TY - JOUR A1 - Wroński, Piotr Andrzej A1 - Wyborski, Paweł A1 - Musiał, Anna A1 - Podemski, Paweł A1 - Sęk, Grzegorz A1 - Höfling, Sven A1 - Jabeen, Fauzia T1 - Metamorphic Buffer Layer Platform for 1550 nm Single-Photon Sources Grown by MBE on (100) GaAs Substrate JF - Materials N2 - We demonstrate single-photon emission with a low probability of multiphoton events of 5% in the C-band of telecommunication spectral range of standard silica fibers from molecular beam epitaxy grown (100)-GaAs-based structure with InAs quantum dots (QDs) on a metamorphic buffer layer. For this purpose, we propose and implement graded In content digitally alloyed InGaAs metamorphic buffer layer with maximal In content of 42% and GaAs/AlAs distributed Bragg reflector underneath to enhance the extraction efficiency of QD emission. The fundamental limit of the emission rate for the investigated structures is 0.5 GHz based on an emission lifetime of 1.95 ns determined from time-resolved photoluminescence. We prove the relevance of a proposed technology platform for the realization of non-classical light sources in the context of fiber-based quantum communication applications. KW - single-photon source KW - quantum dots KW - telecommunication spectral range KW - metamorphic buffer layer Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246145 SN - 1996-1944 VL - 14 IS - 18 ER - TY - THES A1 - Wu, Dong T1 - Aspects of Gender in The Unofficial History of the Scholars T1 - Aspekte des Geschlechts am Roman《Rulin waishi》 N2 - The present study focuses on Rulin waishi 儒林外史 (The Unofficial History of the Scholars), a well-known Qing novel, from the perspective of gender. It attempts to contribute to the discussion about Chinese masculinity by identifying the representation of the scholars' masculinity in Rulin waishi and offer a better understanding of the novel's position regarding femininity and women. This project shows that the novel nevertheless reflects rather than challenges gender ideologies of its time. The ideal manhood showed in the novel comprises real virtues and authentic learning. It goes against the traditional, orthodox Confucian masculinity which advocates that officialdom is the glorious path to fulfill a learned man's masculinity. It is mainly due to Wu Jingzi's own failure in the civil service examinations and official careers. Regarding the relation of masculinity and sexuality, the novel reveals that a masculine man is not tempted by female charm but can enjoy a harmonious and companionate marriage. Besides, scholars show great anxiety about their masculinity since they are in a marginal position in society. Their manliness is challenged by officials, merchants, and even commoners, as well as their colleagues. Through a careful examination of stories of Pinniang, Miss Lu, and Mrs. Wang, it reveals that the novel holds a conventional opinion on women although it criticizes widow suicide and shows an egalitarian husband-wife relationship. It praises Confucian womanly virtues, such as following and serving the husband, managing the household, and keeping chastity. Female sexuality is blamed as an evil temptation to lead men to go astray. Women’s learning gains legitimacy when serving to fulfill domestic responsibilities. It carries the Confucian message that men should take the lead and maintain order in the household and reinforces the rightful patriarchy. In a word, rather than go ahead of its time, Rulin waishi holds a conservative attitude towards gender issues. N2 - Die vorliegende Studie konzentriert sich auf Rulin waishi (Die inoffizielle Geschichte der Gelehrten), einen bekannten Qing-Roman aus der Perspektive des Geschlechts. Es versucht, zur Diskussion über die chinesische Männlichkeit beizutragen, indem es die Repräsentation der Männlichkeit der Gelehrten in Rulin waishi identifiziert und ein besseres Verständnis der Position des Romans in Bezug auf Weiblichkeit und Frauen bietet. Dieses Projekt zeigt, dass der Roman die Geschlechterideologien seiner Zeit widerspiegelt und nicht in Frage stellt. Die im Roman gezeigte ideale Männlichkeit umfasst echte Tugenden und authentisches Lernen. Es widerspricht der traditionellen, orthodoxen konfuzianischen Männlichkeit, die befürwortet, dass das Amt der herrliche Weg ist, um die Männlichkeit eines gelehrten Mannes zu erfüllen. Dies ist hauptsächlich auf Wu Jingzis eigenes Versagen bei den Prüfungen im öffentlichen Dienst und bei offiziellen Karrieren zurückzuführen. In Bezug auf das Verhältnis von Männlichkeit und Sexualität zeigt der Roman, dass ein männlicher Mann nicht vom weiblichen Charme verführt wird, sondern eine harmonische und kameradschaftliche Ehe führen kann. Außerdem zeigen sich Wissenschaftler sehr besorgt über ihre Männlichkeit, da sie in der Gesellschaft eine marginale Position einnehmen. Ihre Männlichkeit wird von Beamten, Kaufleuten und sogar Bürgern sowie ihren Kollegen in Frage gestellt. Durch eine sorgfältige Untersuchung der Geschichten von Pinniang, Miss Lu und Mrs. Wang wird deutlich, dass der Roman eine konventionelle Meinung zu Frauen vertritt, obwohl er den Selbstmord von Witwen kritisiert und eine egalitäre Beziehung zwischen Mann und Frau zeigt. Es lobt konfuzianische weibliche Tugenden, wie dem Ehemann zu folgen und ihm zu dienen, den Haushalt zu führen und die Keuschheit zu bewahren. Weibliche Sexualität wird als böse Versuchung beschuldigt, Männer in die Irre zu führen. Das Lernen von Frauen gewinnt an Legitimität, wenn es dazu dient, häusliche Pflichten zu erfüllen. Es trägt die konfuzianische Botschaft, dass Männer die Führung übernehmen und die Ordnung im Haushalt aufrechterhalten sollten, und stärkt das rechtmäßige Patriarchat. Mit einem Wort, Rulin waishi ist seiner Zeit gegenüber konservativ eingestellt, anstatt seiner Zeit voraus zu sein. KW - Rulin waishi KW - The Unofficial History of the Scholars KW - gender KW - masculinity KW - femininity Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-219202 ER - TY - THES A1 - Wu, Fang T1 - Adding new functions to insulin-like growth factor-I (IGF-I) via genetic codon expansion T1 - Hinzufügen neuer Funktionen zu Insulin-like Growth Factor-I (IGF-I) durch genetische Codon-Erweiterung N2 - Insulin-like growth factor-I (IGF-I) is a 70-amino acid polypeptide with a molecular weight of approximately 7.6 kDa acting as an anabolic effector. It is essential for tissue growth and remodeling. Clinically, it is used for the treatment of growth disorders and has been proposed for various other applications including musculoskeletal diseases. Unlike insulin, IGF-I is complexed to at least six high-affinity binding proteins (IGFBPs) exerting homeostatic effects by modulating IGF-I availability to its receptor (IGF-IR) on most cells in the body as well as changing the distribution of the growth factor within the organism.1-3 Short half-lived IGF-I have been the driving forces for the design of localized IGF-I depot systems or protein modification with enhanced pharmacokinetic properties. In this thesis, we endeavor to present a versatile biologic into which galenical properties were engineered through chemical synthesis, e.g., by site-specific coupling of biomaterials or complex composites to IGF-I. For that, we redesigned the therapeutic via genetic codon expansion resulting in an alkyne introduced IGF-I, thereby becoming a substrate for biorthogonal click chemistries yielding a site-specific decoration. In this approach, an orthogonal pyrrolysine tRNA synthetase (PylRS)/tRNAPyl CUA pair was employed to direct the co-translational incorporation of an unnatural amino acid—¬propargyl-L-lysine (plk)—bearing a clickable alkyne functional handle into IGF-I in response to the amber stop codon (UAG) introduced into the defined position in the gene of interest. We summarized the systematic optimization of upstream and downstream process alike with the ultimate goal to increase the yield of plk modified IGF-I therapeutic, from the construction of gene fusions resulting in (i) Trx-plk-IGF-I fusion variants, (ii) naturally occurring pro-IGF-I protein (IGF-I + Ea peptide) (plk-IGF-I Ea), over the subsequent bacterial cultivation and protein extraction to the final chromatographic purification. The opportunities and hurdles of all of the above strategies were discussed. Evidence was provided that the wild-type IGF-I yields were pure by exploiting the advantages of the pHisTrx expression vector system in concert with a thrombin enzyme with its highly specific proteolytic digestion site and multiple-chromatography steps. The alkyne functionality was successfully introduced into IGF-I by amber codon suppression. The proper folding of plk-IGF-I Ea was assessed by WST-1 proliferation assay and the detection of phosphorylated AKT in MG-63 cell lysate. The purity of plk-IGF-I Ea was monitored with RP-HPLC and SDS-PAGE analysis. This work also showed site-specific coupling an alkyne in plk-IGF-I Ea by copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) with potent activities in vitro. The site-specific immobilization of plk-IGF-I Ea to the model carrier (i.e., agarose beads) resulted in enhanced cell proliferation and adhesion surrounding the IGF-I-presenting particles. Cell proliferation and differentiation were enhanced in the accessibility of IGF-I decorated beads, reflecting the multivalence on cellular performance. Next, we aimed at effectively showing the disease environment by co-delivery of fibroblast growth factor 2 (FGF2) and IGF-I, deploying localized matrix metalloproteinases (MMPs) upregulation as a surrogate marker driving the response of the drug delivery system. For this purpose, we genetically engineered FGF2 variant containing an (S)-2-amino-6-(((2-azidoethoxy)carbonyl)amino)hexanoic acid incorporated at its N-terminus, followed by an MMPs-cleavable linker (PCL) and FGF2 sequence, thereby allowing site-directed, specific decoration of the resultant azide-PCL-FGF2 with the previously mentioned plk-IGF-I Ea to generate defined protein-protein conjugates with a PCL in between. The click reaction between plk-IGF-I Ea and azide-PCL-FGF2 was systematically optimized to increase the yield of IGF-FGF conjugates, including reaction temperature, incubation duration, the addition of anionic detergent, and different ratios of the participating biopharmaceutics. The challenge here was that CuAAC reaction components or conditions might oxidize free cysteines of azide-PCL-FGF2 and future work needs to present the extent of activity retention after conjugation. Furthermore, our study provides potential options for dual-labeling of IGF-I either by the introduction of unnatural amino acids within two distinct positions of the protein of interest for parallel “double-click” labeling of the resultant plk-IGF-I Ea-plk or by using a combination of enzymatic-catalyzed and CuAAC bioorthogonal coupling strategies for sequentially dual-labeling of plk-IGF-I Ea. In conclusion, genetic code expansion in combination with click-chemistry provides the fundament for novel IGF-I analogs allowing unprecedented site specificity for decoration. Considerable progress towards IGF-I based therapies with enhanced pharmacological properties was made by demonstrating the feasibility of the expression of plk incorporated IGF-I using E. coli and retained activity of unconjugated and conjugated IGF-I variant. Dual-labeling of IGF-I provides further insights into the functional requirements of IGF-I. Still, further investigation warrants to develop precise IGF-I therapy through unmatched temporal and spatial regulation of the pleiotropic IGF-I. N2 - Insulin-like growth factor-I (IGF-I) ist ein 70 Aminosäuren langes Polypeptid mit einem Molekuargewicht von 7,5 kDa, dass als anaboler Effektor wirkt und dadurch eine essentielle Rolle in Gewebewachstum und -umbau spielt. Klinisch wird IGF-I für die Behandlung von Wachstumsstörungen verwendet und ist für weitere Anwendungen wie muskuloskelettale Erkrankungen von Interesse. Im Gegensatz zu Insulin wird IGF-I von mindestens sechs hochaffinen Bindungsproteinen (IGFBPs) komplexiert, die homöostatisch regulierend wirken, indem sie die Verfügbarkeit von IGF-I zu seinem Rezeptor (IGF-IR) auf vielen Zellen modulieren und ebenso die Verteilung des Wachstumsfaktors im Körper steuern. Aufgrund der kurzen Halbwertszeit von IGF-I wurde die Entwicklung von lokalen IGF-I Depot-Systemen und von auf Proteinebene modifizierten IGF-I-Varianten mit verbesserten pharmakokinetischen Eigenschaften vorangetrieben. In der vorliegenden Arbeit sind wir bestrebt ein vielseitiges Biopharmazeutikum zu präsentieren, das hinsichtlich seiner galenischen Eigenschaften optimiert wurde, z. B. durch chemische Modifikation, wie ortsspezifische Kopplung von IGF-I an Biomaterialien oder komplexe Verbundstoffe. Für diesen Zweck wurde das Therapeutikum neu entworfen und über die Erweiterung des genetischen Codes eine Alkin-Funktionalität eingefügt. Durch dieses Alkin wird IGF-I zugänglich für die Modifizierung mit bio-orthogonaler, ortsspezifischer „Click-Chemie“. In diesem Ansatz wird ein orthogonales Pyrrolysin tRNA-Synthase (PylRS)/tRNAPyl-CUA – Paar verwendet, um den co-translationalen Einbau einer unnatürlichen Aminosäure — Propargyl-L-lysine (Plk) —, die eine Alkin-Funktionalität für Click-Reaktionen enthält, an Stelle des Amber-Stop-Codons (UAG) im entsprechenden Gen, im IGF-I-Protein zu gewährleisten. Die systematische Optimierung von Up- und Downstream-Prozessen, mit dem Ziel die Ausbeute von Plk-modifiziertem IGF-I-Biopharmazeutikum zu erhöhen, wurden zusammengefasst: von der Konstruktion von Genfusionen, die in (i) einer Trx-plk-IGF-I Fusionsvariant und (ii) natürlich vorkommendem pro-IGF-I Protein (IGF-I + Ea peptide) (Plk-IGF-I Ea) resultierten, über die folgende Expression in Bakterien und Proteinextraktion, bis hin zur finalen chromatographischen Reinigung des Biopharmazeutikums. Die Möglichkeiten und Schwierigkeiten aller oben genannten Strategien wurden diskutiert. Es wurde gezeigt, dass die Wildtyp-IGF-I-Ausbeuten durch den Einsatz des vorteilhaften pHisTrx-Expressionsvektor-Systems zusammen mit dem Enzym Thrombin und seiner hochspezifischen proteolytischen Spaltstelle und mehrfacher chromatographischer Aufreinigung einen hohen Reinheitsgrad aufwiesen. Die Alkin-Funktionalität wurde erfolgreich durch Unterdrückung des Amber-Codons in IGF-I eingeführt. Die richtige Faltung von Plk-IGF-I Ea wurde durch den WST-1 Proliferationsassay und den Nachweis von phosphorylierten Akt in MG-63-Zelllysat nachgewiesen. Die Reinheit von Plk-IGF-I Ea wurde durch RP-HPLC- und SDS-PAGE-Analyse überwacht. In dieser Arbeit konnte auch gezeigt werden, dass die ortspezifische Kopplung von Alkinen an Plk-IGF-I Ea durch Kupfer(I)-katalysierte Azid-Alkin Zykloaddition (CuAAC) in einem Produkt mit hoher in vitro Aktivität resultiert. Die ortspezifische Immobilisierung von Plk-IGF-I Ea an einem Modell-Trägersystem (hier: Agarosepartikel) führte zu einer verbesserten Zellproliferation und Zelladhäsion in der Umgebung der IGF-I-präsentierenden Partikel. Der vielfältige Einfluss von IGF-I auf Zellen wird durch die verbesserte Zellproliferation und -differenzierung durch die Verfügbarkeit von IGF-I präsentierenden Partikeln widergespiegelt. Als Nächstes setzten wir uns zum Ziel den Krankheitseinfluss durch die gleichzeitige Anwendung von Fibroblast-Wachstumsfaktor 2 (FGF2) und IGF-I zu zeigen, indem wir uns der lokalen Hochregulierung von Matrixmetalloproteinasen (MMPS) als Surrogat-Krankheitsmarker bedienten, der die Antwort des Drug Delivery-Systems auslöst. Zu diesem Zweck wurde eine FGF2-Variante genetisch modifiziert, sodass sie am N-Terminus eine (S)-2-amino-6-(((2-azidoethoxy)carbonyl)amino)Hexansäure trägt - gefolgt von einen durch MMPs spaltbaren Verbindungsstück (PCL) und der FGF2 Sequenz - und dadurch die gezielte, spezifische Konjugation des resultierenden Azid-PCL-FGF2 mit dem vorher erwähnten Plk-IGF-I Ea ermöglicht, um definierte Protein-Protein-Konjugate, die mit einem PCL verbunden sind, zu erzeugen. Die Click-Reaktion zwischen Plk-IGF-I Ea und Azid-PCL-FGF2 wurde zur Erhöhung der IGF-FGF Ausbeute systematisch optimiert, indem die Parameter Temperatur, Inkubationsdauer, Zugabe von anionischem Tensid und verschiedene Eduktverhältnisse untersucht wurden. Es gilt zu bedenken, dass die in der CuAAC Reaktion eingesetzten Komponenten oder Reaktionsbedingungen freie Cysteinreste von Azid-PCL-FGF2 oxidieren können und es in Zukunft gilt, die verbleibende Aktivität nach Proteinkonjugation zu bestimmen. Des Weiteren zeigen unsere Untersuchungen potentielle Möglichkeiten für duale Konjugation von IGF-I entweder durch die Einführung einer unnatürlichen Aminosäure an zwei verschiedenen Positionen innerhalb des Proteins (Plk-IGF-I Ea-Plk) für parallele „Doppel-Click“-Konjugation oder durch die Kombination bioorthogonaler Kopplungsreaktionen - einer enzymkatalysierten Reaktion und CuAAC - für sequentielles duales Verknüpfen von Plk-IGF-I Ea. Schlussendlich stellt die Erweiterung des genetischen Codes in Kombination mit Click-Chemie eine Grundlage für neue IGF-I-Analoga dar, die eine noch nie dagewesene Ortsspezifität für Konjugationen besitzen. Ein entscheidender Fortschritt hin zu IGF-I basierten Therapeutika mit verbesserten pharmakologischen Eigenschaften wurde durch die Expression, Reinigung und Konjugation von bioaktivem IGF-I mit Plk sowie konjugierten IGF-I Varianten erreicht. Duales Modifizieren von IGF-I erlaubt weitere Einblicke in die funktionalen Anforderungen an IGF-I. Dennoch sind weitere Untersuchungen nötig, um eine gezielte IGF-I Therapie trotz der unterschiedlichen zeitlichen und räumlichen Regulierung des pleiotropen IGF-I zu ermöglichen. KW - Insulin-like growth factor-I KW - genetic codon expansion KW - site-specific protein modification KW - Insulin-like Growth Factor I KW - Codon Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175330 ER - TY - JOUR A1 - Wu, Hao A1 - Reimann, Sabine A1 - Siddiqui, Sophiya A1 - Haag, Rainer A1 - Siegmund, Britta A1 - Dernedde, Jens A1 - Glauben, Rainer T1 - dPGS Regulates the Phenotype of Macrophages via Metabolic Switching JF - Macromolecular Bioscience N2 - The synthetic compound dendritic polyglycerol sulfate (dPGS) is a pleiotropic acting molecule but shows a high binding affinity to immunological active molecules as L‐/P‐selectin or complement proteins leading to well described anti‐inflammatory properties in various mouse models. In order to make a comprehensive evaluation of the direct effect on the innate immune system, macrophage polarization is analyzed in the presence of dPGS on a phenotypic but also metabolic level. dPGS administered macrophages show a significant increase of MCP1 production paralleled by a reduction of IL‐10 secretion. Metabolic analysis reveals that dPGS could potently enhance the glycolysis and mitochondrial respiration in M0 macrophages as well as decrease the mitochondrial respiration of M2 macrophages. In summary the data indicate that dPGS polarizes macrophages into a pro‐inflammatory phenotype in a metabolic pathway‐dependent manner. KW - infection KW - macrophage polarization KW - MCP1 KW - metabolic switch KW - polyglycerol sulfates Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-212711 VL - 19 IS - 12 ER - TY - JOUR A1 - Wu, Hao A1 - Zhao, Xiufeng A1 - Hochrein, Sophia M. A1 - Eckstein, Miriam A1 - Gubert, Gabriela F. A1 - Knöpper, Konrad A1 - Mansilla, Ana Maria A1 - Öner, Arman A1 - Doucet-Ladevèze, Remi A1 - Schmitz, Werner A1 - Ghesquière, Bart A1 - Theurich, Sebastian A1 - Dudek, Jan A1 - Gasteiger, Georg A1 - Zernecke, Alma A1 - Kobold, Sebastian A1 - Kastenmüller, Wolfgang A1 - Vaeth, Martin T1 - Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming JF - Nature Communications N2 - T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy. KW - cytotoxic T cells KW - infection KW - lymphocyte differentiation KW - translational research Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-358052 VL - 14 ER - TY - THES A1 - Wu, Lingdan T1 - Emotion Regulation in Addicted Smokers T1 - Emotionsregulation bei abhängigen Rauchern N2 - Background: Nicotine addiction is the most prevalent type of drug addiction that has been described as a cycle of spiraling dysregulation of the brain reward systems. Imaging studies have shown that nicotine addiction is associated with abnormal function in prefrontal brain regions that are important for cognitive emotion regulation. It was assumed that addicts may perform less well than healthy nonsmokers in cognitive emotion regulation tasks. The primary aims of this thesis were to investigate emotional responses to natural rewards among smokers and nonsmokers and to determine whether smokers differ from nonsmokers in cognitive regulation of positive and negative emotions. To address these aims, two forms of appraisal paradigms (i.e., appraisal frame and reappraisal) were applied to compare changes in emotional responses of smokers with that of nonsmokers as a function of appraisal strategies. Experiment 1: The aim of the first experiment was to evaluate whether and how appraisal frames preceding positive and negative picture stimuli affect emotional experience and facial expression of individuals. Twenty participants were exposed to 125 pairs of auditory appraisal frames (either neutral or emotional) followed by picture stimuli reflecting five conditions: unpleasant-negative, unpleasant-neutral, pleasant-positive, pleasant-neutral and neutral-neutral. Ratings of valence and arousal as well as facial EMG activity over the corrugator supercilii and the zygomaticus major were measured simultaneously. The results indicated that appraisal frames could alter both subjective emotional experience and facial expressions, irrespective of the valence of the pictorial stimuli. These results suggest and support that appraisal frame is an efficient paradigm in regulation of multi-level emotional responses. 8 Experiment 2: The second experiment applied the appraisal frame paradigm to investigate how smokers differ from nonsmokers on cognitive emotion regulation. Sixty participants (22 nonsmokers, 19 nondeprived smokers and 19 12-h deprived smokers) completed emotion regulation tasks as described in Experiment 1 while emotional responses were concurrently recorded as reflected by self-ratings and psychophysiological measures (i.e., facial EMG and EEG). The results indicated that there was no group difference on emotional responses to natural rewards. Moreover, nondeprived smokers and deprived smokers performed as well as nonsmokers on the emotion regulation task. The lack of group differences in multiple emotional responses (i.e., self-reports, facial EMG activity and brain EEG activity) suggests that nicotine addicts have no deficit in cognitive emotion regulation of natural rewards via appraisal frames. Experiment 3: The third experiment aimed to further evaluate smokers’ emotion regulation ability by comparing performances of smokers and nonsmokers in a more challenging cognitive task (i.e., reappraisal task). Sixty-five participants (23 nonsmokers, 22 nondeprived smokers and 20 12-h deprived smokers) were instructed to regulate emotions by imagining that the depicted negative or positive scenario would become less negative or less positive over time, respectively. The results showed that nondeprived smokers and deprived smokers responded similarly to emotional pictures and performed as well as nonsmokers in down-regulating positive and negative emotions via the reappraisal strategy. These results indicated that nicotine addicts do not have deficit in emotion regulation using cognitive appraisal strategies. In sum, the three studies consistently revealed that addicted smokers were capable to regulate emotions via appraisal strategies. This thesis establishes the groundwork for therapeutic use of appraisal instructions to cope with potential self-regulation failures in nicotine addicts. N2 - Hintergrund: Nikotinsucht ist die am weitesten verbreitete Form von Drogenabhängigkeit und wird beschrieben als eine immer stärker werdende Dysregulation des Belohnungssystems im Gehirn. Bildgebende Studien zeigten, dass Nikotinabhängige eine abnormale Funktion der präfrontalen Gehirnregionen aufweisen, die für die kognitive Emotionsregulation von entscheidender Bedeutung sind. Es wurde angenommen, dass Süchtige bei kognitiven Aufgaben zur Emotionsregulation schlechter abschneiden als gesunde Nichtraucher. Vorrangige Ziele dieser Thesis waren die Untersuchung emotionaler Reaktionen auf natürliche, Raucher-irrelevante Stimuli bei Rauchern und Nichtrauchern. Außerdem sollte herausgefunden werden, ob sich Raucher von Nichtrauchern bezüglich ihrer kognitiven Regulation von positiven und negativen Emotionen unterscheiden. Um diese Veränderungen in der emotionalen Reaktion in Abhängigkeit der Interpretationsstrategie vergleichen zu können, wurden zwei Paradigmen zur Einschätzung emotionaler Stimuli eingesetzt: Eine prospektive Interpretationsstrategie des kommenden Stimulus (appraisal frame) und eine retrospektive Interpretationsstrategie nach der Stimuluspräsentation (reappraisal). Experiment 1: Ziel des ersten Experiments war die Evaluierung ob und wie Interpretationen vor positiven oder negativen Stimulusbildern die emotionale Erfahrung und den Gesichtsausdruck von Personen beeinflussen. 20 Versuchspersonen wurden 125 Paare auditiver Beschreibungen (entweder neutral oder emotional) präsentiert, gefolgt von Stimulusbildern, die zusammen fünf Stimulus-Kategorien bildeten: unangenehm – negativ, unangenehm – neutral, angenehm – positiv, angenehm – neutral und neutral – neutral. Valenz- und Arousal-Ratings wurden abgefragt und die EMG-Aktivität der Gesichtsmuskeln corrugator supercilii und zygomaticus 10 major wurden zeitgleich aufgenommen. Die Ergebnisse zeigten, dass appraisal frames sowohl emotionale Reaktionen einschließlich subjektiver emotionaler Erfahrungen beeinflussen als auch den Gesichtsausdruck verändern können, unabhängig von der Valenz des Bildstimulus. Dies zeigt und beweist die Effizienz des appraisal frame Paradigmas bei der Regulation von emotionalen Reaktionen auf mehreren Verarbeitungsebenen. Experiment 2: Das zweite Experiment bezog sich auf das appraisal frame Paradigma und sollte untersuchen wie sich Raucher von Nichtrauchern in ihrer kognitiven Emotionsregulation unterscheiden. 60 Probanden (22 Nichtraucher, 19 Raucher ohne Entzug und 19 Raucher mit 12 Stunden Zigarettenentzug) führten Emotionsregulationsaufgaben wie in Experiment 1 beschrieben aus, während ihre emotionalen Reaktionen ständig über Selbsteinschätzungen und psychophysiologische Messungen aufgenommen wurden (faziales EMG und EEG). Die Ergebnisse zeigten keine Gruppenunterschieden bei den emotionalen Reaktionen auf natürliche Stimuli, ohne Bezug zum Rauchen; Außerdem schnitten Raucher mit und ohne Zigarettenentzug in der Emotionsregulationsaufgabe genauso gut ab wie Nichtraucher. Die gleichen Ergebnisse in allen Gruppen hinsichtlich emotionaler Reaktionen (Selbsteinschätzung, faziale EMG Aktivität und EEG Aktivität) machten deutlich, dass Nikotinabhängige keine Einschränkungen in der kognitiven Emotionsregulation auf natürliche Stimuli mittels Vorbeurteilungen haben. Experiment 3: Der dritte Versuch wurde durchgeführt, um die Fähigkeiten von Rauchern bei der Emotionsregulation zu untersuchen, indem die Erfolge von Rauchern und Nichtrauchern in einer schwierigeren kognitiven Aufgabe (reappraisal task) verglichen wurden. 65 Versuchspersonen (23 Nichtraucher, 22 Raucher ohne Entzug und 20 Raucher mit 12 Stunden Zigarettenentzug) wurden instruiert ihre Emotionen zu regulieren, indem sie emotionale Bilder 11 mit neutralem Gefühl interpretieren. Die Probanden sollten sich vorstellen, dass die negativen oder positiven Syenarios immer weniger negativ oder weniger positiv werden. Die Ergebnisse stellen heraus, dass Raucher mit und ohne Zigarettenentzug ähnlich auf emotionale Bilder reagierten und ihre positiven und negativen Emotionen mit der reappraisal Strategie genauso gut herunterregulierten wie Nichtraucher. Zusammenfassend machen die drei Studien deutlich, dass Nikotinabhängige mittels Interpretationsstrategien ihre Emotionen regulieren können. Diese Thesis bilden das Fundament für den therapeutischen Nutzen von Interpretationsstrategien, damit Nikotinabhängige mit potenziellen Selbstregulationsstörungen umgehen können. KW - Gefühl KW - Regulation KW - Rauch KW - Elektroencephalogramm KW - Elektromyographie KW - Emotion KW - Regulation KW - Smoke KW - Electroencephalography KW - Electromyography Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85471 ER - TY - JOUR A1 - Wu, Lingdan A1 - Pu, Jie A1 - Allen, John J. B. A1 - Pauli, Paul T1 - Recognition of facial expressions in individuals with elevated levels of depressive symptoms: an eye-movement study JF - Depression Research and Treatment N2 - Previous studies consistently reported abnormal recognition of facial expressions in depression. However, it is still not clear whether this abnormality is due to an enhanced or impaired ability to recognize facial expressions, and what underlying cognitive systems are involved. The present study aimed to examine how individuals with elevated levels of depressive symptoms differ from controls on facial expression recognition and to assess attention and information processing using eye tracking. Forty participants (18 with elevated depressive symptoms) were instructed to label facial expressions depicting one of seven emotions. Results showed that the high-depression group, in comparison with the low-depression group, recognized facial expressions faster and with comparable accuracy. Furthermore, the high-depression group demonstrated greater leftwards attention bias which has been argued to be an indicator of hyperactivation of right hemisphere during facial expression recognition. KW - Depression Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123153 VL - 2012 IS - 249030 ER - TY - JOUR A1 - Wu, Lingdan A1 - Winkler, Markus H. A1 - Wieser, Matthias J. A1 - Andreatta, Marta A1 - Li, Yonghui A1 - Pauli, Paul T1 - Emotion regulation in heavy smokers: experiential, expressive and physiological consequences of cognitive reappraisal JF - Frontiers in Psychology N2 - Emotion regulation dysfunctions are assumed to contribute to the development of tobacco addiction and relapses among smokers attempting to quit. To further examine this hypothesis, the present study compared heavy smokers with non-smokers (NS) in a reappraisal task. Specifically, we investigated whether non-deprived smokers (NDS) and deprived smokers (DS) differ from non-smokers in cognitive emotion regulation and whether there is an association between the outcome of emotion regulation and the cigarette craving. Sixty-five participants (23 non-smokers, 22 NDS, and 20 DS) were instructed to down-regulate emotions by reappraising negative or positive pictorial scenarios. Self-ratings of valence, arousal, and cigarette craving as well as facial electromyography and electroencephalograph activities were measured. Ratings, facial electromyography, and electroencephalograph data indicated that both NDS and DS performed comparably to nonsmokers in regulating emotional responses via reappraisal, irrespective of the valence of pictorial stimuli. Interestingly, changes in cigarette craving were positively associated with regulation of emotional arousal irrespective of emotional valence. These results suggest that heavy smokers are capable to regulate emotion via deliberate reappraisal and smokers' cigarette craving is associated with emotional arousal rather than emotional valence. This study provides preliminary support for the therapeutic use of reappraisal to replace maladaptive emotion-regulation strategies in nicotine addicts. KW - negative affect KW - regulation strategies KW - positive emotions KW - craving KW - late positive potential KW - nicotine addiction KW - smoking KW - emotion regulation KW - reappraisal KW - facial electromyography KW - brain reactivity KW - down regulation KW - unpleasant pictures KW - anxiety sensitivity KW - facial expressions KW - cigarette smokers KW - smoking motives Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145225 VL - 6 IS - 1555 ER - TY - THES A1 - Wu, Rongxue T1 - Integrins and SPARC : potential implications for cardiac remodeling N2 - Der enorme Umbau des Herzgewebes, wie man ihn nach Drucküberlastung des Ventrikels oder MyokardInfarkt beobachten kann, gilt als eine der kausalen Ursachen des Herzversagens. Die Veränderungen in der Architektur des Herzens beeinflussen die mechanischen Eigenschaften des Herzmuskels, begründet sind sie jedoch in Anpassungsprozessen auf der zellulären Ebene vor allem in einer Modulation der Expression bestimmter Gene. Gemeinsam mit Integrinen, den Transmembran-Rezeptoren, welche die extrazelluläre Umgebung mit dem intrazellulären Zytoskelett verbinden, gehören Proteine der extrazellulären Matrix (ECM) und matrizelluläre Proteine zu den Schlüsselkomponenten, die den Umbauprozess im Herzen steuern. Aus diesen Gründen hatte diese Doktorarbeit zum Ziel, die Rolle der Integrine für die Regulation der Genexpression und die Leistungsfähigkeit des Herzmuskels während der durch Drucküberlastung oder myokardialen Infarkt (MI) hervorgerufenen Wundheilungsprozesse zu analysieren. Um die Beteiligung von Integrin Beta 1 zu untersuchen, wurde ein experimentelles Modell der Drucküberlastung im Mausherzen (aortic banding; Konstriktion der Aorta; AB) eingesetzt, wobei Mäuse mit einer konditionalen, Herz-spezifischen Deletion des Integrin Beta 1 Gens untersucht wurden. Ein besonderes Augenmerk wurde dabei auf die physiologischen Unterschiede und eine veränderte Genexpression im gestressten Herzen in An- oder Abwesenheit von Integrin Beta 1 gelegt. Interessanterweise wurden die Mäuse, welche eine Kombination aus Integrin knock-out Allel und dem Kardiomyozyten-spezifischen konditionalen knock-out Allel von Integrin Beta 1 aufwiesen im normalen Mendelschen Verhältnis geboren und wuchsen normal auf. Obwohl diese Tiere immer noch geringe Mengen von Integrin Beta 1 in ihrem Herzen aufwiesen (exprimiert von nicht-Myozyten), besaßen diese Mäuse eine veränderte Herzfunktion und waren sehr sensitiv gegenüber AB. Im Gegensatz zu der kompensatorischen hypertrophischen Reaktion, die in Wildtyp Mäusen zu beobachten war, zeigte sich in den Integrin Beta 1–defizienten Mausherzen kein Gewebeumbau. Auch die erhöhte Expression von verschiedenen ECM Proteinen, insbesondere die verstärkte Expression des matrizellulären Proteins SPARC, unterblieb nach AB in den Integrin Beta 1–defizienten Tieren. Interessanterweise konnte auch eine transiente Erhöhung der SPARC mRNA während der Umbauprozesse im Herzen in Folge von myokardialem Infarkt (MI) mittels cDNA Makroarrays festgestellt werden. In der Tat fanden sich größere Mengen von SPARC bereits 2 Tage (~2,5-fach erhöht), 7 Tage (~4-fach erhöht) und 1 Monat (~2-fach erhöht) nach MI, während ein spezifischer Inhibitor der Integrin alpha v Untereinheit diese Hochregulation von SPARC in vivo verhinderte. Immunfluoreszenz Untersuchungen von Herzgewebe verdeutlichten, dass sich die erhöhte Expression von SPARC auf das Infarktareal beschränkte, dass die Expression von SPARC nach einer anfänglichen Erhöhung im Verlauf von 1 Monaten wieder auf das Anfangsniveau zurückging und dass die verstärkte Expression von der Einwanderung von Fibroblasten in das ischämische Herzgewebe begleitet war. In vitro stimulierten die Wachstumsfaktoren TGF-Beta 1 und PDGF-BB die Expression von SPARC durch Fibroblasten. Wie sich an Hand von ELISA und Western Blot Untersuchungen feststellen ließ, war die Inhibition von Integrin Beta v nicht in der Lage, die durch TGF-Beta 1 oder PDGF induzierte Sekretion von SPARC zu beeinflussen. Jedoch zeigte sich, dass Vitronektin, ein Ligand von Integrin alpha v, sowohl die Sekretion von TGF-Beta 1 als auch von PDGF-BB durch Kardiomyozyten induzierte und diese Reaktion wurde durch den Integrin alpha v Inhibitor komplett unterdrückt. In funktioneller Hinsicht wirkte SPARC auf die durch ECM Proteine induzierte Migration von Fibroblasten ein, so dass man davon ausgehen kann, dass die lokale Freisetzung von SPARC nach myokardialem Infarkt zur Wundheilung im Herzen beiträgt. Zusammenfassend läßt die Kombination der in vivo und in vitro erhobenen experimentellen Daten den Schluss zu, dass mehrere Integrin Untereinheiten eine entscheidende Rolle während der Gewebeumbildung im Herzen spielen. Integrin-abhängige Genexpressionsereignisse wie beispielsweise die erhöhte Expression von SPARC nach MI sind entscheidend an der Koordination der Wundheilung beteiligt. Diese Prozesse scheinen auf einer komplexen Wechselwirkung und Kommunikation zwischen verschiedenen Zelltypen wie Kardiomyozyten und Fibroblasten zu beruhen, um lokal begrenzt eine Heilung und Vernarbung des verletzten Gewebes zu regulieren. Die Aufklärung des fein abgestimmten Wechselspiels zwischen Integrinen matrizellulären Proteinen wie SPARC und Wachstumsfaktoren wird sicherlich zu einem besseren und klinisch nutzbarem Verständnis der molekularen Mechanismen des Gewebeumbaus im Herzen beitragen. N2 - The massive remodeling of the heart tissue, as observed in response to pressure overload or myocardial infarction, is considered to play a causative role in the development of heart failure. Alterations in the heart architecture clearly affect the mechanical properties of the heart muscle, but they are rooted in changes at the cellular level including modulation of gene expression. Together with integrins, the transmembrane receptors linking the extracellular environment to the cytoskeleton, extracellular matrix (ECM) proteins and matricellular proteins are key components of the remodeling process in the heart. Therefore, this thesis was aimed at analysing the role of integrins in the regulation of gene expression and heart muscle performance during cardiac wound repair induced by pressure overload or myocardial infarction (MI). To investigate the contribution of integrin Beta 1, we characterised the response of mice with a conditional, cardiac-specific deletion of the integrin Beta 1 gene in an experimental model of pressure overload by aortic banding (AB). In particular, we measured physiological alterations and gene expression events in the stressed heart in the presence or absence of integrin Beta 1. Interestingly, mice containing a knock-out allele and the ventricular myocyte-specific conditional allele of the integrin Beta 1 gene were born and grew up to adulthood. Though these animals still exhibited minor amounts of integrin Beta1 in the heart (expressed by non-myocytes), these mice displayed abnormal cardiac function and were highly sensitive to AB. Whereas a compensatory hypertrophic response to pressure overload was observed in wildtype mice, the integrin Beta 1-deficient mice were not able to undergo heart tissue remodeling. Furthermore, ECM gene expression was altered and, in particular, the increased expression of the matricellular protein SPARC after AB was abolished in integrin Beta 1–deficient mice. Interestingly, we also found a transient upregulation of SPARC mRNA during heart remodeling after MI using cDNA macroarrays. Indeed, increased SPARC protein levels were observed starting at day 2 (2.55±0.21fold, p<0.01), day 7 (3.72±0.28 fold, p<0.01) and 1 month (1.9±0.16 fold, p<0.01) after MI, which could be abolished by using an integrin alpha v inhibitor in vivo. Immunofluorescence analysis of heart tissue demonstrated that the increased SPARC expression was confined to the infarcted area and occurred together with the influx of fibroblasts into the heart. In vitro, either TGF-Beta 1 or PDGF-BB stimulated SPARC expression by fibroblasts. Inhibition of integrin alpha v did not interfere with TGF-Beta1 or PDGF induced SPARC secretion as determined by ELISA assays or Western blot. However, secretion of TGF-Beta1 and PDGF-BB by cardiomyocytes was induced by vitronectin, a ligand of integrin alpha v, and this response was blocked by the integrin alpga v inhibitor. Functionally, SPARC modulated the migratory response of fibroblasts towards ECM proteins suggesting that the local deposition of SPARC following MI contributes to scar formation. Taken together, our combined in vivo and in vitro data demonstrate that several integrin subunits play critical roles during tissue remodeling in the injured heart. Integrin-dependent gene expression events such as the upregulation of SPARC following MI are critical to orchestrate the healing response. These processes appear to involve complex cross-talk between different cell types such as cardiomyocytes and fibroblasts to allow for locally confined scar formation. The elucidation of the sophisticated interplay between integrins, matricellular proteins such as SPARC, and growth factors will undoubtedly provide us with a better and clinically useful understanding of the molecular mechanisms governing heart remodeling. KW - Integrine KW - Genexpression KW - Herzmuskel KW - Grundsubstanz KW - Extracellular matrix KW - gene expression KW - cardiac remodeling KW - migration Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-17531 ER - TY - THES A1 - Wu, Rongxue T1 - Treatment with integrin alpha v inhibitor abolishes compensatory cardiac hypertrophy due to altered signal transduction and ECM gene expression T1 - xxx N2 - Integrine sind Transmembranrezeptoren, welche mechanische Signale von der extrazellulären Matrix (ECM) zum Zytoskelett übermitteln ("outside-in-signaling"). Viele molekulare Defekte in der Verbindung zwischen Zytoskelett und ECM erzeugen bekanntermaßen Kardiomyopathien. alpha v Integrin scheint eine Hauptrolle in verschiedenen Prozessen der kardialen Reorganisation zu spielen, wie z.B. Regulation der Zellproliferation, -migration und -differenzierung. Unsere Hypothese war, dass alpha v -Integrin-vermittelte Signale notwendig für die kompensatorische Hypertrophie nach Aortenkonstriktion sind und assoziiert mit der Modulation der Expression von ECM-Proteinen. Dazu wurden Mäuse mit einem spezifischen alpha v Integrin-Inhibitor behandelt und einer Aortenkonstriktion (AB) unterzogen. Nach zwei Tagen und nach sieben Tagen wurden die Mäuse echokardiographisch untersucht und eingehende hämodynamische Untersuchungen wurden durchgeführt. Die Behandlung mit dem alpha v -Integrin-Inhibitor führte zu einer dilatativen Kardiomyopathie und Herzinsuffizienz in den AB-Mäusen, gekennzeichnet durch einen dilatierten linken Ventrikel, schlechte linksventrikuläre Funktion und einer Lungenstauung, wohingegen die scheinbehandelten Tiere eine kompensatorische Hypertrophie des linken Ventrikels zeigten. Untersuchungen der beteiligten Signalwege zeigten eine Aktivierung des p38 MAP-Kinase-Signalwegs, von ERK 1 und -2, der Focal Adhesion Kinase FAK und Tyrosin-Phosphorylierung von c-Src in den Kontrollherzen, was in den Inhibitor-behandelten Herzen fehlte. mRNA-Expressionsanalysen für 96 Gene mittels "Micro-Arrays" ermittelten verschiedene genomische Ziele des alpha v -Integrin-aktivierten Signalwegs. 18 für ECM-Proteine codierende Gene wurden mehr als 2-fach hochreguliert, z.B. Kollagen (8,11-fach ± 2,2), Fibronectin (2,32 ± 094), SPARC (3,78 ± 0,12), ADAMTS-1 (3,51 ± 0,81) und TIMP2 (2,23 ± 0,98), wohingegen die Aktivierung dieser Gene in Inhibitor-behandelten Tieren aufgehoben war. Wir folgern daraus, dass Signalwege unterhalb von alpha v -Integrin, mediiert durch MAP-Kinasen, FAK und c-Src, zu einer verstärkten Expression von ECM-Komponenten führt, welche für die kompensatorische Antwort auf Druckbelastung nötig sind. N2 - Integrins are transmembrane receptors transmitting mechanical signals from the extracellular matrix (ECM) to the cytoskeleton (outside-in-signaling). Many molecular defects in the link between cytoskeleton and ECM are known to induce cardiomyopathies. alpha v integrin appears to play a major role in several processes relevant to remodeling, such as binding and activation of matrix metalloproteinases as well as regulation of cell proliferation, migration, and differentiation. We hypothesized that alpha v integrin-mediated signaling is required for the compensatory hypertrophy after aortic banding (AB) and associated with the modulation of ECM protein expression. Mice were treated in vivo with a specific integrin alpha v inhibitor or vehicle via osmotic minipumps starting 1 day prior to aortic banding (AB). At day 2 and day 7 following AB or sham-operation, the mice were examined by echocardiography and hemodynamic analyses were performed. Treatment of alpha v Integrin inhibitor led to a dilated cardiomyopathy and congestive heart failure in AB mice (dilated left ventricle, depressed LV function, and pulmonary congestion), but not to hypertrophy as observed in mice without inhibitor treatment. Investigation of downstream signaling revealed significant activation of the p38 Mitogen-Activated Protein Kinase (MAPK), the Extracellular signal-Regulated Kinases 1 and 2 (Erk 1/2), Focal Adhesion Kinase (FAK) and tyrosine-phosphorylation of c-Src in mice 7 days after AB. This response was blunted in mice treated with integrin alpha v inhibitor. Microarrays probing for a total of 96 cell adhesion and ECM genes identified various genomic targets of integrin alpha v mediated signalling. 7 days after AB 18 ECM genes were up-regulated more than 2-fold (n=6), e.g. collagen (8.11 ± 2.2), fibronectin (2.32 ± 0.94), secreted protein, acidic and rich in cysteine (SPARC, 3.78 ± 0.12), A disintegrin-like and metalloprotease (reprolysin type) with trombospondin type 1 (Adamts-1, 3.51 ± 0.81) and Tissue inhibitor of metalloproteinase 2 (TIMP2, 2.23 ± 0.98), whereas this up-regulation was abolished in mice that were treatd by integrin alpha v inhibitor via mini pumps. We conclude that signaling downstream of integrin alpha v is mediated by the MAPK, FAK and c-Src pathways leading to an up-regulation of extracelluar matrix components necessary for the compensatory response of the heart under a condition of pressure overload. KW - Antigen KW - integrin alpha v KW - cardiac hypertrophy KW - signal transduction KW - ECM KW - gene expression KW - integrin alpha v KW - cardiac hypertrophy KW - signal transduction KW - ECM KW - gene expression Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-21339 ER - TY - JOUR A1 - Wu, Y. S. A1 - Becker, Charles R. A1 - Waag, A. A1 - Bicknell-Tassius, R. N. A1 - Landwehr, G. T1 - Thermal effects on (100) CdZnTe substrates as studied by x-ray photoelectron spectroscopy and reflection high energy electron diffraction N2 - The influence of different CdZnTe substrate treatments prior to II-VI molecular beam epitaxial growth on surface stoichiometry, oxygen, and carbon contamination has been studied using x-ray photoelectron spectroscopy and reflection high energy electron diffraction. Heating the substrate at 300 °C can eliminate oxygen contamination, but cannot completely remove carbon from the surface. Heating at higher temperatures decreases the carbon contamination only slightly, while increasing the Zn-Cd ratio on the surface considerably. The magnitude of the latter effect is surprising and is crucial when one is using lattice matched CdZnTe (Zn 4%) substrates. Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37801 SN - 0003-6951 ER - TY - JOUR A1 - Wu, Y. S. A1 - Becker, Charles R. A1 - Waag, A. A1 - Bicknell-Tassius, R. N. A1 - Landwehr, G. T1 - Removal of oxygen and reduction of carbon contamination on (100) Cd\(_{0.96}\)Zn\(_{0.04}\)Te N2 - No abstract available Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-38014 ER - TY - JOUR A1 - Wu, Y. S. A1 - Becker, Charles R. A1 - Waag, A. A1 - Bicknell-Tassius, R. N. A1 - Landwehr, G. T1 - The effects of laser illumination and high energy electrons on molecular-beam epitaxial growth of CdTe N2 - We report the results of a detailed investigation on the Te-stabilized (2 x 1) and the Cdstabilized c( 2 X 2) surfaces of ( 100) CdTe substrates. The investigation demonstrates for the first time that both laser illumination and, to a greater extent, high-energy electron irradiation increase the Te desorption and reduce the Cd desorption from ( 100) CdTe surfaces. Thus it is possible by choosing the proper growth temperature and photon or electron fluxes to change the surface reconstruction from the normally Te-stabilized to a Cd-stabilized phase. Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-30795 ER - TY - JOUR A1 - Wu, Y. S. A1 - Becker, Charles R. A1 - Waag, A. A1 - Schmiedl, R. A1 - Einfeldt, S. A1 - Landwehr, G. T1 - Oxygen on the (100) CdTe surface N2 - We have investigated oxygen on CdTe substrates by means of x-ray photoelectron spectroscopy (XPS) and reflection high-energy electron diffraction (RHEED). A Te oxide layer that was at least 15 A thick was found on the surface of as-delivered CdTe substrates that were mechanically polished. This oxide is not easily evaporated at temperatures lower than 350°C. Furthermore, heating in air, which further oxidizes the CdTe layer, should be avoided. Etching with HCI acid (15% HCl) for at least 20 s and then rinsing with de-ionized water reduces the Te oxide layer on the surface down to 4% of a monoatomic layer. However, according to XPS measurements of the 0 Is peak, 20%-30% of a monoatomic layer of oxygen remains on the surface, which can be eliminated by heating at temperatures ranging between 300 and 340 cC. The RHEED patterns for a molecular beam epitaxially (MBE)-grown CdTe film on a (lOO) CdTe substrate with approximately one monoatomic layer of oxidized Te on the surface lose the characteristics of the normal RHEED pattems for a MBE-grown CdTe film on an oxygen-free CdTe substrate. Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37869 ER - TY - JOUR A1 - Wu, Y. S. A1 - Becker, Charles R. A1 - Waag, A. A1 - von Schierstedt, K. A1 - Bicknell-Tassius, R. N. A1 - Landwehr, G. T1 - Surface sublimation of zinc blende CdTe N2 - The surface sublimation of Cd and Te atoms from the zinc blende (111)A CdTe surface has been investigated in detail by reflection high energy electron diffraction and x-ray photoelectron spectroscopy. These experiments verify that Te is much easier to evaporate than Cd. The experimental value for the Te activation energy from a Te stabilized (111)A CdTe surface is 1.41 ±0.1O eV, which is apparently inconsistent with recent theoretical results. Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37829 ER - TY - JOUR A1 - Wu, Y.S. A1 - Becker, Charles R. A1 - Waag, A. A1 - Kraus, M. M. A1 - Bicknell-Tassius, R. N. A1 - Landwehr, G. T1 - Correlation of the Cd-to-Te ratio on CdTe surfaces with the surface structure N2 - We report here that reconstruction on (100), (1lIlA, and (1l1lB CdTe surfaces is either C(2X2), (2X2), and (l X I) or (2X I), (l X I), and (l X I) when they are Cd or Te stabilized, respectively. There is a mixed region between Cd and Te stabilization in which the reflected high-energy electron-diffraction (RHEED) patterns contain characteristics of both Cd- and Te-stabilized surfaces. We have also found that the Cd-to-Te ratio of the x-ray photoelectron intensities of their 3d\(_{3/ 2}\) core levels is about 20% larger for a Cd-stabilized (1lIlA, (1lIlB, or (100) CdTe surface than for a Te-stabilized one. According to a simple model calculation, which was normalized by means of the photoelectron intensity ratio of a Cd-stabilized (lll)A and aTe-stabilized (1l1lB CdTe surface, the experimental data for CdTe surfaces can be explained by a linear dependence of the photoelectron-intensity ratio on the fraction of Cd in the uppermost monatomic layer. This surface composition can be correlated with the surface structure, i.e., the corresponding RHEED patterns. This correlation can in turn be employed to determine Te and Cd evaporation rates. The Te reevaporation rate is increasingly slower for the Te-stabilized (Ill) A, (l1l)B, and (100) surfaces, while the opposite is true for Cd from Cd-stabilized (Ill) A and (Ill)B surfaces. In addition, Te is much more easily evaporated from all the investigated surfaces than is Cd, if the substrate is kept at normal molecular-beam-epitaxy growth temperatures ranging from 2oo·C to 300 ·C. KW - Festkörperphysik Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-37789 SN - 0163-1829 ER - TY - JOUR A1 - Wu, Yu A1 - Pons, Valérie A1 - Goudet, Amélie A1 - Panigai, Laetitia A1 - Fischer, Annette A1 - Herweg, Jo-Ana A1 - Kali, Sabrina A1 - Davey, Robert A. A1 - Laporte, Jérôme A1 - Bouclier, Céline A1 - Yousfi, Rahima A1 - Aubenque, Céline A1 - Merer, Goulven A1 - Gobbo, Emilie A1 - Lopez, Roman A1 - Gillet, Cynthia A1 - Cojean, Sandrine A1 - Popoff, Michel R. A1 - Clayette, Pascal A1 - Le Grand, Roger A1 - Boulogne, Claire A1 - Tordo, Noël A1 - Lemichez, Emmanuel A1 - Loiseau, Philippe M. A1 - Rudel, Thomas A1 - Sauvaire, Didier A1 - Cintrat, Jean-Christophe A1 - Gillet, Daniel A1 - Barbier, Julien T1 - ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments JF - Scientific Reports N2 - Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identifed the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efciently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases. KW - biology KW - antimicrobials KW - high-throughput screening KW - infectious diseases Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173170 VL - 7 ER - TY - THES A1 - Wu, Zhu T1 - Room Temperature Phosphorescence (RTP): Experimental And Theoretical Studies on Boron-Containing Materials T1 - Raumtemperaturphosphoreszenz (RTP): Experimentelle und theoretische Studien zu borhaltigen Materialien N2 - Persistent room temperature phosphorescent (RTP) luminophores have gained remarkable interest recently for a number of applications in security printing, OLEDs, optical storage, time-gated biological imaging and oxygen sensors. We report the first persistent RTP with lifetimes up to 0.5 s from simple triarylboranes which have no lone pairs. We also have prepared 3 isomeric (o, m, p-bromophenyl)-bis(2,6-dimethylphenyl)boranes. Among the 3 isomers (o-, m- and p-BrTAB) synthesized, the ortho-one is the only one which shows dual phosphorescence, with a short lifetime of 0.8 ms and a long lifetime of 234 ms in the crystalline state at room temperature. At last, we checked the RTP properties from the boric acid. We found that the pure boric acid does not show RTP in the solid state. N2 - In letzter Zeit haben Luminophore, die eine effiziente Raumtemperatur-Phosphoreszenz (RTP) zeigen, aufgrund ihrer zahlreichen Anwendungen enormes Interesse gefunden. Die meisten phosphoreszierenden Materialien leiten sich jedoch von Übergangsmetallkomplexen ab, da ihr intrinsisches schnelles Intersystem Crossing (ISC) durch die starke Spin Bahn Kopplung (SOC) des Schwermetalls induziert wird. Metallfreie RTP Materialien sind selten und haben sich zu einem vielversprechenden Gebiet entwickelt, da sie kostengünstig und umweltfreundlich sind. Dieses Kapitel fasst organische molekulare Materialien mit langen Triplett Lebensdauern bei Raumtemperatur aus der Perspektive zusammen, ob sie aus einem molekularen oder Mehrkomponentensystem stammen. Bei rein organischen phosphoreszierenden Materialien werden üblicherweise Heteroatome in das Rückgrat eingeführt, um die Singulett Triplett ISC Geschwindigkeitskonstante zu erhöhen. In Mehrkomponentensystemen wird durch nützliche Strategien wie Wirt-Gast, Polymermatrix, Copolymerisation und supramolekulare Anordnung eine starre Matrixanordnung erreicht, durch die nichtstrahlende Pfade eingeschränkt und so ultralange RTP realisiert wird. ... KW - room temperature phosphorescence Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260844 ER - TY - JOUR A1 - Wu, Zhu A1 - Dinkelbach, Fabian A1 - Kerner, Florian A1 - Friedrich, Alexandra A1 - Ji, Lei A1 - Stepanenko, Vladimir A1 - Würthner, Frank A1 - Marian, Christel M. A1 - Marder, Todd B. T1 - Aggregation-Induced Dual Phosphorescence from (o-Bromophenyl)-Bis(2,6-Dimethylphenyl)Borane at Room Temperature JF - Chemistry—A European Journal N2 - Designing highly efficient purely organic phosphors at room temperature remains a challenge because of fast non-radiative processes and slow intersystem crossing (ISC) rates. The majority of them emit only single component phosphorescence. Herein, we have prepared 3 isomers (o, m, p-bromophenyl)-bis(2,6-dimethylphenyl)boranes. Among the 3 isomers (o-, m- and p-BrTAB) synthesized, the ortho-one is the only one which shows dual phosphorescence, with a short lifetime of 0.8 ms and a long lifetime of 234 ms in the crystalline state at room temperature. Based on theoretical calculations and crystal structure analysis of o-BrTAB, the short lifetime component is ascribed to the T\(^M_1\) state of the monomer which emits the higher energy phosphorescence. The long-lived, lower energy phosphorescence emission is attributed to the T\(^A_1\) state of an aggregate, with multiple intermolecular interactions existing in crystalline o-BrTAB inhibiting nonradiative decay and stabilizing the triplet states efficiently. KW - AIE KW - luminescence KW - phosphorescence KW - triarylborane KW - triplet Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318297 VL - 28 IS - 30 ER - TY - JOUR A1 - Wu, Zhu A1 - Nitsch, Jörn A1 - Marder, Todd B. T1 - Persistent room-temperature phosphorence from purely organic molecules and multi-component systems JF - Advanced Optical Materials N2 - Recently, luminophores showing efficient room-temperature phosphorescence (RTP) have gained tremendous interest due to their numerous applications. However, most phosphors are derived from transition metal complexes because of their intrinsic fast intersystem crossing (ISC) induced by strong spin–orbit coupling (SOC) constants of the heavy metal. Metal-free RTP materials are rare and have become a promising field because they are inexpensive and environmentally friendly. This review summarizes organic molecular materials with long triplet lifetimes at room temperature from the perspective of whether they stem from a molecular or multi-component system. Among purely organic phosphors, heteroatoms are usually introduced into the backbone in order to boost the singlet–triplet ISC rate constant. In multi-component systems, useful strategies such as host–guest, polymer matrix, copolymerization, and supramolecular assembly provide a rigid matrix to restrict nonradiative pathways thus realizing ultralong RTP. KW - inorganic chemistry KW - non-radiative decay KW - polymer matrix KW - intersystem crossing KW - luminescence KW - photophysics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256415 VL - 9 IS - 20 ER - TY - JOUR A1 - Wu, Zhu A1 - Roldao, Juan Carlos A1 - Rauch, Florian A1 - Friedrich, Alexandra A1 - Ferger, Matthias A1 - Würthner, Frank A1 - Gierschner, Johannes A1 - Marder, Todd B. T1 - Pure Boric Acid Does Not Show Room-Temperature Phosphorescence (RTP) JF - Angewandte Chemie N2 - Boric acid (BA) has been used as a transparent glass matrix for optical materials for over 100 years. However, recently, apparent room-temperature phosphorescence (RTP) from BA (crystalline and powder states) was reported (Zheng et al., Angew. Chem. Int. Ed. 2021, 60, 9500) when irradiated at 280 nm under ambient conditions. We suspected that RTP from their BA sample was induced by an unidentified impurity. Our experimental results show that pure BA synthesized from B(OMe)\(_{3}\) does not luminesce in the solid state when irradiated at 250–400 nm, while commercial BA indeed (faintly) luminesces. Our theoretical calculations show that neither individual BA molecules nor aggregates would absorb light at >175 nm, and we observe no absorption of solid pure BA experimentally at >200 nm. Therefore, it is not possible for pure BA to be excited at >250 nm even in the solid state. Thus, pure BA does not display RTP, whereas trace impurities can induce RTP. KW - boric acid KW - room-temperature phosphorescence (RTP) KW - optical materials Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-318308 VL - 61 IS - 15 ER - TY - JOUR A1 - Wulf, Maximilian A1 - Barkovits, Katalin A1 - Schork, Karin A1 - Eisenacher, Martin A1 - Riederer, Peter A1 - Gerlach, Manfred A1 - Eggers, Britta A1 - Marcus, Katrin T1 - The proteome of neuromelanin granules in dementia with Lewy bodies JF - Cells N2 - Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson’s disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SN\(_{Surr.}\)) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB. KW - neuromelanin granules KW - neurodegeneration KW - dementia with Lewy bodies KW - proteomics KW - stress granules KW - substantia nigra pars compacta Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297465 SN - 2073-4409 VL - 11 IS - 22 ER - TY - JOUR A1 - Wunder, Juliane A1 - Pemp, Daniela A1 - Cecil, Alexander A1 - Mahdiani, Maryam A1 - Hauptstein, René A1 - Schmalbach, Katja A1 - Geppert, Leo N. A1 - Ickstadt, Katja A1 - Esch, Harald L. A1 - Dankekar, Thomas A1 - Lehmann, Leane T1 - Influence of breast cancer risk factors on proliferation and DNA damage in human breast glandular tissues: role of intracellular estrogen levels, oxidative stress and estrogen biotransformation JF - Archives of Toxicology N2 - Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC- and UHPLC-MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2). KW - metabolic network model KW - estrogens KW - human breast KW - multiple linear regression Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265343 SN - 1432-0738 VL - 96 IS - 2 ER - TY - JOUR A1 - Wunsch, Kathrin A1 - Pfister, Roland A1 - Henning, Anne A1 - Aschersleben, Gisa A1 - Weigelt, Matthias T1 - No Interrelation of Motor Planning and Executive Functions across Young Ages JF - Frontiers in Psychology N2 - The present study examined the developmental trajectories of motor planning and executive functioning in children. To this end, we tested 217 participants with three motor tasks, measuring anticipatory planning abilities (i.e., the bar-transport-task, the sword-rotation-task and the grasp-height-task), and three cognitive tasks, measuring executive functions (i.e., the Tower-of-Hanoi-task, the Mosaic-task, and the D2-attention-endurance-task). Children were aged between 3 and 10 years and were separated into age groups by 1-year bins, resulting in a total of eight groups of children and an additional group of adults. Results suggested (1) a positive developmental trajectory for each of the sub-tests, with better task performance as children get older; (2) that the performance in the separate tasks was not correlated across participants in the different age groups; and (3) that there was no relationship between performance in the motor tasks and in the cognitive tasks used in the present study when controlling for age. These results suggest that both, motor planning and executive functions are rather heterogeneous domains of cognitive functioning with fewer interdependencies than often suggested. KW - anticipatory planning KW - end-state comfort effect KW - developmental disorders KW - child development KW - motor development Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165281 VL - 7 IS - 1031 ER - TY - JOUR A1 - Wunsch, Marie A1 - Caspell, Richard A1 - Kuerten, Stefanie A1 - Lehmann, Paul V. A1 - Sundararaman, Srividya T1 - Serial measurements of apoptotic cell numbers provide better acceptance criterion for PBMC quality than a single measurement prior to the T cell assay JF - Cells N2 - As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay. KW - T cell assay KW - apoptosis KW - acceptance KW - viability KW - ELISPOT Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150213 VL - 4 IS - 1 ER - TY - JOUR A1 - Wunsch, Marie A1 - Hohmann, Christopher A1 - Milles, Bianca A1 - Rostermund, Christina A1 - Lehmann, Paul V. A1 - Schroeter, Michael A1 - Bayas, Antonios A1 - Ulzheimer, Jochen A1 - Mäurer, Mathias A1 - Ergün, Süleyman A1 - Kuerten, Stefanie T1 - The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis JF - Viruses N2 - There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy. KW - B cells KW - CMV KW - EBV KW - ELISPOT KW - MS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146946 VL - 8 IS - 4 ER - TY - JOUR A1 - Wunsch, Marie A1 - Zhang, Wenji A1 - Hanson, Jodi A1 - Caspell, Richard A1 - Karulin, Alexey Y. A1 - Recks, Mascha S. A1 - Kuerten, Stefanie A1 - Sundararaman, Srividya A1 - Lehmann, Paul V. T1 - Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity JF - Viruses N2 - Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood mononuclear cell (PBMC). Using the enzyme-linked Immunospot\(^{®}\) assay (ELISPOT) that excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)-\({\gamma}\) and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN-\({\gamma}\) and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the T\(_{H}\)1, T\(_{H}\)2, and T\(_{H}\)17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails. KW - memory cells KW - hcv infection KW - signature KW - Enzyme-Linked Immunospot assay (ELISPOT) KW - cytokine secretion kinetics KW - chronic viral infection KW - HCMV infection KW - CD4 T cells KW - exhaustion KW - activation KW - human cytomegalovirus (HCMV) KW - B cells KW - cytomegalovirus KW - elispot Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151462 VL - 7 SP - 4414 EP - 4437 ER - TY - JOUR A1 - Wurdack, Matthias A1 - Lundt, Nils A1 - Klaas, Martin A1 - Baumann, Vasilij A1 - Kavokin, Alexey V. A1 - Höfling, Sven A1 - Schneider, Christian T1 - Observation of hybrid Tamm-plasmon exciton-polaritons with GaAs quantum wells and a MoSe\(_{2}\) monolayer JF - Nature Communications N2 - Strong light matter coupling between excitons and microcavity photons, as described in the framework of cavity quantum electrodynamics, leads to the hybridization of light and matter excitations. The regime of collective strong coupling arises, when various excitations from different host media are strongly coupled to the same optical resonance. This leads to a well-controllable admixture of various matter components in three hybrid polariton modes. Here, we study a cavity device with four embedded GaAs quantum wells hosting excitons that are spectrally matched to the A-valley exciton resonance of a MoSe\(_{2}\) monolayer. The formation of hybrid polariton modes is evidenced in momentum resolved photoluminescence and reflectivity studies. We describe the energy and k-vector distribution of exciton-polaritons along the hybrid modes by a thermodynamic model, which yields a very good agreement with the experiment. KW - two-dimensional materials KW - microresonators KW - nanophotonics and plasmonics KW - cavity device KW - strong coupling Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170480 VL - 8 IS - 259 ER - TY - JOUR A1 - Wurm, Michael A1 - Stark, Thomas A1 - Zhu, Xiao Xiang A1 - Weigand, Matthias A1 - Taubenböck, Hannes T1 - Semantic segmentation of slums in satellite images using transfer learning on fully convolutional neural networks JF - ISPRS Journal of Photogrammetry and Remote Sensing N2 - Unprecedented urbanization in particular in countries of the global south result in informal urban development processes, especially in mega cities. With an estimated 1 billion slum dwellers globally, the United Nations have made the fight against poverty the number one sustainable development goal. To provide better infrastructure and thus a better life to slum dwellers, detailed information on the spatial location and size of slums is of crucial importance. In the past, remote sensing has proven to be an extremely valuable and effective tool for mapping slums. The nature of used mapping approaches by machine learning, however, made it necessary to invest a lot of effort in training the models. Recent advances in deep learning allow for transferring trained fully convolutional networks (FCN) from one data set to another. Thus, in our study we aim at analyzing transfer learning capabilities of FCNs to slum mapping in various satellite images. A model trained on very high resolution optical satellite imagery from QuickBird is transferred to Sentinel-2 and TerraSAR-X data. While free-of-charge Sentinel-2 data is widely available, its comparably lower resolution makes slum mapping a challenging task. TerraSAR-X data on the other hand, has a higher resolution and is considered a powerful data source for intra-urban structure analysis. Due to the different image characteristics of SAR compared to optical data, however, transferring the model could not improve the performance of semantic segmentation but we observe very high accuracies for mapped slums in the optical data: QuickBird image obtains 86–88% (positive prediction value and sensitivity) and a significant increase for Sentinel-2 applying transfer learning can be observed (from 38 to 55% and from 79 to 85% for PPV and sensitivity, respectively). Using transfer learning proofs extremely valuable in retrieving information on small-scaled urban structures such as slum patches even in satellite images of decametric resolution. KW - slums KW - FCN KW - convolutional neural networks KW - deep learning KW - transfer learning Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233799 VL - 150 ER - TY - JOUR A1 - Wurmb, T A1 - Schorscher, N A1 - Justice, P A1 - Dietz, S A1 - Schua, R A1 - Jarausch, T A1 - Kinstle, U A1 - Greiner, J A1 - Möldner, G A1 - Müller, J A1 - Kraus, M A1 - Simon, S A1 - Wagenhäuser, U A1 - Hemm, J A1 - Roewer, N A1 - Helm, M T1 - Structured analysis, evaluation and report of the emergency response to a terrorist attack in Wuerzburg, Germany using a new template of standardised quality indicators JF - Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine N2 - Background: Until now there has been a reported lack of systematic reports and scientific evaluations of rescue missions during terror attacks. This however is urgently required in order to improve the performance of emergency medical services and to be able to compare different missions with each other. Aim of the presented work was to report the systematic evaluation and the lessons learned from the response to a terror attack that happened in Wuerzburg, Germany in 2016. Methods: A team of 14 experts developed a template of quality indicators and operational characteristics, which allow for the description, assessment and comparison of civil emergency rescue missions during mass killing incidents. The entire systematic evaluation process consisted of three main steps. The first step was the systematic data collection according to the quality indicators and operational characteristics. Second was the systematic stratification and assessment of the data. The last step was the prioritisation of the identified weaknesses and the definition of the lessons learned. Results: Five important “lessons learned” have been defined. First of all, a comprehensive concept for rescue missions during terror attacks is essential. Furthermore, the establishment of a defined high priority communication infrastructure between the different dispatch centres (“red phone”) is vital. The goal is to secure the continuity of information between a few well-defined individuals. Thirdly, the organization of the incident scene needs to be commonly decided and communicated between police, medical services and fire services during the mission. A successful mission tactic requires continuous flux of reports to the on-site command post. Therefore, a predefined and common communication infrastructure for all operational forces is a crucial point. Finally, all strategies need to be extensively trained before the real life scenario hits. Conclusion: According to a systematic evaluation, we defined the lessons learned from a terror attack in 2016. Further systematic reports and academic work surrounding life threatening rescue missions and mass killing incidents are needed in order to ultimately improve such mission outcomes. In the future, a close international collaboration might help to find the best database to report and evaluate major incidents but also mass killing events. KW - terror attack KW - mass casualties KW - evaluation KW - quality indicators KW - rescue mission Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177054 VL - 26 IS - 87 ER - TY - JOUR A1 - Wurmb, Thomas Erik A1 - Schlereth, Stefan A1 - Kredel, Markus A1 - Muellenbach, Ralf M. A1 - Wunder, Christian A1 - Brederlau, Jörg A1 - Roewer, Norbert A1 - Kenn, Werner A1 - Kunze, Ekkehard T1 - Routine Follow-Up Cranial Computed Tomography for Deeply Sedated, Intubated, and Ventilated Multiple Trauma Patients with Suspected Severe Head Injury JF - BioMed Research International N2 - Background. Missed or delayed detection of progressive neuronal damage after traumatic brain injury (TBI) may have negative impact on the outcome. We investigated whether routine follow-up CT is beneficial in sedated and mechanically ventilated trauma patients. Methods. The study design is a retrospective chart review. A routine follow-up cCT was performed 6 hours after the admission scan. We defined 2 groups of patients, group I: patients with equal or recurrent pathologies and group II: patients with new findings or progression of known pathologies. Results. A progression of intracranial injury was found in 63 patients (42%) and 18 patients (12%) had new findings in cCT 2 (group II). In group II a change in therapy was found in 44 out of 81 patients (54%). 55 patients with progression or new findings on the second cCT had no clinical signs of neurological deterioration. Of those 24 patients (44%) had therapeutic consequences due to the results of the follow-up cCT. Conclusion. We found new diagnosis or progression of intracranial pathology in 54% of the patients. In 54% of patients with new findings and progression of pathology, therapy was changed due to the results of follow-up cCT. In trauma patients who are sedated and ventilated for different reasons a routine follow-up CT is beneficial. KW - Computertomographie Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120084 IS - 361949 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Franke, Axel A1 - Schorscher, Nora A1 - Kowalzik, Barbara A1 - Helm, Matthias A1 - Bohnen, Renate A1 - Helmerichs, Jutta A1 - Grueneisen, Ulrich A1 - Cwojdzinski, Detlef A1 - Jung, Georg A1 - Lücking, Gesa A1 - Weber, Martin T1 - Emergency response to terrorist attacks: results of the federal-conducted evaluation process in Germany JF - European Journal of Trauma and Emergency Surgery N2 - Purpose Rescue missions during terrorist attacks are extremely challenging for all rescue forces (police as well as non-police forces) involved. To improve the quality and safety of the rescue missions during an active killing event, it is obligatory to adapt common rescue mission goals and strategies. Methods After the recent attacks in Europe, the Federal Office of Civil Protection and Disaster Assistance started an evaluation process on behalf of the Federal Ministry of the Interior and the Federal Ministry of Health. This was done to identify weaknesses, lessons learned and to formulate new adapted guidelines. Results The presented bullet point recommendations summarise the basic and most important results of the ongoing evaluation process for the Federal Republic of Germany. The safety of all the rescue forces and survival of the greatest possible number of casualties are the priority goals. Furthermore, the preservation and re-establishment of the socio-political integrity are the overarching goals of the management of active killing events. Strategic incident priorities are to stop the killing and to save as much lives as possible. The early identification and prioritised transportation of casualties with life-threatening non-controllable bleeding are major tasks and the shortest possible on-scene time is an important requirement with respect to safety issues. Conclusion With respect to hazard prevention tactics within Germany, we attributed the highest priority impact to the bullet points. The focus of the process has now shifted to intense work about possible solutions for the identified deficits and implementation strategies of such solutions during mass killing incidents. KW - terror attack KW - mission strategies KW - lessons learned KW - first responders KW - safety Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231777 SN - 1863-9933 VL - 46 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Scholtes, Katja A1 - Kolibay, Felix A1 - Schorscher, Nora A1 - Ertl, Georg A1 - Ernestus, Ralf-Ingo A1 - Vogel, Ulrich A1 - Franke, Axel A1 - Kowalzik, Barbara T1 - Hospital preparedness for mass critical care during SARS-CoV-2 pandemic JF - Critical Care N2 - Mass critical care caused by the severe acute respiratory syndrome corona virus 2 pandemic poses an extreme challenge to hospitals. The primary goal of hospital disaster preparedness and response is to maintain conventional or contingency care for as long as possible. Crisis care must be delayed as long as possible by appropriate measures. Increasing the intensive care unit (ICU) capacities is essential. In order to adjust surge capacity, the reduction of planned, elective patient care is an adequate response. However, this involves numerous problems that must be solved with a sense of proportion. This paper summarises preparedness and response measures recommended to acute care hospitals. KW - Mass critical care KW - Disaster response KW - SARS-CoV-2 KW - Hospital emergency plan Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230201 VL - 24 ER - TY - JOUR A1 - Wurmb, Thomas A1 - Vollmer, Tina A1 - Sefrin, Peter A1 - Kraus, Martin A1 - Happel, Oliver A1 - Wunder, Christian A1 - Steinisch, Andrias A1 - Roewer, Norbert A1 - Maier, Sebastian T1 - Monitoring of in-hospital cardiac arrest events with the focus on Automated External Defibrillators – a retrospective observational study JF - Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine N2 - Background Patients with cardiac arrest have lower survival rates, when resuscitation performance is low. In In-hospital settings the first responders on scene are usually nursing staff without rhythm analysing skills. In such cases Automated External Defibrillators (AED) might help guiding resuscitation performance. At the Wuerzburg University Hospital (Germany) an AED-program was initiated in 2007. Aim of the presented study was to monitor the impact of Automated External Defibrillators on the management of in-hospital cardiac arrest events. Methods The data acquisition was part of a continuous quality improvement process of the Wuerzburg University Hospital. For analysing the CPR performance, the chest compression rate (CCR), compression depth (CCD), the no flow fraction (NFF), time interval from AED-activation to the first compression (TtC), the time interval from AED-activation to the first shock (TtS) and the post schock pause (TtCS) were determined by AED captured data. A questionnaire was completed by the first responders. Results From 2010 to 2012 there were 359 emergency calls. From these 53 were cardiac arrests with an AED-application. Complete data were available in 46 cases. The TtC was 34 (32–52) seconds (median and IQR).The TtS was 30 (28–32) seconds (median and IQR) . The TtCS was 4 (3–6) seconds (median and IQR) . The CCD was 5.5 ± 1 cm while the CCR was 107 ± 11/min. The NFF was calculated as 41 %. ROSC was achieved in 21 patients (45 %), 8 patients (17 %) died on scene and 17 patients (37 %) were transferred under ongoing CPR to an Intensive Care Unit (ICU). Conclusion The TtS and TtC indicate that there is an AED-user dependent time loss. These time intervals can be markedly reduced, when the user is trained to interrupt the AED’s “chain of advices” by placing the electrode-paddles immediately on the patient’s thorax. At this time the AED switches directly to the analysing mode. Intensive training and adaption of the training contents is needed to optimize the handling of the AED in order to maximize its advantages and to minimize its disadvantages. KW - cardio-pulmonary resuscitation KW - team-training KW - chest-compression rate KW - automated external defibrillators KW - in-hospital cardiac arrest Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125756 VL - 23 IS - 87 ER - TY - THES A1 - Wurst, Jan-Eric T1 - Hp-Finite Elements for PDE-Constrained Optimization N2 - Diese Arbeit behandelt die hp-Finite Elemente Methode (FEM) für linear quadratische Optimal-steuerungsprobleme. Dabei soll ein Zielfunktional, welches die Entfernung zu einem angestrebten Zustand und hohe Steuerungskosten (als Regularisierung) bestraft, unter der Nebenbedingung einer elliptischen partiellen Differentialgleichung minimiert werden. Bei der Anwesenheit von Steuerungsbeschränkungen können die notwendigen Bedingungen erster Ordnung, die typischerweise für numerische Lösungsverfahren genutzt werden, als halbglatte Projektionsformel formuliert werden. Folglich sind optimale Lösungen oftmals auch nicht-glatt. Die Technik der hp-Diskretisierung berücksichtigt diese Tatsache und approximiert raue Funktionen auf feinen Gittern, während Elemente höherer Ordnung auf Gebieten verwendet werden, auf denen die Lösung glatt ist. Die erste Leistung dieser Arbeit ist die erfolgreiche Anwendung der hp-FEM auf zwei verwandte Problemklassen: Neumann- und Interface-Steuerungsprobleme. Diese werden zunächst mit entsprechenden a-priori Verfeinerungsstrategien gelöst, mit der randkonzentrierten (bc) FEM oder interface konzentrierten (ic) FEM. Diese Strategien generieren Gitter, die stark in Richtung des Randes beziehungsweise des Interfaces verfeinert werden. Um für beide Techniken eine algebraische Reduktion des Approximationsfehlers zu beweisen, wird eine elementweise interpolierende Funktion konstruiert. Außerdem werden die lokale und globale Regularität von Lösungen behandelt, weil sie entscheidend für die Konvergenzgeschwindigkeit ist. Da die bc- und ic- FEM kleine Polynomgrade für Elemente verwenden, die den Rand beziehungsweise das Interface berühren, können eine neue L2- und L∞-Fehlerabschätzung hergeleitet werden. Letztere bildet die Grundlage für eine a-priori Strategie zum Aufdatieren des Regularisierungsparameters im Zielfunktional, um Probleme mit bang-bang Charakter zu lösen. Zudem wird die herkömmliche hp-Idee, die daraus besteht das Gitter geometrisch in Richtung der Ecken des Gebiets abzustufen, auf die Lösung von Optimalsteuerungsproblemen übertragen (vc-FEM). Es gelingt, Regularität in abzählbar normierten Räumen für die Variablen des gekoppelten Optimalitätssystems zu zeigen. Hieraus resultiert die exponentielle Konvergenz im Bezug auf die Anzahl der Freiheitsgrade. Die zweite Leistung dieser Arbeit ist die Entwicklung einer völlig adaptiven hp-Innere-Punkte-Methode, die Probleme mit verteilter oder Neumann Steuerung lösen kann. Das zugrundeliegende Barriereproblem besitzt ein nichtlineares Optimilitätssystem, das eine numerische Herausforderung beinhaltet: die stabile Berechnung von Integralen über Funktionen mit möglichen Singularitäten in Elementen höherer Ordnung. Dieses Problem wird dadurch gelöst, dass die Steuerung an den Integrationspunkten überwacht wird. Die Zulässigkeit an diesen Punkten wird durch einen Glättungsschritt garantiert. In dieser Arbeit werden sowohl die Konvergenz eines Innere-Punkte-Verfahrens mit Glättungsschritt als auch a-posteriori Schranken für den Diskretisierungsfehler gezeigt. Dies führt zu einem adaptiven Lösungsalgorithmus, dessen Gitterverfeinerung auf der Entwicklung der Lösung in eine Legendre Reihe basiert. Hierbei dient das Abklingverhalten der Koeffizienten als Glattheitsindikator und wird für die Entscheidung zwischen h- und p-Verfeinerung herangezogen. N2 - This thesis deals with the hp-finite element method (FEM) for linear quadratic optimal control problems. Here, a tracking type functional with control costs as regularization shall be minimized subject to an elliptic partial differential equation. In the presence of control constraints, the first order necessary conditions, which are typically used to find optimal solutions numerically, can be formulated as a semi-smooth projection formula. Consequently, optimal solutions may be non-smooth as well. The hp-discretization technique considers this fact and approximates rough functions on fine meshes while using higher order finite elements on domains where the solution is smooth. The first main achievement of this thesis is the successful application of hp-FEM to two related problem classes: Neumann boundary and interface control problems. They are solved with an a-priori refinement strategy called boundary concentrated (bc) FEM and interface concentrated (ic) FEM, respectively. These strategies generate grids that are heavily refined towards the boundary or interface. We construct an elementwise interpolant that allows to prove algebraic decay of the approximation error for both techniques. Additionally, a detailed analysis of global and local regularity of solutions, which is critical for the speed of convergence, is included. Since the bc- and ic-FEM retain small polynomial degrees for elements touching the boundary and interface, respectively, we are able to deduce novel error estimates in the L2- and L∞-norm. The latter allows an a-priori strategy for updating the regularization parameter in the objective functional to solve bang-bang problems. Furthermore, we apply the traditional idea of the hp-FEM, i.e., grading the mesh geometrically towards vertices of the domain, for solving optimal control problems (vc-FEM). In doing so, we obtain exponential convergence with respect to the number of unknowns. This is proved with a regularity result in countably normed spaces for the variables of the coupled optimality system. The second main achievement of this thesis is the development of a fully adaptive hp-interior point method that can solve problems with distributed or Neumann control. The underlying barrier problem yields a non-linear optimality system, which poses a numerical challenge: the numerically stable evaluation of integrals over possibly singular functions in higher order elements. We successfully overcome this difficulty by monitoring the control variable at the integration points and enforcing feasibility in an additional smoothing step. In this work, we prove convergence of an interior point method with smoothing step and derive a-posteriori error estimators. The adaptive mesh refinement is based on the expansion of the solution in a Legendre series. The decay of the coefficients serves as an indicator for smoothness that guides between h- and p-refinement. KW - Finite-Elemente-Methode KW - Optimale Kontrolle KW - Elliptische Differentialgleichung KW - finite elements KW - optimal control KW - higher order methods KW - partial differetial equations Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115027 SN - 978-3-95826-024-5 (print) SN - 978-3-95826-025-2 (online) PB - Würzburg University Press CY - Würzburg ER - TY - JOUR A1 - Wutzler, Alexander A1 - Krogias, Christos A1 - Grau, Anna A1 - Veltkamp, Roland A1 - Heuschmann, Peter U. A1 - Haeusler, Karl Georg T1 - Stroke prevention in patients with acute ischemic stroke and atrial fibrillation in Germany - a cross sectional survey JF - BMC Neurology N2 - Background Atrial fibrillation (AF) is present in 15–20% of patients with acute ischemic stroke. Oral anticoagulation reduces the risk of AF-related recurrent stroke but clinical guideline recommendations are rather vague regarding its use in the acute phase of stroke. We aimed to assess the current clinical practice of medical stroke prevention in AF patients during the acute phase of ischemic stroke. Methods In April 2017, a standardized anonymous questionnaire was sent to clinical leads of all 298 certified stroke units in Germany. Results Overall, 154 stroke unit leads participated (response rate 52%). Anticoagulation in the acute phase of stroke is considered feasible in more than 90% of AF patients with ischemic stroke. Clinicians assume that about two thirds of all AF patients (range 20–100%) are discharged on oral anticoagulation. According to local preferences, acetylsalicylic acid is given orally in the majority of patients with delayed initiation of oral anticoagulation. A non-vitamin K-dependent oral anticoagulant (NOAC) is more often prescribed than a vitamin K-dependent oral anticoagulant (VKA). VKA is more often chosen in patients with previous VKA intake than in VKA naive patients. In the minority of patients, stroke unit leads discuss the prescription of a specific oral anticoagulant with the treating general practitioner. Adherence to medical stroke prevention after hospital discharge is not assessed on a regular basis in any patient by the majority of participating stroke centers. Conclusions Early secondary stroke prevention in AF patients in German stroke units is based on OAC use but prescription modalities vary in clinical practice. KW - Ischemic stroke KW - Secondary stroke prevention KW - Atrial fibrillation KW - Survey KW - Oral anticoagulation KW - Stroke unit Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201078 VL - 19 ER - TY - JOUR A1 - Wußmann, Maximiliane A1 - Groeber-Becker, Florian Kai A1 - Riedl, Sabrina A1 - Alihodzic, Dina A1 - Padaric, Daniel A1 - Gerlitz, Lisa A1 - Stallinger, Alexander A1 - Liegl-Atzwanger, Bernadette A1 - Zweytick, Dagmar A1 - Rinner, Beate T1 - In model, in vitro and in vivo killing efficacy of antitumor peptide RDP22 on MUG-Mel2, a patient derived cell line of an aggressive melanoma metastasis JF - Biomedicines N2 - The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC\(_{50}\) of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo. KW - melanoma metastases KW - NRAS mutation KW - antitumor peptide KW - tumor model systems KW - phosphatidylserine Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297525 SN - 2227-9059 VL - 10 IS - 11 ER - TY - JOUR A1 - Wyborski, Paweł A1 - Musiał, Anna A1 - Mrowiński, Paweł A1 - Podemski, Paweł A1 - Baumann, Vasilij A1 - Wroński, Piotr A1 - Jabeen, Fauzia A1 - Höfling, Sven A1 - Sęk, Grzegorz T1 - InP-substrate-based quantum dashes on a DBR as single-photon emitters at the third telecommunication window JF - Materials N2 - We investigated emission properties of photonic structures with InAs/InGaAlAs/InP quantum dashes grown by molecular beam epitaxy on a distributed Bragg reflector. In high-spatial-resolution photoluminescence experiment, well-resolved sharp spectral lines are observed and single-photon emission is detected in the third telecommunication window characterized by very low multiphoton events probabilities. The photoluminescence spectra measured on simple photonic structures in the form of cylindrical mesas reveal significant intensity enhancement by a factor of 4 when compared to a planar sample. These results are supported by simulations of the electromagnetic field distribution, which show emission extraction efficiencies even above 18% for optimized designs. When combined with relatively simple and undemanding fabrication approach, it makes this kind of structures competitive with the existing solutions in that spectral range and prospective in the context of efficient and practical single-photon sources for fiber-based quantum networks applications. KW - single-photon emitter KW - III–V quantum dot KW - telecommunication spectral range KW - photonic structure KW - extraction efficiency Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228773 SN - 1996-1944 VL - 14 IS - 4 ER - TY - JOUR A1 - Wyborski, Paweł A1 - Podemski, Paweł A1 - Wroński, Piotr Andrzej A1 - Jabeen, Fauzia A1 - Höfling, Sven A1 - Sęk, Grzegorz T1 - Electronic and optical properties of InAs QDs grown by MBE on InGaAs metamorphic buffer JF - Materials N2 - We present the optical characterization of GaAs-based InAs quantum dots (QDs) grown by molecular beam epitaxy on a digitally alloyed InGaAs metamorphic buffer layer (MBL) with gradual composition ensuring a redshift of the QD emission up to the second telecom window. Based on the photoluminescence (PL) measurements and numerical calculations, we analyzed the factors influencing the energies of optical transitions in QDs, among which the QD height seems to be dominating. In addition, polarization anisotropy of the QD emission was observed, which is a fingerprint of significant valence states mixing enhanced by the QD confinement potential asymmetry, driven by the decreased strain with increasing In content in the MBL. The barrier-related transitions were probed by photoreflectance, which combined with photoluminescence data and the PL temperature dependence, allowed for the determination of the carrier activation energies and the main channels of carrier loss, identified as the carrier escape to the MBL barrier. Eventually, the zero-dimensional character of the emission was confirmed by detecting the photoluminescence from single QDs with identified features of the confined neutral exciton and biexciton complexes via the excitation power and polarization dependences. KW - molecular beam epitaxy KW - quantum dot KW - metamorphic buffer layer KW - band structure KW - photoluminescence KW - photoreflectance Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297037 SN - 1996-1944 VL - 15 IS - 3 ER - TY - JOUR A1 - Wyler, Emanuel A1 - Menegatti, Jennifer A1 - Franke, Vedran A1 - Kocks, Christine A1 - Boltengagen, Anastasiya A1 - Hennig, Thomas A1 - Theil, Kathrin A1 - Rutkowski, Andrzej A1 - Ferrai, Carmelo A1 - Baer, Laura A1 - Kermas, Lisa A1 - Friedel, Caroline A1 - Rajewsky, Nikolaus A1 - Akalin, Altuna A1 - Dölken, Lars A1 - Grässer, Friedrich A1 - Landthaler, Markus T1 - Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection JF - Genome Biology N2 - Background Herpesviruses can infect a wide range of animal species. Herpes simplex virus 1 (HSV-1) is one of the eight herpesviruses that can infect humans and is prevalent worldwide. Herpesviruses have evolved multiple ways to adapt the infected cells to their needs, but knowledge about these transcriptional and post-transcriptional modifications is sparse. Results Here, we show that HSV-1 induces the expression of about 1000 antisense transcripts from the human host cell genome. A subset of these is also activated by the closely related varicella zoster virus. Antisense transcripts originate either at gene promoters or within the gene body, and they show different susceptibility to the inhibition of early and immediate early viral gene expression. Overexpression of the major viral transcription factor ICP4 is sufficient to turn on a subset of antisense transcripts. Histone marks around transcription start sites of HSV-1-induced and constitutively transcribed antisense transcripts are highly similar, indicating that the genetic loci are already poised to transcribe these novel RNAs. Furthermore, an antisense transcript overlapping with the BBC3 gene (also known as PUMA) transcriptionally silences this potent inducer of apoptosis in cis. Conclusions We show for the first time that a virus induces widespread antisense transcription of the host cell genome. We provide evidence that HSV-1 uses this to downregulate a strong inducer of apoptosis. Our findings open new perspectives on global and specific alterations of host cell transcription by viruses. KW - Virology KW - Herpes KW - Virus KW - Antisense KW - Transcription KW - IncRNA KW - ICP4 KW - BBC3 KW - NFKB Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173381 VL - 18 ER - TY - JOUR A1 - Wächtler, Maria A1 - Kübel, Joachim A1 - Barthelmes, Kevin A1 - Winter, Andreas A1 - Schmiedel, Alexander A1 - Pascher, Torbjörn A1 - Lambert, Christoph A1 - Schubert, Ulrich S. A1 - Dietzek, Benjamin T1 - Energy transfer and formation of long-lived \(^3\)MLCT states in multimetallic complexes with extended highly conjugated bis-terpyridyl ligands JF - Physical Chemistry Chemical Physics N2 - Multimetallic complexes with extended and highly conjugated bis-2,2':6',2''-terpyridyl bridging ligands, which present building blocks for coordination polymers, are investigated with respect to their ability to act as light-harvesting antennae. The investigated species combine Ru(II)- with Os(II)- and Fe(II)-terpyridyl chromophores, the latter acting as energy sinks. Due to the extended conjugated system the ligands are able to prolong the lifetime of the \(^3\)MLCT states compared to unsubstituted terpyridyl species by delocalization and energetic stabilization of the \(^3\)MLCT states. This concept is applied for the first time to Fe(II) terpyridyl species and results in an exceptionally long lifetime of 23 ps for the Fe(II) \(^3\)MLCT state. While partial energy (>80%) transfer is observed between the Ru(II) and Fe(II) centers with a time-constant of 15 ps, excitation energy is transferred completely from the Ru(II) to the Os(II) center within the first 200 fs after excitation. KW - polypyridyl complexes KW - bis-terpyridyl ligands KW - multimetallic complexes KW - metal-to-ligand charge transfer (MLCT) KW - RU-(II) complexes KW - Ru(II)–Fe(II)–Ru(II) complex Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191041 VL - 18 IS - 4 ER - TY - THES A1 - Wäldchen, Felix T1 - 3D Single Molecule Imaging In Whole Cells Enabled By Lattice Light-Sheet Illumination T1 - 3D Einzelmolekülbildgebung in ganzen Zellen ermöglicht durch Gitterlichtblattbeleuchtung N2 - Single molecule localization microscopy has seen a remarkable growth since its first experimental implementations about a decade ago. Despite its technical challenges, it is already widely used in medicine and biology and is valued as a unique tool to gain molecular information with high specificity. However, common illumination techniques do not allow the use of single molecule sensitive super-resolution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) for whole cell imaging. In addition, they can potentially alter the quantitative information. In this thesis, I combine dSTORM imaging in three dimensions with lattice lightsheet illumination to gain quantitative molecular information from cells unperturbed by the illumination and cover slip effects. Lattice light-sheet illumination uses optical lattices for beam shaping to restrict the illumination to the detectable volume. I describe the theoretical background needed for both techniques and detail the experimental realization of the system as well as the software that I developed to efficiently evaluate the data. Eventually, I will present key datasets that demonstrate the capabilities of the developed microscope system with and without dSTORM. My main goal here was to use these techniques for imaging the neural cell adhesion molecule (NCAM, also known as CD56) in whole cells. NCAM is a plasma membrane receptor known to play a key role in biological processes such as memory and learning. Combining dSTORM and lattice light-sheet illumination enables the collection of quantitative data of the distribution of molecules across the whole plasma membrane, and shows an accumulation of NCAM at cell-cell interfaces. The low phototoxicity of lattice light-sheet illumination further allows for tracking individual NCAM dimers in living cells, showing a significant dependence of its mobility on the actin skeleton of the cell. N2 - Die Einzelmoleküllokalisationsmikroskopie hat seit der ersten experimentellen Umsetzung vor etwa 10 Jahren einen bemerkenswerten Aufschwung erfahren. Trotz des hohen technischen Anspruchs findet sie bereits weite Verbreitung in der Biologie und Medizin und wird als einzigartiges Werkzeug geschätzt, um molekulare Information mit hoher Spezifität zu erlangen. Dennoch erschweren die gebräuchlichen Beleuchtungsmethoden die Anwendung von Methoden der Einzelmoleküllokalisationsmikroskopie wie dSTORM (engl. direct stochastic optical reconstruction microscopy) auf das Volumen ganzer Zellen, denn hier kann die Beleuchtung selbst die quantitativen Daten beeinflussen. In dieser Arbeit kombiniere ich dreidimensionale dSTORM-Bildgebung mit Gitterlichtblattbeleuchtung (engl. lattice light-sheet illumination) um quantitative, molekulare Information ohne durch die Beleuchtung verursachte Störungen zu gewinnen. Die Gitterlichtblattbeleuchtung nutzt optische Gitter zur Strahlformung, um das beleuchtete Volumen auf das detektierbare Volumen zu beschränken. Ich stelle den nötigen, theoretischen Hintergrund für beide Methoden dar und beschreibe die experimentelle Umsetzung sowie die von mir zur effizienten Datenauswertung entwickelte Software. Schließlich präsentiere ich verschiedene Datensätze, die die Fähigkeiten des Systems mit und ohne dSTORM demonstrieren. Mein Hauptziel war hierbei, beide Methoden zu nutzen, um das neuronale Zelladhäsionsmolekül (NCAM, engl. neural cell adhesion molecule) in ganzen Zellen abzubilden. NCAM (auch bekannt als CD56) ist ein Rezeptor auf der Plasmembran, der für seine Schlüsselrolle im Zusammenhang mit biologischen Prozessen wie Lernen und Gedächtnis bekannt ist. Die Kombination von dSTORM und Gitterlichtblattbeleuchtung ermöglicht das sammeln quantitativer Daten der Verteilung über die komplette Plasmamembran, wobei sich eine Akkumulation an Zell-Zell Kontaktflächen zeigt. Die niedrige Photoschädigung der Gitterlichtblattbeleuchtung ermöglicht weiterhin das Verfolgen von einzelnen NCAM-Dimeren in lebenden Zellen. Dort zeigt sich eine signifikante Abhängigkeit ihrer Mobilität vom Aktinskelett der Zelle. KW - Einzelmolekülmikroskopie KW - Optik KW - Light-Sheet KW - Lattice Light-Sheet KW - dSTORM KW - Single Molecule Imaging KW - Localization Microscopy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-207111 ER - TY - JOUR A1 - Wäldchen, Sina A1 - Lehmann, Julian A1 - Klein, Teresa A1 - van de Linde, Sebastian A1 - Sauer, Markus T1 - Light-induced cell damage in live-cell super-resolution microscopy JF - Scientific Reports N2 - Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of similar to 1 kW cm\(^{-2}\) at 640 nm for several minutes, the maximum dose at 405 nm is only similar to 50 J cm\(^{-2}\), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. KW - optical reconstruction microscopy KW - tag fusion proteins KW - localization microscopy KW - photodynamic therapy KW - diffraction limit KW - illumination microscopy KW - structured illumination KW - fluorescent probes KW - in vitro KW - dynamics Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145207 VL - 5 IS - 15348 ER - TY - JOUR A1 - Wäschke, Nicole A1 - Hardge, Kerstin A1 - Hancock, Christine A1 - Hilker, Monika A1 - Obermaier, Elisabeth A1 - Meiners, Torsten T1 - Odour Environments: How Does Plant Diversity Affect Herbivore and Parasitoid Orientation? JF - PlOS ONE N2 - Plant diversity is known to affect success of host location by pest insects, but its effect on olfactory orientation of non-pest insect species has hardly been addressed. First, we tested in laboratory experiments the hypothesis that non-host plants, which increase odour complexity in habitats, affect the host location ability of herbivores and parasitoids. Furthermore, we recorded field data of plant diversity in addition to herbivore and parasitoid abundance at 77 grassland sites in three different regions in Germany in order to elucidate whether our laboratory results reflect the field situation. As a model system we used the herb Plantago lanceolata, the herbivorous weevil Mecinus pascuorum, and its larval parasitoid Mesopolobus incultus. The laboratory bioassays revealed that both the herbivorous weevil and its larval parasitoid can locate their host plant and host via olfactory cues even in the presence of non-host odour. In a newly established two-circle olfactometer, the weevils capability to detect host plant odour was not affected by odours from non-host plants. However, addition of non-host plant odours to host plant odour enhanced the weevils foraging activity. The parasitoid was attracted by a combination of host plant and host volatiles in both the absence and presence of non-host plant volatiles in a Y-tube olfactometer. In dual choice tests the parasitoid preferred the blend of host plant and host volatiles over its combination with non-host plant volatiles. In the field, no indication was found that high plant diversity disturbs host (plant) location by the weevil and its parasitoid. In contrast, plant diversity was positively correlated with weevil abundance, whereas parasitoid abundance was independent of plant diversity. Therefore, we conclude that weevils and parasitoids showed the sensory capacity to successfully cope with complex vegetation odours when searching for hosts. KW - dentichasmias busseolae KW - nonhost plant KW - volatiles KW - selection KW - invertebrate herbivory KW - location behavior KW - foraging behavior KW - background odor KW - natural enemies Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117687 SN - 1932-6203 VL - 9 IS - 1 ER - TY - JOUR A1 - Wölfel, Angela A1 - Sättele, Mathias A1 - Zechmeister, Christina A1 - Nikolaev, Viacheslov O. A1 - Lohse, Martin J. A1 - Boege, Fritz A1 - Jahns, Roland A1 - Boivin-Jahns, Valérie T1 - Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure JF - ESC Heart Failure N2 - Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine–serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211–214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211–214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF. KW - antibody/autoantibody KW - β1-adrenoceptor/β1-adrenergic receptor KW - chronic heart failure KW - conformational auto-epitope KW - cyclic peptides/cyclopeptides KW - cyclopeptide therapy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235974 VL - 7 IS - 4 ER - TY - JOUR A1 - Wölfling, Mirko A1 - Becker, Mira C. A1 - Uhl, Britta A1 - Traub, Anja A1 - Fiedler, Konrad T1 - How differences in the settling behaviour of moths (Lepidoptera) may contribute to sampling bias when using automated light traps JF - European Journal of Entomology N2 - Quantitative community-wide moth surveys frequently employ flight-interception traps equipped with UV-light emitting sources as attractants. It has long been known that moth species differ in their responsiveness to light traps. We studied how the settling behaviour of moths at a light trap may further contribute to sampling bias. We observed the behaviour of 1426 moths at a light tower. Moths were classified as either, settling and remaining still after arrival, or continually moving on the gauze for extended periods of time. Moths that did not move after settling may not end up in the sampling container of the light trap and therefore are under-represented in automated trap samples relative to their true proportions in the community. Our analyses revealed highly significant behavioural differences between moths that differed in body size. Small moths were more likely to remain stationary after settling. As a corollary, representatives of three taxa, which in Europe are predominantly small species (Nolidae, Geometridae: Eupitheciini, Erebidae: Lithosiini), usually settled down immediately, whereas most other moths remained active on or flying around the trap for some time. Moth behaviour was also modulated by ambient temperature. At high temperatures, they were less likely to settle down immediately, but this behavioural difference was most strongly apparent among medium-sized moths. These results indicate the likely extent of the sampling bias when analysing and interpreting automated light-trap samples. Furthermore, to control for temperature modulated sampling bias temperature should always be recorded when sampling moths using flight-interception traps. KW - Lepidoptera KW - moths KW - biodiversity assessment KW - sampling method KW - light-trapping KW - sampling bias Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191154 VL - 113 ER - TY - JOUR A1 - Wölk, Julian A1 - Sütterlin, Stefan A1 - Koch, Stefan A1 - Vögele, Claus A1 - Schulz, Stefan M. T1 - Enhanced cardiac perception predicts impaired performance in the Iowa Gambling Task in patients with panic disorder JF - Brain and Behavior N2 - OBJECTIVE: Somatic marker theory predicts that somatic cues serve intuitive decision making; however, cardiovascular symptoms are threat cues for patients with panic disorder (PD). Therefore, enhanced cardiac perception may aid intuitive decision making only in healthy individuals, but impair intuitive decision making in PD patients. METHODS: PD patients and age-and sex-matched volunteers without a psychiatric diagnosis (n=17, respectively) completed the Iowa Gambling Task (IGT) as a measure of intuitive decision making. Interindividual differences in cardiac perception were assessed with a common mental-tracking task. RESULTS: In line with our hypothesis, we found a pattern of opposing associations (Fisher's Z=1.78, P=0.04) of high cardiac perception with improved IGT-performance in matched control-participants (r=0.36, n=14) but impaired IGT-performance in PD patients (r=-0.38, n=13). CONCLUSION: Interoceptive skills, typically assumed to aid intuitive decision making, can have the opposite effect in PD patients who experience interoceptive cues as threatening, and tend to avoid them. This may explain why PD patients frequently have problems with decision making in everyday life. Screening of cardiac perception may help identifying patients who benefit from specifically tailored interventions. KW - panic disorder KW - somatic marker hypothesis KW - cardiac perception KW - Iowa Gambling Task KW - decision making KW - interoception Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119865 SN - 2162-3279 VL - 4 IS - 2 ER - TY - JOUR A1 - Wörsdörfer, Philipp A1 - Dalda, Nahide A1 - Kern, Anna A1 - Krüger, Sarah A1 - Wagner, Nicole A1 - Kwok, Chee Keong A1 - Henke, Erik A1 - Ergün, Süleyman T1 - Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells JF - Scientific Reports N2 - Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and disease. We propose, that the directed incorporation of mesodermal progenitor cells (MPCs) into organoids will overcome the aforementioned limitations. In order to demonstrate the feasibility of the method, we generated complex human tumor as well as neural organoids. We show that the formed blood vessels display a hierarchic organization and mural cells are assembled into the vessel wall. Moreover, we demonstrate a typical blood vessel ultrastructure including endothelial cell-cell junctions, a basement membrane as well as luminal caveolae and microvesicles. We observe a high plasticity in the endothelial network, which expands, while the organoids grow and is responsive to anti-angiogenic compounds and pro-angiogenic conditions such as hypoxia. We show that vessels within tumor organoids connect to host vessels following transplantation. Remarkably, MPCs also deliver Iba1\(^+\) cells that infiltrate the neural tissue in a microglia-like manner. KW - Developmental biology KW - Stem cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202681 VL - 9 ER - TY - JOUR A1 - Wörsdörfer, Philipp A1 - Ergün, Süleyman T1 - “Organoids”: insights from the first issues JF - Organoids N2 - No abstract available KW - organoids KW - editorial Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313694 SN - 2674-1172 VL - 2 IS - 2 SP - 79 EP - 81 ER - TY - JOUR A1 - Wörsdörfer, Philipp A1 - I, Takashi A1 - Asahina, Izumi A1 - Sumita, Yoshinori A1 - Ergün, Süleyman T1 - Do not keep it simple: recent advances in the generation of complex organoids JF - Journal of Neural Transmission N2 - 3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models. KW - organoid KW - stroma KW - sasculature KW - neural KW - microglia KW - blood vessel Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235628 SN - 0300-9564 VL - 127 ER - TY - JOUR A1 - Wünsch, Anna Chiara A1 - Ries, Elena A1 - Heinzelmann, Sina A1 - Frabschka, Andrea A1 - Wagner, Peter Christoph A1 - Rauch, Theresa A1 - Koderer, Corinna A1 - El-Mesery, Mohamed A1 - Volland, Julian Manuel A1 - Kübler, Alexander Christian A1 - Hartmann, Stefan A1 - Seher, Axel T1 - Metabolic silencing via methionine-based amino acid restriction in head and neck cancer JF - Current Issues in Molecular Biology N2 - In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines. KW - amino acid restriction KW - caloric restriction KW - methionine KW - HNSCC KW - SCCHN KW - cisplatin KW - amino acid transporter KW - SLC-family KW - cell vitality KW - low energy metabolism Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-319257 SN - 1467-3045 VL - 45 IS - 6 SP - 4557 EP - 4573 ER - TY - THES A1 - Würtemberger-Pietsch, Sabrina T1 - Anionic and Neutral Lewis-Base Adducts of Diboron(4) Compounds T1 - Anionische und Neutrale Lewis-Basen Addukte von Diboran(4)-Verbindungen N2 - Anionic Adducts Sp2-sp3 tetraalkoxy diboron compounds have gained attention due to the development of new, synthetically useful catalytic reactions either with or without transition-metals. Lewis-base adducts of the diboron(4) compounds were suggested as possible intermediates in Cu catalyzed borylation reactions some time ago. However, intermolecular adducts of tetraalkoxy diboron compounds have not been studied yet in great detail. In preliminary studies, we have synthesized a series of anionic sp2-sp3 adducts of B2pin2 with alkoxy-groups (L = [OMe]–, [OtBu]–), a phenoxy-group (L = [4-tBuC6H4O]–) and fluoride (L = [F]–, with [nBu4N]+ as the counter ion) as Lewis-bases. Neutral Adducts Since their isolation and characterization, applications of N-heterocyclic carbenes (NHCs) and related molecules, e.g., cyclic alkylaminocarbenes (CAACs) and acyclic diaminocarbenes (aDCs), have grown rapidly. Their use as ligands in homogeneous catalysis and directly in organocatalysis, including recently developed borylation reactions, is now well established. Recently, several examples of ring expansion reactions (RER) involving NHCs were reported to take place at elevated temperatures, involving Be, B, and Si. Furthermore, preliminary studies in the group of Marder et al. showed the presence of neutral sp2-sp3 diboron compounds with B2pin2 and the NHC Cy2Im. In this work, we focused on the synthesis and characterization of further neutral sp2-sp3 as well as sp3-sp3 diboron adducts with B2cat2 and B2neop2 and different NHCs. Whereas the mono-NHC adduct is stable for several hours at temperatures up to 60 °C, the bis-NHC adducts undergo thermally induced rearrangement to form the ring expanded products compound 26 and 27. B2neop2 is much more reactive than B2cat2 giving ring expanded product 29 at room temperature in quantitative yields, demonstrating that NHC ring expansion and B–B bond cleavage can be very facile processes. Whereas the mono-NHC adduct is stable for several hours at temperatures up to 60 °C, the bis-NHC adducts undergo thermally induced rearrangement to form the ring expanded products compound 26 and 27. B2neop2 is much more reactive than B2cat2 giving ring expanded product 29 at room temperature in quantitative yields, demonstrating that NHC ring expansion and B–B bond cleavage can be very facile processes. N2 - Im Rahmen der vorliegenden Arbeit wurde die Synthese und das Reaktionsverhalten Lewis-Säuren/Lewis-Basen-Addukte von Diboran(4)-Verbindungen als Lewis-Säuren untersucht. Als Lewis-Basen dienten zum einem das Fluorid-Ion, zum anderen N-Heterozyklische Carbene. Ein Ziel der vorliegenden Arbeit war somit die Synthese und Charakterisierung anionischer sp2-sp3-Diboran-Verbindungen des Typs [B2(OR)4F][NMe4] (OR2 = Pinakol, Catechol und Neopentyl), die auf ihre Eigenschaft als „Boryl-Übertragungsreagenz“ gegenüber Diazoniumsalzen überprüft wurden. Der zweite Teil der Arbeit untersucht die Reaktion von Diboranen (B2cat2 und B2neop2) mit gesättigten und ungesättigten N-Heterozyklischen Carbenen (NHCs). Die neutralen, einfach- und zweifach-substituierten NHC-Addukte des Typs B2(OR)4•NHC und B2(OR)4•(NHC)2 wurden anschließend auf ihre thermische Stabilität untersucht. Die Ergebnisse dieser Arbeit zeigen zum einem, dass anionische Addukte des Typs [B2(OR)4F][NMe4] 4, 7 und 9 als „Boryl-Übertragungsreagenzien“ eingesetzt werden können. Ferner lassen sich ausgehend von Diboran(4)-Verbindungen durch die Umsetzung mit N Heterozyklischen Carbenen die einfach- und zweifach-substituierten NHC-Addukte B2(OR)4•NHC und B2(OR)4•(NHC)2 synthetisieren. Diese sind zum Teil instabil gegenüber einer Ringerweiterungsreaktion unter Insertion einer Boryleinheit in die C–N-Bindung des Carbens. Untersuchungen an NHC-Addukten von Boranen BR3 und HB(OR)2 zeigen weiterhin, dass die Addukte Ph3B•NHC gegenüber solchen Ringerweiterungen stabil sind. Die Addukte HB(OR)2•NHC sind je nach eingesetztem Carben und Boran entweder stabil oder reagieren unter B–H-Bindungsaktivierung zur Ringerweiterung des Carbens. KW - Addukt KW - Diborane KW - carben KW - Lewis-Base Adducts KW - Diboron(4) Compounds KW - Ring Expansion Reaction Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-136321 ER - TY - JOUR A1 - Würthner, Frank A1 - Meza-Chincha, Ana-Lucia A1 - Schindler, Dorothee A1 - Natali, Mirco T1 - Effects of Photosensitizers and Reaction Media on Light‐Driven Water Oxidation with Trinuclear Ruthenium Macrocycles JF - ChemPhotoChem N2 - Photocatalytic water oxidation is a promising process for the production of solar fuels and the elucidation of factors that influence this process is of high significance. Thus, we have studied in detail light‐driven water oxidation with a trinuclear Ru(bda) (bda: 2,2’‐bipyridine‐6,6’‐dicarboxylate) macrocycle MC3 and its highly water soluble derivative m‐CH\(_2\)NMe\(_2\)‐MC3 using a series of ruthenium tris(bipyridine) complexes as photosensitizers under varied reaction conditions. Our investigations showed that the catalytic activities of these Ru macrocycles are significantly affected by the choice of photosensitizer (PS) and reaction media, in addition to buffer concentration, light intensity and concentration of the sensitizer. Our steady‐state and transient spectroscopic studies revealed that the photocatalytic performance of trinuclear Ru(bda) macrocycles is not limited by their intrinsic catalytic activities but rather by the efficiency of photogeneration of oxidant PS\(^+\) and its ability to act as an oxidizing agent to the catalysts as both are strongly dependent on the choice of photosensitizer and the amount of employed organic co‐solvent. KW - photosenitizers KW - water oxidation KW - ruthenium complexes KW - macrocycles KW - trinuclear KW - homogenous catalysis KW - photocatalysis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230116 VL - 5 IS - 2 ER - TY - JOUR A1 - Würthner, Frank A1 - Noll, Niklas T1 - A Calix[4]arene‐Based Cyclic Dinuclear Ruthenium Complex for Light‐Driven Catalytic Water Oxidation JF - Chemistry - A European Journal N2 - A cyclic dinuclear ruthenium(bda) (bda: 2,2’‐bipyridine‐6,6’‐dicarboxylate) complex equipped with oligo(ethylene glycol)‐functionalized axial calix[4]arene ligands has been synthesized for homogenous catalytic water oxidation. This novel Ru(bda) macrocycle showed significantly increased catalytic activity in chemical and photocatalytic water oxidation compared to the archetype mononuclear reference [Ru(bda)(pic)\(_2\)]. Kinetic investigations, including kinetic isotope effect studies, disclosed a unimolecular water nucleophilic attack mechanism of this novel dinuclear water oxidation catalyst (WOC) under the involvement of the second coordination sphere. Photocatalytic water oxidation with this cyclic dinuclear Ru complex using [Ru(bpy)\(_3\)]Cl\(_2\) as a standard photosensitizer revealed a turnover frequency of 15.5 s\(^{−1}\) and a turnover number of 460. This so far highest photocatalytic performance reported for a Ru(bda) complex underlines the potential of this water‐soluble WOC for artificial photosynthesis. KW - water KW - oxidation KW - ruthenium KW - dinuclear KW - catalytic KW - artificial photosynthesis KW - homogenous catalysis KW - photocatalysis KW - ruthenium complexes KW - water oxidation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230030 UR - https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202004486 VL - 27 IS - 1 ER - TY - THES A1 - Xavier de Souza, Aline T1 - Ecophysiological adaptations of the cuticular water permeability within the Solanaceae family T1 - Ökophysiologische Anpassungen der kutikulären Wasserpermeabilität innerhalb der Solanaceae Familie N2 - The cuticle, a complex lipidic layer synthesized by epidermal cells, covers and protects primary organs of all land plants. Its main function is to avoid plant desiccation by limiting non-stomatal water loss. The cuticular properties vary widely among plant species. So far, most of the cuticle-related studies have focused on a limited number of species, and studies addressing phylogenetically related plant species are rare. Moreover, comparative studies among organs from the same plant species are still scarce. Thus, this study focus on organ-specificities of the cuticle within and between plant species of the Solanaceae family. Twenty-seven plant species of ten genera, including cultivated and non- cultivated species, were investigated to identify potential cuticular similarities. Structural, chemical and functional traits of fully expanded leaves, inflated fruiting calyces, and ripe fruits were analyzed. The surface morphology was investigated by scanning electron microscopy. Leaves were mainly amphistomatic and covered by an epicuticular wax film. The diversity and distribution of trichomes varied among species. Only the leaves of S. grandiflora were glabrous. Plant species of the Leptostemonum subgenus had numerous prickles and non-glandular stellate trichomes. Fruits were stomata-free, except for S. muricatum, and a wax film covered their surface. Last, lenticel- like structures and remaining scars of broken trichomes were found on the surface of some Solanum fruits. Cuticular water permeability was used as indicators of the cuticular transpiration barrier efficiency. The water permeability differed among plant species, organs and fruit types with values ranging up to one hundred-fold. The minimum leaf conductance ranged from 0.35 × 10-5 m s-1 in S. grandiflora to 31.54 × 10-5 m s-1 in S. muricatum. Cuticular permeability of fruits ranged from 0.64 × 10-5 m s-1 in S. dulcamara (fleshy berry) to 34.98 × 10-5 m s-1 in N. tabacum (capsule). Generally, the cuticular water loss of dry fruits was about to 5-fold higher than that of fleshy fruits. Interestingly, comparisons between cultivated and non-cultivated species showed that wild species have the most efficient cuticular transpiration barrier in leaves and fruits. The average permeability of leaves and fruits of wild plant species was up to three-fold lower in comparison to the cultivated ones. Moreover, ripe fruits of P. ixocarpa and P. peruviana showed two-times lower cuticular transpiration when enclosed by the inflated fruiting calyx. The cuticular chemical composition was examined using gas chromatography. Very-long-chain aliphatic compounds primarily composed the cuticular waxes, being mostly dominated by n- alkanes (up to 80% of the total wax load). Primary alkanols, alkanoic acids, alkyl esters and branched iso- and anteiso-alkanes were also frequently found. Although in minor amounts, sterols, pentacyclic triterpenoids, phenylmethyl esters, coumaric acid esters, and tocopherols were identified in the cuticular waxes. Cuticular wax coverages highly varied in solanaceous (62- fold variation). The cuticular wax load of fruits ranged from 0.55 μg cm−2 (Nicandra physalodes) to 33.99 μg cm−2 (S. pennellii), whereas the wax amount of leaves varied from 0.90 μg cm−2 (N. physalodes) to 28.42 μg cm−2 (S. burchellii). Finally, the wax load of inflated fruiting calyces ranged from 0.56 μg cm−2 in P. peruviana to 2.00 μg cm−2 in N. physalodes. For the first time, a comparative study on the efficiency of the cuticular transpiration barrier in different plant organs of closely related plant species was conducted. Altogether, the cuticular chemical variability found in solanaceous species highlight species-, and organ-specific wax biosynthesis. These chemical variabilities might relate to the waterproofing properties of the plant cuticle, thereby influencing leaf and fruit performances. Additionally, the high cuticular water permeabilities of cultivated plant species suggest a potential existence of a trade-off between fruit organoleptic properties and the efficiency of the cuticular transpiration barrier. Last, the high cuticular water loss of the solanaceous dry fruits might be a physiological adaptation favouring seed dispersion. N2 - Die Kutikula, eine von Epidermiszellen gebildete, komplexe Lipidschicht, bedeckt und schützt die primären Organe niederer und höherer Landpflanzen. Ihre Hauptfunktion besteht darin, die Austrocknung der Pflanzen zu vermeiden, indem der nicht-stomatäre Wasserverlust an die Atmosphäre begrenzt wird. Ihre Eigenschaften können je nach Pflanzenart stark variieren, dennoch wurden kutikuläre Charakteristika unter phylogenetisch nah verwandten Pflanzenarten bisher wenig diskutiert. Die meisten Studien im Zusammenhang mit der Kutikula fokussierten sich auf eine begrenzte Anzahl von Pflanzenarten. Vergleichsstudien zwischen Organen derselben Pflanzenart sind kaum vorhanden. Die vorliegende Studie konzentriert sich daher auf die Kutikula von Pflanzenarten aus der Familie der Solanaceae und deren Organe. Die kutikulären Eigenschaften von 27 Pflanzenarten aus zehn verschiedenen Pflanzengattungen wurden untersucht, einschließlich Wild- und Kulturarten. Strukturelle, chemische und funktionelle Merkmale wurden für vollständig entwickelte Blätter, vergrößerte Blütenkelche und reife Früchte vergleichend analysiert. Die Oberflächenmorphologie wurde mit Hilfe der Rasterelektronenmikroskopie untersucht. Strukturell wiesen die meisten Blätter eine amphistomatische Oberfläche auf, die mit einem epikutikulären Wachsfilm bedeckt war, wobei einfache Trichome und Trichome mit Drüsen eine artspezifische Verteilung und Vielfalt aufzeigten. Bei den meisten Blättern der Untergattung Leptostemonum wurden Stacheln und zahlreiche sternenförmige Trichome beobachtet. Allein Solandra grandiflora hatte eine Blattoberfläche ohne Trichome. Früchte zeichneten sich hauptsächlich durch einen epikutikulären Wachsfilm aus, der ihre Oberfläche bedeckte. Als einzige Pflanzenart besaß Solanum muricatum auf der Fruchtoberfläche Stomata, dennoch wurden Lentizellen und Fragmente von Trichomen auf der Fruchtoberfläche von Solanum tuberosum, Solanum quitoense und Solanum lycopersicum gefunden. Für die Effizienzbestimmung der kutikulären Transpirationsbarriere von Oberflächen mit und ohne Stomata wurden die minimale Wasserleitfähigkeit unter Bedingungen des maximalen Stomaschlusses beziehungsweise die kutikuläre Wasserpermeabilität untersucht. Dieses ergab ein art-, organ- und fruchttypspezifisches Muster. Die Werte variierten zwischen den Pflanzenarten bis zu hundertfach und lagen zwischen 10-6 m s-1 und 10-4 m s-1. Im Gegensatz zu den Ergebnissen früherer Studien zeigte der Vergleich der Wasserpermeabilität von verschiedenen Organen derselben Pflanzenarten, dass eine höhere Wasserpermeabilität für Blätter oder für Früchte gefunden werden kann oder dass sie für beide Organe nahezu gleich sein kann. Die minimale Wasserleitfähigkeit der Blätter lag im Bereich von 0.35 × 10–5 m s–1 für S. grandiflora bis 31.54 × 10–5 m s–1 für S. muricatum. Die kutikuläre Wasserpermeabilität lag im Bereich von 0.64 × 10–5 m s –1 für fleischige Früchte von Solanum dulcamara bis 34.98 × 10–5 m s–1 für Kapselfrüchte von Nicotiana tabacum. Allgemein zeigte sich, dass trockene Früchte eine etwa fünffach höhere kutikuläre Wasserpermeabilität als fleischige Früchte besaßen. Interessanterweise zeigten Vergleiche zwischen Wild- und Kulturarten, dass Wildarten eine wirksamere kutikuläre Transpirationsbarriere der Blätter und Früchte aufwiesen, da ihre Wasserpermeabilität etwa zwei- bis dreifach niedriger war als die der kultivierten Pflanzenarten. Des Weiteren zeigten Physalis ixocarpa und Physalis peruviana, deren Früchte von einem vergrößerten Blütenkelch umschlossen waren, einen schützenden Einfluss dieses Blütenkelches auf die reife Frucht. Eine Reduktion der kutikulären Wasserpermeabilität um den Faktor zwei wurde nachgewiesen. Die chemische Zusammensetzung der kutikulären Transpirationsbarriere wurde mit Hilfe der Gaschromatographie detektiert. Die Analysen ergaben art- und organspezifische Mengen und Zusammensetzungen der kutikulären Wachse, die vor allem aus sehr langkettigen aliphatischen Verbindungen bestanden. Bis zu 80% der kutikulären Wachszusammensetzung bildete die Stoffklasse der n-Alkane. Andere häufig identifizierte Stoffklassen waren primäre Alkanole, Alkansäuren, Alkylester sowie iso- und anteiso-Alkane. Obwohl in geringen Mengen, wurden in den meisten kutikulären Wachsen auch alicyclische und aromatische Stoffklassen gefunden. Hauptsächlich handelte es sich um Phytosterole, pentacyclische Triterpenoide, Phenylmethylester, Cumarsäureester, Tocopherole und Flavonoide. Die kutikuläre Wachsschicht variierte zwischen den Pflanzenarten bis zu 62-fach und betrug zwischen 0.55 μg cm-2 für Nicandra physalodes und 33.99 μg cm-2 für Solanum pennellii, wobei sowohl niedrigste als auch höchste kutikuläre Wachsmenge für Früchte gefunden wurde. Die kutikulären Wachse der Blätter reichten von 0.90 μg cm-2 für N. physalodes bis 28.42 μg cm-2 für Solanum burchellii. Die kutikuläre Wachsmenge der vergrößerten Blütenkelche lag zwischen 0.56 μg cm-2 für P. peruviana und 2.00 μg cm-2 für N. physalodes. Zum ersten Mal wurde eine umfangreiche Studie zur Effizienz der kutikulären Transpirationsbarriere verschiedener Pflanzenorgane von phylogenetisch nah verwandten Pflanzenarten durchgeführt. Insgesamt zeigt die vergleichende Untersuchung innerhalb der Familie der Solanaceae die funktionelle und chemische Variabilität der kutikulären Wasserpermeabilität und der kutikulären Wachsbiosynthese. Die art- und organspezifische Divergenz kann dabei einen Einfluss auf die hydrophoben Eigenschaften der Kutikula haben und wichtige Konsequenzen für die Blatt- und Fruchtleistung mit sich führen. Darüber hinaus deuten diese Ergebnisse auf einen Kompromiss zwischen Fruchteigenschaften und Oberflächenschutz bei den Kulturarten hin. Es wird auch vermutet, dass die verringerte kutikuläre Barriereleistung der trockenen Früchte eine physiologische Anpassung an die Samenausbreitung dieser Pflanzenarten ist. KW - Kutikula KW - Transpiration KW - Kutikularwachs KW - Permeabilität KW - Nachtschattengewächse KW - permeability KW - water loss KW - permeance KW - cuticular waxes KW - minimum leaf conductance KW - fruit cuticle KW - leaf cuticle KW - Solanaceae KW - Solanum species KW - cuticular transpiration barrier Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225395 ER - TY - THES A1 - Xian, Yibo T1 - Identification of essential genes and novel virulence factors of Neisseria gonorrhoeae by transposon mutagenesis T1 - Identifizierung von essentiellen Genen und neuen Virulenzfaktoren von Neisseria gonorrhoeae durch Transposonmutagenese N2 - Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea. It is defined as a super bacterium by the WHO due to the emergence of gonococci that are resistant to a variety of antibiotics and a rapidly increasing infection incidence. Genome-wide investigation of neisserial gene essentiality and novel virulence factors is urgently required in order to identify new targets for anti-neisserial therapeutics. To identify essential genes and new virulence factors, a high-density mutant library in N. gonorrhoeae MS11 was generated by in vitro transposon mutagenesis. The transposon library harbors more than 100,000 individual mutants, a density that is unprecedented in gonococcal research. Essential genes in N. gonorrhoeae were determined by enumerating frequencies of transposon insertion sites (TIS) with Illumina deep sequencing (Tn-seq). Tn-seq indicated an average distance between adjacent TIS of 25 bp. Statistical analysis unequivocally demonstrated 781 genes that were significantly depleted in TIS and thus are essential for Neisseria survival. A subset of the genes was experimentally verified to comprise essential genes and thus support the outcome of the study. The hereby identified candidate essential genes thus may constitute excellent targets for the development of new antibiotics or vaccines. In a second study, the transposon mutant library was applied in a genome-scale “negative-selection strategy” to identify genes that are involved in low phosphate-dependent invasion (LPDI). LPDI is dependent on the Neisseria porin subtype PorBIA which acts as an epithelial cell invasin in absence of phosphate and is associated with severe pathogenicity in disseminated gonococcal infections (DGI). Tn-seq demonstrated 98 genes, which were involved in adherence to host cells and 43 genes involved in host cell invasion. E.g. the hypothetical protein NGFG_00506, an ABC transporter ATP-binding protein NGFG_01643, as well as NGFG_04218 encoding a homolog of mafI in N. gonorrhoeae FA1090 were experimentally verified as new invasive factors in LPDI. NGFG_01605, a predicted protease, was identified to be a common factor involved in PorBIA, Opa50 and Opa57-mediated neisserial engulfment by the epithelial cells. Thus, this first systematic Tn-seq application in N. gonorrhoeae identified a set of previously unknown N. gonorrhoeae invasive factors which demonstrate molecular mechanisms of DGI. N2 - Neisseria gonorrhoeae ist ein human-spezifisches Pathogen, das die Krankheit Gonorrhoe verursacht. Aufgrund der steigenden Anzahl antibiotikaresistenter Gonokokken und der damit verbundenen, rapide zunehmenden Anzahl von Infektionen erklärte die WHO Gonokokken 2012 zum Superbakterium. Daher ist eine genomweite Untersuchung der neisseriellen Genessentiatialität und neuer Virulenzfaktoren dringend erforderlich, um neue Ziele für die antineisserielle Therapie zu identifizieren. Hierzu wurde eine high-density Mutantenbibliothek in N. gonorrhoeae MS11 durch in vitro Transposonmutagenese generiert. Die Transposonbibliothek enthält mehr als 100.000 individuelle Mutanten - eine Dichte, die in der Gonokokken-Forschung beispiellos ist. Essentielle Gene von N. gonorrhoeae wurden durch die Ermittlung der Häufigkeit von Transposon insertion sites (TIS) mit Hilfe von Illumina deep sequencing (Tn-seq) bestimmt. Tn-seq ergab eine durchschnittliche Distanz von 25 Basenpaaren zwischen benachbarten TIS. Die statistische Analyse zeigte eindeutig 781 Gene, die signifikant weniger TIS aufwiesen und deshalb als essentiell für das Überleben der Neisserien verstanden werden können. Für ausgewählte Gene wurde experimentell bestätigt, dass sie essentielle Gene beinhalten, wodurch das Ergebnis der Tn-seq unterstützt wird. Die hierbei identifizierten essentiellen Gene könnten exzellente Targets für die Entwicklung neuer Antibiotika oder Impfstoffe darstellen. In einer zweiten Studie wurde die Transposon Mutanten Bibliothek für eine genomweite „negative Selektionsstrategie“ bereitgestellt. Es sollten Gene identifiziert werden, die an der phosphatfreien Invasion (low phosphate-dependent invasion = LPDI) beteiligt sind. Die LPDI ist vom neisseriellen Porin Subtyp PorBIA abhängig, welches bei Epithelzellen in Abwesenheit von Phosphat als Invasin fungiert und mit einer schweren Pathogenität in disseminierenden Gonokokkeninfektionen (DGI) assoziiert ist. Tn-seq ergab 98 Gene, die an der Adhärenz an die Wirtszelle, und 43 Gene, die an der Wirtszellinvasion beteiligt waren. Zum Beispiel wurden das hypothetische Protein NGFG_00506, ein ABC Transporter, das ATP-bindende Protein NGFG_01643, wie auch NGFG_04218, das für ein Homolog von mafI in N. gonorrhoeae FA1090 kodiert, experimentell als neue Invasionsfaktoren in der LPDI verifiziert. NGFG_01605, bei dem angenommen wird, dass es sich um eine Protease handelt, wurde als ein allgemeiner Faktor identifiziert, der an der PorBIA-, Opa50- and Opa57-vermittelten Einstülpung der Membran von Epithelzellen beteiligt ist. Die erste systematische Anwendung von Tn-seq in N. gonorrhoeae identifizierte eine Reihe bisher unbekannter Invasionsfaktoren von N. gonorrhoeae, die molekulare Mechanismen der DGI zeigen. KW - Neisseria gonorrhoeae KW - transposon mutagenesis KW - essential genes KW - virulence factors KW - Virulenzfaktor KW - Transposon KW - Mutagenese Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-102659 ER - TY - THES A1 - Xiang, Chaomei T1 - The role of B-RAF in embryonic development of mouse forebrain T1 - Die Rolle von B-RAF in der Embryonalentwicklung des Maus-Vorderhirns N2 - Die Familie der RAF-Kinasen umfasst drei Mitglieder, A-RAF, B-RAF und C-RAF. Nur für die B-RAF-Isoform wurde eine wichtige Funktion für die Entwicklung des Zentralen Nervensystems (ZNS) gefunden. Das Fehlen von B-RAF führt bei neu generierten embryonalen Neuronen zum Zelltod, weil sie in vitro nicht auf überlebensfaktoren reagieren können. Bei einer zweiten Zelllinie, die durch die Abwesenheit von B-RAF beeinträchtigt ist, handelt es sich um endotheliale Zellen. Ihr Zelltod führt zu inneren Blutungen und zu Letalität von B-RAF-/--Mäusen zwischen Tag 10.5 (E10.5) und 12.5 (E12.5) der Embryonalentwicklung. Dies verhinderte bisher weitere Untersuchungen der neuralen B-RAF-Funktion bei späteren Stadien. Im Gegensatz zu B-RAF-/--Mäusen überleben B-RAFKIN/KIN-Mäuse die Mitte der Embryonalentwicklung, da ihre Endothelzellen vor Apoptose geschätzt sind. Diese Tiere besitzen kein B-RAF, stattdessen wird im B-RAF-Locus ein chimäres Protein exprimiert, das den N-Terminus von B-RAF sowie alle Domänen von A-RAF umfasst. Der Schutz vor abnormaler neuraler Apoptose im Vorderhirn macht diese Tiere zu einem potentiellen Modell zur Untersuchung der Proliferations- und Differenzierungsfunktion von B-RAF, die die Kinase neben der Überlebensfunktion in der ZNS-Entwicklung ausübt. Die detaillierte Untersuchung der B-RAFKIN/KIN-Tiere konzentrierte sich auf die Entwicklung der Hirnrinde. Augenscheinlich waren kortikale Defekte im B-RAFKIN/KIN Vorderhirn: Der Verlust von B-RAF führte zu einer starken Reduzierung von Brn-2 exprimierenden pyramidalen Projektions-Neuronen begleitet von einer Störung der Dendritenbildung mit weniger und dünneren Dendriten in diesen oberen Schichten. Weitere Untersuchungen mit BrdU-Markierungsexperimenten zeigten in der ventrikulären Schicht reduzierte Zellproliferation für E14.5-E16.5 der Mutantenembryonen und ein Migrationsdefizit der spätgebideten kortikalen Neuronen. Während der Proliferationsdefekt der Hirnrinden-Vorläuferzellen mit einer reduzierten ERK-Aktivierung einherging, bleibt der Mechanismus der gestörten neuralen Migration zu erklären. Unsere Hypothese ist, dass die subzelluläre Lokalisation von Phospho-ERK in den wandernden Hirnrinden-Neuronen der B-RAFKIN/KIN-Mäuse verändert sein könnte. Zur Bestäigung der in vivo-Funktion von B-RAF und weiteren Studien zu ihrer unbekannten Rolle in der embryonalen Neurogenese sowie anderen Morphogenesen wäre die konditionale B-RAF Inaktivierung erforderlich. Durch die Deletion des genetischen Materials bzw. die Inaktivierung der Genfunktion in ausgew�hlten Zellen zu einem bestimmten Zeitpunkt ließen sich die Embryo-Letalität sowie unerwünschte pleiotrope Nebeneffekte vermeiden und akkumulierende, kompensierende Entwicklungsveränderungen von Beginn an ausschließen. Um die Cre Rekombinase-Methode einsetzen zu können, wurden floxed B-RAF embryonale Stammzell (ES)-Zelllinien generiert. Außerdem wurde ein auf dem Tetrazyklin Operator basierendes Schaltallel in den B-RAF Genort von embryonalen Stammzellen integriert, so dass die B-RAF Expression konditional und reversibel durch die Zugabe von Doxyzyklin angeschaltet werden konnte. Bisher wurden hochgradige chimäre Mäuse nach Blastozysten-Injektion geboren. Die Keimbahnübertragung dieser chimären Mäuse wird momentan untersucht. Wenn beide konditionale Mauslinien bereit sind, k�nnte die Entwicklung ihres Zentralnervensystems untersucht werden, um die Rolle von B-RAF in der Entwicklung des Nervensystems herauszufinden. N2 - The RAF family of protein kinases consists of three members, A-RAF, B-RAF and C-RAF. Unlike the other isotypes, B-RAF has been found to have an important function for normal development of the central nervous system (CNS), because newly generated embryonic neurons lacking B-RAF cannot respond to survival factors and undergo cell death in vitro. A second cell lineage affected by the absence of B-RAF are endothelial cells and their death leads to internal bleedings and lethality of B-RAF-/- mice between embryonic day 10.5 (E10.5) and E12.5 precluding an opportunity to further analyze neural B-RAF function at a later stage. In contrast to B-RAF-/- mice, B-RAFKIN/KIN mice, which are B-RAF deficient but express a chimeric protein consisting of the unique N terminus of B-RAF and all the domains of A-RAF in the B-RAF gene locus, survive after midgestation because their endothelial cells are protected from apoptosis. More importantly, overall prevention of abnormal neural apoptosis in the forebrain allows us to study proliferation- or differentiation-oriented function of B-RAF other than its survival effects in CNS development. The detailed investigation of B-RAFKIN/KIN animals was concentrated on cortical development. There were apparent cortical defects in B-RAFKIN/KIN forebrain: Loss of B-RAF led to severe reduction of Brn-2 expressing pyramidal projection neurons accompanied by a disruption of dendrite formation in the upper layers. In further analysis, BrdU labelling experiments showed that from E14.5 to E16.5 cell proliferation in the ventricular zone of the mutant mice was reduced and that the late-born cortical neurons failed to migrate properly. While the proliferation defect of cortical progenitors was associated with reduced ERK activation, the mechanism causing impaired neuronal migration remains to be determined. Our hypothesis is that the subcellular localization of phospho-ERK may be altered in migrating cortical neurons in B-RAFKIN/KIN mice. To confirm in vivo function of B-RAF and further study unknown roles in embryonic neurogenesis as well as other morphogenesis, conditional B-RAF knockouts would be the ideal models, which can efficiently avoid embryonic lethality, prevent unwanted pleiotropic side effects and exclude accumulative compensatory developmental changes from the earliest developmental stage on, through the deletion of genetic material/gene function in selected cells at a specific time. The use of site-specific recombinases such as Cre and the successful development of the reversible tetracycline-based switch have provided powerful venues for creating conditional loss-of-function mouse models. Generation of tetracycline-regulated B-RAF and floxed B-RAF mouse embryonic stem (ES) cell lines was performed. Up to now, high-grade chimeric mice were obtained after blastocyst injection of the modified ES cell clones. The germline transmission from these chimeric mice is currently under investigation. When either of conditional mouse lines is ready, detailed examination in their CNS development would be done to reveal how B-RAF plays a real role for normal development of the nervous system. KW - Maus KW - Embryonalentwicklung KW - Protein-Serin-Threonin-Kinasen KW - Vorderhirn KW - B-RAF KW - neurale Migration KW - Hirnrinden KW - Vorderhirn KW - B-RAF KW - neuronal migration KW - neocortex KW - forebrain Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-18326 ER - TY - THES A1 - Xiao, Yin T1 - Lack of NFATc1 SUMOylation prevents autoimmunity and alloreactivity T1 - Fehlende NFATc1-SUMOylierung verhindert Autoimmunität und Alloreaktivität N2 - SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis. NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs. Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases. Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses. N2 - SUMOylierung als posttranslationale Modifikation spielt bei mehreren biologischen Prozessen eine entscheidende Rolle. Small Ubiquitin-like Modifier (SUMO) Proteine können reversibel mit den Lysinresten innerhalb spezifischer Motive auf zahlreichen Zielproteinen verknüpft werden, was zu einer Veränderung der Stabilität, Lokalisation und Aktivität von Zielproteinen führt, insbesondere durch Interaktionsveränderungen mit anderen Molekülen. Folglich kann es die Gentranskription, Migration, das Fortschreiten des Zellzyklus, zelluläre Reaktionen auf Stress sowie die Tumorentstehung regulieren. NFATc1 ist ein Mitglied der Transkriptionsfaktorfamilie Nuclear Factor of Activated T-Cells (NFAT), welches in Folge von Zellstimulation und einer intrazellulären Konzentrationserhöhung von Ca2+ dephosphoryliert wird, was wiederum die Translokation in den Zellkern ermöglicht. Grundsätzlich spielt NFAT eine entscheidende Rolle bei der Entwicklung und Funktion des Immunsystems. NFATc1 hat drei SUMOylierungsstellen an den Positionen aa 349, 702 und 914. In unserer vorherigen Studie hatten wir gezeigt, dass Punktmutationen innerhalb aller SUMOylierungsstellen, aber auch nur an den Lysinresten K702 und K914 in vitro zu einer verstärkten Expression von IL-2 führten. Um die Funktion der SUMOylierung von NFATc1 auf die T-zellvermittelte Immunität in vivo zu untersuchen, haben wir nicht nur einen transgenen Mausstamm (NFATc1/ΔS-Maus) durch (Lysin zu Arginin) Punktmutationen an den zwei SUMOylierungsstellen auf dem Exon 10 von NFATc1 erzeugt, sondern auch einen zweiten Mausstamm (NFATc1/ΔBC-Maus), bei welchem das Exon 10 unter Verwendung des LoxP/Cre-Systems vollständig ausschaltet wurde. In den NFATc1/ΔS+ T-Zellen beobachteten wir eine erhöhte IL-2-Produktion und eine geringere IL-17A- und IFN-γ-Expression. NFATc1/ΔBC-Mäuse verhielten sich ähnlich zu NFATc1/ΔS-Mäusen. In den CD4+ T-Zellen mit diesen Genotypen wirkte die erhöhte IL-2 Sekretion der Expression von IL-17A und IFN-γ über Stat5- bzw. Blimp-1-Induktion entgegen. Desweiteren unterdrückte Blimp-1 auch IL-2 und reprimierte die Expression von Bcl2A1. Als nächstes evaluierten wir die NFATc1/ΔS Mäuse in zwei verschiedenen Krankheitsmodellen, der akuten Transplantat-gegen-Wirt-Reaktion (graft-versus-host disease; GvHD) und der Experimentellen autoimmunen Enzephalomyelitis (EAE). Wir stellten fest, dass bei der Induktion einer aGvHD durch Transplantation von Knochenmarkzellen zusammen mit NFATc1/ΔS+ CD3+ T-Zellen die Empfängertiere geschützter waren als in Anwesenheit von WT T-Zellen. Auch aufgrund einer erhöhten Anzahl von NFATc1/ΔS+ Tregs proliferierten die NFATc1/ΔS+ T Zellen weniger. Desgleichen war unter Verwendung von 2D2.NFATc1/ΔS+ T-Helfer 1 und 17 war eine Transfer-EAE in Rag Mäusen wesentlich schwächer induziert als bei der Verwendung von WT T-Zellen. Auch in diesem Modell konnten wie einen schützenden Effekt u.a. in Folge einer erhöhten Anzahl an Tregs feststellen. Zusammenfassend konnte gezeigt werden, dass die Vermeidung einer NFATc1-SUMOylierung zu einer erhöhten IL-2 Sekretion in T-Zellen führt und somit STAT5 aktiviert. STAT5 kompetiert mit STAT3 und hemmt so die IL-17A Produktion. STAT5 beeinflusste auch die Höhe der Blimp-1-Expression, wodurch IFN-γ sowie auf Dauer IL-2 supprimiert wurde. Darüber hinaus fördert IL-2 die Differenzierung und Expansion von Tregs. So kann trotz einer nur kurzen Phase an erhöhter IL-2-Sekretion eine fehlende NFATc1-SUMOylierung vor autoreaktiven und alloreaktiven Krankheiten schützen. Um die Funktion der SUMOylierung von NFATc1 beim Menschen zu verstehen, haben wir zunächst ein In vitro-3D-Kultursystem entwickelt. Unter der Verwendung von Organoiden aus Tonsillen, in Gegenwart von Feeder-Zellen, IL-4 und IL-7. Um zu bestätigen, dass 3D-Tonsillen-Organoide auf echte Antigene reagieren, wurden CMV-Peptide und Peptide von Covid-19 Spike-Proteinen verwendet. Wir konnten zeigen, dass wir durch die Co-Kultivierung der Peptide mit den Organoiden tatsächlich in der Lage sind, Gedächtniszellen und Plasmablasten zu generieren. Schließlich wurden die 3D und 2D Kulturen miteinander verglichen. Trotz der limitierten Gesamtzahl an B-Zellen in der 3D-Kultur, verglichen zur 2D-Kultur, konnten wir äquivalente Tendenzen der Erzeugung von Plasmablasten und Gedächtniszellen feststellen. Dies deutet darauf hin, dass die Verwendung dieses 3D-Kultursystems ein geeignetes in vitro-model darstellt, um NFATc1/ΔS+ oder NFATc1/ΔBC+ TFH- und TFR-Zellen in der Dynamik menschlicher GC-Antworten zu untersuchen. KW - NFATc1 KW - SUMOylation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-321054 ER - TY - THES A1 - Xiao, Zheng T1 - Blimp-1 Regulates Terminal Differentiation of T Cells T1 - Regulation der terminalen Differenzierung von T-Zellen durch Blimp-1 N2 - The transcriptional repressor-Blimp-1 terminates differentiation of B lymphocytes as well as myeloid cells. Our data show that Blimp-1 is highly expressed in freshly isolated murine primary T lymphocytes, particularly its minor splice variant. Ectopic expression of Blimp-1 by retroviral transduction neither dramatically altered secretion of IFN-ã or IL-4 nor did it induce the ability to suppress as regulatory T cells. However, induction of Blimp-1 resulted in not only a significant reduction in the production of IL-2 but also an inability to proliferate as well as in the reduced viability. These results demonstrate that Blimp-1 might mark end stages of lineage differentiation in T cells. KW - Blimp-1 KW - T- Zelle KW - Regluation KW - Blimp-1 KW - T cell KW - Regulation Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-10530 ER - TY - JOUR A1 - Xiu, Daiming A1 - Geiger, Maximilian J. A1 - Klaver, Peter T1 - Emotional face expression modulates occipital-frontal effective connectivity during memory formation in a bottom-up fashion JF - Frontiers in Behavioral Neuroscience N2 - This study investigated the role of bottom-up and top-down neural mechanisms in the processing of emotional face expression during memory formation. Functional brain imaging data was acquired during incidental learning of positive ("happy"), neutral and negative ("angry" or "fearful") faces. Dynamic Causal Modeling (DCM) was applied on the functional magnetic resonance imaging (fMRI) data to characterize effective connectivity within a brain network involving face perception (inferior occipital gyrus and fusiform gyrus) and successful memory formation related areas (hippocampus, superior parietal lobule, amygdala, and orbitofrontal cortex). The bottom-up models assumed processing of emotional face expression along feed forward pathways to the orbitofrontal cortex. The top-down models assumed that the orbitofrontal cortex processed emotional valence and mediated connections to the hippocampus. A subsequent recognition memory test showed an effect of negative emotion on the response bias, but not on memory performance. Our DCM findings showed that the bottom-up model family of effective connectivity best explained the data across all subjects and specified that emotion affected most bottom-up connections to the orbitofrontal cortex, especially from the occipital visual cortex and superior parietal lobule. Of those pathways to the orbitofrontal cortex the connection from the inferior occipital gyrus correlated with memory performance independently of valence. We suggest that bottom-up neural mechanisms support effects of emotional face expression and memory formation in a parallel and partially overlapping fashion. KW - medial temporal lobe KW - human orbitofrontal cortex KW - subsequent memory KW - recognition memory KW - fMRI KW - event-related fMRI KW - posterior parietal cortex KW - short-term-memory KW - human brain KW - prefrontal activity KW - neural mechanisms KW - Dynamic Causal Modeling KW - facial affect KW - memory formation Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143211 VL - 9 IS - 90 ER - TY - THES A1 - Xu, Jiajia T1 - A high-complexity lentiviral shRNA screen identifies synthetic lethal interactions with deregulated N-Myc in neuroblastoma cells T1 - Ein hoch-Komplexität Genom-weit RNAi Screen für synthetisch letale Interaktion mit dereguliertem N-Myc in Neuroblastomzellen N2 - In contrast to c-Myc, a deregulated expression of the MYCN gene is restricted to human neuroendocrine tumours. In most cases, the excessive activity of N-Myc results from a MYCN amplification. In neuroblastoma, amplification of MYCN is a predictor of poor prognosis and resistance to therapy. The inability to target the N-Myc protein directly necessitates the search for alternative targets. This project aimed at identifying genes specifically required for growth and survival of cells that express high levels of N-Myc using high-throughput shRNA screening combined with next generation sequencing. The identification and analysis of these genes will shed light on functional interaction partners of N-Myc. We screened a shRNA library containing 18,327 shRNAs and identified 148 shRNAs, which were selectively depleted in the presence of active N-Myc. In addition, shRNAs targeting genes that are involved in p53 and ARF turnover and apoptosis were depleted in the cell population during the screen. These processes are known to affect N-Myc-mediated apoptosis. Consequently, these results biologically validated the screen. The 148 shRNAs that showed a significant synthetic lethal interaction with high levels of N-Myc expression were further analysed using the bioinformatics program DAVID. We found an enrichment of shRNAs that target genes involved in specific biological processes. For example, we validated synthetic lethal interactions for genes such as, THOC1, NUP153 and LARP7, which play an important role in the process of RNA polymerase II-mediated transcription elongation. We also validated genes that are involved in the neddylation pathway. In the screen we identified Cullin 3, which is a component of the BTB-CUL3-Rbx1 ubiquitin ligase that is involved in the turnover of Cyclin E. Depletion of cullin 3 and activation of N-Myc was found to synergistically increase Cyclin E expression to supraphysiological levels, inducing S-phase arrest and a strong DNA damage response. Together with results from a proteomics analysis of N-Myc associated proteins, our results lead us to the following hypothesis: In a neuroblastoma cell, the high levels of N-Myc result in a conflict between RNA polymerase II and the replication machinery during S-phase. The newly identified interaction partners of N- Myc are required to solve this conflict. Consequently, loss of the interaction leads to a massive DNA damage and the induction of apoptosis. In addition, inhibition or depletion of the essential components of the neddylation pathway also results in an unresolvable problem during S-phase. N2 - 6.2 Zusammenfassung Im Gegensatz zu c-Myc findet man eine Deregulation von N-Myc nur in einer begrenzten Anzahl maligner Tumore die neuroektodermalen Ursprungs sind. Die übermäßige Aktivität ist dabei fast immer durch eine genomische Amplifikation von N-Myc begründet. Im Neuroblastom korreliert eine MYCN-Amplifikation mit einer schlechten Prognose. Da es auf Grund einer fehlenden katalytischen Domäne nicht möglich ist N-Myc direkt zu inhibieren, ist die Suche nach alternativen Targets notwendig. Das Ziel dieser Arbeit war es neue Gene zu identifizieren, die notwendig für das Wachstum und Überleben von MYCN amplifizierten Zellen sind. Dies wurde durch eine Kombination von Hochdurchsatz-RNAi-Screens und Next-Generation-Sequenzierung erreicht. Durch das Screenen einer shRNA-Bibliothek, die insgesamt 18327 shRNAs beinhaltet, konnten 148 shRNAs identifiziert werden, die selektiv nachteilig für das Überleben N-Myc überexpremierender Zellen sind. Die statistische Auswertung der Ergebnisse des Screens zeigte zusätzlich eine Anreichung von shRNAs gegen Gene, die p53-und ARF-abhängig Apoptose vermitteln. Da es bekannt ist, dass diese Gene in der N-Myc-vermittelten Apoptose involviert sind, konnte dadurch der Screen validiert werden. Die weitere Auswertung mit dem bioinformatischen Programm DAVID ergab, dass unter den 148 als synthetisch letal identifizierten shRNAs solche angereichert waren, die gegen Gene spezifischer biologischer Prozesse gerichtet sind. Zum einen wurden Gene wie THOC1, NUP153 und LARP7 validiert, die eine Rolle im Prozeß der Elongation der RNA Polymerase II spielen. Zum anderen konnten Gene validiert werden die einen Beitrag bei der Neddylierung von Proteinen leisten. Durch die Depletion von Cullin 3, ein Bestandteil des BTB-CUL3-Rbx1 Ubiquitin-Ligase-Komplexes, der am Abbau von Cyclin E beteiligt ist, konnte gezeigt werden, dass zusammen mit der Aktivierung von N-Myc eine supraphysiologische Erhöhung von Cyclin E induziert wird. Dies führt zu einem S-Phase Arrest in der Zelle, der die DNA-Schadens-Signalkaskade auslöst. Zusammen mit den Ergebnissen einer Proteomanalyse, bei der neue N-Myc-assoziierte Proteine identifiziert wurden, konnte folgende Hypothese aufgestellt werden: In einer Neuroblastomzelle helfen diese neuen Interaktionspartner den durch die N-Myc Überexpression in der S-phase entstehenden Konflikt zwischen RNA-Polymerase II und Replikationsmaschinerie zu lösen. Der Verlust dieser Interaktion führt zu einer massiven Schädigung der DNA, worauf in der Zelle Apoptose ausgelöst wird. Des Weiteren führen auch die Inhibition oder Ausschaltung wesentlicher Komponenten des Neddylierungs-Signalwegs zu unlösbaren Problemen in der S-Phase des Zellzyklus. KW - Neuroblastom KW - synthetic lethality KW - apoptosis KW - cul3 ring ligase KW - replicative stress KW - N-Myc KW - Deregulierung KW - RNS-Interferenz KW - synthetische Letalität Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-103157 ER - TY - JOUR A1 - Xu, Jietao A1 - Fahmy-Garcia, Shorouk A1 - Wesdorp, Marinus A. A1 - Kops, Nicole A1 - Forte, Lucia A1 - De Luca, Claudio A1 - Misciagna, Massimiliano Maraglino A1 - Dolcini, Laura A1 - Filardo, Giuseppe A1 - Labberté, Margot A1 - Vancíková, Karin A1 - Kok, Joeri A1 - van Rietbergen, Bert A1 - Nickel, Joachim A1 - Farrell, Eric A1 - Brama, Pieter A. J. A1 - van Osch, Gerjo J. V. M. T1 - Effectiveness of BMP-2 and PDGF-BB adsorption onto a collagen/collagen-magnesium-hydroxyapatite scaffold in weight-bearing and non-weight-bearing osteochondral defect bone repair: in vitro, ex vivo and in vivo evaluation JF - Journal of Functional Biomaterials N2 - Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects. KW - tissue engineering KW - regenerative medicine KW - osteochondral lesion KW - biocompatible materials KW - bone morphogenetic proteins KW - platelet-derived growth factor KW - animal model KW - weight-bearing Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304019 SN - 2079-4983 VL - 14 IS - 2 ER - TY - JOUR A1 - Xu, K. A1 - Näveri, L. A1 - Frerichs, K. A1 - Hallenbeck, J. M. A1 - Feuerstein, G. A1 - Davis, J. N. A1 - Sirén, Anna-Leena T1 - Extracellular catecholamine levels in rat hippocampus after a selective alpha2-adrenoceptor antagonist or a selective dopamnie uptake inhibitor: Evidence for dopamine release from local dopaminergic nerve terminals N2 - The effect of 6-chloro-2,3,4,5-tetrahydro-3-methyi-1-H-3-benzazepine (SKF 86466), a selectlve nonimldazoline alpha-2 adrenoceptor antagonlst, on hippocampal re1ease of norepinephrine and dopamlne in conscious rats was lnvestigated by /n vlvo mlcrodialysis and high-pressure liquid chromatography. Additionally, extracellular concentrations of hippocampal dopamine (DA) and norepinephrtne (NE), durtng Infusion of selective monoamine uptake Inhibitors, were determined in freely moving rats. The basal concentration of NE in the dialysate was 4.9 ± 0.3 pg/20 pl. lntravenous admlnistratlon of 5 or 10 mgJkg of SKF 86466 was associated wlth a transierlt inc:rease (30 min) of 2-fold (12 ± 1 pg/20 ,d; p < .05) and 8-fold (39 ± 3 pg/20 pl; p < .05), respectlvely, in dlalysate NE, whereas a 1-mgfkg dose had no effect. DA was not detected in basal dlalysates, but after the adminlstratlon of 5 or 10 mgJkg of SKF 86466, 3.9 ± 0.4 and 6.4 ± 0.6 pg/20 pl, respectlvely, was present in the dialysates. The rnaxlmum increase in dialysate DA was reached 60 to 90 min after SKF 86466. The DA was not derived from plasma because plasma NE was elevated after the 5 mgJkg dose of SKF 86466 whereas no plasma DA was detected. ln order to determlne whether DA was present in noradrenergic nerve termlnals, the dopamine ß-hydroxylase Inhibitor SKF 1 02698 was administered (50 mgJkg i.p.). The Inhibitor decreased dialysate NE but DA was stin not detected in the dialysate. When SKF 86466 (5 mgJkg t.v.) was adminlstered 4 hr after SKF 102698, DA appeared in the dialysate but there was no lncrease in dialysate NE. Administration through the dialysis probe of the DA uptake Inhibitor, GBR-12909 (0.1 and 1 pM), dose-dependently lnaeased DA Ieveis to 5.7 ± 1.2 and 9.6 ± 2.8 pg/20 pl, respectively. GBR-12909 had no effect on hippocampal NE. Desipramine (5 and 10 pM) lncreased dose-dependently dialysate NE and lncreased DA concentrations to detectable Ieveis (2.7 ± 0.5 and 3.5 ± 0.7 pg/20 ,d, respectively). These results suggest that the a/pha-2 adrenoceptors modulate both NE and DA release in the rat hlppocampus and that DA detected in the hlppocampal dialysate might be released from dopaminergic neurons. KW - Neurobiologie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62997 ER - TY - JOUR A1 - Xu, Li A1 - He, Jianzheng A1 - Kaiser, Andrea A1 - Gräber, Nikolas A1 - Schläger, Laura A1 - Ritze, Yvonne A1 - Scholz, Henrike T1 - A Single Pair of Serotonergic Neurons Counteracts Serotonergic Inhibition of Ethanol Attraction in Drosophila JF - PLoS ONE N2 - Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. Here, we dissect the function of the key regulator of serotonin signaling—the serotonin transporter–in innate olfactory attraction to ethanol in Drosophila melanogaster. We generated a mutated version of the serotonin transporter that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. We identified four serotonergic neurons that inhibit the olfactory attraction to ethanol and two additional neurons that counteract this inhibition by strengthening olfactory information. Our results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior. KW - attraction KW - ethanol KW - Drosophila melanogaster KW - serotonin transporter Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166762 VL - 11 IS - 12 ER - TY - JOUR A1 - Xu, Shanjia A1 - Sheng, Xinqing A1 - Greiner, P. A1 - Becker, Charles R. A1 - Geick, R. T1 - High-order finite-element analysis of scattering properties of II-VI semiconductor materials N2 - The sattering characteristics ot the n-VI semiconductors were analyzed by a method which combines the second-order finite-element method with the rigorous mode matching procedure. The method avolds the difficulty of solving the complex transcendental equation introduced in the multimode network method and calculates all the eigenvalues and eigenfunctions simultaneously which are needed for the mode matching treatment in the longitudinal direction. As a result, the whole solution procedure is significantly simplified. A comparison is given between the experimental data and the calculated results obtained with this analysis and tbe network method. Very good agreement has been achieved, the accuracy and efficiency of the present method are thus verified. KW - Halbleiter KW - II-VI semiconductor KW - scattering characteristics KW - high-order finite element KW - mode matching method Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86283 ER - TY - THES A1 - Xu, Wenshan T1 - Regulation of the DNA Damage Response by the Ubiquitin System T1 - Regulierung der DNA-Schadensreaktion durch das Ubiquitin System N2 - DNA damage occurs frequently during normal cellular progresses or by environmental factors. To preserve the genome integrity, DNA damage response (DDR) has evolved to repair DNA and the non-properly repaired DNA induces human diseases like immune deficiency and cancer. Since a large number of proteins involved in DDR are enzymes of ubiquitin system, it is critical to investigate how the ubiquitin system regulates cellular response to DNA damage. Hereby, we reveal a novel mechanism for DDR regulation via activation of SCF ubiquitin ligase upon DNA damage. As an essential step for DNA damage-induced inhibition of DNA replication, Cdc25A degradation by the E3 ligase β-TrCP upon DNA damage requires the deubiquitinase Usp28. Usp28 deubiquitinates β-TrCP in response to DNA damage, thereby promotes its dimerization, which is required for its activity in substrate ubiquitination and degradation. Particularly, ubiquitination at a specific lysine on β-TrCP suppresses dimerization. The key mediator protein of DDR, 53BP1, forms oligomers and associates with β-TrCP to inhibit its activity in unstressed cells. Upon DNA damage, 53BP1 is degraded in the nucleoplasm, which requires oligomerization and is promoted by Usp28 in a β-TrCP-dependent manner. Consequently, 53BP1 destruction releases and activates β-TrCP during DNA damage response. Moreover, 53BP1 deletion and DNA damage promote β-TrCP dimerization and recruitment to chromatin sites that locate in the vicinity of putative replication origins. Subsequently, the chromatin-associated Cdc25A is degraded by β-TrCP at the origins. The stimulation of β-TrCP binding to the origins upon DNA damage is accompanied by unloading of Cdc45, a crucial component of pre-initiation complexes for replication. Loading of Cdc45 to origins is a key Cdk2-dependent step for DNA replication initiation, indicating that localized Cdc25A degradation by β-TrCP at origins inactivates Cdk2, thereby inhibits the initiation of DNA replication. Collectively, this study suggests a novel mechanism for the regulation of DNA replication upon DNA damage, which involves 53BP1- and Usp28-dependent activation of the SCF(β-TrCP) ligase in Cdc25A degradation. N2 - DNA-Schäden treten häufig in Folge zellulären Fortschrittes oder durch externe Faktoren auf. Um die Integrität des Genoms zu bewahren und DNA Schäden zu reparieren, die Ursache für viele Autoimmunkrankheiten und Krebs sind, hat sich ein durch DNA Schäden getriggertes Geflecht aus Reparaturprozessen (englisch: “DNA damage response (DDR)”) entwickelt. Hierbei ist es von großem Interesse zu verstehen, wie das Ubiquitin-Proteasom-System die zelluläre Antwort auf DNA-Schäden reguliert. Wir konnten zeigen, dass die SCF Ubiquitin Ligase β-TrCP durch geschädigte DNA aktiviert wird, was einen bisher unbekannten Mechanismus für die Regulation der DDR darstellt. Für den grundlegenden Schritt der durch DNA Schäden ausgelösten Inhibition der DNA Replikation – der Abbau von Cdc25A durch die E3 Ligase β-TrCP – wird die Deubiquitinase Usp28 benötigt. Diese deubiquitiniert β-TrCP als Antwort auf DNA-Schäden und fördert dadurch seine Dimerisierung, die für die Substrat-Ubiquitinierung und dem anschließenden Abbau erforderlich ist. Hierbei unterdrückt die Ubiquitinierung eines spezifischen Lysin-Rests von β-TrCP dessen Dimerisierung. Das Schlüsselprotein vom DDR, 53BP1, oligomerisiert und assoziiert mit β-TrCP, was seine Aktivität in gesunden Zellen inhibiert. Auf DNA-Schäden hin oligomerisiert 53BP1 und wird mit Hilfe von Usp28 abhängig von β-TrCP im Nukleoplasma abgebaut. Durch den Abbau von 53BP1 wird β-TrCP freigesetzt, aktiviert und kann auf DNA Schäden reagieren. Die Deletion von 53BP1 fördert die Dimerisierung von β-TrCP. Die Reparaturmaschinerie wird daraufhin an Stellen des Chromatins rekrutiert, die in der Nähe von vermeintlichen Replikationsursprüngen liegen. Chromatin-assoziiertes Cdc25A wird dann durch β-TrCP ubiquitiniert. Die Bindung von β-TrCP an die Replikationsursprünge in Folge von DNA Schädigung wird begleitet von der Freisetzung von Cdc45, das eine entscheidende Komponente des Präinitiationskomplexes darstellt. Das Beladen von Cdc45 an die Replikationsursprünge stellt eine Schlüsselfunktion der Cdc25A-abhängigen DNA Replikationsinititation dar. Gezielter Abbau von Cdc25A durch β-TrCP an den Replikationsursprüngen inaktiviert Cdk2 und inhibiert dadurch DNA Replikation. Zusammenfassend lässt sich konstatieren, dass unsere Studien einen neuartigen Mechanismus für die Regulation der DNA Replikation auf DNA Schäden hin aufgezeigt haben, der die 53BP1- und Usp28-abhängige Aktivierung der SCF(β-TrCP) Ubiquitin Ligase im Abbau von Cdc25A beinhaltet. KW - DNS-Schädigung KW - DNS-Reparatur KW - Ubiquitin KW - DNA damage KW - Ubiquitin system Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-160064 ER - TY - THES A1 - Xu, Zhihao T1 - Cooperative Formation Controller Design for Time-Delay and Optimality Problems T1 - Reglerentwurf der kooperativen Formation für Zeitverzögerungs- und Optimalitäts Probleme N2 - This dissertation presents controller design methodologies for a formation of cooperative mobile robots to perform trajectory tracking and convoy protection tasks. Two major problems related to multi-agent formation control are addressed, namely the time-delay and optimality problems. For the task of trajectory tracking, a leader-follower based system structure is adopted for the controller design, where the selection criteria for controller parameters are derived through analyses of characteristic polynomials. The resulting parameters ensure the stability of the system and overcome the steady-state error as well as the oscillation behavior under time-delay effect. In the convoy protection scenario, a decentralized coordination strategy for balanced deployment of mobile robots is first proposed. Based on this coordination scheme, optimal controller parameters are generated in both centralized and decentralized fashion to achieve dynamic convoy protection in a unified framework, where distributed optimization technique is applied in the decentralized strategy. This unified framework takes into account the motion of the target to be protected, and the desired system performance, for instance, minimal energy to spend, equal inter-vehicle distance to keep, etc. Both trajectory tracking and convoy protection tasks are demonstrated through simulations and real-world hardware experiments based on the robotic equipment at Department of Computer Science VII, University of Würzburg. N2 - Diese Dissertation handelt sich um Controller Entwurfsmethoden für eine Bildung von kooperativen mobile Roboter auf Bahnverfolgung und Konvoischutz Aufgaben. Zwei große Probleme zu Multi-Agenten-Formation Steuerung werden angesprochen, nämlich die Zeitverzögerung und Optimalitätsprobleme. Für die Aufgabe der Bahnverfolgung wird eine Leader-Follower-basiertes System für den Reglerentwurf angenommen, in dem die Auswahlkriterien für die Reglerparameter durch Analysen von charakteristischen Polynome abgeleitet werden. Die herauskommenden Parameter sorgen für die Stabilität des Systems und vermeiden die Steady-State Fehler sowie das Schwingungsverhalten unter Zeitverzögerungseffekt. In der Konvoischutz Szenario wird eine dezentrale Koordinationsstrategie zur ausgewogenen Einsatz mobiler Roboter zum ersten Mal vorgeschlagen. Auf der Grundlage dieser Abstimmungsschema werden optimale Reglerparameter in zentrale und dezentrale Weise für den dynamischen Konvoischutz in einem einheitlichen Rahmen erzeugt, in dem verteilten Optimierungstechnik in der dezentralen Strategie angewendet ist. Diese einheitlichen Rahmen berücksichtigt die Bewegung des zuschützenden Ziels, und die gewünschte Systemleistung, zum Beispiel, einen minimalen ausgegebene Energie, und einen gleichen Zwischenfahrzeugabstand, usw. Die Aufgaben der Bahnverfolgung und des Konvoischutzes werden durch Simulationen und realen Hardware-Experimente auf der Basis der Roboterausrüstungen am Institut für Informatik VII, Universität Würzburg demonstriert. T3 - Forschungsberichte in der Robotik = Research Notes in Robotics - 9 KW - Optimalwertregelung KW - Robotik KW - Autonomer Roboter KW - Geleitzug KW - Optimal control KW - Decentralized formation control KW - Trajectory tracking KW - Convoy Protection KW - Optimale Regelung KW - Konvoi Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-105555 SN - 978-3-923959-96-9 ER - TY - THES A1 - Yadav, Preeti T1 - Studying Neuronal Cytoskeleton Defects and Synaptic Defects in Mouse Model of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy T1 - Die Analyse des neuronalen Zytoskeletts und synaptischer Defekte im Mausmodel der Amyotrophen Lateralsklerose und der Spinalen Muskelatrophie N2 - Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature. Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMA. N2 - Die Amyotrophe Lateralsklerose und die spinale Muskelatrophie sind die beiden häufigsten Formen der Motoneuronerkrankungen. Sie sind charakterisiert durch eine Destabilisierung der Axonendigungen, durch Axondegeneration und durch Änderungen im neuronalen Zytoskelett. Eine Anhäufung von Neurofilamenten konnte in einigen neurodegenerativen Erkrankungen beobachtet werden. Der genaue Mechanismus, welcher zu einer Destabilisierung des Axons führt, ist bis heute jedoch unklar. Hiermit zeige ich, dass eine gesteigerte Expression von Neurofilamenten in motorischen Nerven von pmn mutierten Mäusen zu einer Störung der Mikrotubuli – Dynamik führt. Ein Neurofilamentabbau durch Nefl knockout steigert die Anzahl an neu wachsenden Mikrotubuli in pmn mutierten Motoneuronen und führt zu erneutem Axonwachstum. Dieser Effekt wird durch eine Interaktion zwischen dem Neurofilament und dem Stathmin Komplex vermittelt. Ein Abbau des Neurofilaments führt zu einer Erhöhung der Stathmin-Stat3 Interaktion und zu einer Stabilisierung der Mikrotubuli. Demzufolge ist die Versorgung der Axone verbessert und die pmn mutierten Mäuse überleben länger. Wir vermuten, dass dieser Mechanismus auch für andere neurodegenerative Erkrankungen, bei denen Neurofilamentanhäufung ein dominantes Merkmal ist, relevant sein könnte. Des Weiteren studierte ich mit Hilfe des Smn-/-;SMN2 Mausmodels, den molekularen Mechanismus der sich hinter dem Synapsenverlust bei SMA verbirgt. SMA ist charakterisiert durch eine Degeneration der unteren -Motoneuronen im Rückenmark. Es ist jedoch unklar, wie ein Verlust des ubiquitär exprimierten SMN Proteins zu einer MN-spezifischen Degeneration führt. Smn ist involviert in den Prozess des pre-mRNA Splicing (Pellizzoni, Kataoka et al. 1998) und ein Verlust des Proteins führt zu einer Störung des Splicing. Eine Denervierung der motorischen Endplatte führt zu einer Neurodegeneration in SMA. Es gibt jedoch keinen Hinweis auf eine kausale Verbindung zwischen anomalem Splicen von stromabwärts gelegenen Transkripten des Smn und einer Reduktion präsynaptischer Axone, wie man es bei SMA beobachten kann. In dieser Studie konnten wir beobachten, dass Expression und Splicing von Nrxn2, welches für ein präsynaptisches Protein kodiert, in SMA Mäusen betroffen ist und dass Nrxn2 ein Kandidat sein könnte, der eine Verbindung zwischen Störungen im Splice Prozess und synaptischen Motoneuron-Defekten in der SMA herstellen könnte. KW - Neurofilament KW - Neurofilament KW - Zellskelett KW - Spinale Muskelatrophie KW - Cytoskeleton KW - Spinal muscular Atrophy KW - Pmn mutant mouse Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-138093 ER - TY - JOUR A1 - Yadav, Preeti A1 - Selvaraj, Bhuvaneish T. A1 - Bender, Florian L. P. A1 - Behringer, Marcus A1 - Moradi, Mehri A1 - Sivadasan, Rajeeve A1 - Dombert, Benjamin A1 - Blum, Robert A1 - Asan, Esther A1 - Sauer, Markus A1 - Julien, Jean-Pierre A1 - Sendtner, Michael T1 - Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling JF - Acta Neuropathologica N2 - In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features. KW - Amyotrophic-lateral-sclerosis KW - Transgenic mice KW - Mouse model KW - Alzheimers disease KW - Neurofilament KW - Progressive motor neuronopathy KW - Axonal transport KW - Intermediate filaments KW - Motoneuron disease KW - Lacking neurofilaments KW - Missense mutation KW - Axon degeneration KW - Microtubules KW - Stathmin KW - Stat3 Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188234 VL - 132 IS - 1 ER - TY - JOUR A1 - Yamakawa, Hisanori A1 - Fukushima, Yoshimasa A1 - Itoh, Shigeru A1 - Heber, Ulrich T1 - Three different mechanisms of energy dissipation of a desiccation-tolerant moss serve one common purpose: to protect reaction centres against photo-oxidation JF - Journal of Experimental Botany N2 - Three different types of non-photochemical de-excitation of absorbed light energy protect photosystem II of the sun- and desiccation-tolerant moss Rhytidium rugosum against photo-oxidation. The first mechanism, which is light-induced in hydrated thalli, is sensitive to inhibition by dithiothreitol. It is controlled by the protonation of a thylakoid protein. Other mechanisms are activated by desiccation. One of them permits exciton migration towards a far-red band in the antenna pigments where fast thermal deactivation takes place. This mechanism appears to be similar to a mechanism detected before in desiccated lichens. A third mechanism is based on the reversible photo-accumulation of a radical that acts as a quencher of excitation energy in reaction centres of photosystem II. On the basis of absorption changes around 800 nm, the quencher is suggested to be an oxidized chlorophyll. The data show that desiccated moss is better protected against photo-oxidative damage than hydrated moss. Slow drying of moss thalli in the light increases photo-protection more than slow drying in darkness. KW - reaction centre KW - photoprotection KW - energy dissipation KW - energy conservation KW - chlorophyll fluorescence KW - photosystem II Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126897 VL - 63 IS - 10 ER - TY - JOUR A1 - Yan, Yan A1 - Hong, Ni A1 - Chen, Tiansheng A1 - Li, Mingyou A1 - Wang, Tiansu A1 - Guan, Guijun A1 - Qiao, Yongkang A1 - Chen, Songlin A1 - Schartl, Manfred A1 - Li, Chang-Ming A1 - Hong, Yunhan T1 - p53 Gene Targeting by Homologous Recombination in Fish ES Cells JF - PLoS One N2 - Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology. KW - mouse KW - in-vitro KW - drug selection KW - chimera formation KW - medakafish oryzias latipes KW - embryonic stem-cells KW - zebrafish KW - differentiation KW - cultures KW - pluripotency Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133416 VL - 8 IS - 3 ER - TY - JOUR A1 - Yan, Zhe T1 - “I tried to control my emotions”: nursing home care workers’ experiences of emotional labor in China JF - Journal of Cross-Cultural Gerontology N2 - Despite dramatic expansions in the Chinese nursing home sector in meeting the increasing care needs of a rapidly aging population, direct care work in China remains largely devalued and socially unrecognized. Consequently, scant attention has been given to the caregiving experiences of direct care workers (DCWs) in Chinese nursing homes. In particular, given the relational nature of care work, there is little knowledge as to how Chinese DCWs manage emotions and inner feelings through their emotional labor. This article examines the emotional labor of Chinese DCWs through ethnographic data collected with 20 DCWs in one nursing home located in an urban setting in central China. Data were analyzed using conventional content analysis and constant comparison. Participants’ accounts of sustaining a caring self, preserving professional identity, and hoping for reciprocity revealed implicit meanings about the often-conflicting nature of emotional labor and the nonreciprocal elements of care work under constrained working conditions. Importantly, the moral-cultural notion of bao (报 norm of reciprocity) was found to be central among DCWs in navigating strained resources and suggested their agency in meaning-construction. However, their constructed moral buffers may be insufficient if emotional labor continues to be made invisible by care organizations. KW - Gerontologie KW - Care-Arbeit KW - Emotionsregulation KW - China KW - Altenpflege KW - China KW - Long-term care KW - Direct care workers KW - Emotional labor KW - Filial piety/xiao KW - Professionalism KW - Reciprocity Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324295 VL - 37 IS - 1 ER - TY - THES A1 - Yang, Manli T1 - \(Chlamydia\) \(trachomatis\) metabolism during infection and metatranscriptome analysis in \(Neisseria\) \(gonorrhoeae\) coinfected STD patients T1 - \(Chlamydia\) \(trachomatis\) Metabolismus während der Infektion sowie die Analyse des Metatranskriptoms bei \(Neisseria\) \(gonorrhoeae\) koinfizierten STD-Patienten N2 - Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen. It causes blinding trachoma and sexually transmitted disease such as chlamydia, pelvic inflammatory disease and lymphogranuloma venereum. Ct has a unique biphasic development cycle and replicates in an intracellular vacuole called inclusion. Normally it has two forms: the infectious form, elementary body (EB); and the non-infectious form, reticulate body (RB). Ct is not easily amenable to genetic manipulation. Hence, to understand the infection process, it is crucial to study how the metabolic activity of Ct exactly evolves in the host cell and what roles of EB and RB play differentially in Ct metabolism during infection. In addition, Ct was found regularly coinfected with other pathogens in patients who got sexually transmitted diseases (STDs). A lack of powerful methods to culture Ct outside of the host cell makes the detailed molecular mechanisms of coinfection difficult to study. In this work, a genome-scale metabolic model with 321 metabolites and 277 reactions was first reconstructed by me to study Ct metabolic adaptation in the host cell during infection. This model was calculated to yield 84 extreme pathways, and metabolic flux strength was then modelled regarding 20hpi, 40hpi and later based on a published proteomics dataset. Activities of key enzymes involved in target pathways were further validated by RT-qPCR in both HeLa229 and HUVEC cell lines. This study suggests that Ct's major active pathways involve glycolysis, gluconeogenesis, glycerolphospholipid biosynthesis and pentose phosphate pathway, while Ct's incomplete tricarboxylic acid cycle and fatty acid biosynthesis are less active. EB is more activated in almost all these carbohydrate pathways than RB. Result suggests the survival of Ct generally requires a lot of acetyl-CoA from the host. Besides, both EB and RB can utilize folate biosynthesis to generate NAD(P)H but may use different pathways depending on the demands of ATP. When more ATP is available from both host cell and Ct itself, RB is more activated by utilizing energy providing chemicals generated by enzymes associated in the nucleic acid metabolism. The forming of folate also suggests large glutamate consumption, which is supposed to be converted from glutamine by the glutamine-fructose-6-phosphate transaminase (glmS) and CTP synthase (pyrG). Then, RNA sequencing (RNA-seq) data analysis was performed by me in a coinfection study. Metatranscriptome from patient RNA-seq data provides a realistic overview. Thirteen patient samples were collected and sequenced by our collaborators. Six male samples were obtained by urethral swab, and seven female samples were collected by cervicovaginal lavage. All the samples were Neisseria gonorrhoeae (GC) positive, and half of them had coinfection with Ct. HISAT2 and Stringtie were used for transcriptomic mapping and assembly respectively, and differential expression analysis by DESeq2, Ballgown and Cuffdiff2 are parallelly processed for comparison. Although the measured transcripts were not sufficient to assemble Ct's transcriptome, the differential expression of genes in both the host and GC were analyzed by comparing Ct positive group (Ct+) against Ct-uninfected group. The results show that in the Ct+ group, the host MHC class II immune response was highly induced. Ct infection is associated with the regulation of DNA methylation, DNA double-strand damage and ubiquitination. The analysis also shows Ct infection enhances host fatty acid beta oxidation, thereby inducing mROS, and the host responds to reduce ceramide production and glycolysis. The coinfection upregulates GC's own ion transporters and amino acid uptake, while it downregulates GC's restriction and modification systems. Meanwhile, GC has the nitrosative and oxidative stress response and also increases the ability for ferric uptake especially in the Ct+ group compared to Ct-uninfected group. In conclusion, methods in bioinformatics were used here in analyzing the metabolism of Ct itself, and the responses of the host and GC respectively in a coinfection study with and without Ct. These methods provide metabolic and metatranscriptomic details to study Ct metabolism during infection and Ct associated coinfection in the human microbiota. N2 - Chlamydia trachomatis (Ct) ist ein obligater intrazellulärer Pathogen des Menschen. Er verursacht Trachoma und sexuell übertragbare Krankheiten, wie Chlamydiose, Unterleibsentzündung und Lymphogranuloma venereum. Ct besitzt einen biphasischen Entwicklungszyklus und vermehrt sich in intrazellulären Vakuolen, sogenannten Einschlusskörperchen. Normalerweise können zwei Formen beobachtete werden: Die infektiöse Form, Elementarkörperchen (EK); und die nicht-infektiöse Form, Retikularkörperchen (RK). Ct ist nicht einfach genetisch zu manipulieren. Um den Infektionsablauf besser zu verstehen, ist es wichtig, zu untersuchen, wie sich genau die metabolische Aktivität von Ct in der Wirtszelle entwickelt und welche Rolle EK und RK im Metabolismus von Ct während der Infektion spielen. Zusätzlich wurde Ct häufig bei Patienten mit sexuell übertragbaren Krankheiten (STD) in Co-Infektion mit anderen Erregern gefunden. Ein Mangel an leistungsfähigen Methoden zur Kultivierung von Ct außerhalb der Wirtszelle macht es schwierig die genauen molekularen Mechanismen von Co-Infektionen zu untersuchen. In dieser Arbeit wurde erstmals ein genomweites metabolisches Model mit 321 Metaboliten und 277 Reaktionen aufgebaut, um die metabolische Adaption von Ct in der Wirtzelle während der Infektion zu untersuchen. Dieses Model wurde erstellt und umfasst 84 „extreme pathways“ (Grenz-Stoffwechselwege). Darauf aufbauend wurde die metabolische Fluss-Stärke berechnet. Die Zeitpunkte 20 hpi (20 Stunden nach der Infektion), 40 hpi und die anschließende Infektionsphase wurden durch Nutzung von Proteom-Daten modelliert. Die Aktivitäten von Schlüsselenzymen, welche in wichtigen Stoffwechselwegen involviert sind, wurden zusätzlich durch RT-qPCR überprüft. Dabei wurden die Ergebnisse sowohl für HeLA229- als auch HUVEC-Zellen nachgemessen. Diese Untersuchungen zeigten, dass Ct’s wichtigste aktive Stoffwechselwege die Glykolyse, die Gluconeogenese und der Pentosephosphatweg sind, während der unvollständige Zitronensäurezyklus und die Fettsäuresynthese weniger aktiv sind. Gegenüber RK sind bei EK fast alle diese Kohlenhydratwege stärker aktiviert. Im Allgemeinen benötigt Ct eine größere Menge an Acetyl-CoA. Außerdem können sowohl EK, als auch RK die Folsäurebiosynthese nutzen, um NAD(P)H zu generieren. Dabei werden möglicherweise unterschiedliche Pathways genutzt, abhängig vom Bedarf an ATP. Sobald mehr ATP sowohl durch die Wirtszellen als auch von der Ct-Zelle selbst zur Verfügung steht, wird die Nutzung von Energieträgern, produziert durch Enzyme des Nukleinsäurestoffwechsels, in RK stärker aktiviert. Die Bildung von Folsäure lässt den Schluss zu, dass große Mengen von Glutamat umgesetzt werden, welches vermutlich aus der Umwandlung von Glutamin durch die Glutamine-fructose-6-phosphate-transaminase (glmS) und CTP-Syntase (pyrG) stammt. Anschließend wurde eine Analyse von RNA-Sequenzierungsdaten (RNA-seq) aus einer Co-Infektions-Studie (Chlamydien und andere Keime, insbesondere Gonokokken (GC)) durchgeführt. Dafür wurden Proben von dreizehn Patienten gesammelt und von Kollaborationspartnern sequenziert. Sechs Proben männlicher Patienten wurden durch Abstrich der Harnröhre und sieben Proben weiblicher Patientinnen durch cervicovaginale Lavage gewonnen. Alle Proben waren Neisseria gonorrhoeae (GC) positiv, wobei die Hälfte eine Co-Infektion mit Ct aufwies. Die Programme HISAT2 and Stringtie wurden zum Abbilden der transgenomischen Reads beziehungsweise zur Assemblierung des Genoms verwendet, und eine Analyse der differentiellen Expression wurde jeweils mit DESeq2, Ballgown und Cuffdiff2 durchgeführt und die Ergebnisse verglichen. Obwohl nicht ausreichend viele Transkripte von Ct gewonnen werden konnten, um das Transkriptom komplett assemblieren zu können, wurde die differentielle Expression der Gene sowohl von Wirt als auch von GC durch den Vergleich zwischen der Gruppe der Ct-positiven (Ct+) der Gruppe der Ct-unifizierten Patienten analysiert. Die Ergebnisse zeigten, dass in der (Ct+)-Gruppe die auf der MHC-Klasse-II basierte Immunantwort stark induziert war. Die Infektion von Ct ist mit der Regulation der DNA-Methylierung, DNA-Doppel-Strang-Schädigung und Ubiquitinierung verbunden. Die Analyse zeigte zusätzlich, dass die Infektion mit Ct die Fettsäure β-Oxidation des Wirts steigert, dadurch mROS induziert, und sowohl die Ceramid-Produktion als auch die Glycolyse reduziert. Die Co-Infektion reguliert GC’s eigene Eisentransporter und Aminosäureaufnahme hoch, während Restriktions- und Modifikationssysteme herunterreguliert werden. Gleichzeitig zeigt GC sowohl eine stickstoffsensitve Stress Antwort als auch eine oxidative. Dies verstärkt zusätzlich die Fähigkeit für die Aufnahme von Eisen, insbesondere in der (Ct+)-Gruppe. Zusammenfassend wurden Methoden der Bioinformatik genutzt, um den Metabolismus von Ct selbst, und die Antwort des Wirtes respektive GC‘s in einer Co-Infektionsstudie mit und ohne Ct zu analysieren. Diese Methoden lieferten wichtige metabolische und metatranskriptomische Details, um den Metabolismus von Ct während der Infektion, aber auch das Mikrobiom während einer Ct assoziierter Co-Infektion zu untersuchen. KW - chlamydia trachomatis KW - Neisseria gonorrhoeae KW - metabolic modelling KW - metatranscriptome KW - STD patients Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-184993 ER - TY - JOUR A1 - Yang, Manli A1 - Rajeeve, Karthika A1 - Rudel, Thomas A1 - Dandekar, Thomas T1 - Comprehensive Flux Modeling of Chlamydia trachomatis Proteome and qRT-PCR Data Indicate Biphasic Metabolic Differences Between Elementary Bodies and Reticulate Bodies During Infection JF - Frontiers in Microbiology N2 - Metabolic adaptation to the host cell is important for obligate intracellular pathogens such as Chlamydia trachomatis (Ct). Here we infer the flux differences for Ct from proteome and qRT-PCR data by comprehensive pathway modeling. We compare the comparatively inert infectious elementary body (EB) and the active replicative reticulate body (RB) systematically using a genome-scale metabolic model with 321 metabolites and 277 reactions. This did yield 84 extreme pathways based on a published proteomics dataset at three different time points of infection. Validation of predictions was done by quantitative RT-PCR of enzyme mRNA expression at three time points. Ct’s major active pathways are glycolysis, gluconeogenesis, glycerol-phospholipid (GPL) biosynthesis (support from host acetyl-CoA) and pentose phosphate pathway (PPP), while its incomplete TCA and fatty acid biosynthesis are less active. The modeled metabolic pathways are much more active in RB than in EB. Our in silico model suggests that EB and RB utilize folate to generate NAD(P)H using independent pathways. The only low metabolic flux inferred for EB involves mainly carbohydrate metabolism. RB utilizes energy -rich compounds to generate ATP in nucleic acid metabolism. Validation data for the modeling include proteomics experiments (model basis) as well as qRT-PCR confirmation of selected metabolic enzyme mRNA expression differences. The metabolic modeling is made fully available here. Its detailed insights and models on Ct metabolic adaptations during infection are a useful modeling basis for future studies. KW - metabolic modeling KW - metabolic flux KW - infection biology KW - elementary body KW - reticulate body KW - Chlamydia trachomatis Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189434 SN - 1664-302X VL - 10 IS - 2350 ER - TY - THES A1 - Yang, Mengshi T1 - Synthesis, solubility and optical activity of chiral poly(2,4- disubstituted-2-oxazoline)s T1 - Synthese, Löslichkeit und optische Aktivität von chiralen Poly(2,4-disubstituierten-2-oxazolin)en N2 - Motivated by the perceived great potential of chiral polymers, the presented work aimed at the investigation of synthesis, solubility and optical activity of chiral poly(2,4-disubstituted-2-oxazoline)s. A novel polymeric carrier based on ABA-type triblock copolymers poly(2-oxazoline)s with chiral and racemic hydrophobic blocks was developed for the formulation of chiral and achiral drugs (Fig. 5.1). Poly(2-methyl-2-oxazoline) (pMeOx) was used as hydrophilic A block, and poly(2-ethyl-4-ethyl-2-oxazoline) (pEtEtOx) and poly(2-propyl-4-methyl-2-oxazoline) (pPrMeOx) were used as hydrophobic B blocks. Curcumin (CUR), paclitaxel (PTX) and chiral/racemic ibuprofen (R/S/RS-IBU) were applied as model drugs. Nanoformulations were prepared consisting of these triblock copolymers and model drugs. ... N2 - Motiviert durch das wahrgenommene große Potential chiraler Polymere zielte die vorliegende Arbeit auf die Untersuchung der Synthese, Löslichkeit und optischen Aktivität von chiralen Poly(2,4-disubstituierten-2-oxazolin)en ab. Für die Formulierung von chiralen und achiralen Arzneimitteln wurde ein neuartiger polymerer Träger auf der Basis von ABA-Typ Triblock-Copolymeren aus Poly(2-oxazolin)en mit chiralen und racemischen hydrophoben Blocken entwickelt (Abbildung 5.1). Poly(2-methyl-2-oxazolin) (pMeOx) wurde als hydrophiler A Block und Poly(2-ethyl-4-ethyl-2-oxazolin) (pEtEtOx) und Poly(2-propyl-4-methyl-2-oxazolin) (pPrMeOx) als hydrophober B Block verwendet. Als Modellarzneimittel wurden Curcumin (CUR), Paclitaxel (PTX) und chirales/racemisches Ibuprofen (R/S/RS-IBU) eingesetzt. Es wurden Nanoformulierungen hergestellt, die aus diesen Triblock-Copolymeren und Modellarzneimitteln bestehen. ... KW - poly(2-oxazoline) KW - chiral KW - drug delivery KW - secondary structure Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322429 ER - TY - THES A1 - Yang, Shang T1 - Characterization and engineering of photoreceptors with improved properties for optogenetic application T1 - Charakterisierung und Entwicklung von Photorezeptoren mit verbesserten Eigenschaften für die optogenetische Anwendung N2 - Optogenetics became successful in neuroscience with Channelrhodopsin-2 (ChR2), a light-gated cation channel from the green alga Chlamydomonas reinhardtii, as an easy applicable tool. The success of ChR2 inspired the development of various photosensory proteins as powerful actuators for optogenetic manipulation of biological activity. However, the current optogenetic toolbox is still not perfect and further improvements are desirable. In my thesis, I engineered and characterized several different optogenetic tools with new features. (i) Although ChR2 is the most often used optogenetic actuator, its single-channel conductance and its Ca2+ permeability are relatively low. ChR2 variants with increased Ca2+ conductance were described recently but a further increase seemed possible. In addition, the H+ conductance of ChR2 may lead to cellular acidification and unintended pH-related side effects upon prolonged illumination. Through rational design, I developed several improved ChR2 variants with larger photocurrent, higher cation selectivity, and lower H+ conductance. (ii) The light-activated inward chloride pump NpHR is a widely used optogenetic tool for neural silencing. However, pronounced inactivation upon long time illumination constrains its application for long-lasting neural inhibition. I found that the deprotonation of the Schiff base underlies the inactivation of NpHR. Through systematically exploring optimized illumination schemes, I found illumination with blue light alone could profoundly increase the temporal stability of the NpHR-mediated photocurrent. A combination of green and violet light eliminates the inactivation effect, similar to blue light, but leading to a higher photocurrent and therefore better light-induced inhibition. (iii) Photoactivated adenylyl cyclases (PACs) were shown to be useful for light-manipulation of cellular cAMP levels. I developed a convenient in-vitro assay for soluble PACs that allows their reliable characterization. Comparison of different PACs revealed that bPAC from Beggiatoa is the best optogenetic tool for cAMP manipulation, due to its high efficiency and small size. However, a residual activity of bPAC in the dark is unwanted and the cytosolic localization prevents subcellular precise cAMP manipulation. I therefore introduced point mutations into bPAC to reduce its dark activity. Interestingly, I found that membrane targeting of bPAC with different linkers can remarkably alter its activity, in addition to its localization. Taken together, a set of PACs with different activity and subcellular localization were engineered for selection based on the intended usage. The membrane-bound PM-bPAC 2.0 with reduced dark activity is well-tolerated by hippocampal neurons and reliably evokes a transient photocurrent, when co-expression with a CNG channel. (iv) Bidirectional manipulation of cell activity with light of different wavelengths is of great importance in dissecting neural networks in the brain. Selection of optimal tool pairs is the first and most important step for dual-color optogenetics. Through N- and C-terminal modifications, an improved ChR variant (i.e. vf-Chrimson 2.0) was engineered and selected as the red light-controlled actuator for excitation. Detailed comparison of three two-component potassium channels, composed of bPAC and the cAMP-activated potassium channel SthK, revealed the superior properties of SthK-bP. Combining vf-Chrimson 2.0 and improved SthK-bP “SthK(TV418)-bP” could reliably induce depolarization by red light and hyperpolarization by blue light. A residual tiny crosstalk between vf-Chrimson 2.0 and SthK(TV418)-bP, when applying blue light, can be minimized to a negligible level by applying light pulses or simply lowering the blue light intensity. N2 - Die Optogenetik wurde in den Neurowissenschaften mit Channelrhodopsin-2 (ChR2), einem lichtgesteuerten Kationenkanal aus der Grünalge Chlamydomonas reinhardtii, als leicht anwendbares Werkzeug erfolgreich. Der Erfolg von ChR2 inspirierte die Entwicklung verschiedener photosensorischer Proteine als leistungsstarke Aktuatoren für die optogenetische Manipulation der biologischen Aktivität. Die derzeitige optogenetische Toolbox ist jedoch immer noch nicht perfekt und weitere Verbesserungen sind wünschenswert. In meiner Arbeit habe ich verschiedene optogenetische Werkzeuge mit neuen Funktionen entwickelt und charakterisiert. (i) Obwohl ChR2 der am häufigsten verwendete optogenetische Aktuator ist, sind seine Einzelkanal-Leitfähigkeit und seine Ca2 + -Permeabilität relativ gering. Kürzlich wurden ChR2-Varianten mit erhöhter Ca2 + -Leitfähigkeit beschrieben, eine weitere Verbesserung schien jedoch möglich. Darüber hinaus kann die H+-Leitfähigkeit von ChR2 bei längerer Beleuchtung zu einer Ansäuerung der Zellen und zu unbeabsichtigten Nebenwirkungen im Zusammenhang mit dem pH-Wert führen. Durch rationales Design entwickelte ich mehrere verbesserte ChR2-Varianten mit größerem Photostrom, höherer Kationenselektivität und geringerer H+-Leitfähigkeit. (ii) Die lichtaktivierte Chloridpumpe NpHR ist ein weit verbreitetes optogenetisches Werkzeug für die neuronale Inhibierung. Eine ausgeprägte Inaktivierung bei längerer Beleuchtung schränkt jedoch die Anwendung für eine lang anhaltende neuronale Hemmung ein. Ich konnte zeigen, dass die Deprotonierung der Schiffschen Base der Inaktivierung von NpHR zugrunde liegt. Durch die systematische Untersuchung optimierter Beleuchtungsschemata fand ich heraus, dass die Beleuchtung mit blauem Licht allein die zeitliche Stabilität des NpHR-vermittelten Photostroms erheblich verbessern kann. Eine Kombination aus grünem und violettem Licht eliminiert den Inaktivierungseffekt, ähnlich wie blaues Licht, führt jedoch zu einem höheren Photostrom und deswegen effektiverer Licht-induzierter Inhibierung. (iii) Photoaktivierte Adenylylcyclasen (PACs) erwiesen sich als nützlich für die Lichtmanipulation der zellulären cAMP-Spiegel. Ich habe einen praktischen in-vitro-Test für lösliche PACs entwickelt, der deren zuverlässige Charakterisierung ermöglicht. Ein Vergleich verschiedener PACs ergab, dass bPAC von Beggiatoa aufgrund seiner hohen Effizienz und geringen Größe das beste optogenetische Werkzeug für die cAMP-Manipulation ist. Eine Restaktivität von bPAC im Dunkeln ist jedoch unerwünscht und die cytosolische Lokalisierung verhindert eine subzellulär präzise cAMP-Manipulation. Ich habe daher Punktmutationen in bPAC eingeführt, um dessen Dunkelaktivität zu reduzieren. Interessanterweise fanden Ich heraus, dass das Membrantargeting von bPAC mit verschiedenen „Linkern“ zusätzlich zu seiner Lokalisierung seine Aktivität erheblich verändern kann. Zusammengenommen wurde eine Reihe von PACs mit unterschiedlicher Aktivität und subzellulärer Lokalisation für unterschiedliche Anwendungen konstruiert. Das membrangebundene PM-bPAC 2.0 mit reduzierter Dunkelaktivität wird von Hippocampus-Neuronen gut vertragen und erlaubt, bei gleichzeitiger Expression mit einem CNG-Kanal, zuverlässig einen Licht-induzierten Strom auszulösen. (iv) Die bidirektionale Manipulation der Zellaktivität mit Licht unterschiedlicher Wellenlänge ist für die Dissektion neuronaler Netze im Gehirn von großer Bedeutung. Die Auswahl der optimalen Werkzeugpaare ist der erste und wichtigste Schritt für die zweifarbige Optogenetik. Durch N- und C-terminale Modifikationen wurde eine verbesserte ChR-Variante (d. h. Vf-Chrimson 2.0) entwickelt und als Rotlicht-gesteuerter Aktuator zur Anregung ausgewählt. Ein detaillierter Vergleich von drei Zweikomponenten-Kaliumkanälen, bestehend aus bPAC und dem cAMP-aktivierten Kaliumkanal SthK, ergab die überlegenen Eigenschaften von SthK-bP. Die Kombination von vf-Chrimson 2.0 und verbessertem SthK-bP „SthK(TV418)-bP“ konnte zuverlässig eine Depolarisation durch rotes Licht und eine Hyperpolarisation durch blaues Licht induzieren. Ein restliches, kleines Übersprechen zwischen vf-Chrimson 2.0 und SthK(TV418)-bP kann beim Anlegen von blauem Licht durch Anlegen von Lichtimpulsen oder durch einfaches Verringern der Intensität von blauem Licht auf ein vernachlässigbares Maß minimiert werden. KW - Optogenetics KW - ChR2 KW - NpHR KW - PAC KW - Bidirectional manipulation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-205273 ER - TY - THES A1 - Yang, Shaoxian T1 - The role of NFAT proteins in Rag and Nfatc1a Gene Regulation in Murine Thymus T1 - Die Rolle von NFAT Proteinen in Rag und Nfatc1a Gen-Anordnung in Murine Thymus N2 - In this thesis we have investigated the effect of NFAT (Nuclear Factor of Activated T Cell) transcription factors on the expression of Rag-(Recombination Activating Genes) genes in murine thymus. The protein products of Rag genes, RAG1 and RAG2, are critical for the recombination and generation of the TCR (T Cell Receptor) repertoire during thymocyte development, and their expression can be suppressed by the activity of NFAT factors. In thymus, the expression of Rag1 and Rag2 genes is induced at the double-negative (DN, CD4-8-) 3 stage, down-regulated at the DN4 stage, re-induced at the double-positive (DP, CD4+8+) stage, and suppressed again at the single-positive (SP, CD4+8- or CD4-8+) stage. Although it is known that TCR signaling suppresses the expression of Rag1 and Rag2 at the SP stage, the signals that mediate the Rag gene down-reulation remain elusive. Here we report that both the calcineurin-NFAT-signaling and MAPKinase signaling pathways, which are activated by TCR signaling during positive selection, mediate the Rag gene down-regulation in DP thymocytes. The calcineurin-NFAT pathway suppresses both the Rag1 and the Rag2 gene expression. This pathway has a stronger suppressive effect on the Rag1 than the Rag2 gene. A synergistic activity between the two NFAT factors NFATc2 and NFATc3 is essential for calcineurin-NFAT signaling to efficiently suppress the Rag gene expression in DP thymocytes. It is likely that the calcineurin-NFAT signaling down-regulates Rag gene expression by suppressing both the Rag anti-silencer element (ASE) activity and the Rag promoter activity. Similarly, MEK-ERK signaling of MAPK signaling pathway mediates the Rag gene suppression in DP thymocytes although the mechanism through which MEK-ERK mediates the Rag gene down-regulation has to be elucidated. In DN thymocytes, it appears that neither the calcineurin-NFAT signaling nor MAPK signaling is involved in the Rag gene down-regulation. However, a role for these two signaling pathways in the Rag gene up-regulation in DN thymocytes is not excluded. In DN thymocytes, pre-TCR signaling stimulates the expression both Nfatc1 and Nfatc2 genes but has no effect on Nfatc3 gene expression. In DN thymocytes, pre-TCR signaling activates Nfatc1α expression but not Nfatc1ß expression, i.e. the two promoters controling Nfatc1 gene xpression are differently controled by pre-TCR signals. Nfatc1α gene expression in DN thymocytes is mainly regulated by the MAPK signaling pathway because activation of Nfatc1α is mediated by MEK-ERK signaling but opposed by JNK signaling. Calcineuirn-NFAT and p38 signaling pathways are not involved in Nfatc1α promoter regulation in DN thymocytes. In DP thymocytes, TCR signaling up-regulates Nfatc1 and Nfatc2 expression but down-regulates Nfatc3 expression. In DP thymocytes, TCR signaling activates Nfatc1α expression. The activation of Nfatc1α in DP thymocytes is mediated by NFATc1, but not or to a less degree by NFATc2 and NFATc3. MEK-ERK, JNK, and p38 signaling pathways are involved in Nfatc1α gene activation in DP thymocytes, probably by activating NFAT trans-activation activity. All these findings illustrate that in thymocytes the expression of NFAT transcription factors – which are essential for thymic development - is controled at multiple levels. N2 - Wir haben in den experimentellen Arbeiten zu dieser Dissertation den Effekt der NFAT (Nuclear Factor of Activated T Cell)-Transkriptionsfaktoren auf die Expression der Rag (Recombination Activating)-Gene im Thymus der Maus untersucht. Die Proteine der beiden Rag-Gene, RAG1 und RAG2, sind entscheidend für die Bildung des TCR (T Zell-Rezeptor)-Repertoires, und ihre Expression wird durch die NFATs supprimiert. Während der Thymozyten-Entwicklung wird die Expression der Rag1- und Rag2-Gene in DN (double negative, CD4-8-) 3-Thymozyten induziert, in DN4-Thymozyten „herunterreguliert“, re-induziert in DP (double positive, CD4+8+)-Thymozyten und supprimiert in SP (single positive, CD4+8- oder CD4-8+) Thymozyten. Obwohl bekannt ist, dass der TCR-Signalweg die Expression von Rag1 und Rag2 in SP-Thymozyten supprimiert, sind die daran beteiligten Signale weitgehend unbekannt. In der vorliegenden Arbeit wurde gezeigt, dass sowohl der Calcineurin-NFAT-Signalweg als auch der MAP Kinase-Signalweg die Rag-Gen- „Herunterregulierung“ in DP-Thymozyten vermitteln. Beide Wege werden über TCR-Signale während der positiven Selektion aktiviert. Der Calcineurin-NFAT-Signalweg supprimiert die Genexpression von Rag1 und Rag2, wobei Rag1 stärker betroffen ist. Dabei ist eine synergistische Aktiviät zwischen den beiden NFAT-Transkriptionsfaktoren NFATc2 und NFATc3 im Calcineurin-NFAT-Signalweg notwendig, um die Rag Genexpression in DP Thymozyten „herunterzuregulieren“. Der Calcineurin-NFAT-Signalweg reguliert offensichtlich die Rag-Genexpression durch eine Unterdrückung der Rag anti-silencer-element-(ASE)-Aktivität und der Rag-Promotoraktivität. Auch die MEK-ERK-Signalkaskade des MAPK-Signalwegs ist an der Suppression des Rag- Gens in DP Thymozyten beteiligt, wobei der Mechanismus zu untersuchen bleibt. In DN-Thymozyten hingegen scheinen weder der Calcineurin-NFAT-Signalweg noch der MAPK-Signalweg an der Hemmung der Rag-Genaktivität beteiligt zu sein. Eine Beteiligung dieser beiden Signalwege bei der Rag-Gen-Aktivierung in DN-Thymozyten kann hingegen nicht ausgeschlossen werden. In DN-Thymozyten regulieren Prä-TCR-Signale eine stärkere Expression der beiden NFAT-Faktoren Nfatc1 und Nfatc2, wohingegen Nfatc3 unbeeinflusst bleibt. In DN-Thymozyten aktivieren die Prä-TCR-Signale die Expression von Nfatc1α, aber nicht von Nfatc1ß. Die Nfatc1α Genexpression wird vermutlich hauptsächlich über den MAPK-Signalweg reguliert, da eine Aktivierung von Nfatc1α über MEK-ERK Signale vermittelt wird und JNK Signale gegensätzlich wirken. Der Calcineurin-NFAT- und der p38-Signalweg spielen keine Rolle bei der Regulation von Nfatc1α in DN-Thymozyten. In DP-Thymozyten erfolgt durch TCR-Signale eine Aktivierung der Nfatc1- und Nfatc2- sowie eine Represson der Nfatc3-Genexpression. In DP-Thymozyten aktivieren die TCR-Signale die Nfatc1α Expression. Die Aktivierung von Nfatc1α in DP Thymozyten wird über NFATc1 autoreguliert. NFATc2 und NFATc3 sind daran wenig oder gar nicht beteiligt. Hingegen sind MEK-ERK-, JNK- und p38-Signalwege bei der Nfatc1α−Genaktivierung in DP-Thymozyten - wahrscheinlich durch die Aktivierung der NFAT Transaktivierungsaktivität - beteiligt. All diese Daten zeigen, dass die NFAT-Transkriptonsfaktoren einer komplexen Regulation in Thymozyten unterzogen sind, deren Entwicklung sie andererseits – wie z.B. durch die Suppression der Rag-Gene – maßgeblich beeinflussen. KW - NFAT KW - rag KW - thymus KW - Gen-Anordnung KW - NFAT KW - rag KW - thymus KW - gene regulation Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-23691 ER - TY - THES A1 - Yang, Tao T1 - Functional insights into the role of a bacterial virulence factor and a host factor in Neisseria gonorrhoeae infection T1 - Funktionelle Einblicke in die Rolle eines bakteriellen Virulenzfaktors und eines Wirtsfaktors bei der Infektion mit Neisseria gonorrhoeae N2 - Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing. This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration. The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration. In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection. N2 - Neisseria gonorrhoeae (GC) ist ein humanpathogenes Bakterium. Gegen jedes kommerziell erhältliche Antibiotikum konnten bereits Resistenzen entdeckt werden. Die Infektion von Neisserien startet mit der Kolonisierung der Zelloberfläche, gefolgt von der Invasion in die Zelle, intrazellulärer Persistenz, Transzytose und Verlassen des subepithelen Raums. Subepithele Bakterien können den Blutfluss erreichen und sich somit auf andere Gewebe verbreiten und dadurch schwerwiegende Erkrankungen wie Arthritis und Lungenentzündungen verursachen. Zahlreiche Studien haben die Interaktion zwischen Wirtszelle und Pathogen zwischen Adhärenz und Invasion gut charakterisiert. Allerdings ist der Mechanismus des intrazellulären Überlebens und der Transmigration weitestgehend unbekannt. Um neue therapeutische Ansätze zu definieren ist es deshalb wichtig weitere bakterielle und zelluläre Faktoren verantwortlich für Interaktionen zwischen Wirt und Pathogen zu identifizieren. Außerdem wurden Studien über N. gonorrhoeae bisher in konventioneller Zellkultur durchgeführt. Auch wenn diese weitere Einsichten in Wirt-Pathogen Interaktionen geben, ist viel Information in der nativen Infektionsumgebung, wie Zellpolarisierung oder Barrierefunktionen, bisher nicht bekannt. Diese Arbeit fokussiert sich auf die Bestimmung eines neuen bakteriellen Virulenzfaktors, NGFG_01605, und Wirtsfaktoren (FLCN) in der Infektion von Gonokokken. NGFG_01605 wurde über Screening einer Transposonsammlung identifiziert und ist eine vermeintliche U32 Protease. Im Gegensatz zu anderen Proteinen dieser Familie, wird diese Protease nicht sekretiert und hat keine ex vivo Proteaseaktivität. Der Knockout von NGFG_01605 vermindert die Überlebensrate von Gonokokken in Epithelzellen. 3D-Modelle basierend auf T84-Zellen wurden für die Analyse der bakteriellen Transmigration entwickelt. Der Knockout von NGFG_01605 hat keinen Einfluss auf die Transmigration. Bei der Suche neuer Wirtsfaktoren, die für die Adhärenz und/oder Internalisierung wichtig sind, wurde FLCN durch ein Screening einer shRNA Sammlung entdeckt. Wir konnten zeigen, dass dieser Faktor zwar nicht für die Adhärenz oder Invasion von N. gonorrhoeae, aber für das Überleben der Bakterien in der Wirtszelle essenziell ist. Da der programmierte Zelltod ein typischer Abwehrmechanismus gegen intrazelluläre Bakterien ist, haben wir Apoptose und Autophagie weiter untersucht und konnten zeigen, dass FLCN zwar keinen Effekt auf Apoptose hat, aber Autophagie inhibiert. Außerdem konnten wir zeigen, dass FLCN die Expression von E-cadherin inhibiert. Auch der Knockdown von E-cadherin verringerte Autophagie und erhöhte das Überleben von N. gonorrhoeae. Im Gebärmutterhals sind sowohl polarisierte als auch nicht-polarisierte Zellen präsent. E-cadherin hat zudem unterschiedliche Einflüsse auf diese unterschiedlichen Zellen. Aus diesem Grund haben wir ein 3D-Modell entwickelt, um die Funktionen von FLCN besser zu verstehen. Wir entdeckten, dass FLCN zwar auch im 3D Modell wichtig für das Überleben ist, aber nicht durch Inhibition von Autophagie. Außerdem inhibiert FLCN die Expression und Verteilung von E-cadherin in 3D-Modellen. Da N. gonorrhoeae die Epithelzellbarierre durch sowohl Zell-Zell-Kontakte als auch transzelluläre Migration überwinden kann, untersuchten wir daraufhin die Rollen von FLCN und E-cadherin in der transzellulären Migration. Diese wird durch FLCN verspätet und durch Knockdown von E-cadherin erhöht. Zusammengefasst, haben wir die Rolle von NGFG_01605 und den Zusammenhang von FLCN und E-cadherin während der Infektion von N. gonorrhoeae untersucht. Unser Fokus war hierbei das intrazelluläre Überleben sowie zelluläre Transmigration. Diese Arbeit ist die Erste, die FLCN und humanpathogenspezifische Infektion miteinander in Verbindung bringt. KW - Folliculin KW - autophagy KW - polarized epithelium KW - protease KW - Gonococcal invasion KW - autophagocytose Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-208959 ER - TY - JOUR A1 - Yang, Tao A1 - Heydarian, Motaharehsadat A1 - Kozjak-Pavlovic, Vera A1 - Urban, Manuela A1 - Harbottle, Richard P. A1 - Rudel, Thomas T1 - Folliculin Controls the Intracellular Survival and Trans-Epithelial Passage of Neisseria gonorrhoeae JF - Frontiers in Cellular and Infection Microbiology N2 - Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers. KW - gonococcal invasion KW - folliculin KW - autophagy KW - polarized epithelium KW - polarized cell culture Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-211372 SN - 2235-2988 VL - 10 IS - 422 ER - TY - THES A1 - Yang, Weiyue T1 - Population Policy and Governance at the Local Level - A Qualitative Research of the Implementation of the Universal Two-Child Policy in China T1 - Bevölkerungspolitik und Governance auf lokaler Ebene – Eine qualitative Untersuchung der Durchführung der Zwei-Kind-Politik in China N2 - The universal two-child policy was introduced by the central government of China in 2016 to respond to the country’s deteriorating population problems, but it was soon replaced by a three-child policy in 2021 given that it failed to continuously boost fertility in Chinese society. This dissertation empirically investigates the implementation of universal two-child policy in three Chinese major cities. Based on the data collected through semi-structured interviews with leaders of local family planning agencies, it finds that local officials are primarily devoted to coping with the discontent of the bereaved single-child parents (shidu families), which is an unexpected consequence of the historical one-child policy, rather than working on the tasks regarding birth encouragement. The dissertation suggests understanding the implementation of China’s population policy within the framework of both historical and rational choice institutionalism. The target responsibility system as an effective tool of the central authority drives local agents to fix their attention at tasks that have larger impact on their career. The shifted focus in the implementation of the universal two-child policy is a result of local officials’ emphasis on the task of maintaining social stability. Shidu families are deemed as a salient threat to social order because their discontent with the state support has incurred continuous petitions at both the national and local level, which would severely undermine local officials’ career advancement. However, in the meantime, stability maintenance is found to have become alienated as reflected by the rising costs and that it replaced birth support to be the focus of local family planning agents in the universal two-child policy era. Since the conflict between the shidu group and the state is unlikely to be resolved, the future population policy design and enforcement will continue to be constrained by the shidu problem. N2 - Die Zwei-Kind-Politik wurde 2016 von der chinesischen Zentralregierung als Reaktion auf die sich verschlechternden Bevölkerungsprobleme des Landes eingeführt, aber 2021 durch eine Drei-Kind-Politik ersetzt, da sie die Fruchtbarkeitsrate der chinesischen Gesellschaft nicht kontinuierlich steigern konnte. Diese Dissertation untersucht empirisch die Durchführung der Zwei-Kind-Politik in drei chinesischen Großstädten. Basierend auf den Daten, die durch halbstrukturierte Interviews mit Leitern lokaler Familienplanungsagenturen gesammelt wurden, stellt es fest, dass lokale Beamte sich in erster Linie der Bewältigung der Unzufriedenheit der hinterbliebenen Ein-Kind-Familien (Shidu-Familien) widmen, anstatt die Aufgaben der Geburtenförderung zu bearbeiten. Diese Dissertation schlägt vor, die Durchführung der chinesischen Bevölkerungspolitik sowohl im Rahmen des historischen als auch des Rational-Choice-Institutionalismus zu verstehen. Das System der Zielverantwortung als wirksames Instrument der zentralen Behörde treibt die lokalen Agenten dazu, ihre Aufmerksamkeit auf Aufgaben zu richten, die einen größeren Einfluss auf ihre Karriere haben. Der verschobene Fokus bei der Durchführung der Zwei-Kind-Politik ist ein Ergebnis der Betonung der Aufrechterhaltung der sozialen Stabilität bei lokalen Beamten. Shidu-Familien sind als eine seriöse Bedrohung für die soziale Stabilität betrachted geworden, da ihre Unzufriedenheit mit der staatlichen Unterstützung nicht nur zu ständigen Petitionen auf nationaler und lokaler Ebene geführt hat, als auch den beruflichen Aufstieg lokaler Beamter ernsthaft untergraben würde. Inzwischen hat sich jedoch herausgestellt, dass die Aufrechterhaltung der sozialen Stabilität, was sich in den steigenden Kosten widerspiegelt, entfremdet wurde und die Geburtsförderung als der Arbeitsschwerpunk lokaler Familienplanungsbehörden in der Ära der Zwei-Kind-Politik ersetzt hat. Da es unwahrscheinlich ist, dass der Konflikt zwischen der Shidu-Gruppe und dem Staat gelöst wird, wird die zukünftige Gestaltung und Durchsetzung der Bevölkerungspolitik weiterhin durch das Shidu-Problem eingeschränkt. KW - Chinese Population Policy KW - Universal Two-Child Policy KW - Policy Implementation in China KW - Stability Maintenance KW - Bereaved Parents Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313052 ER - TY - JOUR A1 - Yang, Xuting A1 - Yao, Wanqiang A1 - Li, Pengfei A1 - Hu, Jinfei A1 - Latifi, Hooman A1 - Kang, Li A1 - Wang, Ningjing A1 - Zhang, Dingming T1 - Changes of SOC content in China's Shendong coal mining area during 1990–2020 investigated using remote sensing techniques JF - Sustainability N2 - Coal mining, an important human activity, disturbs soil organic carbon (SOC) accumulation and decomposition, eventually affecting terrestrial carbon cycling and the sustainability of human society. However, changes of SOC content and their relation with influential factors in coal mining areas remained unclear. In the study, predictive models of SOC content were developed based on field sampling and Landsat images for different land-use types (grassland, forest, farmland, and bare land) of the largest coal mining area in China (i.e., Shendong). The established models were employed to estimate SOC content across the Shendong mining area during 1990–2020, followed by an investigation into the impacts of climate change and human disturbance on SOC content by a Geo-detector. Results showed that the models produced satisfactory results (R\(^2\) > 0.69, p < 0.05), demonstrating that SOC content over a large coal mining area can be effectively assessed using remote sensing techniques. Results revealed that average SOC content in the study area rose from 5.67 gC·kg\(^{−1}\) in 1990 to 9.23 gC·kg\(^{−1}\) in 2010 and then declined to 5.31 gC·Kg\(^{−1}\) in 2020. This could be attributed to the interaction between the disturbance of soil caused by coal mining and the improvement of eco-environment by land reclamation. Spatially, the SOC content of farmland was the highest, followed by grassland, and that of bare land was the lowest. SOC accumulation was inhibited by coal mining activities, with the effect of high-intensity mining being lower than that of moderate- and low-intensity mining activities. Land use was found to be the strongest individual influencing factor for SOC content changes, while the interaction between vegetation coverage and precipitation exerted the most significant influence on the variability of SOC content. Furthermore, the influence of mining intensity combined with precipitation was 10 times higher than that of mining intensity alone. KW - loess plateau KW - coal mining area KW - SOC content prediction KW - human disturbance KW - vegetation restoration KW - climate change Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278939 SN - 2071-1050 VL - 14 IS - 12 ER - TY - THES A1 - Yang, Zhenghong T1 - A systematic study of learned helplessness in Drosophila melanogaster T1 - Eine systematische Untersuchung der erlernten Hilflosigkeit in Drosophila melanogaster N2 - The learned helplessness phenomenon is a specific animal behavior induced by prior exposure to uncontrollable aversive stimuli. It was first found by Seligman and Maier (1967) in dogs and then has been reported in many other species, e.g. in rats (Vollmayr and Henn, 2001), in goldfishes (Padilla, 1970), in cockroaches (Brown, 1988) and also in fruit flies (Brown, 1996; Bertolucci, 2008). However, the learned helplessness effect in fruit flies (Drosophila melanogaster) has not been studied in detail. Thus, in this doctoral study, we investigated systematically learned helplessness behavior of Drosophila for the first time. Three groups of flies were tested in heatbox. Control group was in the chambers experiencing constant, mild temperature. Second group, master flies were punished in their chambers by being heated if they stopped walking for 0.9s. The heat pulses ended as soon as they resumed walking again. A third group, the yoked fly, was in their chambers at the same time. However, their behavior didn’t affect anything: yoked flies were heated whenever master flies did, with same timing and durations. After certain amount of heating events, yoked flies associated their own behavior with the uncontrollability of the environment. They suppressed their innate responses such as reducing their walking time and walking speed; making longer escape latencies and less turning around behavior under heat pulses. Even after the conditioning phase, yoked flies showed lower activity level than master and control flies. Interestingly, we have also observed sex dimorphisms in flies. Male flies expressed learned helplessness not like female flies. Differences between master and yoked flies were smaller in male than in female flies. Another interesting finding was that prolonged or even repetition of training phases didn’t enhance learned helplessness effect in flies. Furthermore, we investigated serotonergic and dopaminergic nervous systems in learned helplessness. Using genetic and pharmacological manipulations, we altered the levels of serotonin and dopamine in flies’ central nervous system. Female flies with reduced serotonin concentration didn’t show helpless behavior, while the learned helplessness effect in male flies seems not to be affected by a reduction of serotonin. Flies with lower dopamine level do not display the learned helplessness effect in the test phase, suggesting that with low dopamine the motivational change in learned helplessness in Drosophila may decline faster than with a normal dopamine level. N2 - Das „learned helplessness“ Phänomen ist ein spezifisches Verhalten nach vorheriger Exposition von unkontrollierbaren aversiven Stimuli induziert. Es wurde zuerst von Seligman und Maier (1967) bei Hunden und dann in vielen anderen Tierarten berichtet, z.B. in Ratten (Vollmayr und Henn, 2001), in Goldfische (Padilla , 1970), in Kakerlaken (Brown, 1988) sowie in Fruchtfliegen (Brown, 1996; Bertolucci, 2008). Allerdings wurde das learned helplessness Phänomen in Fruchtfliegen (Drosophila melanogaster) noch nicht genau erforscht. Somit wird in dieser Doktorarbeit haben wir erlernten learned helplessness von Drosophila zum ersten Mal systematisch untersucht. Drei Gruppen von Fliegen wurden in Heatbox getestet. Die Kontrollgruppe war in den Kammern erlebter konstant milder Temperatur. Die zweite Master Gruppe wurde in ihren Kammern erhitzt, wenn sie blieb stehen für 0,9 s. Die Hitze endete, sobald sie sich wieder bewegten. Eine dritte Gruppe, die Yoked Fliegen, war in ihren Kammern gleichzeitig. Doch ihr Verhalten keine Auswirkungen auf die Hitze hatte: Yoked Fliegen wurden erhitzt, wenn Master Fliegen wurden, mit gleichen Zeitpunkt und Dauer. Nach gewissen Hitze Veranstaltungen, Yoked Fliegen assoziierten ihre eigenen Verhalten mit der Unkontrollierbarkeit der Umwelt. Sie unterdrückte ihre angeborene Reaktionen, wie die Verringerung ihrer Laufaktivität; verlängerte mehr Fluchtlatenzzeiten und weniger Umdrehen Verhalten unter Hitzen. Auch nach der Konditionierungsphase zeigte Yoked Fliegen niedrigeren Aktivität als Master und Kontrolle Fliegen. Interessanterweise haben wir auch Sex Dimorphismus in Fliegen beobachtet. Male Fliegen haben learned helplessness nicht wie weibliche Fliegen ausgedrückt. Die Unterschiede zwischen den Master und Yoked Fliegen waren bei männlichen kleiner als bei weiblichen Fliegen. Ein weiteres interessantes Ergebnis war, dass längere oder sogar wiederholte Trainingsphasen die lerned helplessness Wirkung bei Fliegen nicht verstärken könnten. Darüber hinaus haben wir serotonergen und dopaminerge Nervensysteme in learned helplessness erforscht. Mit genetischen und pharmakologischen Manipulationen, haben wir das Niveau von Serotonin und Dopamin im zentralen Nervensystem der Fliegen geändert. Weibliche Fliegen mit reduzierten Serotoninkonzentration zeigten kein hilflos Verhalten, während die learned helplessness Wirkung in männlichen Fliegen schien nicht durch eine Reduktion von Serotonin beeinflusst zu werden. Fliegen mit niedrigerer Dopamin Konzentration zeigten keine learned helplessness Wirkung in der Testphase an, was darauf hindeutet, dass mit niedrigen Dopamin die Motivationsänderung in learned helplessness in Drosophila kann schneller als mit einem normalen Dopaminspiegel sinken. KW - Taufliege KW - Gelernte Hilflosigkeit KW - Drosophila KW - learned helplessness KW - depression KW - learning and memory Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112424 ER - TY - JOUR A1 - Yanku, Yifat A1 - Bitman-Lotan, Eliya A1 - Zohar, Yaniv A1 - Kurant, Estee A1 - Zilke, Norman A1 - Eilers, Martin A1 - Orian, Amir T1 - Drosophila HUWE1 ubiquitin ligase regulates endoreplication and antagonizes JNK signaling during salivary gland development JF - Cells N2 - The HECT-type ubiquitin ligase HECT, UBA and WWE Domain Containing 1, (HUWE1) regulates key cancer-related pathways, including the Myc oncogene. It affects cell proliferation, stress and immune signaling, mitochondria homeostasis, and cell death. HUWE1 is evolutionarily conserved from Caenorhabditis elegance to Drosophila melanogaster and Humans. Here, we report that the Drosophila ortholog, dHUWE1 (CG8184), is an essential gene whose loss results in embryonic lethality and whose tissue-specific disruption establishes its regulatory role in larval salivary gland development. dHUWE1 is essential for endoreplication of salivary gland cells and its knockdown results in the inability of these cells to replicate DNA. Remarkably, dHUWE1 is a survival factor that prevents premature activation of JNK signaling, thus preventing the disintegration of the salivary gland, which occurs physiologically during pupal stages. This function of dHUWE1 is general, as its inhibitory effect is observed also during eye development and at the organismal level. Epistatic studies revealed that the loss of dHUWE1 is compensated by dMyc proeitn expression or the loss of dmP53. dHUWE1 is therefore a conserved survival factor that regulates organ formation during Drosophila development. KW - HECT KW - HUWE1 KW - ubiquitin KW - salivary gland KW - endoreplication KW - JNK KW - dMyc KW - dmP53 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197630 SN - 2073-4409 VL - 7 IS - 10 ER - TY - THES A1 - Yarali, Ayse T1 - Aspects of predictive learning in the fruit fly T1 - Aspekte des assoziatives Lernens bei Taufliegen N2 - Past experience contributes to behavioural organization mainly via learning: Animals learn otherwise ordinary cues as predictors for biologically significant events. This thesis studies such predictive, associative learning, using the fruit fly Drosophila melanogaster. I ask two main questions, which complement each other: One deals with the processing of those cues that are to be learned as predictors for an important event; the other one deals with the processing of the important event itself, which is to be predicted. Do fruit flies learn about combinations of olfactory and visual cues? I probe larval as well as adult fruit flies for the learning about combinations of olfactory and visual cues, using a so called ‘biconditional discrimination’ task: During training, one odour is paired with reinforcement only in light, but not in darkness; the other odour in turn is reinforced only in darkness, but not in light. Thus, neither the odours nor the visual conditions alone predict reinforcement, only combinations of both do. I find no evidence that either larval or adult fruit flies were to solve such task, speaking against a cross-talk between olfactory and visual modalities. Previous studies however suggest such cross-talk. To reconcile these results, I suggest classifying different kinds of interaction between sensory modalities, according to their site along the sensory-motor continuum: I consider an interaction ‘truly’ cross-modal, if it is between the specific features of the stimuli. I consider an interaction ’amodal’ if it instead engages the behavioural tendencies or ‘values’ elicited by each stimulus. Such reasoning brings me to conclude that different behavioural tasks require different kinds of interaction between sensory modalities; whether a given kind of interaction will be found depends on the neuronal infrastructure, which is a function of the species and the developmental stage. Predictive learning of pain-relief in fruit flies Fruit flies build two opposing kinds of memory, based on an experience with electric shock: Those odours that precede shock during training are learned as predictors for punishment and are subsequently avoided; those odours that follow shock during training on the other hand are learned as signals for relief and are subsequently approached. I focus on such relief learning. I start with a detailed parametric analysis of relief learning, testing for reproducibility as well as effects of gender, repetition of training, odour identity, odour concentration and shock intensity. I also characterize how relief memories, once formed, decay. In addition, concerning the psychological mechanisms of relief learning, first, I show that relief learning establishes genuinely associative conditioned approach behaviour and second, I report that it is most likely not mediated by context associations. These results enable the following neurobiological analysis of relief learning; further, they will form in the future the basis for a mathematical model; finally, they will guide the researchers aiming at uncovering relief learning in other experimental systems. Next, I embark upon neurogenetic analysis of relief learning. First, I report that fruit flies mutant for the so called white gene build overall more ‘negative’ memories about an experience with electric shock. That is, in the white mutants, learning about the painful onset of shock is enhanced, whereas learning about the relieving offset of shock is diminished. As they are coherently affected, these two kinds of learning should be in a balance. The molecular mechanism of the effect of white on this balance remains unresolved. Finally, as a first step towards a neuronal circuit analysis of relief learning, I compare it to reward learning and punishment learning. I find that relief learning is distinct from both in terms of the requirement for biogenic amine signaling: Reward and punishment are respectively signalled by octopamine and dopamine, for relief learning, either of these seem dispensible. Further, I find no evidence for roles for two other biogenic amines, tyramine and serotonin in relief learning. Based on these findings I give directions for further research. N2 - Vergangene Ereignisse beeinflussen die Organisation des Verhaltens hauptsächlich durch das Lernen: Tiere lernen natürlich vorkommende neutrale Reize als Signal für biologisch relevante Ereignisse zu nutzen. Diese Dissertation befasst sich mit derartigen assoziativen Lernvorgängen bei der Taufliege Drosophila melanogaster. Ich stelle zwei, sich ergänzende, grundlegende Fragen: Die eine Frage beschäftigt sich mit der Verarbeitung von Reizen, die als Signal für ein wichtiges Ereignis erlernt werden. Die andere Frage behandelt die Verarbeitung des Ereignisses selbst. Lernen Taufliegen etwas über Kombinationen von olfaktorischen und visuellen Reizen? Sowohl bei larvalen, als auch bei adulten Taufliegen wird das Lernen von Kombinationen aus olfaktorischen und visuellen Stimuli untersucht. Ich verwende einen sogenannten „bikonditionalen Diskriminierungs-Versuchsaufbau“: Während des Trainings wird ein Duft nur im Licht und nicht im Dunkeln mit Reinforcement kombiniert, während ein anderer Duft nur im Dunkeln und nicht im Licht mit Reinforcement kombiniert wird. Somit signalisieren weder die Düfte, noch die visuellen Bedingungen allein das Reinforcement, sondern nur eine Kombination aus Beiden. Ich finde keine Beweise dafür, dass larvale oder adulte Taufliegen eine solche Aufgabe lösen können. Dies spricht gegen eine Interaktion zwischen olfaktorischen und visuellen Modalitäten. Allerdings weisen frühere Studien auf derartige Interaktionen hin. Um meine Ergebnisse mit den bekannten Studien in Einklang zu bringen, ordne ich die unterschiedlichen Interaktionen zwischen den sensorischen Modalitäten nach ihrer Lage entlang des sensorisch-motorischen Kontinuums: Ich bezeichnen eine Interaktion für „echt“ cross-modal, wenn sie zwischen den spezifischen Eigenschaften der beiden Reize stattfindet. Ich halte eine Interaktion für „amodal“, wenn sie zwischen den von den Reizen induzierten Verhaltenstendenzen und „Werten“ stattfindet. Aufgrund dieser Argumentation komme ich zu der Schlussfolgerung, dass unterschiedliche Verhaltensaufgaben unterschiedliche Interaktionen zwischen den sensorischen Modalitäten erfordern. Ob eine Art von Interaktion gefunden wird oder nicht hängt von der neuronalen Vernetzung ab, welche charakteristisch für Art und Entwicklungsstadium ist. Assoziatives Lernen von Schmerz-Erleichterung bei Taufliegen Taufliegen entwickeln zwei unterschiedliche Arten von Gedächtnissen basierend auf Erfahrung mit Elektro-Schock: Düfte, die während des Trainings dem Schock vorausgehen, werden als Bestrafungssignale gelernt und deshalb vermieden. Düfte, die während des Trainings auf den Schock folgen, werden als Erleichterungssignale gelernt und deshalb bevorzugt. Ich beschäftige mich mit der zweiten Art dieses assoziativen Lernens, das ich als „Erleichterungslernen“ bezeichne. Ich beginne mit einer detaillierten parametrischen Analyse des Erleichterungslernens. Die Reproduzierbarkeit, sowie die Einflüsse des Geschlechts, der Anzahl an Trainingswiederholungen, der Duftintensität, der Duftkonzentration und der Schockintensität werden geprüft. Ich teste, wie das Erleichterungsgedächtnis, nachdem es gebildet wurde, wieder gelöscht wird. Des Weiteren gehe ich zwei wichtigen Fragen zu den psychologischen Mechanismen des Erleichterungslernen nach: Zum einen zeige ich, dass das Erleichterungslernen echtes assoziativ konditioniertes Annäherungsverhalten etabliert. Zum anderen zeige ich, dass vorausgegangenes Kontext-Schock Training das folgende Erleichterungslernen nicht beeinflusst. Das Erleichterungslernen wird also nicht durch Kontextassoziation vermittelt. Diese Ergebnisse erlauben die folgende neurobiologische Analyse des Erleichterungslernens. Außerdem werden sie in Zukunft als Grundlage für ein mathematisches Modell des Erleichterungslernens dienen. Schließlich werden die Forscher/innen, die das Erleichterungslernen in anderen experimentellen Systemen untersuchen, von diesen parametrischen Erkenntnissen profitieren. In einer neurobiologischen Analyse des Erleichterungslernens zeige ich, dass der Verlust der Funktion des sogenannten white Gens die beiden unterschiedlichen Arten von Schock-Induziertem Lernen zusammenhängend beeinflusst: Das Bestrafungslernen wird verstärkt und das Erleichterungslernen wird abgeschwächt. Auf Grund dieses Ergebnisses schlagen ich vor, dass sich diese zwei Arten von Lernen in einem Gleichgewicht befinden sollen, welches vom white Gen beeinflusst wird. Die zugrunde liegenden molekularen Mechanismen eines solchen Gleichgewichts sind noch nicht bekannt. Schließlich vergleiche ich das Erleichterungslernen mit dem Belohnungslernen und dem Bestrafungslernen. Ich zeige, dass das Erleichterungslernen anders ist als beide: Bestrafung und Belohnung werden entsprechend von Dopamin und Octopamin vermittelt. Für das Erleichterungslernen sind beide diese biogenen Aminen unnötig. Ebenso finde ich beim Erleichterungslernen keinen Beleg für die Rolle von zwei weiteren Aminen: Tyramin und Serotonin. Aufgrund dieser Ergebnisse schlage ich vor weitere Forschungsrichtungen. KW - Lernen KW - Drosophila KW - Neurogenetik KW - Lernverhalten KW - olfaktorik KW - sehen KW - erleichterungslernen KW - associative learning KW - drosophila KW - neurogenetic analyses KW - behavioural analyses KW - relief Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-28741 ER - TY - THES A1 - Yazdani Rashvanlouei, Kourosh T1 - Developing a Framework for International Projects of ERP Implementation T1 - Entwicklung eines Rahmens für internationale Projekte der ERP-Implementierung N2 - Enterprise Systeme werden immer mehr von Bedeutung, was sie in die Mitte der Aufmerksamkeit und der Berücksichtigung durch Organisationen in verschiedensten Formen rückt – seien es Unternehmen oder Industrien von riesigen öffentlichen oder privaten Organisationen bis hin zu mittleren und kleinen Dienstleistungsunternehmen. Diese Systeme verbessern sich ständig, sowohl funktionell, als auch technologisch und sie sind unumgänglich für Unternehmen, um ihre Produktivität zu vergrößern und um in dem nationalen und globalen Wettbewerb mitzuhalten. Da lokale Softwarelösungen die Bedingungen, speziell von großen Betrieben, funktionell und technologisch nicht erfüllen konnten und da riesige globale Softwarehersteller, wie SAP, Oracle und Microsoft ihre Lösungen rapide verbessern und sie ihren Markt immer mehr über den Globus expandieren, nimmt die Nachfrage für diese globalen Marken und deren nahezu einwandfreien Softwarelösungen täglich zu. Die Zustimmung für internationale ERP Unternehmensberatungsanwendungen nimmt deswegen exponentiell zu, während die Forschung der beeinflussenden Faktoren und des Fachwissens wenig verbreitet ist. Deswegen ist es so dringlich, dieses Gebiet zu erforschen. Das schlussendliche fünf-in-fünf Framework dieser Studie sammelt zum ersten Mal in der Geschichte alle historisch erwähnten, kritischen Erfolgsfaktoren und Projektaktivitäten. Diese wurden in fünf Phasen unterteilt und nach den fünf Schwerpunkten der internationalen ERP Projektdurchführung kategorisiert. Dieses Framework bietet einen Überblick und bildet einen umfassenden Fahrplan für solche Projekte. N2 - The importance of enterprise systems is increasingly growing and they are in the center of attention and consideration by organizations in various types of business and industries from extra-large public or private organizations to small and medium-sized service sector business. These systems are continuously advancing functionally and technologically and are inevitable and ineluctable for the enterprises to maximize their productivity and integration in current competitive national and global business environments. Also, since local software solutions could not meet the requirements of especially large enterprises functionally and technically, and as giant global enterprise software producers like SAP, Oracle and Microsoft are improving their solutions rapidly and since they are expanding their market to more corners of the globe, demand for these globally branded low-defect software solutions is daily ascending. The agreements for international ERP implementation project consultancy are, therefore, exponentially increasing, while the research on the influencing factors and know-hows is scattered and rare, and thus, a timely urgency for this field of research is being felt. The final developed five-in-five framework of this study, for the first time, collects all mentioned-in-the-history critical success factors and project activities, while sequencing them in five phases and categorizing them in five focus areas for international ERP implementation projects. This framework provides a bird’s-eye view and draws a comprehensive roadmap or instruction for such projects. KW - ERP KW - International IT Projects KW - ERP Implementation KW - Framework Develpment KW - ERP CSF Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154000 ER - TY - THES A1 - Ye, Fang T1 - The role of DNA supercoiling in the coordinated regulation of gene expression in Helicobacter pylori N2 - Summary Mechanisms of global gene regulation in bacteria are not well characterized yet. Changes in global or local supercoiling of chromosomal DNA are thought to play a role in global gene silencing and gene activation. In Helicobacter pylori, a bacterium with few dedicated transcriptional regulators, the structure of some promoters indicates a dependency on DNA topology. For example, the promoter of the major flagellar subunit gene flaA (ó28-dependent) has a shorter spacing of 13 nucleotides (nt) in comparison to the consensus promoter (15 nt). Supercoiling changes might be a mechanism of gene-specific and global transcriptional regulation in this bacterium. The aim of this study was to elucidate, if changes in global supercoiling have an influence on global gene regulation in H. pylori, and on the temporal regulation of the flagellar biosynthesis pathway in this organism. In the present work, global DNA supercoiling in H. pylori was visualized for the first time, by determining the supercoiling state of plasmids under different growth conditions. Using this method, we showed that cellular supercoiling was clearly growth phase-dependent in H. pylori. Coinciding with increased supercoiling during the growth phases, transcription of the flaA gene was increased, while the transcription of a second ó28-dependent gene with regular promoter spacing (HP0472) was reduced, supporting the hypothesis that growth phase-dependency of promoters might be mediated by changes of DNA topology. Supercoiling in H. pylori could be influenced in a reproducible fashion by inhibition of gyrase using novobiocin, which led to DNA relaxation and to a concomitant decrease of flaA transcript levels. Promoter spacer mutagenesis of the flaA promoter was performed. With flaA promoters of increased or reduced length, transcription of flaA was reduced, less susceptible to supercoiling changes, and, under specific conditions, inverted as compared to the wild type promoter. Transcriptional interdependence between the coupled topA-flaB genes and flaA was found by analysis of the flaA promoter mutants. Chromosomally linked gyrA-flgR, and topA-flaB genes were all dependent on supercoiling and coregulated with each other. Comprehensive transcript profiling (DNA microarrays) of wildtype H. pylori with and without novobiocin treatment identified a number of genes (10% of total genes), including flagellin, virulence and housekeeping genes, which were strongly dependent on and appeared to be synchronized by supercoiling changes (transcriptional up- or downregulation). These findings indicate a tightly coupled temporal regulation of flagellar biogenesis and metabolism in H. pylori, dependent on global supercoiling. A specific group of genes was also regulated in H. pylori by overexpression of Topoisomerase I, as detected by genome-wide analysis (DNA microarray). The DNA-bending protein HU is thought to be responsible for influencing the negative supercoiling of DNA, through its ability to wrap DNA. HU is encoded by the hup single gene in H. pylori, and constitutively expressed during the whole growth curve. An H. pylori hup mutant was constructed. H. pylori cells lacking HU protein were viable, but exhibited a severe growth defect. Our data indicate that the lack of HU dramatically changes global DNA supercoiling, indicating an important function of HU in chromosome structuring in H. pylori. Transcriptome analyses were performed and demonstrated that a total of 66 genes were differentially transcribed upon hup deletion, which include virulence genes and many other cell functions. The data indicate that HU might act as further important global regulator in H. pylori. Increased gene expression of heat shock proteins and a decreased transcription of the urease gene cluster may indicate a co-ordinated response of H. pylori to changes of environmental conditions in its specific ecological niche, mediated by HU. After the whole genomic sequences of H. pylori strains 26695 and J99 were published, two ORFs (HP0116 and HP0440) were presumptively annotated as topoisomerase I orthologs. HP0116 is the functional H. pylori topoisomerase I (TopA). HP0440 (topA2) was found in only few (5 of 43) strains. Western blot analysis indicated that TopA2 is antigenically different from TopA. TopA2 is transcribed in H. pylori, but the protein must be functionally different from TopA, since it is lacking one functionally essential zinc finger motif, and was not able to functionally complement a TopA-deficient E. coli. Like topA, topA2 was also transcribed in a growth phase-dependent manner. We did not find a function of TopA2 in DNA structuring or topology, but, in the present study, we were able for the first time to establish a unique function for TopA2 in global gene regulation, by comprehensive transcriptome analysis (DNA microarray). Transcriptome analysis showed that a total of 46 genes were differentially regulated upon topA2 deletion, which included flagellar genes and urease genes. These results suggest that TopA2 might act as a novel important regulator of both flagellar biosynthesis and urease in H. pylori. N2 - Zusammenfassung Die Mechanismen der globalen Kontrolle der Genregulation bei Bakterien sind bisher noch wenig charakterisiert. Unterschiede in der globalen oder lokalen Topologie der chromosomalen DNA spielen wahrscheinlich eine Rolle bei der globalen Kontrolle der Genexpression. Bei Helicobacter pylori, einem Bakterium mit wenigen funktionell definierten Transkriptionsregulatoren, spricht die Struktur einiger Promotoren dafür, daß sie durch die DNA-Topologie kontrolliert werden. Der Promotor des Hauptflagellingens flaA, ein Sigma-28 abhängiger Promotor, hat ein gegenüber dem Konsensuspromotor (15 Nukleotide) verkürztes Spacing von 13 Nukleotiden. Veränderungen der DNA-Superhelizität könnten ein Mechanismus der genspezifischen und globalen transkriptionellen Kontrolle in diesem Bakterium sein. Ziel dieser Untersuchungen war es zu zeigen, ob Veränderungen des globalen Supercoiling-Niveaus einen Einfluß auf die globale Genregulation von H. pylori haben und ob sie sich auf die zeitlich gesteuerte Regulation der Geißelbiosynthese (Beweglichkeitsorganell; essenzieller Virulenz- und Persistenzfaktor von H. pylori) in diesem Organismus auswirkt. In der vorliegenden Arbeit wurde das Niveau der DNA-Superspiralisierung bei H. pylori erstmals durch Visualisierung des Supercoilingzustands von Plasmiden unter unterschiedlichen Wachstumsbedingungen dargestellt. Wir konnten mit dieser Methode zeigen, daß das zelluläre Supercoiling-Niveau bei H. pylori in Abhängigkeit von der Wachstumsphase stark variiert. In Wachstumsphasen mit erhöhtem Supercoiling war auch die Transkription des flaA-Gens erhöht, während die Transkription eines zweiten Sigma-28-abhängigen Gens mit normalem Promotorabstand (HP0472) reduziert war. Dieser Befund stützte die Hypothese, daß die wachstumsphasenabhängige Aktivität dieser Promotoren durch Veränderungen der DNA-Topologie bewirkt wird. Das Supercoiling-Niveau konnte reproduzierbar durch Hemmung der Gyrase mit Novobiocin beeinflusst werden. Die Gegenwart von Novobiocin führte zur DNA-Relaxation und zu einem gleichzeitigen Absinken der Transkription von flaA. Es wurde eine gerichtete Mutagenese der Promotor-Spacer-Region des flaA-Promotors durchgeführt. Die Verlängerung oder Verkürzung des H.pylori flaA-Promotors führte zu einer verminderten Transkription von flaA, sowie zu reduzierter Empfindlichkeit der Promotoraktivität gegenüber Veränderungen des Supercoiling-Niveaus. Unter spezifischen Bedingungen war die Supercoiling-Abhängigkeit umgekehrt im Vergleich zum Wildtyppromotor. Es konnte weiterhin eine inverse transkriptionelle Abhängigkeit zwischen dem gekoppelten Genpaar topA-flaB und flaA durch Analyse der flaA-Promotormutanten nachgewiesen werden. Auch die chromosomal gekoppelten Gene gyrA und flgR sowie topA und flaB waren abhängig vom Supercoiling-Zustand und miteinander koreguliert. Die Analyse des Transkriptoms von H.pylori-Wildtypbakterien mit DNA-Microarrays mit und ohne Novobiocinbehandlung führte zur Identifizierung von zahlreichen Genen (etwa 10% des Gesamttranskriptoms), deren Expression Supercoiling-abhängig war und durch Veränderungen des Supercoilings synchronisiert verändert werden konnte. Unter diesen waren Flagellin-, andere Virulenz-, sowie Grundstoffwechsel-Gene. Diese Befunde weisen auf eine enge Verbindung zwischen der chronologischen Kontrolle der Flagellen-Biogenese und des Metabolismus bei H. pylori, die gemeinsam durch das Supercoiling-Niveau gesteuert werden. Eine definierte Gruppe von Genen konnte bei H.pylori durch Überexpression von Topoisomerase-1 reguliert werden. Das Protein HU beeinflusst ebenfalls das Supercoiling-Niveau von DNA durch seine Fähigkeit, DNA zu biegen. HU wird bei H. pylori durch das Gen hup kodiert und ist während sämtlicher Wachstumsphasen konstitutiv exprimiert. Eine HU-defiziente Mutante wurde konstruiert. Zellen, die kein HU-Protein exprimierten, waren lebensfähig, zeigten aber einen deutlichen Wachstumsdefekt. Unsere Daten weisen daraufhin, daß der Mangel von HU sich dramatisch auf das globale DNA-Supercoiling-Niveau auswirkt, und sprechen für eine wichtige Funktion von HU bei der Kontrolle der DNA-Struktur von H. pylori. Mittels DNA-Microarray-Hybridisierung wurden die Transkriptome von H. pylori-Wildtyp und HU-Mutante miteinander verglichen. Die Ergebnisse zeigen, daß insgesamt 66 Gene in der HU-Mutante differentiell transkribiert werden, darunter Virulenzgene und Gene für viele andere Zellfunktionen. Diese Daten deuten darauf hin, daß auch HU eine wichtige Rolle in der Kontrolle der globalen Genexpression bei H. pylori spielt. Die erhöhte Expression von Hitzestress-Proteinen, verbunden mit einer verminderten Transkription des Ureasegenclusters, könnte auf eine koordinierte Antwort der Bakterien auf Veränderungen der Umweltbedingungen in ihrer spezifischen ökologischen Nische hinweisen. Nach der Publikation der Gesamtgenomsequenzen von H.pylori 26695 und J99 wurden 2 ORFs (HP 0116 und HP 0440) als Topoisomerase-1-Orthologe annotiert. HP 0116 ist das funktionelle H.pylori Topoisomerase-1-Gen. HP 0442 (topA2) wurde nur in wenigen (5 aus 43) Stämmen nachgewiesen. topA2 ist trotz seines seltenen Vorkommens kein Pseudogen und wird in H.pylori transkribiert. Westernblot-Analysen sprechen dafür, daß TopA2 sich antigenetisch von TopA unterscheidet. Das TopA2-Protein unterscheidet sich ebenfalls funktionell von TopA, da ihm ein funktionell essentielles Zinkfingermotiv fehlt. TopA2 konnte außerdem eine TopA-defiziente E.coli-Mutante nicht funktionell komplementieren. Wie bei topA war auch die Transkription von topA2 von der Wachstumsphase abhängig. Eine Funktion von TopA2 bei der Kontrolle der DNA-Topologie konnte bisher nicht nachgewiesen werden, Transkriptomanalysen zeigten aber, dass TopA2 eine klare Regulationsfunktion hat, da die topA2-Mutante gravierende Veränderungen des Transkriptoms gegenüber dem Wildtyp aufwies. Diese Untersuchungen zeigten, daß 46 Gene in der TopA2-Mutante differentiell reguliert wurden, darunter Flagellengene und Ureasegene. Die Ergebnisse sprechen dafür, daß TopA2 ein weiterer wichtiger Regulator von sowohl Flagellenbiosynthese als auch Ureasebildung bei H.pylori sein könnte. KW - Helicobacter pylori KW - Genexpression KW - Flagelline KW - Supercoiling KW - regulation KW - flagella Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-9878 ER - TY - THES A1 - Ye, Liqing T1 - RNA-RNA interactions in viral genome packaging T1 - RNA-RNA-Interaktionen bei der viralen Genomverpackung N2 - RNA is one of the most abundant macromolecules and plays essential roles in numerous biological processes. This doctoral thesis consists of two projects focusing on RNA structure and RNA-RNA interactions in viral genome packaging. In the first project I developed a method called Functional Analysis of RNA Structure (FARS-seq) to investigate structural features regulating genome dimerization within the HIV-1 5’UTR. Genome dimerization is a conserved feature of retroviral replication and is thought to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. It also plays a role in genome integrity and evolution through recombination, and is linked to a structural switch that may regulate genome packaging and translation within cells. Despite its importance for HIV-1 replication, the RNA signals regulating genome dimerization, and the molecular mechanism leading to the selection of the genome dimer over the monomer for packaging are incompletely understood. The FARS-seq method combines RNA structural information obtained by chemical probing with single nucleotide resolution profiles of RNA function obtained by mutational interference. In this way, we found nucleotides that were critical for dimerization, especially within the well-characterized dimerization motif within stem-loop 1 (SL1). We also found stretches of nucleotides that enhanced genome dimerization upon mutation, suggesting their role in negatively regulating dimerization. A structural analysis identified distinct structural signatures within monomeric and dimeric RNA. The dimeric conformation displayed the canonical transactivation response (TAR), PolyA, primer binding site (PBS), and SL1-SL3 stem-loops, and contained a long range U5-AUG interaction. Unexpectedly, in monomeric RNA, SL1 was reconfigured into long- and short-range base-pairings with PolyA and PBS, respectively. Intriguingly, these base pairings concealed the palindromic sequence needed for dimerization and disrupted the internal loop in SL1 previously shown to contain the major packaging motif for Pr55Gag. We therefore rationally introduced mutations into PolyA and PBS, and showed how these regions regulate genome dimerization, and the binding of Pr55Gag in vitro, as well as genome packaging into virions. These findings give insights into late stages of the HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging. In the second project, I developed a proximity ligation and high-throughput sequencing-based method, RNA-RNA seq, which can measure direct (RNA-RNA) and indirect (protein-mediated) interactions. In contrast to existing methods, RNA-RNA seq is not limited by specific protein or RNA baits, nor to a particular crosslinking reagent. The genome of influenza A virus contains eight segments, which assemble into a “7+1” supramolecular complex. However, the molecular details of genome assembly are poorly understood. Our goal is to use RNA-RNA seq to identify the sites of interaction between the eight genomic RNAs of influenza, and to use this information to define the quaternary RNA architecture of the genome. We showed that RNA-RNA seq worked on model substrates, like the HIV-1 Dimerization Initiation Site (DIS) RNA and purified ribosome, as well as influenza A virus infected cells. N2 - RNA ist eines der am häufigsten vorkommenden Makromoleküle und spielt bei allen biologischen Prozessen eine wesentliche Rolle. Diese Doktorarbeit besteht aus zwei Projekten, die sich auf die RNA-Struktur und RNA-RNA-Interaktionen bei der viralen Genomverpackung konzentrieren. Im ersten Projekt habe ich eine Methode namens Functional Analysis of RNA Structure (FARS-seq) entwickelt, um strukturelle Merkmale zu untersuchen, die die Genom-Dimerisierung innerhalb des HIV-1 5'UTR regulieren. Die Genomdimerisierung ist ein konserviertes Merkmal der retroviralen Replikation und gilt als Voraussetzung für die Bindung an das virale Strukturprotein Pr55Gag während der Genomverpackung. Sie spielt auch eine Rolle bei der Genomintegrität und -evolution durch Rekombination und ist mit einem strukturellen Schalter verbunden, der die Genomverpackung und -translation in Zellen regulieren kann. Trotz der Bedeutung für die HIV-1-Replikation sind die RNA-Signale welche die Genom-Dimerisierung regulieren, und der molekulare Mechanismus der zur Auswahl des Genom-Dimers gegenüber dem Monomer für die Verpackung führt, nur unvollständig verstanden. FARS-seq kombiniert RNA-Strukturinformationen, die durch chemisches Sondieren gewonnen werden, mit Profilen der RNA-Funktion in Einzelnukleotid-Auflösung, die durch Mutationsinterferenz gewonnen werden. Auf diese Weise fanden wir Nukleotide, die für die Dimerisierung kritisch sind, insbesondere innerhalb des gut charakterisierten Dimerisierungsmotivs von stem-loop 1 (SL1). Wir fanden auch Nukleotidabschnitte, die bei Mutation die Dimerisierung des Genoms verstärkten, was auf ihre Rolle bei der negativen Regulierung der Dimerisierung hindeutet. Eine Strukturanalyse ergab zudem unterschiedliche strukturelle Signaturen innerhalb der RNA von Monomeren und Dimeren. Die dimere Konformation wies die kanonische Transaktivierungsantwort (TAR), PolyA, die Primerbindestelle (PBS) und SL1-SL3-stem loops auf und enthielt eine weitreichende U5-AUG-Interaktion. Unerwarteterweise interagierte SL1 in monomerer RNA mit dem weit entfernten PolyA Signal und der nahegelegenen PBS. Interessanterweise verbargen diese Basenpaarungen die für die Dimerisierung erforderliche palindromische Sequenz und unterbrachen die interne Schleife in SL1, von der zuvor gezeigt wurde, dass sie das Hauptverpackungsmotiv für Pr55Gag enthält. Wir haben daher auf rationale Weise Mutationen in PolyA und PBS eingeführt und gezeigt, wie diese Regionen die Dimerisierung des Genoms und die Bindung von Pr55Gag in vitro sowie die Verpackung des Genoms in Virionen regulieren. Diese Ergebnisse geben Einblicke in späte Stadien des HIV-1-Lebenszyklus und eine mechanistische Erklärung für die Verbindung zwischen RNA-Dimerisierung und Verpackung. Im zweiten Projekt entwickelte ich eine auf Proximity Ligation und Hochdurchsatz-Sequenzierung basierende Methode, RNA-RNA seq, mit der direkte (RNA-RNA) und indirekte (proteinvermittelte) Wechselwirkungen gemessen werden können. Im Gegensatz zu bestehenden Methoden ist RNA-RNA seq nicht durch spezifische Protein- oder RNA Crosslink-Reagenzien eingeschränkt. Das Genom des Influenza-A-Virus besteht aus acht Segmenten, die zu einem supramolekularen "7+1"-Komplex assemblieren. Die molekularen Details des Genomaufbaus sind jedoch kaum bekannt. Unser Ziel ist es, mit Hilfe von RNA-RNA seq die Interaktionsstellen zwischen den acht genomischen RNAs des Influenza-Virus zu identifizieren und somit die quaternäre RNA-Architektur des Genoms zu definieren. Wir haben gezeigt, dass RNA-RNA seq an Modellsubstraten wie der HIV Dimerization Initiation Site (DIS) RNA und gereinigtem Ribosom sowie an mit dem Influenza-A-Virus infizierten Zellen funktioniert. KW - RNS-Viren KW - RNA-RNA interactions KW - Virus infection KW - viral genome packaging Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-296361 ER - TY - JOUR A1 - Ye, Liqing A1 - Ambi, Uddhav B. A1 - Olguin-Nava, Marco A1 - Gribling-Burrer, Anne-Sophie A1 - Ahmad, Shazeb A1 - Bohn, Patrick A1 - Weber, Melanie M. A1 - Smyth, Redmond P. T1 - RNA structures and their role in selective genome packaging JF - Viruses N2 - To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution. KW - RNA virus KW - RNA KW - RNA structure KW - genome packaging KW - viral assembly KW - evolution Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246101 SN - 1999-4915 VL - 13 IS - 9 ER - TY - THES A1 - Ye, Mingyu T1 - Immunotherapy with Vaccinia virus co-expressing tumor-associated antigens and mouse IL-2 cytokine in mice with mammary cancer T1 - Immuntherapie von Brustkrebs in tumortragenden Mäusen mit genetisch modifizierten Vaccinia Viren, die simultan Interleukin-2 und tumorassoziierte Antigene exprimieren N2 - Interleukin 2 (IL-2) was the first cytokine applied for cancer treatment in human history. It has been approved as monotherapy for renal cell carcinoma and melanoma by the FDA and does mediate the regression of the tumors in patients. One of the possible mechanisms is that the administration of IL-2 led to T lymphocytes expansion, including CD4+ and CD8+ T cells. In addition, a recent study demonstrated that antigen-specific T cells could also be expanded through the induction of IL-2, which plays a crucial role in mediating tumor regression. However, despite the long-term and extensive use of IL-2 in the clinic, the ratio of patients who get a complete response was still low, and only about one-fifth of patients showed objective tumor regression. Therefore, the function of IL-2 in cancer treatment should continue to be optimized and investigated. A study by Franz O. Smith et al. has shown that the combination treatment of IL-2 and tumor-associated antigen vaccine has a strong trend to increased objective responses compared to patients with melanoma receiving IL-2 alone. Peptide vaccines are anti-cancer vaccines able to induce a powerful tumor antigenspecific immune response capable of eradicating the tumors. According to the type of antigens, peptide vaccines can be classified into two distinct categories: Tumor-associated antigens (TAA) vaccine and tumor-specific neoantigens (TSA) vaccine. Currently, Peptide vaccines are mainly investigated in phase I and phase II clinical trials of human cancer patients with various advanced cancers such as lung cancer, gastrointestinal tumors, and breast cancers. Vaccinia virus (VACV) is one of the safest viral vectors, which has been wildly used in cancer treatment and pathogen prevention. As an oncolytic vector, VACV can carry multiple large foreign genes, which enable the virus to introduce diagnostic and therapeutic agents without dramatically reducing the viral replication. Meanwhile, the recombinant vaccinia virus (rVACV) can be easily generated by homologous recombination. Here, we used the vaccinia virus as the therapeutic cancer vector, expressing mouse Interleukin 2 (IL-2) and tumor-associated antigens simultaneously to investigate the combined effect of anti-tumor immune response in the 4T1 mouse tumor model. As expected, the VACV driven mIL-2 expression remarkably increased both CD4+ and CD8+ populations in vivo, and the virus-expressed tumor-associated peptides successfully elicited theantigen-specific T cell response to inhibit the growth of tumors. Furthermore, the experiments with tumor-bearing animals showed that the mIL-2 plus tumor antigens expressing VACV vector gave a better anti-cancer response than the mIL-2 alone expressing vector. The combinations did significantly more inhibit tumor growth than mIL-2 treatment alone. Moreover, the results confirmed our previous unpublished data that the mIL-2 expression driven by synthetic early/late promoter in the Lister strain VACV could enhance the tumor regression in the 4T1 mouse model. N2 - Interleukin 2 (IL-2) war das erste Zytokin in der Geschichte des Menschen, das zur Krebsbehandlung eingesetzt wurde. Es ist von der FDA als Monotherapie für Nierenzellkarzinome und Melanome zugelassen und kann bei Patienten die Rückbildung von Tumorerkrankungen fördern. Einer der möglichen Mechanismen ist, dass die Verabreichung von IL-2 zu einer T-Zell- Expansion führte. Darüber hinaus zeigte eine aktuelle Studie, dass auch antigenspezifische T- Zellen vermehrt werden können, was eine entscheidende Rolle bei der Vermittlung der Tumorregression spielt. Trotz des langjährigen und umfangreichen Einsatzes von IL-2 in der Klinik war der Anteil der Patienten, die eine komplette Antwort Zeigten, jedoch immer noch gering, und nur etwa ein Fünftel der Patienten weist eine objektive Tumorregression auf. Daher sollte die Funktion von IL-2 in der Krebsbehandlung weiter optimiert und untersucht werden. Eine Studie von Franz O. Smith et al. hat gezeigt, dass die Kombinationsbehandlung von IL-2 und tumorassoziiertem Antigenimpfstoff im Vergleich zu Melanomapatienten, die IL-2 allein erhalten, einen starken Trend zu verstärkten objektiven Reaktionen aufweist. Peptidimpfstoff ist ein Anti- Krebs-Impfstoff, der in der Lage ist, eine starke tumorantigenspezifische Immunantwort zu induzieren, die die Tumore ausrotten kann. Je nach Art der Antigene kann es in zwei verschiedene Kategorien eingeteilt werden: Impfstoff gegen tumorassoziierte Antigene (TAA) und Impfstoff gegen tumorspezifische Neoantigene (TSA). Derzeit werden Peptidimpfstoffe hauptsächlich in klinischen Studien in Phasen I und II an Patienten mit verschiedenen fortgeschrittenen Krebsarten wie Lungenkrebs, Magen-Darm-Tumoren und Brustkrebs untersucht ... KW - Immunotherapy KW - Vaccinia virus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-253095 ER - TY - JOUR A1 - Ye, Mingyu A1 - Keicher, Markus A1 - Gentschev, Ivaylo A1 - Szalay, Aladar A. T1 - Efficient selection of recombinant fluorescent vaccinia virus strains and rapid virus titer determination by using a multi-well plate imaging system JF - Biomedicines N2 - Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte\(^®\)S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer. KW - fluorescent recombinant vaccinia virus KW - plaque isolation KW - IncuCyte\(^®\)S3 KW - plaque assay Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245104 SN - 2227-9059 VL - 9 IS - 8 ER - TY - JOUR A1 - Ye, Mingyu A1 - Wilhelm, Martina A1 - Gentschev, Ivaylo A1 - Szalay, Aladár T1 - A modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: A practical workflow and advanced applications JF - Methods and Protocols N2 - Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte\(^®\)S3. KW - monoclonal stable cell KW - limiting dilution cloning KW - ncuCyte\(^®\)S3 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228896 VL - 4 IS - 1 ER - TY - THES A1 - Ye, Qing T1 - Synthesis and Investigation of Borylene Complexes: from Borylene Transfer to Borylene Catenation T1 - Synthese und Untersuchung von Borylenkomplexen: von Borylentransfer zu Borylen-Verkettung N2 - Im Rahmen dieser Arbeit wurde das Spektrum des Borylentransfers ausgeweitet, indem Übergangsmetall Alkinylkomplexe und Metall-Kohlenstoff-Doppelbindungen als Borylen-Akzeptoren eingeschlossen wurden. Neben der Salzeliminierung, Halogenidabstraktion und Dehydrierung, wurde eine neuartige Syntheseroute zu terminalen Borylenkomplexen durch Salz- und Silylhalogenideliminierung etabliert. Mithilfe dieser Strategie gelang die Darstellung von [(OC)3(Me3P)Fe=BDur], ein seltenes Beispiel für einen neutralen Arylborylenkomplex. Im Speziellen hat diese Verbindung ein großes Anwendungspotenzial für Metathesereaktionen und die Funktionalisierung von polycyclischen aromatischen Kohlenwasserstoffen, wie z. B. Naphthalin, gezeigt. Außerdem konnte ein Eisen-Bis(borylen)-Komplex [(OC)3Fe(BDur){BN(SiMe3)2}] durch einen Phosphan-Borylen-Austausch dargestellt werden. Ausgehend von diesem Komplex gelang die Darstellung von 1,4-Diboracyclohexadien bzw. des ersten 1,4-Dibora-1,3-Butadien-Komplexes, wodurch eine neue Art von Borylentransfer etabliert werden konnte. Höchst interessant ist es, dass der Transfer von weiteren Borylen-Einheiten in die Koordinationssphäre des Eisenatoms zu einer kontrollierten Borylen-Verkettung geführt hat. N2 - Within the scope of this thesis, the area of borylene transfer has been broadened by including transition-metal alkynyl complexes and metal-carbon double bonds as borylene acceptors. In addition to double salt elimination, halide abstraction and dehydrogenation processes, a novel high-yield synthetic procedure for terminal borylene complexes was established, i.e. salt elimination and subsequent silylhalogenide liberation. Accordingly, it was possible to prepare [(OC)3(Me3P)Fe=BDur] as a rare example of a neutral arylborylene species. Moreover, this compound has been demonstrated to possess great potential for metathesis reactions and the functionalization of polycyclic aromatic hydrocarbons such as naphthalene. Moreover, it could undergo a phosphine-borylene exchange reaction, yielding the iron bis(borylene) complex [(OC)3Fe(BDur){BN(SiMe3)2}], which has turned out to be applicable for preparation of 1,4-diboracyclohexadiene and unprecedented 1,4-dibora-1,3-butadiene complexes, thus establishing a new type of borylene transfer. Most interestingly, upon transfer of further borylene moieties into the coordination sphere of iron, borylene-catenation was accomplished in a highly controlled manner. KW - Borylene KW - Übergangsmetallkomplexe KW - Borylenkomplexe KW - Bor KW - Photochemie KW - Borheterocyclen KW - Boracumulen KW - Borylene complexes KW - Boron KW - Photochemistry KW - Boraheterocycles KW - Boracumulene Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71443 ER - TY - THES A1 - Ye, Yuxiang T1 - Molecular and Cellular Magnetic Resonance Imaging of Myocardial Infarct Healing T1 - Molekulare und zelluläre Magnetresonanztomographie der Wundheilung nach Myokardinfarkt N2 - Myokardinfarkte (MI) sind eine der häufigsten Todesursachen weltweit. Eine rechtzeitige Wiederherstellung des koronaren Blutflusses im ischämischen Myokard reduziert signifikant die Sterblichkeitsrate akuter Infarkte und vermindert das ventrikuläre Remodeling. Überlebende MI-Patienten entwickeln jedoch häufig eine Herzinsuffizienz, die mit einer reduzierten Lebensqualität, hohen Sterblichkeitsrate (10% jährlich), sowie hohen Kosten für das Gesundheitssystem einhergeht. Die Entwicklung der Herzinsuffizienz nach einem MI ist auf den hohen Verlust kontraktiler Kardiomyozyten, während der Ischämie-Reperfusion zurückzuführen. Anschließende komplexe strukturelle und funktionelle Veränderungen resultieren aus Modifikationen des infarzierten und nicht infarzierten Myokards auf molekularer und zellulärer Ebene. Die verbesserte Überlebensrate von Patienten mit akutem MI und das Fehlen effizienter Therapien, die die Entwicklung und das Fortschreiten des ventrikulären Remodelings verhindern, führen zu einer hohen Prävalenz der Herzinsuffizienz. Die kardiale Magnetresonanztomographie (MRT) ist eine wichtige Methode zur Diagnose und Beurteilung des Myokardinfarktes. Mit dem technologischen Fortschritt wurden die Grenzen der MRT erweitert, so dass es heute möglich ist, auch molekulare und zelluläre Ereignisse in vivo und nicht-invasiv zu untersuchen. In Kombination mit kardialer Morphologie und Funktion könnte die Visualisierung essentieller molekularer und zellulärer Marker in vivo weitreichende Einblicke in den Heilungsprozess infarzierter Herzen liefern, was zu neuen Erkenntnissen für ein besseres Verständnis und bessere Therapien des akuten MI führen könnte. In dieser Arbeit wurden Methoden für die molekulare und zelluläre kardiale MRT-Bildgebung der Inflammation und des Kalziumstroms im Heilungsprozess des akuten Myokardinfarktes in vivo in einem Rattenmodel mit klinischer Relevanz etabliert. N2 - Myocardial infarction (MI) is a leading cause of death worldwide. Timely restoration of coronary blood flow to ischemic myocardium significantly reduces acute infarct mortality and attenuates ventricular remodeling. However, surviving MI patients frequently develop heart failure, which is associated with reduced quality of life, high mortality rate (10% annually), as well as high healthcare expenditures. The main processes involved in the evolution of heart failure post-MI are the great loss of contractile cardiomyocytes during ischemia-reperfusion and the subsequent complex structural and functional alterations, which are rooted in modifications at molecular and cellular levels in both the infarcted and non-infarcted myocardium. However, we still lack efficient treatments to prevent the development and progression of left ventricular remodeling. The improved survival rate of acute MI patients combined with the lack of effective therapy for post-MI remodeling contributes to the high prevalence of heart failure. Cardiac Magnetic Resonance Imaging (MRI) is an important tool for diagnosis and assessment of MI. With the advancement of this technology, the frontier of MRI has been extended to probing molecular and cellular events in vivo and non-invasively. In combination with assessment of morphology and function, the visualization of essential molecular and cellular markers in vivo could provide comprehensive, multifaceted views of the healing process in infarcted hearts, which might give new insight for the treatment of acute MI. In this thesis, molecular and cellular cardiac MRI methods were established to visualize and investigate inflammation and calcium flux in the healing process of acute MI in vivo, in a clinically relevant rat model. KW - Kernspintomografie KW - Herzinfarkt KW - magnetic resonance imaging KW - myocardial infarction KW - Wundheilung KW - NMR-Tomographie Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-72514 ER - TY - JOUR A1 - Yilmaz, Ali A1 - Rösch, Sabine A1 - Klingel, Karin A1 - Kandolf, Reinhard A1 - Helluy, Xavier A1 - Hiller, Karl-Heinz A1 - Jakob, Peter M A1 - Sechtem, Udo T1 - Molecular magnetic resonance imaging (MRI) of inflamed myocardium using ferucarbotran in patients with acute myocardial infarction JF - Journal of Cardiovascular Magnetic Resonance N2 - Introduction: Superparamagnetic iron oxide nanoparticle (SPIO)-based molecular imaging agents targeting macrophages have been developed and successfully applied in animal models of myocardial infarction. KW - Acute Myocardial Infarction KW - Cardiovascular Magnetic Resonance KW - Iron Oxide Nanoparticle KW - Superparamagnetic Iron Oxide KW - Infarct Zone Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140991 VL - 13 IS - Suppl. 1 ER - TY - JOUR A1 - Yilmaz, Ayse A1 - Aksoy, Volkan A1 - Camlitepe, Yilmaz A1 - Giurfa, Martin T1 - Eye structure, activity rhythms, and visually-driven behavior are tuned to visual niche in ants JF - Frontiers in Behavioral Neuroscience N2 - Insects have evolved physiological adaptations and behavioral strategies that allow them to cope with a broad spectrum of environmental challenges and contribute to their evolutionary success. Visual performance plays a key role in this success. Correlates between life style and eye organization have been reported in various insect species. Yet, if and how visual ecology translates effectively into different visual discrimination and learning capabilities has been less explored. Here we report results from optical and behavioral analyses performed in two sympatric ant species, Formica cunicularia and Camponotus aethiops. We show that the former are diurnal while the latter are cathemeral. Accordingly, F. cunicularia workers present compound eyes with higher resolution, while C. aethiops workers exhibit eyes with lower resolution but higher sensitivity. The discrimination and learning of visual stimuli differs significantly between these species in controlled dual-choice experiments: discrimination learning of small-field visual stimuli is achieved by F. cunicularia but not by C. aethiops, while both species master the discrimination of large-field visual stimuli. Our work thus provides a paradigmatic example about how timing of foraging activities and visual environment match the organization of compound eyes and visually-driven behavior. This correspondence underlines the relevance of an ecological/evolutionary framework for analyses in behavioral neuroscience. KW - visual learning KW - ant KW - activity rhythm KW - camponotus aethiops KW - formica cunicularia KW - compound eye Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119595 VL - 8 ER - TY - THES A1 - Yin, Jing T1 - Progressive alterations of pro- and antidegeneration markers in the nigrostriatal tract of the AAV1/2-A53T-α synuclein rat model of Parkinson’s disease T1 - Progressive Veränderungen von Pro- und Antidegenerationsmarkern im Nigrostriataltrakt des AAV1/2-A53T-α-Synuclein-Rattenmodells der Parkinson-Krankheit N2 - Neurodegeneration plays an essential role in Parkinson’s disease (PD). Several crucial neuronal pro-and antidegeneration markers were described to be altered in disease models accompanied by neurodegeneration. In the AAV1/2-A53T-aSyn PD rat model progressive time-dependent motor impairment and neurodegeneration in the nigrostriatal tract starting from 2 weeks after PD model induction could be found. Downregulation of Nrf2 in SN and nigrostriatal axon localization, a trend of Tau downregulation in SN and upregulation in axon localization in the AAV1/2-A53T-aSyn PD rat model were observed, indicating potential therapeutic value of these two molecular targets in PD. No alterations of SARM1 and NMNAT2 could be detected, indicating little relevance of these two molecules with our AAV1/2-A53T-aSyn rat model. N2 - Die Neurodegeneration spielt eine wesentliche Rolle bei der Parkinson-Krankheit (PD). Es wurde beschrieben, dass mehrere entscheidende neuronale Pro- und Antidegenerationsmarker in Krankheitsmodellen, die von Neurodegeneration begleitet werden, verändert sind. Im AAV1/2-A53T-aSyn PD-Rattenmodell konnte eine fortschreitende zeitabhängige motorische Beeinträchtigung und Neurodegeneration im Nigrostriataltrakt ab 2 Wochen nach PD-Modellinduktion gefunden werden. Herunterregulierung von Nrf2 in SN und nigrostriataler Axonlokalisierung, ein Trend der Tau-Herunterregulierung in SN und Hochregulierung in Axonlokalisierung im AAV1/2-A53T-aSyn-PD-Rattenmodell wurden beobachtet, was auf einen potenziellen therapeutischen Wert dieser beiden molekularen Ziele bei PD hinweist. Es konnten keine Veränderungen von SARM1 und NMNAT2 nachgewiesen werden, was auf eine geringe Relevanz dieser beiden Moleküle mit unserem AAV1/2-A53T-aSyn-Rattenmodell hinweist. KW - Parkinson's disease KW - Neurodegeneration Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260645 ER - TY - JOUR A1 - Yin, Jun A1 - Brocher, Jan A1 - Fischer, Utz A1 - Winkler, Christoph T1 - Mutant Prpf31 causes pre-mRNA splicing defects and rod photoreceptor cell degeneration in a zebrafish model for Retinitis pigmentosa JF - Molecular neurodegeneration N2 - Background: Retinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects. Results: We have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors. Conclusion: Our data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects. KW - Factor gene PRPF31 KW - TRI-SNRNP KW - Transgenic zebrafish KW - Homebox gene KW - Chinese family KW - Mutations KW - RP11 KW - Expression KW - Disease KW - Protein KW - Retinitis pigmentosa (RP) KW - PRPF31 KW - AD5 mutation KW - SP117 mutation KW - haploinsufficiency KW - dominant-negative KW - rod degeneration KW - apoptosis KW - splicing defect Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141090 VL - 6 IS - 56 ER - TY - INPR A1 - Yin, Yafu A1 - Werner, Rudolf A. A1 - Higuchi, Takahiro A1 - Lapa, Constantin A1 - Pienta, Kenneth J. A1 - Pomper, Martin G. A1 - Gorin, Michael A. A1 - Rowe, Steven P. T1 - Follow-Up of Lesions with Equivocal Radiotracer Uptake on PSMA-Targeted PET in Patients with Prostate Cancer: Predictive Values of the PSMA-RADS-3A and PSMARADS- 3B Categories T2 - Journal of Nuclear Medicine N2 - Purpose: Prostate-specific membrane antigen (PSMA)-targeted positron emission tomography (PET) imaging has become commonly utilized in patients with prostate cancer (PCa). The PSMA reporting and data system version 1.0 (PSMA-RADS version 1.0) categorizes lesions on the basis of the likelihood of PCa involvement, with PSMA-RADS-3A (soft tissue) and PSMA-RADS-3B (bone) lesions being indeterminate for the presence of disease. We retrospectively reviewed the imaging follow-up of such lesions to determine the rate at which they underwent changes suggestive of underlying PCa. Methods: PET/CT imaging with \(^{18}\)F-DCFPyL was carried out in 110 patients with PCa and lesions were categorized according to PSMA-RADS Version 1.0. 56/110 (50.9%) patients were determined to have indeterminate PSMA-RADS-3A or PSMA-RADS-3B lesions and 22/56 (39.3%) patients had adequate follow-up to be included in the analysis. The maximum standardized uptake values (SUV\(_{max}\)) of the lesions were obtained and the ratios of SUV\(_{max}\) of the lesions to SUV\(_{mean}\) of blood pool (SUV\(_{max}\)-lesion/SUV\(_{mean}\)-bloodpool) were calculated. Pre-determined criteria were used to evaluate the PSMA-RADS-3A and PSMA-RADS-3B lesions on follow-up imaging to determine if they demonstrated evidence of underlying malignancy. Results: A total of 46 lesions in 22 patients were considered indeterminate for PCa (i.e. PSMA-RADS-3A (32 lesions) or PSMA-RADS-3B (14 lesions)) and were evaluable on follow-up imaging. 27/46 (58.7%) lesions demonstrated changes on follow-up imaging consistent with the presence of underlying PCa at baseline. These lesions included 24/32 (75.0%) PSMA-RADS-3A lesions and 3/14 (21.4%) lesions categorized as PSMA-RADS-3B. The ranges of SUVmax and SUVmax-lesion/SUVmean-bloodpool overlapped between those lesions demonstrating changes consistent with malignancy on follow-up imaging and those lesions that remained unchanged on follow-up. Conclusion: PSMA-RADS-3A and PSMA-RADS-3B lesions are truly indeterminate in that proportions of findings in both categories demonstrate evidence of malignancy on follow-up imaging. Overall, PSMA-RADS-3A lesions are more likely than PSMA-RADS-3B lesions to represent sites of PCa and this information should be taken into when guiding patient therapy. KW - PSMA-RADS-3B KW - Positronen-Emissions-Tomografie KW - prostate-specific membrane antigen KW - prostate cancer KW - PSMA-targeted PET KW - PSMA-RADS-3A Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-167594 SN - 0161-5505 N1 - This research was originally published in JNM. Yafu Yin, Rudolf A. Werner, Takahiro Higuchi, Constantin Lapa, Kenneth J. Pienta, Martin G. Pomper, Michael A. Gorin, Steven P. Rowe. Follow-Up of Lesions with Equivocal Radiotracer Uptake on PSMA-Targeted PET in Patients with Prostate Cancer: Predictive Values of the PSMA-RADS-3A and PSMA-RADS-3B Categories. J Nucl Med. 2019;60:511-516 © SNMMI. ER - TY - JOUR A1 - Yong, Liu A1 - Jacobowitz, David M. A1 - Barone, Frank A1 - McCarron, Richard A1 - Spatz, Maria A1 - Feuerstein, Giora A1 - Hallenbeck, John M. A1 - Sirén, Anna-Leena T1 - Quantitation of perivascular monocyte / macrophages around cerebral blood vessels of hypertensive and aged rats N2 - The numbers of monocytes and macrophages in the walls of cerebral blood vessels were counted on perfusion-fixed frozen brain sections (16 JLffi) of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHR-SP), normotensive Wistar-Kyoto (WKY) rats, and young (16-week-old) and old (2-year-old) normotensive Sprague-Dawley rats (SD-l6w and SD-2y, respectively) using monoclonal antiborlies against rat macrophages (ED2). The staining was visualized with fluoresceinlabeled second antiborlies. The ED2-specific staining in brain sections was restricted to macrophages in a perivascular location. The number of perivascular cells per square millimeter of high-power field was significantly greater in SHR-SP (8.6 ± 2.1; n = 4) and SHR (6. 7 ± 0.9; n = 6) than in normotensive WKY (4.0 ± 0.5; n = 6; p <0.01). The number of perivascular macrophages was also greater in SD-2y (7.5 ± 2.7; n = 9) than in SD-l6w (2.9 ± 1.8; n = 8; p < 0.01). No ED2 staining was found in the resident microglia or in the endothelial cells, which were identified by double staining with rhodamine-labeled anti-factor VIII-related antigen antiborlies. The results suggest that the stroke risk factors hypertension and advanced age are associated with increased subendothelial accumulation of monocytes and macrophages. This accumulation could increase the tendency for the endothelium to convert from an anticoagulant to a procoagulant surface in response to mediators released from these subendothelial cells. KW - Willebrand-Faktor KW - immunofluorescence KW - ED2 KW - Von Willebrand factor KW - rats KW - brain Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86800 ER - TY - JOUR A1 - Young, Joanna C. A1 - Clements, Abigail A1 - Lang, Alexander E. A1 - Garnett, James A. A1 - Munera, Diana A1 - Arbeloa, Ana A1 - Pearson, Jaclyn A1 - Hartland, Elizabeth L. A1 - Matthews, Stephen J. A1 - Mousnier, Aurelie A1 - Barry, David J. A1 - Way, Michael A1 - Schlosser, Andreas A1 - Aktories, Klaus A1 - Frankel, Gad T1 - The Escherichia coli effector EspJ blocks Src kinase activity via amidation and ADP ribosylation JF - Nature Communications N2 - The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector Tir by cellular Src and Abl family tyrosine kinases. This leads to recruitment of the Nck-WIP-N-WASP complex that triggers Arp2/3-dependent actin polymerization in the host cell. The same phosphorylation-mediated signalling network is also assembled downstream of the Vaccinia virus protein A36 and the phagocytic Fc-gamma receptor FcγRIIa. Here we report that the EPEC type-III secretion system effector EspJ inhibits autophosphorylation of Src and phosphorylation of the Src substrates Tir and FcγRIIa. Consistent with this, EspJ inhibits actin polymerization downstream of EPEC, Vaccinia virus and opsonized red blood cells. We identify EspJ as a unique adenosine diphosphate (ADP) ribosyltransferase that directly inhibits Src kinase by simultaneous amidation and ADP ribosylation of the conserved kinase-domain residue, Src E310, resulting in glutamine-ADP ribose. Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-121157 VL - 5 IS - 5887 ER -