@phdthesis{Grob2022,
  author    = {Grob, Robin},
  title     = {The Function of Learning Walks of \({Cataglyphis Ants}\): Behavioral and Neuronal Analyses},
  doi       = {10.25972/OPUS-29017},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-290173},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Humans and animals alike use the sun, the moon, and the stars to guide their ways. However, the position of celestial cues changes depending on daytime, season, and place on earth. To use these celestial cues for reliable navigation, the rotation of the sky has to be compensated. While humans invented complicated mechanisms like the Antikythera mechanism to keep track of celestial movements, animals can only rely on their brains. The desert ant Cataglyphis is a prime example of an animal using celestial cues for navigation. Using the sun and the related skylight polarization pattern as a compass, and a step integrator for distance measurements, it can determine a vector always pointing homewards. This mechanism is called path integration. Since the sun's position and, therefore, also the polarization pattern changes throughout the day, Cataglyphis have to correct this movement. If they did not compensate for time, the ants' compass would direct them in different directions in the morning and the evening. Thus, the ants have to learn the solar ephemeris before their far-reaching foraging trips. To do so, Cataglyphis ants perform a well-structured learning-walk behavior during the transition phase from indoor worker to outdoor forager. While walking in small loops around the nest entrance, the ants repeatedly stop their forward movements to perform turns. These can be small walked circles (voltes) or tight turns about the ants' body axes (pirouettes). During pirouettes, the ants gaze back to their nest entrance during stopping phases. These look backs provide a behavioral read-out for the state of the path integrator. The ants "tell" the observer where they think their nest is, by looking back to it. Pirouettes are only performed by Cataglyphis ants inhabiting an environment with a prominent visual panorama. This indicates, that pirouettes are performed to learn the visual panorama. Voltes, on the other hand, might be used for calibrating the celestial compass of the ants. In my doctoral thesis, I employed a wide range of state-of-the-art techniques from different disciplines in biology to gain a deeper understanding of how navigational information is acquired, memorized, used, and calibrated during the transition phase from interior worker to outdoor forager. I could show, that celestial orientation cues that provide the main compass during foraging, do not guide the ants during the look-backbehavior of initial learning walks. Instead Cataglyphis nodus relies on the earth's magnetic field as a compass during this early learning phase. While not guiding the ants during their first walks outside of the nest, excluding the ants from perceiving the natural polarization pattern of the skylight has significant consequences on learning-related plasticity in the ants' brain. Only if the ants are able to perform their learning-walk behavior under a skylight polarization pattern that changes throughout the day, plastic neuronal changes in high-order integration centers are induced. Especially the mushroom bogy collar, a center for learning and memory, and the central complex, a center for orientation and motor control, showed an increase in volume after learning walks. This underlines the importance of learning walks for calibrating the celestial compass. The magnetic compass might provide the necessary stable reference system for the ants to calibrate their celestial compass and learn the position of landmark information. In the ant brain, visual information from the polarization-sensitive ocelli converge in tight apposition with neuronal afferents of the mechanosensitive Johnston's organ in the ant's antennae. This makes the ants' antennae an interesting candidate for studying the sensory bases of compass calibration in Cataglyphis ants. The brain of the desert navigators is well adapted to successfully accomplish their navigational needs. Females (gynes and workers) have voluminous mushroom bodies, and the synaptic complexity to store large amount of view-based navigational information, which they acquire during initial learning walks. The male Cataglyphis brain is better suited for innate behaviors that support finding a mate. The results of my thesis show that the well adapted brain of C. nodus ants undergoes massive structural changes during leaning walks, dependent on a changing celestial polarization pattern. This underlies the essential role of learning walks in the calibration of orientation systems in desert ants.},
  subject      = {Cataglyphis},
  language  = {en}
}
@phdthesis{Sbiera2022,
  author    = {Sbiera, Iuliu},
  title     = {Possible role of epithelial to mesenchymal transition and its associated FGF/FGFR pathway in adrenocortical carcinoma},
  doi       = {10.25972/OPUS-27745},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-277454},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Recent studies have hinted to an involvement of epithelial to mesenchymal transition, a mechanism often associated with metastasis in epithelial cancers, in adrenocortical carcinoma. In addition, the knowledge about the FGF/FGFR pathway in pathogenesis of the adrenal gland, a pathway often associated with the epithelial to mesenchymal transition, is sparse and fragmented. We assessed, in a large number of normal, benign and malignant adrenocortical tissues (a total of 181 different samples), the expression of canonical and novel epithelial and mesenchymal markers and compared it with their expression in typical epithelial and mesenchymal tissues. In addition, we also quantified the expression of most members of the FGF/FGFR pathway in adrenocortical tissues and compared it against well-studied epithelial and mesenchymal tissues as well as between malignant and not malignant adrenocortical tissues, in order to assess the possible connection to epithelial to mesenchymal transition and find possible drug targets. Surprisingly, both normal and neoplastic adrenocortical tissues lacked expression of epithelial markers (e.g. E-Cadhering or EpCAM) but strongly expressed mesenchymal markers (e.g. N-Cadherin or SLUG), suggesting a higher similarity of adrenocortical tissues to mesenchymal compared to epithelial tissues, reminiscent of the adrenocortical origin from the intermediate mesoderm. Despite their ubiquitous expression in all adrenocortical tissues, mesenchymal markers had a variable expression in adrenocortical carcinoma, associating either directly or inversely with different clinical markers of tumor aggressiveness. Lymph node infiltration was associated with high expression of SLUG (p = 0.04), and at the same time low expression of N-cadherin (p = 0.001), and the same pattern was observed for venous infiltration of tumoral tissue, Weiss score of tumor malignancy or Ki67 proliferation marker. In malignant compared to benign adrenal tumors, we found significant differences in the expression of 16 out of the 94 studied FGF receptor pathway related genes. Genes involved in tissue differentiation and metastatic spread through epithelial to mesenchymal transition were most strongly altered. The therapeutically targetable FGF receptors 1 and 4 were upregulated 4.6- and 6-fold, respectively, in malignant compared to benign adrenocortical tumors, which was confirmed by using two different quantification methods in both frozen and paraffin embedded tissue material. High expression of FGFR1 and 4 was significantly associated with worse patient prognosis (High FGFR1 expression was associated with a shorter overall patient survival of 84 vs 148 months (HR=1.8, 95\% CI: 1.01-3.25) as well as a shorter resection free survival of 25 vs 75 months ((HR=2.93, 95\% CI: 1.25-6.84), while high FGFR4 was associated with a much shorter overall survival of 50 vs 155 months (HR=2.44, 95\% CI: 1.41-4.22). In conclusion, epithelial to mesenchymal transition does not seem to play a role in adrenocortical carcinoma tumor progression, and the FGF/FGFR pathway, even if it is probably not related to EMT, is nonetheless associated with tumor aggressiveness. Furthermore, quantification of FGF receptors may enable a stratification of adrenocortical carcinoma for the use of FGFR inhibitors in future clinical trials.},
  subject      = {Nebennierenrindenkrebs},
  language  = {en}
}
@phdthesis{Diebold2023,
  author    = {Diebold, Mathias},
  title     = {Virtuelles Screening und Entwicklung selektiver Liganden des Aurora-A - MYCN Komplexes und computergest{\"u}tzte Methoden zur Analyse und Design von PROTACs},
  doi       = {10.25972/OPUS-31759},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317594},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Die Interaktion des onkogenen Transkriptionsfaktors MYCN mit der Ser/Thr Kinase Aurora-A verhindert dessen Abbau {\"u}ber das Ubiquitin Proteasomsystem indem die Rekrutierung des SCF FbxW7 Komplexes verhindert wird. Die Kinase nimmt mit der Bindung an MYCN eine aktive Konformation ein und erh{\"a}lt somit die F{\"a}higkeit zur Kinaseaktivit{\"a}t ohne die sonst notwendige Phosphorylierung von Thr288 oder die Anwesenheit eines Aktivators wie TPX2. Da hohe MYCN Konzentrationen Tumore wie Neuroblastome antreiben, ist die St{\"o}rung der Komplexbildung mit Aurora-A eine valide Strategie zur Entwicklung von Chemotherapeutika. Einige Inhibitoren von Aurora-A wie Alisertib (MLN8237) sind in der Lage, eine Konformations{\"a}nderung in der Kinase zu verursachen, die mit der Bindung von MYCN inkompatibel ist und auf diese Weise den Abbau des Transkriptionsfaktors induziert. Da Aurora-A wichtige Funktionen in der Mitose {\"u}bernimmt, k{\"o}nnte eine direkte Adressierung des Komplexes anstelle einer systemischen Inhibition der Kinase vielversprechender sein. Ziel des Projektes war die Identifizierung von Molek{\"u}len, die selektiv an das Interface des Aurora-A - MYCN Komplexes binden und weiter optimiert werden k{\"o}nnen, um einen gezielten Abbau des Transkriptionsfaktors {\"u}ber einen PROTAC Ansatz zu erm{\"o}glichen. Virtuelle Screenings und molekulardynamische Simulationen wurden durchgef{\"u}hrt, um kommerziell erh{\"a}ltliche Verbindungen zu identifizieren, welche mit einer Bindetasche des Komplexes interagieren, die nur zustande kommt, wenn beide Proteine miteinander interagieren. Aus einem ersten Set von zehn potentiellen Liganden wurde f{\"u}r vier eine selektive Interaktion mit dem Protein - Protein Komplex gegen{\"u}ber Aurora-A oder MYCN alleine in STD-NMR Experimenten best{\"a}tigt. Zwei der Hits besaßen ein identisches Grundger{\"u}st und wurden als Ausganspunkt f{\"u}r die Optimierung zu potenteren Liganden genutzt. Das Ger{\"u}st wurde fragmentweise vergr{\"o}ßert und in Richtung besserer in-silico Ergebnisse und Funktionalisierung zur Anbringung von E3-Ligase-Liganden optimiert. Neun dieser Liganden der zweiten Generation wurden synthetisiert. Um quantitative Bindungsdaten zu erhalten, wurde ein kovalent verkn{\"u}pftes Aurora-A - MYCN Konstrukt entworfen. Die strukturelle und funktionale Integrit{\"a}t wurde in STD-NMR und BLI Experimenten mit bekannten Aurora-A Inhibitoren best{\"a}tigt, sowie in NMR-basierten ATPase Assays. Zus{\"a}tzlich konnte die Kristallstruktur des Konstrukts gel{\"o}st und damit die Validit{\"a}t des Designs best{\"a}tigt werden. Quantitative Messungen der synthetisierten Molek{\"u}le identifizierten HD19S als Hit mit einer zehnfach h{\"o}heren Affinit{\"a}t f{\"u}r das Aurora-A - MYCN Konstrukt im Vergleich zu der Kinase allein. Zus{\"a}tzlich wurden in-silico Untersuchungen zu PROTACs der Aurora-A Kinase durchgef{\"u}hrt. Interaktionen zwischen Aurora-A, der E3-Ligase Cereblon und den Liganden wurden modelliert und f{\"u}r die Erkl{\"a}rung unterschiedlicher Aktivit{\"a}ten der eingesetzten PROTACs verwendet. Zudem zeigte das aktivste PROTAC eine hohe Selektivit{\"a}t f{\"u}r Aurora-A gegen{\"u}ber Aurora-B, obwohl die verwendete Erkennungseinheit (Alisertib) an beide Aurora-Proteine bindet. Dieser Umstand konnte durch energetische Analysen von molekulardynamischen Simulationen der tern{\"a}ren Komplexe erkl{\"a}rt werden. Optimierungsm{\"o}glichkeiten f{\"u}r eine effizientere Degradation von Aurora-A durch die PROTACs wurden basierend auf modifizierten Erkennungseinheiten und verbesserten Linkern untersucht.},
  subject      = {Arzneimitteldesign},
  language  = {de}
}
@phdthesis{KayisogluKaya2022,
  author    = {Kayisoglu-Kaya, {\"O}zge},
  title     = {Analysis of gastrointestinal epithelial innate immune barrier using human and murine organoids as a model},
  doi       = {10.25972/OPUS-27749},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-277497},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The epithelial layer of the gastrointestinal (GI) tract provides a barrier between the environment and the body. Dysfunction of the epithelium, including changes of the innate immune response facilitated by pattern recognition receptors (PRRs), plays a major role in the development of GI disorders. However, the organization of innate immune sensing, the expression and activity of PRRs and the factors contri¬buting to such possible organization along the GI tract are unclear. In recent years, stem cell-derived organoids gained increasing attention as promising tissue models. Here, a biobank of human and murine organoids comprising three lines from each GI segment; corpus, pylorus, duodenum, jejunum, ileum, colon was generated. RNA sequencing of 42 lines confirmed the preservation of tissue identity and revealed an extensive organization of innate immune signaling components along the cephalocaudal axis, giving each segment a specific innate immune profile. Comple-menting the region-specific expression analysis, several PRRs in human and murine organoids showed region- and species-specific function. To investigate the factors contributing to the patterning of innate immunity in the GI tract, the impact of microbial components was analyzed using murine embryo-derived, never colonized gastric and proximal intestinal organoids. Transcriptional profiling of embryo-derived organoids showed that while expression of some PRRs may depend on environmental cues as expected, an unexpectedly large part of segment-specific expression of PRR signaling components is independent of prior contact with microbial products. Further, analysis of published RNA-seq data as well as in vitro experiments using directed differentiation of organoids into specific cell types showed that expression of innate immune gene also depended on cellular differentiation along the crypt-villus axis. This underlined the importance of cellular differentiation rather than contact to microbial compounds for expression of PRRs. Lastly, analysis of published datasets of RNA-seq and ATAC-seq after knockout of the intestinal transcription factor Cdx2 demonstrated that Cdx2 is likely important for the expression of Nlrp6 and Naip1 in the murine intestine. Future experiments have to support these preliminary findings. Taken together, the expression of a large part of epithelial innate immunity is develop¬mentally defined and conserved in tissue-resident stem cells. The identification of mechanisms governing expression of genes related to immunity will provide further insights into the mechanisms that play a role in the progress of inflammatory diseases.},
  subject      = {Organoid},
  language  = {en}
}
@phdthesis{Jannasch2019,
  author    = {Jannasch, Maren Annika},
  title     = {In vitro Fremdk{\"o}rpermodellsysteme zur Vorhersage von biomaterialinduzierten Immunreaktionen},
  doi       = {10.25972/OPUS-16289},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162893},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die Implantation eines Medizinprodukts in den menschlichen K{\"o}rper ruft eine Immunreaktion hervor, die zur fibr{\"o}sen Einkapselung f{\"u}hren kann. Makrophagen in direktem Kontakt mit der Oberfl{\"a}che des Implantats erfassen sensorisch den Fremdk{\"o}rper und {\"u}bersetzten das Signal in die Freisetzung zahlreicher l{\"o}slicher Mediatoren. Das generierte Entz{\"u}ndungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erh{\"o}hung der Matrixsyntheserate in der Wundumgebung f{\"u}hren. Eine dichte fibr{\"o}se Kapsel um ein Medizinprodukt beeintr{\"a}chtigt den Ersatz von K{\"o}rperstrukturen, das Unterst{\"u}tzen physiologischer K{\"o}rperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterst{\"u}tzt werden. Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begr{\"u}ndet die Komplexi-t{\"a}t der ablaufenden Fremdk{\"o}rperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verf{\"u}gbarkeit validierter, verl{\"a}sslicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen w{\"a}hrend einer Fremdk{\"o}rperreaktion abbildet und sich zur Testung {\"u}ber l{\"a}ngere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch k{\"o}nnen in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine h{\"o}here {\"U}bertragbarkeit auf den Menschen aufweisen. Zus{\"a}tzlich adressiert die Pr{\"a}ferenz zu in vitro Testmethoden den Aspekt „Reduzierung" der 3R-Prinzipien „Replacement, Reduction, Refinement" (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begr{\"u}ndeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium f{\"u}r die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellul{\"a}rer Matrix in der Wundumgebung stellen wichtige Teilprozesse w{\"a}hrend einer Fremdk{\"o}rperreaktion dar. F{\"u}r beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von l{\"o}slichen Mediatoren durch materialadh{\"a}rente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden. Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialst{\"a}ndige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberfl{\"a}chenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix erm{\"o}glichte die {\"U}bersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Dar{\"u}ber hinaus konnte in einem dreidimensionalen Fremdk{\"o}rpermodell die komplexe dreidimensionale Struktur der extrazellul{\"a}ren Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdk{\"o}rpermodelle erm{\"o}glichten die Testung von Biomaterialien {\"u}ber l{\"a}ngere Testphasen und k{\"o}nnen in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen erm{\"o}glichen, (II) die Identifizierung verl{\"a}sslicher Testbedingungen unterst{\"u}tzen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verl{\"a}ssliche Unterscheidungskapazit{\"a}t zwischen Biomaterialien aufweist.},
  subject      = {Biomaterial},
  language  = {de}
}
@phdthesis{Kauk2018,
  author    = {Kauk, Michael},
  title     = {Investigating the Molecular Mechanism of Receptor Activation at Muscarinic Receptors by Means of Pathway-Specific Dualsteric Ligands and Partial Agonists},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173729},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology. Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored. In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 \% could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  language  = {en}
}
@phdthesis{Bemm2018,
  author    = {Bemm, Felix Mathias},
  title     = {Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157109},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: S{\"o}nke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.},
  subject      = {B{\"a}rtierchen},
  language  = {en}
}
@phdthesis{Weigel2019,
  author    = {Weigel, Tobias Maximilian},
  title     = {Entwicklung von 3D-Herzschrittmacher-Elektroden auf Basis von Kohlenstoffnanofasern},
  doi       = {10.25972/OPUS-17636},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176362},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Herzschrittmachersysteme sind eine weitverbreitete M{\"o}glichkeit Herz-Kreislauf-Erkrankungen zu behandeln. Wegen der nat{\"u}rlichen Reaktion des Immunsystems auf Fremdk{\"o}rper, erfolgt aber eine fortschreitende Verkapselung der Herzschrittmacherelektrode. Die Folge ist eine ansteigende Verminderung der Stimulationseffizienz durch Erh{\"o}hung der Anregungsschwelle. Die Integration der Elektrode in das Gewebe ist dabei mangelhaft und wird bestimmt durch Implantateigenschaften wie Gr{\"o}ße, Flexibilit{\"a}t und Dimensionalit{\"a}t. Um die Integration zu verbessern, stellen dreidimensionale (3D) bzw. gewebeartige Elektroden eine Alternative zu den derzeit verwendeten planaren Metallelektroden dar. Zur Entwicklung einer leitf{\"a}higen, 3D und faserf{\"o}rmigen Elektrode wurden in dieser Arbeit Kohlenstoff-Nanofaser-Scaffolds {\"u}ber Elektrospinnen hergestellt. Durch die Modifikation des Faserger{\"u}stes mit Natriumchlorid (NaCl) w{\"a}hrend der Scaffoldherstellung, konnte das Fasernetzwerk aufgelockert und Poren generiert werden. Die Kohlenstofffaser-Elektroden zeigten einen effizienten Energie{\"u}bertrag, welcher vergleichbar mit heutigen Titannitrid (TiN) -Elektroden ist. Die Auflockerung des Fasergewebes hatte eine verbesserte Flexibilit{\"a}t des Faserscaffolds zu Folge. Neben der Flexibilit{\"a}t, konnte auch die Infiltration von Zellen in das por{\"o}se Faserscaffold erheblich verbessert werden. Dabei konnten Fibroblasten durch das gesamte Scaffold migrieren. Die Kompatibilit{\"a}t mit kardialen Zellen, die Grundvoraussetzung von Herzschrittmacherelektroden, wurde in vitro nachgewiesen. Durch die Kombination aus dem 3D-Elektrodenger{\"u}st mit einer Co-Kultur aus humanen Kardiomyozyten, mesenchymalen Stammzellen und Fibroblasten, erfolgte eine Einbettung der Elektrode in funktionelles kardiales Gewebe. Dadurch konnte ein lebender Gewebe-Elektroden-Hybrid generiert werden, welcher m{\"o}glicherweise die Elektrode vor Immunzellen in vivo abschirmen kann. Eine Zusammenf{\"u}hrung der hybriden Elektrode mit einen Tissue-Engineerten humanen kardialen Patch in vitro, f{\"u}hrte zu Bildung einer nahtlosen Elektronik-Gewebe-Schnittstelle. Die fusionierte Einheit wurde abschließend auf ihre mechanische Belastbarkeit getestet und konnte {\"u}ber einen Elektroden-Anschluss elektrisch stimuliert werden.},
  subject      = {Herzschrittmacher},
  language  = {de}
}
@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{RiechelmannverhSteinbacher2021,
  author    = {Riechelmann [verh. Steinbacher], Eva Katharina},
  title     = {Gaze interaction: Cognitive mechanisms of oculomotor action control},
  doi       = {10.25972/OPUS-21527},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-215279},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Humans use their eyes not only as visual input devices to perceive the environment, but also as an action tool in order to generate intended effects in their environment. For instance, glances are used to direct someone else's attention to a place of interest, indicating that gaze control is an important part of social communication. Previous research on gaze control in a social context mainly focused on the gaze recipient by asking how humans respond to perceived gaze (gaze cueing). So far, this perspective has hardly considered the actor's point of view by neglecting to investigate what mental processes are involved when actors decide to perform an eye movement to trigger a gaze response in another person. Furthermore, eye movements are also used to affect the non-social environment, for instance when unlocking the smartphone with the help of the eyes. This and other observations demonstrate the necessity to consider gaze control in contexts other than social communication whilst at the same time focusing on commonalities and differences inherent to the nature of a social (vs. non-social) action context. Thus, the present work explores the cognitive mechanisms that control such goal-oriented eye movements in both social and non-social contexts. The experiments presented throughout this work are built on pre-established paradigms from both the oculomotor research domain and from basic cognitive psychology. These paradigms are based on the principle of ideomotor action control, which provides an explanatory framework for understanding how goal-oriented, intentional actions come into being. The ideomotor idea suggests that humans acquire associations between their actions and the resulting effects, which can be accessed in a bi-directional manner: Actions can trigger anticipations of their effects, but the anticipated resulting effects can also trigger the associated actions. According to ideomotor theory, action generation involves the mental anticipation of the intended effect (i.e., the action goal) to activate the associated motor pattern. The present experiments involve situations where participants control the gaze of a virtual face via their eye movements. The triggered gaze responses of the virtual face are consistent to the participant's eye movements, representing visual action effects. Experimental situations are varied with respect to determinants of action-effect learning (e.g., contingency, contiguity, action mode during acquisition) in order to unravel the underlying dynamics of oculomotor control in these situations. In addition to faces, conditions involving changes in non-social objects were included to address the question of whether mechanisms underlying gaze control differ for social versus non-social context situations. The results of the present work can be summarized into three major findings. 1. My data suggest that humans indeed acquire bi-directional associations between their eye movements and the subsequently perceived gaze response of another person, which in turn affect oculomotor action control via the anticipation of the intended effects. The observed results show for the first time that eye movements in a gaze-interaction scenario are represented in terms of their gaze response in others. This observation is in line with the ideomotor theory of action control. 2. The present series of experiments confirms and extends pioneering results of Huestegge and Kreutzfeldt (2012) with respect to the significant influence of action effects in gaze control. I have shown that the results of Huestegge and Kreutzfeldt (2012) can be replicated across different contexts with different stimulus material given that the perceived action effects were sufficiently salient. 3. Furthermore, I could show that mechanisms of gaze control in a social gaze-interaction context do not appear to be qualitatively different from those in a non-social context. All in all, the results support recent theoretical claims emphasizing the role of anticipation-based action control in social interaction. Moreover, my results suggest that anticipation-based gaze control in a social context is based on the same general psychological mechanisms as ideomotor gaze control, and thus should be considered as an integral part rather than as a special form of ideomotor gaze control.},
  subject      = {Verhaltenskontrolle},
  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{MonyNair2021,
  author    = {Mony Nair, Rahul},
  title     = {Elucidating ubiquitin recognition by the HECT-type ubiquitin ligase HUWE1},
  doi       = {10.25972/OPUS-22103},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221030},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The small protein modifier ubiquitin is at the heart of an immensely versatile posttranslational modification system that orchestrates countless physiological and disease-associated cellular processes. Key to this versatility are the manifold modifications that can be assembled from ubiquitin "building blocks" and are associated with specific functional outcomes for the modified substrates. In particular, ubiquitin molecules can form polymeric chains of distinct lengths and linkage types that give rise to distinct chain conformations, thereby providing recognition sites for specific signaling receptors/effectors. The class of E3 enzymes (ubiquitin ligases) provides critical specificity determinants in ubiquitin linkage formation; it is therefore crucial to unravel precisely how E3 enzymes operate in order to understand the structural basis of ubiquitin signaling and exploit these insights for therapeutic benefit. Overexpression and deregulation of the HECT-type ubiquitin ligase HUWE1 is implicated in several different cancer types and neurodegenerative disorders. It is largely unknown which factors control the ubiquitin modifications formed by HUWE1, how the catalytic HECT domain interacts with functionally distinct ubiquitin molecules (donor, acceptor and regulatory ubiquitin molecules) and which conformational transitions enable these interactions during ubiquitin chain formation. One aim of this study was to structurally elucidate the recognition of donor ubiquitin by the HECT domain of HUWE1. To this end I utilized a ubiquitin activity-based probe to reconstitute a proxy for a donor ubiquitin-linked conjugate of the HECT domain of HUWE1 and determined its structure by X-ray crystallography. This structure reveals that the donor ubiquitin binds to the C-lobe of HUWE1 in the same way as NEDD4-type ligases, corroborating the idea that HECT ligases utilize a conserved mode of donor ubiquitin recognition. independent of their linkage and substrate specificities. With the help of biochemical analyses, I also validated specific features of the structure, in particular the positioning of the C-terminal tail of the ligase, which was known to be critical for activity. In the newly determined structure, which reflects an "L-shaped", active state of the HECT domain, this tail is fully resolved and coordinated at the N-lobe-C-lobe interface. I defined residues that are critical for this coordination and showed that they are also essential for the activity of HUWE1, including auto-ubiquitination, free ubiquitin chain formation, and substrate ubiquitination. Furthermore, I discovered that the N-lobe of HUWE1 harbors a ubiquitin-binding exosite similar to NEDD4-type ligases and E6AP. My in-vitro activity and binding assays show that HUWE1 uses the exosite for isopeptide bond formation, but that it is dispensable for thioester bond formation. The binding assays further show that the donor ubiquitin loaded HECT domain binds an additional ubiquitin molecule at the exosite more tightly than the apo HECT domain, which possibly suggests allosteric communication between the two sites. Finally, I showed that the ubiquitin activity-based probe (ubiquitin-propargylamine) can label the catalytic cysteine of HUWE1 and NEDD4-type with close to quantitative turn- over, while it does not react with the HECT domain of the evolutionarily more divergent E6AP. The determinants underlying these differential reactivities remain to be explored. Taken, together my results significantly enhance our mechanistic understanding of the catalytic domain of HUWE1 and pinpoint linchpins for therapeutic interventions with the activity of this disease-relevant enzyme.},
  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{Herrmann2019,
  author    = {Herrmann, Johannes Bernd},
  title     = {Rolle des Komplement C5a-Rezeptors 1 in der Pathophysiologie der Meningokokken-Sepsis},
  doi       = {10.25972/OPUS-18453},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184533},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das bekapselte, Gram-negative, diplokokkenf{\"o}rmige Bakterium Neisseria meningitidis (Nme) ist ein asymptomatischer Kommensale des oberen Nasenrachenraums im Men-schen. Gerade bei Kindern ist es dem humanspezifischen Pathogen in seltenen F{\"a}llen m{\"o}glich, in den Blutstrom einzuwandern und lebensbedrohliche Krankheitsbilder wie Meningoenzephalitis und Sepsis auszul{\"o}sen, welche als „Invasive Meningokokkener-krankung" (IMD) zusammengefasst werden. J{\"a}hrlich ereignen sich weltweit bis zu 1,2 Mio F{\"a}lle von IMD, welche aufgrund des fulminanten Verlaufs und der hohen Letalit{\"a}t gef{\"u}rchtet sind. In der Bek{\"a}mpfung der Nme-Sepsis ist das humane Komplementsystem von entscheidender Bedeutung. Vor diesem Hintergrund ist die protektive Rolle des lytischen (Membranangriffskomplex MAK) und opsonisierenden Arms (Opsonine iC3b und C1q) der Komplementkaskade gut dokumentiert. Dagegen ist der Beitrag des in-flammatorischen Arms (Anaphylatoxine C3a und C5a) in der Nme-Sepsis bisher unklar. Aus diesem Grunde wurde mit dieser Arbeit die Rolle des inflammatorischen Arms an-hand des Komplement C5a-Rezeptors 1 (C5aR1) in der Pathophysiologie der Nme-Sepsis am Mausmodell untersucht. Nach Etablierung des murinen, intraperitonealen In-fektionsmodells konnte ein sch{\"a}dlicher Effekt des C5aR1 in der Nme-Sepsis beobachtet werden. Aus der Abwesenheit des C5aR1 resultierte eine h{\"o}here {\"U}berlebensrate, ein besserer klinischer Zustand, eine niedrigere Bakteri{\"a}mie und niedrigere Konzentrationen der pro-inflammatorischen Mediatoren IL-6, CXCL-1 und TNF-α. Im Hinblick auf den zellul{\"a}ren Pathomechanismus sprechen Ergebnisse dieser Arbeit daf{\"u}r, dass der C5aR1 prim{\"a}r eine gesteigerte Freisetzung inflammatorischer Mediatoren durch verschiedene Zellpopulationen triggert (Zytokinsturm), wodurch sekund{\"a}r Zellparalyse, steigende Bakteri{\"a}mie und h{\"o}here Letalit{\"a}t bedingt sind. Durch Depletionsversuche und Immun-fluoreszenzf{\"a}rbungen konnte, unabh{\"a}ngig vom C5aR1, eine allgemein protektive Rolle von neutrophilen Granulozyten und Monozyten/Makrophagen in der Nme-Sepsis beo-bachtet werden. Dar{\"u}ber hinaus pr{\"a}sentierte sich der zyklische C5aR1-Antagonist PMX205 als erfolgsversprechende Therapieoption, um Parameter einer murinen Nme-Sepsis zu verbessern. Weitere Untersuchungen sind n{\"o}tig, um die Wirksamkeit dieser Substanz in der humanen Nme-Sepsis zu erforschen. Zudem k{\"o}nnte das murine, intrape-ritoneale Infektionsmodell zur Kl{\"a}rung der Rolle des C5aR2 in der Nme-Sepsis genutzt werden.},
  subject      = {Komplement C5a},
  language  = {de}
}
@phdthesis{Koenig2019,
  author    = {K{\"o}nig, Eva-Maria},
  title     = {Pathogenese von Kraniosynostosen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175181},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das humane Sch{\"a}deldach besteht aus f{\"u}nf Sch{\"a}delplatten, die durch intramembran{\"o}se Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Sch{\"a}deln{\"a}hte aus, die eine Fusion der Sch{\"a}delplatten verhindern und damit ein Sch{\"a}delwachstum parallel zu Gehirnentwicklung erm{\"o}glichen. F{\"u}r diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung n{\"o}tig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gew{\"a}hrleistet wird. St{\"o}rungen in diesem regulatorischen System k{\"o}nnen zu einer vorzeitigen Fusion der Sch{\"a}delplatten, Kraniosynostose genannt, f{\"u}hren. Die Kraniosynostose ist eine der h{\"a}ufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Sch{\"a}deldeformationen, die sekund{\"a}r einen erh{\"o}hten intrakranialen Druck zur Folge haben k{\"o}nnen. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auff{\"a}lligkeiten vorliegen. Bisher sind {\"u}ber 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30\% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil {\"u}berlappen, ist eine klare klinische Diagnose h{\"a}ufig erschwert. Sowohl Umwelteinfl{\"u}sse als auch genetische Ver{\"a}nderungen k{\"o}nnen die Ursache f{\"u}r Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Ver{\"a}nderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Dar{\"u}ber hinaus wurden chromosomale Ver{\"a}nderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in {\"u}ber 50\% der F{\"a}lle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollst{\"a}ndig gekl{\"a}rt. Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufkl{\"a}rung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30\% der Patienten konnte eine potentiell pathogene Ver{\"a}nderung identifiziert werden. Davon waren 23\% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass m{\"o}glicherweise eine MSX2 {\"U}berexpression vorliegt. F{\"u}r ein normales Sch{\"a}delwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert f{\"u}r einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgef{\"u}hrt. Eine simulierte {\"U}berexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium f{\"u}hrte zu schweren Gehirn-, Herz- und Augendefekten. Mittels NGS wurden 77\% der potentiell pathogenen genetischen Ver{\"a}nderungen identifiziert. Hierf{\"u}r wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von f{\"u}nf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zus{\"a}tzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. F{\"u}r diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine {\"A}nderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenit{\"a}t dieser spezifischen Varianten zu bekr{\"a}ftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zuk{\"u}nftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenz{\"a}nderungen bei Kraniosynostose Patienten sinnvoll ist.},
  subject      = {Kraniosynostose},
  language  = {de}
}
@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{Reutter2019,
  author    = {Reutter, Mario},
  title     = {Biologische Marker f{\"u}r Aufmerksamkeitsverzerrungen bei sozialer {\"A}ngstlichkeit und deren Modifikation},
  doi       = {10.25972/OPUS-17870},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178706},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Diese Dissertationsschrift besch{\"a}ftigt sich mit biologischen Korrelaten von Aufmerksamkeits-verzerrungen und eruiert deren Modifikation in einem l{\"a}ngsschnittlich angelegten Experiment. Hierf{\"u}r wurden {\"u}ber 100 sozial-{\"a}ngstliche Teilnehmer mit Hilfe einer Screening-Prozedur gewonnen und hinsichtlich der Auspr{\"a}gung einer ereigniskorrelierten Lateralisation namens „N2pc" untersucht. W{\"a}hrend der ersten Labormessung indizierte die N2pc bei der Bearbeitung eines Dot Probe Paradigmas einen mittelgroßen, statistisch hochbedeutsamen Attentional Bias hin zu w{\"u}tenden Gesichtern im Vergleich zu neutralen. Das hierf{\"u}r klassischerweise verwendete Maß von Reaktionszeitunterschieden hingegen konnte diese Verzerrung der Aufmerksamkeit nicht abbilden. Ferner zeigten weder die elektrophysiologische noch die behaviorale Messgr{\"o}ße einen Zusammenhang mit Frageb{\"o}gen sozialer Angst, was teilweise auf ein Fehlen interner Konsistenz zur{\"u}ckgef{\"u}hrt werden kann. Im weiteren Verlauf absolvierten die {\"u}berwiegend weiblichen Teilnehmer an acht unterschiedlichen Terminen {\"u}ber zwei bis vier Wochen fast 7000 Durchg{\"a}nge eines Aufmerksamkeitsverzerrungsmodifikationstrainings oder einer aktiven Kontrollprozedur. Daraufhin zeigte sich eine Ausl{\"o}schung der ereigniskorrelierten Lateralisation, allerdings in einem sp{\"a}teren Zeitfenster als erwartet. Dieses Verschwinden des Attentional Bias blieb bis elf Wochen nach Ende der Trainingsprozedur stabil. Außerdem trat dieselbe Modifikation ebenfalls f{\"u}r die Kontrollgruppe auf. Die selbstberichtete Schwere der Symptomauspr{\"a}gung ver{\"a}nderte sich zwar nicht, allerdings konnte eine Reduktion des Pers{\"o}nlichkeitsmerkmals Neurotizismus verzeichnet werden, welches konzeptuell mit dem Begriff der {\"A}ngstlichkeit eng verwoben ist. Durch explorative Folgeanalysen konnte eine st{\"a}rkere Modulation der rechten Großhirnh{\"a}lfte, also durch Reize im linken visuellen Halbfeld aufgedeckt werden. Eine Neuberechnung des Attentional Bias separat f{\"u}r jede Hemisph{\"a}re scheint daher auch f{\"u}r k{\"u}nftige Untersuchungen angebracht. Ferner wurde als Tr{\"a}ger der Modifikation {\"u}ber die Zeit eine Ver{\"a}nderung der Hyperpolarisation nach der N2-Komponente identifiziert. Ob durch eine Anpassung der Prozedur eine Modulation einer fr{\"u}heren ereigniskorrelierten Komponente erzielt werden kann, bleibt zum aktuellen Zeitpunkt unbeantwortet.},
  subject      = {Attention},
  language  = {de}
}