats' hind paw elicited thermal and mechanical hypersensitivity. Genetic and pharmacological evidence in vivo confirmed the role of TRPA1 in OxPLs-induced hypersensitivity. OxPLs formation increased in complete Freund's adjuvant (CFA)-induced inflamed rats' paw. E06 mAb and D-4F prevented OxPAPC-induced mechanical and thermal hypersensitivity (hyperalgesia) as well as CFA-induced mechanical hypersensitivity. Also, all irritants induced thermal and mechanical hypersensitivity as well as affective-emotional responses and spontaneous nocifensive behaviors. E06 mAb blocked prolonged mechanical hypersensitivity by all but hydrogen peroxide. In parallel, D-4F prevented mechanical hypersensitivity induced by all irritants as well as thermal hypersensitivity induced by capsaicin and 4-HNE. In addition, competitive binding assays showed that all TRPA1/V1 agonists induced prolonged formation of OxPLs in the paw tissue explaining the anti-nociceptive properties of E06 mAb and D-4F. Finally, the potential of gait analysis as a readout for non-provoked pain behavioral measurements were examined. Conclusion and implications: OxPLs were characterized as novel targets in inflammatory pain. Treatment with the monoclonal antibody E06 or apolipoprotein A-I mimetic peptide D-4F are suggested as potential inflammatory pain medications. OxPLs' role in neuropathic pain is yet to be investigated.},
  language  = {en}
}