Super-resolution microscopy enables the observation of structures below the diffraction limit of light. A relatively new method, Expansion Microscopy (ExM) involves physically expanding a hydrogel-embedded facsimile of a biological structure by tearing it apart. The resultant larger replica permits sub-diffraction imaging with a typical fluorescent microscope. However, there are challenges, such as retaining the fluorescent signal after expansion and the isotropic nature of the expansion procedure, which this study will address. The signal loss is due to the loss of labels, which is mainly caused by gel formation and digestion. To address this issue, an agent was developed in this study that combines targeting, fluorescent labeling, and gel linkage into a single small molecule. The findings presented here suggest that label loss is due to inadequate surface grafting of fluorophores into the hydrogel. In order to gain a mechanistic understanding of dye retention, it is crucial to understand the function of surface grafting in ExM. By delivering monomers directly to the target of interest via nanobodies, the amount of polymerizable units bound to the surface was increased, resulting in retention of the signal. Consequently, the density of local monomers is crucial for retaining signals. In addition to signal retention, it is essential to verify that the expansion was uniform in all directions, i.e. isotropic, since only a facsimile of the original biological structure is detected after expansion. This is necessary to draw accurate conclusions from the results. Therefore, my goal was to develop a ruler that measures the isotropy of the expansion. For this experiment, tobacco mosaic virus (TMV) particles were fluorescently labeled and introduced as an internal standard into cells by coupling them with microtubules. However, the distribution of particle lengths and the loss of signal after expansion proved to be problematic. These obstacles can be resolved in future research. A uniform virus length could be achieved through size exclusion chromatography. Secondly, signal retention could be improved by fluorescently labeling additional amino acids of TMV coat proteins (CPs) and providing particles with polymerizable groups.
Weniger anzeigenMit dem Ziel der Theoriebildung gehe ich der Fragestellung nach, welchen Einfluss politische Steuerung auf die Versorgungssicherheit mit Strom hat, um die Auswirkungen der Privatisierung und Liberalisierung der Elektrizitätswirtschaft abzuschätzen. Mittels multipler Regression kontrolliere ich auf die Bedeutung technischer Merkmale der physischen Elektrizitätsinfrastruktur, sozioökonomischer Rahmenbedingungen, struktureller und prozeduraler Merkmale des politischen Systems, um den Effekt der Energiewirtschafts- und Infrastrukturpolitik zu isolieren.
Die Versorgungssicherheit mit Strom steigt allgemein an. Die Widerstandsfähigkeit der Elektrizitätsinfrastruktur gegen Störereignisse ist langfristig konstant. Veränderungen der durchschnittlichen Unterbrechungsdauer SAIDI gehen meist auf die Zeit bis zur Wiederversorgung nach einem Stromausfall CAIDI zurück. Dies deutet auf einen Einmaleffekt des Alters der langlebigen Basiskomponenten und einen hohen Einfluss von Investitionsentscheidungen in kurzlebige Komponenten wie die Mess-, Steuerungs- und Regelungstechnik hin.
Weder die durch natürliche Widrigkeiten bedingten Versorgungskosten, die langfristige Stabilität der wirtschaftlichen oder politischen Rahmenbedingungen, die Kapitalverfügbarkeit noch die instrumentelle Energiewirtschafts- oder Infrastrukturpolitik erklären die internationalen Unterschiede bei der Versorgungssicherheit mit Strom. Vielmehr erklärt die Korruptionswahrnehmung knapp die Hälfte der international zu beobachtenden Varianz.
Mit der Theorie der Determinanten der Versorgungssicherheit mit Strom interpretiere ich anschließend an die Capture-Theorie zur Regulierungsgenese in konzentrierten Wirtschaftssektoren das Energiesystem als ein von konkurrierenden Interessen geprägtes, selbstregelndes soziotechnisches System. Die Verbreitung von Korruption bestimmt, ob die Stromversorgung als öffentliches Gut im Bereich des gesellschaftlichen Optimums zwischen Preisgünstigkeit und Zuverlässigkeit oder als privates Gut erbracht wird. Vereinnahmen Interessengruppen die Energiewirtschaftspolitik illegal als privates Gut, sinkt die Versorgungssicherheit mit Strom durch das soziale Dilemma der Korruption und die Erosion des Staatswesens, selbst wenn diese Gruppen sich für eine höhere Versorgungssicherheit mit Strom einsetzen. Aufgrund der Bedeutung der prozessualen Dimension von Politik bewerte ich den Einfluss politischer Steuerung trotz der fehlenden Effekte der instrumentellen Energiewirtschafts- und Infrastrukturpolitik auf die Versorgungssicherheit mit Strom als sehr hoch.
Ich prognostiziere, dass die Privatisierung und Liberalisierung der Energiewirtschaft keinen feststellbaren Effekt auf die Versorgungssicherheit mit Strom haben werden. Erstens können angesichts der schlechten Qualität der Daten zur Quantifizierung der Versorgungssicherheit mit Strom nur große Effekte identifiziert werden. Auf einen substantiellen Effekt dieser Reformen gibt der im internationalen Vergleich fehlende Effekt der instrumentellen Energiewirtschaftspolitik aber keinen Hinweis. Zweitens erwarte ich aus der prozeduralen Konzeption des Energiesystems als selbstregelndes soziotechnisches System, dass mögliche substanzielle Effekte auf die Versorgungssicherheit mit Strom anderweitig über das System der Interessenvermittlung kompensiert würden. Drittens sehe ich keine wesentliche Veränderung der Interessengruppenstruktur durch die Privatisierung und Liberalisierung vormals staatlicher EVU unter dem Gesichtspunkt der Versorgungssicherheit mit Strom. Energieversorgungsunternehmen haben durch ihr natürliches Monopol im Bereich der Stromnetze geringe Anreize, der gesellschaftlichen Nachfrage nach einer preisgünstigen und zuverlässigen Stromversorgung zu entsprechen. Auch bei einer Entflechtung der Bereiche Stromerzeugung, ‑vertrieb und Netzbetrieb sind Strategien der Koexistenz die kosteneffizienteste Art der Interessenwahrnehmung, die auf einen Regulierungsrahmen hinwirken, der zuverlässigkeitssteigernde Investitionen des Netzbetreibers anregt und damit verbundene Kosten auf unorganisierte gesellschaftliche Gruppen abwälzt. Deshalb bedarf es unabhängig vom eigentumsrechtlichen Status der Elektrizitätswirtschaft einer staatlichen Korrektur der Anreizstrukturen.
Weniger anzeigenAll video coding standards of practical importance, such as Advanced Video Coding (AVC), its successor High Efficiency Video Coding (HEVC), and the state-of-the-art Versatile Video Coding (VVC), follow the basic principle of block-based hybrid video coding. In such an architecture, the video pictures are partitioned into blocks. Each block is first predicted by either intra-picture or motion-compensated prediction, and the resulting prediction errors, referred to as residuals, are compressed using transform coding. This thesis deals with the entropy coding of quantization indices for transform coefficients, also referred to as transform coefficient levels, as well as the entropy coding of directly quantized residual samples. The entropy coding of quantization indices is referred to as level coding in this thesis. The presented developments focus on both improving the coding efficiency and reducing the complexity of the level coding for HEVC and VVC. These goals were achieved by modifying the context modeling and the binarization of the level coding.
The first development presented in this thesis is a transform coefficient level coding for variable transform block sizes, which was introduced in HEVC. It exploits the fact that non-zero levels are typically concentrated in certain parts of the transform block by partitioning blocks larger than \square{4} samples into \square{4} sub-blocks. Each \square{4} sub-block is then coded similarly to the level coding specified in AVC for \square{4} transform blocks. This sub-block processing improves coding efficiency and has the advantage that the number of required context models is independent of the set of supported transform block sizes.
The maximum number of context-coded bins for a transform coefficient level is one indicator for the complexity of the entropy coding. An adaptive binarization of absolute transform coefficient levels using Rice codes is presented that reduces the maximum number of context-coded bins from 15 (as used in AVC) to three for HEVC. Based on the developed selection of an appropriate Rice code for each scanning position, this adaptive binarization achieves virtually the same coding efficiency as the binarization specified in AVC for bit-rate operation points typically used in consumer applications. The coding efficiency is improved for high bit-rate operation points, which are used in more advanced and professional applications.
In order to further improve the coding efficiency for HEVC and VVC, the statistical dependencies among the transform coefficient levels of a transform block are exploited by a template-based context modeling developed in this thesis. Instead of selecting the context model for a current scanning position primarily based on its location inside a transform block, already coded neighboring locations inside a local template are utilized. To further increase the coding efficiency achieved by the template-based context modeling, the different coding phases of the initially developed level coding are merged into a single coding phase. As a consequence, the template-based context modeling can utilize the absolute levels of the neighboring frequency locations, which provides better conditional probability estimates and further improves coding efficiency.
This template-based context modeling with a single coding phase is also suitable for trellis-coded quantization (TCQ), since TCQ is state-driven and derives the next state from the current state and the parity of the current level. TCQ introduces different context model sets for coding the significance flag depending on the current state. Based on statistical analyses, an extension of the state-dependent context modeling of TCQ is presented, which further improves the coding efficiency in VVC. After that, a method to reduce the complexity of the level coding at the decoder is presented. This method separates the level coding into a coding phase exclusively consisting of context-coded bins and another one consisting of bypass-coded bins only. For retaining the state-dependent context selection, which significantly contributes to the coding efficiency of TCQ, a dedicated parity flag is introduced and coded with context models in the first coding phase. An adaptive approach is then presented that further reduces the worst-case complexity, effectively lowering the maximum number of context-coded bins per transform coefficient to 1.75 without negatively affecting the coding efficiency.
In the last development presented in this thesis, a dedicated level coding for transform skip blocks, which often occur in screen content applications, is introduced for VVC. This dedicated level coding better exploits the statistical properties of directly quantized residual samples for screen content. Various modifications to the level coding improve the coding efficiency for this type of content. Examples for these modifications are a binarization with additional context-coded flags and the coding of the sign information with adaptive context models.
Weniger anzeigenProlongation of the light period causes photoperiod stress in plants. During the night following the prolonged light treatment, stress marker gene expression is induced, and stress hormones and ROS accumulate. The next day, the experienced strong photoperiod stress leads to the formation of water-soaked lesions in leaves and eventually programmed cell death ensues. In this study, the impact of light intensity and light quality on the photoperiod stress response has been investigated. A threshold light intensity of about 50 μmol m-2 s-1 was found to be necessary for the induction of photoperiod stress, hinting at the involvement of chloroplasts. Lower photoperiod stress symptoms in gun4, gun5, rcd1, and glk1 glk2 mutants revealed a possible role of retrograde signaling in photoperiod stress and corroborated the involvement of chloroplasts. Furthermore, starch and sugar content were higher in photoperiod stressed plants and starch biosynthesis mutants developed less photoperiod stress symptoms. This indicated that the starch and sugar metabolism might be affecting a plants’ response to photoperiod stress, strengthening the argument for the importance of chloroplast in photoperiod stress. Both monochromatic red and blue light caused a photoperiod stress response, but the response provoked by red light was stronger. Mutant analysis revealed the photoreceptors phyB and CRY2 as probable sensors of photoperiod stress. Among the downstream light signaling components, HY5 and PIF1 have demonstrated potential for involvement in sensing photoperiod stress. Although cry2 mutation did not rescue the strong photoperiod stress phenotype of cca1 lhy, some clock genes were differentially regulated in cry2. This indicates a possible involvement of CRY2 in photoperiod stress through its role in regulating the circadian rhythm. Overall, these results support that both plastid-dependent and photoreceptor-dependent signaling pathways are involved in sensing light conditions causing photoperiod stress and governing the response to it. Since co and ft tsf mutants demonstrated less photoperiod stress symptoms, participation of the photoperiodic flowering pathway in sensing and responding to photoperiod stress has been considered a possibility. Most ecotypes other than Columbia showed low sensitivity to photoperiod stress, suggesting that photoperiod stress sensitivity might be a rare trait in nature. The low photoperiod stress sensitivity of the F1 generation of crosses between Col-0 and some of the ecotypes that show low photoperiod stress sensitivity is evidence of the recessive nature of the photoperiod stress sensitivity trait.
Weniger anzeigenThe necessity to decrease greenhouse gas (GHG) emissions, mainly methane (CH4), is underscored in diverse national and international regulations and conventions. Agriculture, particularly livestock enteric fermentation and manure management, is a significant source of these emissions. In pig and dairy cattle farming, liquid-manure systems are the most prevalent and responsible for an upsurge in CH4 emissions. This study further investigates the potential of CH4 production from manure as a renewable energy source and an effective strategy to decrease GHG emissions, emphasizing the importance of better understanding the factors that regulate CH4 potential loss from manure storage. This study scrutinized the effect of storage conditions and changes in chemical composition on CH4 formation and emissions from dairy and fattening pig manure. Two different experimental techniques were designed to verify the influence of storage temperature on CH4 production and the effects of manure storage on biochemical methane production (BMP). In the first method, samples were stored without inoculum at varying temperatures, and CH4 production was measured. In pig manure stored at 25°C and 20°C, CH4 production amounted to 69.3% and 50.3% of the BMP, respectively. The highest CH4 production for dairy slurry were observed at 25°C but remained low. The production of CH4 in dairy manure was found to be inhibited during storage. The study suggested several potential reasons for this inhibition, though further research is recommended to confirm these hypotheses. The second set of experiments, evaluate the BMP of manure samples collected year-round from different stages on the manure storage in dairy cow and fattening pig farms. The findings indicated a 20.5% reduction in the BMP of dairy manure due to alterations in chemical composition during the transfer from the barn to outdoor storage. Similarly, there was a 39.5% drop in the BMP for samples of fattening pig manure in the transition from intermediate to outdoor storage. When examining BMP relative to the age of the manure, it was observed that pig manure decomposes quicker than dairy manure. Mathematical models were developed to predict BMP from the chemical composition, and the most effective predictors for each livestock category were identified. Improvements to predict BMP for less aged samples with reduced variability in their chemical compositions would be necessary. Overall, this study highlights the necessity of promptly transporting manure to either a biogas plant or outdoor storage facilities. Such measures can help prevent significant CH4 emissions and avoid a decrease in energy production capacity.
Weniger anzeigenEquines Asthma ist eine häufig vorkommende chronische Erkrankung der Atemwege und kann Pferde jeden Alters betreffen. Neben einer speziellen klinischen Untersuchung des Atemtrakts spielt die zytologische Untersuchung von Tracheobronchialsekret (TBS) und Bronchoalveolärer Lavage (BAL) eine wichtige Rolle bei der Diagnosestellung von equinem Asthma. Ziel dieser Arbeit war der Vergleich zytologischer Ergebnisse und Verdachtsdiagnosen zwischen und innerhalb verschiedener Labore, um die Zuverlässigkeit der zytologischen Untersuchung von TBS und BAL als diagnostisches Mittel zur Diagnosestellung von equinem Asthma zu überprüfen. Die Hypothesen dieser Arbeit waren die Annahmen, dass bei einem intra- und interindividuellen Vergleich zytologischer Ergebnisse von TBS und BAL innerhalb und zwischen mehreren Laboren nur eine geringe oder gar keine Varianz der Ergebnisse und Verdachtsdiagnosen besteht und die Zytologie als zuverlässiges Kriterium zur Atemwegsdiagnostik herangezogen werden kann. Als Studienpopulation standen 15 Pferde zur Verfügung, die zur Abklärung respiratorischer Symptome vorgestellt wurden. Zunächst wurden an allen Pferden Atemwegsuntersuchungen durchgeführt und schließlich die Befunde der klinischen, endoskopischen und zytologischen Untersuchung zur endgültigen Diagnosestellung in der Klinik herangezogen. Auf diese Weise erfolgte die Einteilung der Studienpferde in fünf gesunde, fünf Pferde mit mildem bis moderatem und fünf Pferde mit schwerem equinem Asthma. Im Anschluss erfolgte der Versand von luftgetrockneten Ausstrichen von TBS und BAL der Studienpferde an vier Fremdlabore. Zusätzlich wurden die Ausstriche durch zwei klinikinterne Untersucher beurteilt. Durch einen doppelt verblinden Versand der Proben untersuchte jedes Labor und jeder Untersucher zwei Mal die Proben des gleichen Pferdes. Bei den Ergebnissen der Fremdlabore fiel auf, dass die Angaben zur Differentialzellzählung der zytologischen Auswertung im TBS und in der BAL z.T. erhebliche Unterschiede aufwiesen. Zum einen wurden semiquantitative Angaben, zum anderen Prozentzahlen bei der Differenzialzellzählung angegeben. Um die interindividuelle Varianz der Ergebnisse verschiedener Labore zu ermöglichen, wurde ein vereinheitlichtes Scoring System zur zytologischen Befundung von TBS und BAL erarbeitet. Beim Vergleich der intraindividuellen Übereinstimmung der Ergebnisse innerhalb der Fremdlabore und der Untersucher der Klinik für Pferde konnte bestätigt werden, dass eine gute Übereinstimmung der Ergebnisse der neutrophilen Granulozyten bei fast allen Laboren in der BAL und im TBS erreicht wurde (ICC > 0,75, gewichtetes Kappa >0,6). Darüber hinaus erreichten die Labore, die Prozentangaben zur Differentialzellzählung machten bei Makrophagen, Lymphozyten, eosinophilen Granulozyten und Mastzellen ebenfalls einige gute Ergebnisse der Übereinstimmung. Die semiquantitativen Angaben resultieren in einer geringeren Reproduzierbarkeit der Ergebnisse zu den Zellarten der Differentialzellzählung innerhalb anderer Labore. Somit hatte die Art und Weise der zytologischen Auswertung Einfluss auf die Reproduzierbarkeit der Ergebnisse. Für die BAL variierten die Ergebnisse insgesamt weniger, sodass diese Probenart für validere zytologische Ergebnisse empfohlen wird. Beim interindividuellen Vergleich der Ergebnisse zwischen allen Laboren konnte ebenfalls eine gute Reproduzierbarkeit der Ergebnisse bei den neutrophilen Granulozyten in der BAL und im TBS erreicht werden (Krippendorff’s alpha >0,7). Diese Zellart hat den größten Einfluss auf die zytologische Diagnosestellung. Die Ergebnisse zu allen weiteren Zellarten der Differentialzellzählung variierten deutlicher zwischen den Laboren. Bei den lungengesunden Pferden wurde durch die Fremdlabore häufig mildes-moderates equines Asthma diagnostiziert. Bei den erkrankten Pferden wurde meistens equines Asthma diagnostiziert, allerdings variierte der Schweregrad der Erkrankung zwischen und innerhalb der Labore. Ein Labor stellte darüber hinaus bei einigen Pferden die Diagnose einer bakteriellen Bronchitis. Die Hypothese, dass die TBS- und BAL-Zytologie als zuverlässiges Kriterium zur Atemwegsdiagnostik herangezogen werden kann und einen wichtigen Teil der Diagnosestellung ausmacht, muss aufgrund von einigen intra- und interindividuellen Unterschieden der zytologischen Ergebnisse und bei den Verdachtsdiagnosen zwischen und innerhalb der Labore eingeschränkt werden. Die Unterschiede können z.T. auf die variable Auswertung der Proben zurückgeführt werden. Demnach kann geschlussfolgert werden, dass die zytologische Untersuchung nur zu einem kleinen Teil zur endgültigen Diagnosestellung herangezogen werden sollte und die klinischen Symptome den größten Einfluss auf die Diagnose und damit die weitere Therapie haben sollten. Ohne genaue Angaben der klinischen Symptome und des Vorberichts sind valide zytologische Diagnosen nur eingeschränkt möglich. Das Zusammenspiel aller Befunde sollte als Goldstandard für die Diagnosestellung gelten. Als Empfehlung sollte für valide zytologische Ergebnisse besonders bei Verlaufsuntersuchungen immer das gleiche Labor für die zytologische Auswertung herangezogen werden. Darüber hinaus besteht die Notwendigkeit einer einheitlichen zytologischen Beurteilung von TBS und BAL, um Ergebnisse zwischen verschiedenen Laboren vergleichen zu können. Als Vorschlag für eine vereinheitlichte Befundung soll das in dieser Studie erstellte Scoring System dienen, das in weiteren Studien seine Validität zeigen könnte.
Weniger anzeigenAls Enzephalitis bezeichnet man die Entzündung des Hirngewebes. Jedes Jahr werden etwa 1,5-20/100.000 Einwohner mit Verdacht auf Enzephalitis hospitalisiert. In den meisten Fällen kann keine Ursache identifiziert werden. Mehr als 100 verschiedene Viren können eine Enzephalitis verursachen. Die große Anzahl und Vielfalt der möglichen Erreger erschweren die Diagnostik. Die Routinediagnostik konzentriert sich daher in der Regel auf die häufigsten Erreger und wird gegebenenfalls iterativ erweitert, wenn die initialen Tests keinen Erreger nachweisen. Das (Wieder-)Auftauchen neuer und seltener Viren wird wahrscheinlich durch Faktoren wie Klimawandel, Entwaldung, Globalisierung und Urbanisierung weiter zunehmen und ist in den letzten Jahren durch die Zika-Virus-Ausbrüche in Lateinamerika, das Auftreten autochthoner West-Nil-Virus (WNV) Infektionen in Deutschland und die COVID-19-Pandemie eindrücklich belegt worden. Daher ist es wichtig, eine breite Diagnostik zur schnellen und zuverlässigen Identifizierung von Viren zu entwickeln. Diese muss in der Lage sein, bekannte Krankheitserreger, aber auch unbekannte oder ungewöhnliche, zum Beispiel reiseassoziierte Erreger, nachzuweisen. Das initiale Ziel dieser Arbeit war daher die Entwicklung einer Hochdurchsatz-Sequenziermethode mit möglichst kurzer Zeitspanne von der Probenentnahme bis zur Erreger-Identifikation. Dies ist von entscheidender Bedeutung für eine schnelle Diagnose, Behandlung und somit für das Überleben der Patienten. Mit dem etablierten Arbeitsablauf können die Ergebnisse innerhalb von drei Tagen nach dem Probeneingang bereitgestellt werden. Kritisch ist dabei die Erkennung klinisch relevanter Nachweise, die Abgrenzung von möglichen Virusnachweisen ohne ursächliche Bedeutung für die Enzephalitis sowie das Erfassen von Kontaminationen. Die hier etablierte Methode wurde bei der retrospektiven Untersuchung von 1676 Liquorproben von Enzephalitis-/Meningitis-Patienten ungeklärter Ursache angewendet und in der Routinediagnostik angewendet. Bei den untersuchten Personen wurden bereits bekannte Infektionen festgestellt, aber auch zahlreiche seltene, zuvor nicht diagnostizierte Erreger. Dazu gehören ein humanes Cosavirus bei einem Kind mit akuter schlaffer Lähmung und ein Dobrava-Belgrad-Hantavirus im Liquor eines Patienten mit Enzephalitis. Besonders erwähnenswert sind der mehrfache Nachweis des Hepatitis-E-Virus (HEV), die Identifikation zuvor unbekannter WNV-Infektionen und der Nachweis von humanen Pegiviren (HPgV) im zentralen Nervensystem (ZNS) bei Patienten mit Enzephalitis. Die Etablierung von Methoden zur Vollgenomsequenzierung und der mehrfache Nachweis von HEV und HPgV in ZNS-Proben ermöglichten eine vertiefende Untersuchung dieser Erreger. So wurde die Virus-Diversität in einer chronischen HEV-Infektion mit neurologischen Symptomen untersucht. Dabei konnten distinkte virale Populationen in verschiedenen Kompartimenten festgestellt werden. Diese zeigten eine unterschiedliche Komposition von Mutationen, die zuvor im Zusammenhang mit einer Ribavirin-Resistenz beschrieben wurden. Ausgelöst durch WNV-Nachweise in der retrospektiven Kohorte wurde ein weiteres Ziel dieser Dissertation definiert: Die Untersuchung der Rolle des WNV bei Berliner Enzephalitis-Fällen. Nach dem Auftreten erster WNV-Fälle bei Vögeln in Deutschland war es wichtig zu verstehen, ob es sich bei humanen Fällen um reiseassoziierte oder lokal erworbene Infektionen handelt. Zu diesem Zweck wurden verschiedene Strategien für die Vollgenomsequenzierung entwickelt. In dieser Dissertation wurden insgesamt 13 menschliche WNV-Fälle untersucht. Eine phylogenetische Studie zeigte eine enge Verwandtschaft zwischen den WNV-Sequenzen von Menschen, Vögeln sowie Stechmücken. Dies deutet darauf hin, dass sich in Berlin eine isolierte und stabile WNV-Population etabliert hat. Die serologische Untersuchung von 105 Freiwilligen aus einer Kleingartenkolonie führte zur Entdeckung einer weiteren möglichen WNV-Infektion. Da WNV nur in etwa 1% der Fälle neurologische Symptome hervorruft, ist auf Grundlage unserer Nachweise im Liquor davon auszugehen, dass es eine hohe Zahl nicht diagnostizierter Infektionen in Berlin gibt. Ausgelöst durch den häufigen Nachweis von HPgV im Liquor stellte sich die Frage nach deren Bedeutung als Auslöser einer Enzephalitis. Humane Pegiviren wurden bisher nicht mit einer Erkrankung in Verbindung gebracht. Sie wurden aber in den letzten Jahren vereinzelt in Liquorproben von Enzephalitis-Patienten nachgewiesen. Analysen der Virusdiversität zeigten das Vorhandensein von Viruspopulationen in verschiedenen Kompartimenten und somit eine unabhängige Replikation im ZNS. Intensive Untersuchungen zeigten einen anhaltenden Nachweis von HPgV-RNA im Serum und Liquor über teilweise mehr als einem Jahr und führten zur Klassifizierung des Bildes einer HPgV-Enzephalitis. Die Sensitivität der etablierten Hochdurchsatz-Sequenziermethode ist fast mit der einer quantitativen Polymerase-Kettenreaktion vergleichbar. Aufgrund der zeitnahen Ergebnisse des nicht-zielgerichteten Arbeitsablaufs könnte der Einsatz dieser Methodik die Diagnostik in Zukunft erheblich erleichtern und weitere Daten zum Tropismus verschiedener Erreger bereitstellen. Eine verbleibende Herausforderung bleibt aber die Frage der Qualitätssicherung einer solchen breiten Diagnostik. Es stellt sich besonders die Frage nach der klinischen Signifikanz bei der Detektion einzelner Viren.
Weniger anzeigenWith its significant contribution to sociology, political science, and critical race theory, this doctoral research addresses two sets of questions by unfolding two contrasting discourses about race and racism, as it took place in the USA. What defines the dominant interpretation of race-related injustice in the USA? What are the shortcomings of the hegemonic view of social justice in the 1954-1968 Civil Rights Era to remedy injustice entangled with racial discrimination? Drawing on those two questions, the dissertation seeks to grasp two understandings of injustice in the color line and its historical and critical repercussions in public spheres. The first view, the so-called race liberalism, consolidates as the hegemonic standpoint along segments of the Civil Rights movement and subsequent legislation in the 1954-1968 civil rights era. It furnishes numerous channels and actors, such as government institutions, educational systems, and media platforms, with the ideological contours of the interpretation of the causes of the problem of the color line and the institutional problem-solving to injustices and inequalities. In short, race liberal view shapes the blueprint for laws and social engineering responsive to the demands of the Civil Rights Movement segments in the United States. Race Liberalism mesmerizes policy-making with political implications on the normative compass and policy-making strategies put into practice in the 1954-1968 Civil Rights Era to remedy race-related injustice. Contrasting to that belief, a philosophical trait present in radical black visions spells out racism interwoven with capitalist exploitation whose economic injustices insistently harm racial and ethical groups. The opposing discourse of black radical visions from social movements and political activism carried out by the Black Panthers up to organic intellectuals of the Critical Race Theory views racism as a far more complicated matter amalgamated with other inequalities in the existing social stratification. The radical view challenges disentailing racism from complex structures at the bottom of a web of existing institutions, the economy, and social and cultural practices. The radical vision of revolutionary sectors in social movements and academia sets up to critically reevaluate consolidated patterns in the US American understanding of the legitimate democratic organization and fair economic structures and prompt emancipatory thinking and social changes for racial and ethnic minorities. This doctoral research seeks to develop a 'descriptive overview' that provides a comprehensive and detailed account of two discursive matrixes about racism and a 'normative conceptualization' that offers a theoretical framework for understanding and critiquing these discourses. These critical theories of race and racism prompt social critique and paradigmatically point towards a blueprint to tackle racism and interrelated injustices. Initially, it outlines a social and historical analysis of how pervasive injustices and inequalities harm Blacks and Browns. Furthermore, this dissertation aims to underline the normative force of counter-discourse critical race theories, pinpointing a few elements in the making of countervailing public spheres. In this spirit, it stresses how the powerful spirit of critique of racial hierarchy has endured the hegemonic ideology of race liberalism in distinct channels within civil society and political activism and electrifyingly reanimates political debate today. This doctoral study draws on the unfolding of these two contrasting discourses and their implications for understanding and envisioning problem-solving for the problem of the color line: Race liberalism and radical black visions or critique of racial hierarchy. The dissertation proceeds in three parts. Each, in its turn, draws on how these two discursive matrixes – race liberalism and critique of racial hierarchy –, despite their 'heterogenous inflections' which refer to the various interpretations and applications of these discourses, have shaped distinct views of the permanence of inequality allocated in racial and ethical groups and the perceptions of foundational institutions in coping with the crucial matter of injustices tied with race in the US. The first part (Chapters 1-2) concentrates on the conceptual presuppositions and political implications underlying the hegemonic view and the critique of how racial differentiation has been crucial in configuring the existing social stratification and hierarchy in the racial line. The conceptual distinctions are essential for the discursive landscape in civil-rights policy-making and radical visions of the quest for equal citizenship and black liberation. The second part (Chapters 3-4) outlines race-sensitive affirmative programs as a problem-solving ideal and equity-enhancing policy in higher education. It revises the normative presuppositions and the moral and legal disputes underlying race-based college admissions to shed light on how the hegemonic view illustrated in the Bakke landmark decision (1978) and reiterated by many liberals (Chap. 4) has favored market-led interests to the detriment of members of racialized and ethnic minorities. Finally, the third part (chapters 5-7) spells out critical race theories in challenging the dominant race liberal view and pinpointing the normative force of countervailing public spheres in two directions: (i) by raising awareness of the intricacies of racism in institutional, structural and systemic levels and by urging public spheres to rethink the problem of the color line today; (ii) concurrently, as elaborated by radical visions in organic practices of intellectual activities and social movements, by reassessing resources far beyond legal entitlements to reach feasible social mobility and empowerment in redesigning alternatives to cope with the pervasive problem of inequality in the color line. These findings hold the potential to inspire transformative change in our understanding and approach to racial inequality. Critical Race scholarship elaborates a rich conceptual framework to examine the range of intersecting forces and dynamics reproducing race-related disadvantages that operate through entrenched values and political economy within the liberal system. More significantly, for this doctorate's normative purpose and appreciation of the political effects of the unfolding of counteracting discourse, the last chapters stress how the social critique of critical race theories gives vigor to diverse public spheres in ongoing attempts to contest the current status quo in matters about race and beyond. With its rigorous and comprehensive approach, this research calls attention to how critical race theories have been organically tied with political engagement to encourage discursive practices about the hegemonic explanation of racial matters and race-based policies in the US and abroad.
Weniger anzeigenThis thesis introduces the novel hybrid algorithm DisCOptER for globally optimal flight planning. DisCOptER (Discrete-Continuous Optimization for Enhanced Resolution) com- bines discrete and continuous optimization in a two-stage approach to find optimal trajectories up to arbitrary precision in finite time. In the discrete phase, a directed auxiliary graph is created in order to define a set of candidate paths that densely covers the relevant part of the trajectory space. Then, Yen’s algorithm is employed to identify a set of promising candidate paths. These are used as starting points for the subsequent stage in which they are refined with a locally convergent optimal control method. The correctness, accuracy, and complexity of DisCOptER are intricately linked to the choice of the switch-over point, defined by the discretization coarseness. Only a sufficiently dense graph enables the algorithm to find a path within the convex domain surrounding the global minimizer. Initialized with such a path, the second stage rapidly converges to the optimum. Conversely, an excessively dense graph poses the risk of overly costly and redundant computations. The determination of the optimal switch-over point necessitates a profound understanding of the local behavior of the problem, the approximation properties of the graph, and the convergence characteristics of the employed optimal control method. These topics are explored extensively in this thesis. Crucially, the density of the auxiliary graph is solely dependent on the en- vironmental conditions, yet independent of the desired solution accuracy. As a consequence, the algorithm inherits the superior asymptotic convergence properties of the optimal control stage. The practical implications of this computational efficiency are demonstrated in realistic environments, where the DisCOptER algorithm consistently delivers highly accurate globally optimal trajectories with exceptional computational efficiency. This notable improvement upon existing approaches underscores the algorithm’s significance. Beyond its technical prowess, the DisCOptER algorithm stands as a valuable tool contributing to the reduction of costs and the overall enhancement of flight operations efficiency.
Weniger anzeigenPolymeric Core-Multishell-Nanocarriers (CMS) are a family of molecules designed to function as universal drug carriers. Their architecture resembles unimolecular micelles/liposomes, with shell-like, hydrophobic, and hydrophilic domains. Studies using ex vivo/in vitro conditions have shown that they can be used to topically deliver drugs to the skin, including strongly hydrophobic drugs in water-based formulations. Interestingly, these studies reported that CMS not only successfully delivered cargo substances, but in fact increased their concentrations in the target area, the viable skin. Along with the relatively good biocompatibility reported, these properties make them interesting as tools for the treatment of inflammatory skin conditions and other topical applications. The work described here is part of a project that aimed to reproduce and further investigate these findings under conditions of inflammatory skin diseases in vivo. The project was part of a Collaborative Research Center of the German Research Foundation that aimed to develop and investigate a range of nanocarriers for topical delivery to the skin. The specific CMS architectures investigated here were hPG-C18-mPEG CMS (C18CMS) as well as hPG-PCL-mPEG CMS (bCMS). Both architectures have been developed at the Institute of Chemistry and Biochemistry, Freie Universität Berlin. C18CMS represent the prototypical CMS architecture, best characterized for topical delivery. bCMS are easily cleaved by esterases to improve long-term biocompatibility. As a model for a prototypical inflammatory skin condition, an oxazolone-induced mouse model with characteristics of atopic dermatitis (AD) was used. The cargo investigated was tacrolimus (TAC). TAC is a potent anti-inflammatory drug and one of the two main pharmacological treatment options for AD. With a relatively large molecular mass of 822 Da, it is considered at the threshold of substances that can penetrate into the skin in relevant amounts for topical treatment. In the first part of the project, the penetration of C18CMS into the skin, their potential systemic distribution, and the effect of oxazolone-induced inflammation on the penetration were investigated by fluorescence microscopy. Furthermore, the potential effects of the carriers on clinical and histological parameters were evaluated. In the second part of the project, the delivery of TAC by bCMS into inflamed skin, resulting systemic drug concentrations, as well as the clinical and histologic anti-inflammatory efficacy, were investigated. Under the conditions used, bCMS did not seem to increase the skin concentrations of the cargo drug in the viable skin compared to the standard ointment formulation. What is more, concentrations measured in the systemic circulation were significantly lower. This in fact suggests that bCMS decreased overall skin penetration. This differs from previous reports obtained using ex vivo/in vitro conditions, which consistently found penetration enhancement for TAC in bCMS and for various other cargo substances in multiple CMS architectures. Nevertheless, drug delivery to the skin and the anti-inflammatory efficacy of the formulation could be demonstrated. Although C18CMS penetrated into the stratum corneum, no penetration was observed into the viable layers of healthy skin. This penetration behavior of the carrier was not affected by oxazolone-induced dermatitis. The same was observed for bCMS in inflamed skin. These results seem to be partially in line with previous ex vivo/in vitro results under certain conditions, such as relatively short incubation times and relatively mild barrier alterations but differ from results under other conditions, such as longer incubation times and models of more severe barrier alterations. When modeling complete penetration of C18CMS through the viable skin, the fluorescently labeled carriers were found in the main sites of the mononuclear phagocyte system and particle clearance, i.e., the local lymph nodes, spleen, lung, liver, and kidney. No adverse effects were observed histologically. Overall, the results seem to confirm that CMS can be used for topical delivery and treatment of inflammatory skin conditions with TAC. However, CMS may not necessarily enhance penetration or efficacy. Moreover, the results suggest a potential systematic difference between ex vivo/in vitro and in vivo conditions. On the one hand, this cautions that murine models may be unsuitable to investigate the penetration enhancement effect of CMS. On the other hand, it suggests parameters that could potentially be optimized in ex vivo/in vitro models to increase predictability. Of these, particularly relevant parameters seem to be incubation times, exposure periods with subsequent removal of formulations, occlusion/hydration status, effects of repeated applications, steady-state conditions and drug depots, effects that depend on tissue layers other than the stratum corneum, and time-resolved kinetics. This may be particularly important when comparing water-based, volatile formulations with less volatile ointment formulations, and for drugs with comparatively high steady-state concentrations in the skin. With respect to the 3R principles, the results strongly suggest that no further in vivo studies should be performed to investigate the penetration enhancement effects of CMS or similar delivery solutions before 1.) a systematic analysis has shown what caused these discrepancies, 2.) all relevant aspects discussed have been modeled in vitro for the specific nanocarrier-drug combination in question, and 3.) an evidence-based estimate is available on whether the expected penetration enhancement would lead to a relevant benefit for the specific use case.
Weniger anzeigenLithium-sulfur (Li-S) batteries have the potential to become one of the energy storage systems of the future due to their ability to store significantly more energy than current battery types today. Furthermore, sulfur is an abundant element that is cheap and environmentally friendly in its processing, making it an optimal material for sustainable energy systems. However, this type of battery also has some disadvantages, mainly related to the sulfur-containing cathode. This dissertation comprises the results of three research projects that investigated the structural and electronic properties of a polymer-based cathode material. The individual studies employ a combination of different theoretical multiscale ap- proaches, which are further compared and discussed with experimental measure- ments. In the first project, the novel fabrication route for the polymer cathode, which combines the processes of electro-polymerization and vulcanization, is presented. The structural evolution of the material during the synthesis is theoretically ra- tionalized, and experiments show that using this material helps to overcome the typical problems associated with Li-S batteries. The second project focuses on the structure of the vulcanized polymer representing the initial stage of a charged cathode. A combination of electronic structure theory and statistical mechanics is used to bridge between the microscopic description of the vulcanization of polymer and sulfur and the derivation of the macroscopic properties of the cathode. This study reveals that the electronic stability on a microscopic level most likely leads to a sulfur cross-linking of neighboring polymer chains, which is experimentally supported. Furthermore, we find that the extent of cross-linking on a macroscopic scale can be controlled by the amount of sulfur and the temperature during vul- canization. The third project covers the interplay between the polymer’s structural morphol- ogy and its electronic properties. Here, we focus on the influence of regiochemistry on the polymer’s aggregation behavior and charge transport. A multiscale ap- proach comprising classical molecular dynamic simulation, electronic band struc- ture calculations, and statistical charge transport theory demonstrates the poly- mers’ ability to form well-ordered crystalline phases, allowing fast charge trans- port. The comparison of experimental and simulated X-ray diffractograms con- firms the presence of crystalline phases in the electro-polymerized polymer. Overall, this thesis’s findings help advance the understanding of a polymer mate- rial in the context of its application as a Li-S battery cathode. Furthermore, this work demonstrates the benefit of using multiscale approaches to investigate such a system’s structural and electronic properties.
Weniger anzeigenJosephson junctions are formed by a non-superconducting weak link between two superconducting electrodes. The dissipation-less superconducting tunnel current flowing across the Josephson junction is carried by superconducting charge carriers which are called Cooper pairs. The tunnel junction is characterized by the overlap of the macroscopic wavefunctions of the two superconducting electrodes. Josephson junctions are highly sensitive to changes of the phase difference between the two wavefunctions. In this thesis Josephson junctions between a superconducting tip and superconducting sample were investigated in a scanning tunneling microscope (STM). The STM enables atomic resolution of the sample surface as well the formation and manipulation of atomic structures on the surface. Josephson spectroscopy in an STM allows the investigation of the influence of the junction composition on the phase coherence between the macroscopic wavefunctions of the superconducting electrodes on the atomic scale. Josephson junctions can be interpreted as an oscillating circuit with distinct damping properties. The damping behaviour due to energy losses caused by the interaction with the environment can be frequency dependent. Biasing the Josephson junction by either an applied current or voltage influences the frequency-dependent impedance that couples the system to the electromagnetic environment. Phase coherence is strongly influenced by the energy exchange of the junction with the environment. In this study, the Josephson junction is exposed to a high frequency (HF) electromagnetic field in order to further investigate the phase coherence of the system. Generally, tunneling charge carriers can absorb quantised energy from the photons of the HF field (photon-assisted tunneling). However, in Josephson contacts coherent absorption of energy from the external field is expected (Shapiro steps). The investigated junctions show resonant absorption at the expected energies in the presence of the HF field. Additionally the current-biased V(I)-curves show a hysteresis depending on the direction of the applied current. This hysteresis indicates phase coherence between tunneling Cooper pairs. However, other features observed in the V(I)-characteristics are correlated to dissipative processes. For that reason a clear identification of Shapiro steps is not possible in this setup. Furthermore, Josephson spectroscopy was performed on single magnetic adatoms on the Pb(111) surface. The magnetic moment of the atom’s unpaired electrons couples to the superconducting condensate and induces localized bound states within the energy gap of the superconductor (YSR states). The tunneling probability for electrons and holes into the YSR states is known to vary due to potential scattering on the surface (electron-hole asymmetry). In a Josephson junction magnetic adatoms were found to induce a diode-like behaviour, i.e., the transition from the resistive single-particle conductance into the Cooper-pair tunneling regime depends on the direction of the applied current. In collaboration with the theory group of Felix von Oppen at Freie Universität Berlin the observed non-reciprocity was correlated to the damping properties of the Josephson junction and explained by the electron-hole asymmetry of the induced YSR bound states.
Weniger anzeigenThis dissertation is concerned with the representation of Iranian films and filmmakers at the Berlinale during the Dieter Kosslick’s tenure as festival director (2001–2019). Since this representation unfolded not only on the silver screen, but especially on the various stages of the festival, from the red carpet to the press conference to the awards ceremony, my analysis is dedicated to the performative dimension of the phenomenon. Given the methodological approach of theater studies that enhances my background in Islamic Studies, I consequently examine the staging of Iranian cinema at the Berlinale.
To comprehend the genealogy of these festival stages and work out a regarding methodological framework, I initially address the phenomenon of late 19th century world exhibitions, from which the film festival format evolved in the 1930s. Here, crucial elements of cultural representation at urban mass events were established, from the form of the national competition crowned by the host to the exhibition of the non-West and the exotic at separate colonial exhibitions. Since the performances of the Berlinale take place in the scenery of Berlin, a look into the recent history and the character of the city is also necessary. Taking cue from Martina Löw’s proposal to examine cities in terms of their specific “inherent logic,” my dissertation subsequently addresses the trope of the divided city. Soon after the war, West Berlin was framed as a beacon of liberalism through spectacularly staged efforts like the Berlin Airlift of 1948/49. The origin of the Berlinale, which in 1951 was founded as a “Schaufenster der Freien Welt” (Showcase of the Free World), is to be understood in this context, too. In later years, the Berlinale established itself as an explicitly political festival and a “bridge between East and West,” which in the 1980s started an effort to showcase East German and the wider East Bloc cinema.
Many of these representational traditions and visual tropes, the genealogy of which I trace in the first half of my work, impacted the staging of Iranian films and filmmakers at the Berlinale from 2006 onwards. Iranian cinema’s wave of success at international film festivals had already ebbed down in the late 1990s, but under the new festival director Dieter Kosslick, the Berlinale began to invite films from the Islamic Republic extensively in the 2000s to offer its stages to the filmmakers, which were framed as restricted and repressed. The paradigms of censorship, repression, and rebellion, which impacted the presentation and reading of these films from the beginning, were seamlessly embedded into the brand of the “political festival” as well as into Kosslick’s understanding of the political as a spectacular counterweight to entertainment and glamour.
The most emblematic case of the festival’s branding as a platform for the cinematic rebellion of Iranian filmmakers is Jafar Panahi, with whom the last two chapters of my dissertation are concerned. After the director was convicted to a 20-year occupational ban and a prohibition to travel to foreign film festivals in 2010, he emerged as the poster boy of political cinema at the Berlinale. In 2011, the festival invited him into the international jury and dedicated large parts of its 61st edition to the absent and allegedly imprisoned filmmaker. Two years later, this performance evolved onto the silver screen, when his film Pardeh (Closed Curtain) was shown in the festival competition. While this cinematic therapy session presented Panahi as a depressive and restricted filmmaker, he returned in good sprits in 2015. In his film Taxi, he can be seen as a witty rebel who is back behind the driving wheel and in control of his creative process, breaking his chains with the help of the Berlinale live in front of the audiences.
Given the myth of the suffering and isolated city of Berlin, which after 1945 with international support regained its status as a global metropolis, Panahi’s three-part stage play of the absent jury member to the silenced filmmaker who finally celebrates his comeback live in front of a global audience on the stage of the Berlinale thus turned out to be extremely suitable for the festival, which accordingly crowned him with the Golden Bear in 2015. Following an analysis of his staging in Berlin as well as his films Pardeh and Taxi, I conclude that the performances of Iranian films and filmmakers at the Berlinale are far more telling of the character and the needs of the festival (and its host city) than about the actual state of Iranian cinema.
Weniger anzeigenAntiestrogen therapy is an integral part of the treatment of estrogen receptor α (ERα)-positive breast cancer, since it inhibits ERα-dependent tumor growth. In addition, over recent years, ERα signaling has been implicated in the mechanisms of breast cancer metastasis, which might add a new mode of action to antiestrogen therapy. Data from in vitro studies suggest that ERα-dependent downregulation of the expression of the cell-cell adhesion protein E-cadherin crucially promotes breast cancer metastasis. However, clinical studies show that E-cadherin expression only poorly predicts the risk of breast cancer metastasis in vivo. In addition to E-cadherin expression, cell-cell adhesion and thus the metastatic potential of breast cancer cells are essentially regulated by the complex interaction between E-cadherin and the cortical actomyosin cytoskeleton at the adherens junction (AJ) protein complex. In my thesis, I studied the impact of the clinical antiestrogen Fulvestrant on AJs using the MCF-7/vBOS breast cancer cell line. ERα inhibition by Fulvestrant did not change E-cadherin expression but induced a distinct reorganization of E-cadherin-mediated AJs. The effect of Fulvestrant and other antiestrogens on AJ organization could be specifically attributed to ERα inhibition. Furthermore, AJ reorganization depended on the cortical actomyosin cytoskeleton which might be the primary target of ERα inhibition. Investigating the functional consequences, I could show that the reorganization of AJs does not represent an impairment of AJ-mediated cell-cell adhesion. On the contrary, reorganized AJs were more stable as indicated by decreased cleavage of E-cadherin. Moreover, cells in a confluent cell monolayer were less motile, suggesting an increase in cell-cell adhesion. Studying breast cancer tissue sections, I observed that AJs were diversely organized in vivo. Here, an irregular organization of AJs correlated with low ERα activity, substantiating the data I obtained from the in vitro experiments with MCF-7/vBOS cells. Taken together, these results suggest that antiestrogen therapy might affect AJ organization, thereby increasing cell-cell adhesion and preventing the metastatic spread of breast cancer cells. However, further studies are required to assess the clinical relevance of AJ organization in vivo. Altering the organization of AJs could represent a novel mode of action of antiestrogen therapy. Moreover, AJ organization might serve as a diagnostic tool to predict the metastatic risk of breast cancer. Consequently, this could improve the diagnosis, therapy, and prognosis of breast cancer patients.
Weniger anzeigenCervical cancer is one of the most common cancers affecting women, and the mortality-to incidence ratio (MIR) of cervical cancer varies between developed and developing regions, North America had the lowest MIR (0.36), followed by MIR in Europe (0.40), while Africa had the highest MIR (0.68). Infection with human high-risk papillomaviruses (HR-HPV) is the most important driver of cervical cancer, but after persistent HR-HPV infection, infected cervix cells still need many years to transform into cancer. Since 90% of failures in current treatments are related to drug resistance, the development of more effective therapies is urgently required. However, many new therapies have to be terminated because of the unsatisfactory drug efficacy at clinical phases, which reflects the current flaws of preclinical drug models. In this context, establishing precise in vitro models to screen out the therapeutic targets and to assess the new drug efficacy against cervical cancer is vital. In this study, based on patient-derived cervical organoids, multi-omic analysis including RNA sequencing and HLA-restricted peptidome sequencing was used to investigate dysregulated genes, biology processes, and HLA-presented epitopes in HPV-transformed and cancerous cervical organoids. Totally 6,515 HLA class I and 719 HLA class II restricted peptides were yielded, thereafter the immunogenicity of the epitopes was predicted with public machine learning-based tools. Interestingly, a large number of upregulated genes and highly immunogenic epitopes in HPV+ and cancer samples were associated with DNA repair. Thus, the role DNA repair-related molecules appear to play in cervical cancer therapies and prognosis was further explored. I first constructed a DNA repair-related gene score (DRGscore) with the public cervical cancer dataset of the Cancer Genome Atlas Program (TCGA-CESE). In the following, I observed a negative correlation between DRGscore and the prognosis of cervical cancer patients. The tumor microenvironment of patients with lower DRGscore contained more immune effectors and less immune suppressors, which suggested the negative correlation between DRGscore and sensitivity to immunotherapy. This finding could provide clues for patient medication guidance: high DRGscore patients might benefit from DNA repair inhibitors to reduce chemoradiotherapy resistance and gain a better prognosis. In contrast, low DRGscore patients rather might be sensitive to different immunotherapy including immune checkpoint inhibitors, and adoptive T cell therapy. Due to HPV infection and viral genome integration, HPV+ and cervical cancer cells are generally under stress conditions, which induces the intracellular accumulation of phosphoantigens (pAgs) that can be recognized by Vγ9Vδ2 T cells in an HLA-independent manner. The DNA repair activities in host cells are exploited by HPV for replication, which may provide some potential ligands recognized by Vγ9Vδ2 T cell receptors. To figure out our hypotheses, I investigated the Vγ9Vδ2 T cell cytotoxic effect against HPV-transformed and cervical cancer cells based on our cervical organoid model. Importantly, the cytotoxic effect was significantly enhanced against HPV+ and cancer organoids compared to healthy organoids in the presence or the absence of bromohydrin pyrophosphate (BrHPP), a synthetic phosphoantigen which activates Vγ9Vδ2 T. Moreover, the BrHPP dependent cytotoxic effect was found strikingly to be reduced to the level in non-activated Vγ9Vδ2 T group with the blockade of butyrophilin-subfamily members (BTN3A, BTN2A1). Besides, CD107a over expression was also associated with BrHPP stimulation. Both pieces of evidence indicated the BTN-subfamily members were mainly responsible for the cytotoxic effect with BrHPP activation and the self-active Vγ9Vδ2 T cells induced indiscriminate killing against HPV+, cancer cells but also healthy cells via degranulation including granzyme B (GzmB), platelet rich fibrin (Prf), interferon gamma (IFNγ) secretion. While the mechanism of the specific cytotoxicity against HPV+ and cancer organoids in the BrHPP absence condition still remained vague. I concluded based on the bioinformatic analysis of multi-omic data that the differential recognition of non-healthy cells by Vγ9Vδ2 T cells depends on multiple-ligand interactions, which should be further explored in the future. Overall, within my thesis project, stable patient-derived cervical organoid lines were established, which acted as a promising in vitro model for screening cervical cancer associated epitopes and for exploring T cell-induced cytotoxic effects. Thus, the organoid-T cell coculture model that I established could prove useful for testing additional modulators (e.g. small molecules, antibodies, tumor sensitizers, etc.). Consequently, my investigations on the Vγ9Vδ2 T-mediated killing effect against HPV-transformed cervical cancer cells have shed light on the potential future utilization of Vγ9Vδ2 T in clinical settings.
Weniger anzeigenKidney transplantation (KTx) stands as the sole curative treatment option for individuals afflicted with end-stage renal disease, providing them a renewed opportunity for kidney function. However, the success of KTx is hindered by immune-mediated graft rejection, occurring both in the early postoperative phase and over the long term. These rejections episodes profoundly affect graft function and survival, posing significant challenges to successful transplantation outcomes. Lipocalin-2 (Lcn2), a member of lipocalin family, has received substantial recognition in the field of kidney transplantation due to its pivotal role in diverse physiological processes, such as iron transport, bacterial defence, and regulation of immune responses. Studies have shown that Lcn2 expression is upregulated in renal allografts during acute rejection episodes, suggesting its involvement in the immune response against the graft. Furthermore, preclinical studies have demonstrated the potential of exogenously administered recombinant Lcn2 (rLcn2) in ameliorating kidney injury and promoting graft survival. The primary objective of this research was to explore the intricate molecular and cellular mechanisms that underlie the renoprotective effects of rLcn2 in a mouse model of kidney transplantation. Treatment with rLcn2 during the perioperative period led to significant alterations in both adaptive and innate immune cell populations, particularly impacting the functionality and degranulation capacities of immune cells within the kidney graft. Moreover, an effective method for isolating and culturing primary proximal tubular epithelial cells (PTEC) was established to investigate their response to treatment with rLcn2. However, the PTEC exhibited diverse gene expression patterns during the culturing process and early on signs of beginning dedifferentiation processes, indicating two possible cellular fates for the cultured cells: a mesenchymal-like and an epithelial-like state. Furthermore, our investigations identified injured proximal straight tubule cells and stressed intermediate state dendritic cells/macrophages as the principal sources of endogenous Lcn2, providing insights into the cellular dynamics involved in Lcn2 production during allograft rejection. By elucidating the effects of rLcn2 on immune cells and uncovering the cellular origins of Lcn2 expression, this study enhances our understanding of how rLcn2 exerts its renoprotective effects in kidney transplantation. These findings contribute to the growing body of knowledge surrounding Lcn2 as a promising therapeutic target for mitigating immune-mediated damage to the kidney grafts and improving their outcomes.
Weniger anzeigenSevere-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), rapidly became a pandemic and has caused 6.6 million deaths since the outbreak started. Although the development and administration of vaccines provided a substantial improvement by decreasing both the number of transmission events and disease severity of infected individuals, emerging viral variants still pose a threat due to their potentially increased transmissibility, immune evasion and virulence. Therefore, there is an ongoing need to better characterize SARS-CoV-2 pathogenicity and the underlying mechanisms to develop alternative strategies for prevention and treatment of COVID-19. This dissertation is based on two publications investigating interferon (IFN) autoantibodies in patients with autoimmune polyendocrine type I (APS-1) and hospitalized COVID-19 patients, respectively. Despite the known production of neutralizing IFN autoantibodies (IFN-AABs) by patients with autoimmune disorders -like APS-1- and well-characterized, key role of IFNs in immune responses during viral infections, the contribution of IFN-AABs to disease in viral infections have been rarely addressed. With the emergence of the COVID-19 pandemic, several studies revealed presence of IFN-AABs in a proportion of patients with severe COVID-19 and pointed towards a link between IFN-AABs and disease severity. Firstly, we conducted a prospective study on a small cohort of patients with APS-1. We hypothesized that these patients have a disposition to develop severe COVID-19 in case of a SARS-CoV-2 infection due to pre-existing IFN-AABs. Contrary to our hypothesis, patients within our cohort that reported an infection with SARS-CoV-2, reported mild disease, showing an incomplete clinical penetrance of IFN-AABs to severe COVID-19. Secondly, we performed a large scale, cross-sectional, multi-cohort study on hospitalized patients with COVID-19. Here, we aimed to identify clinical parameters that co-present with neutralizing IFN-AABs. We propose a novel clinical algorithm for rapid identification of neutralizing IFN-AAB-positive patients that can benefit from specific alternative therapies.
Weniger anzeigenGuanine-rich sequence binding factor 1 (Grsf1) is a heteronuclear ribonucleoprotein that binds to RNA and thus, takes part in the regulation of gene expression. Gene expression is regulated at different levels and involves transcriptional, post-transcriptional, translational and post-translational mechanisms. These regulatory networks enable cells and whole organisms to adapt to changing environmental conditions. Grsf1 has previously been reported to upregulate translation of glutathione peroxidase 4 (Gpx4) mRNA[1]. Since this enzyme plays an important role in embryonic brain development and as anti-oxidative enzyme, Grsf1 has also been implicated in these processes. In fact, studies on in vitro mouse embryogenesis have shown that RNAi-induced knockdown of Grsf1 expression leads to hypoplastic brain development but the molecular basis for these observations remains elusive. Although it has been hypothesized that Grsf1 might interact with other regulatory proteins, identification of Grsf1 binding proteins has been still pending. Moreover, due to the lack of Grsf1 knockout mice, it has not been explored whether the reported in vitro data on the biological functions of Grsf1 could be reproduced in vivo. This situation resulted in two major questions for the present dissertation, which have been worked on simultaneously. 1. Which intracellular proteins might function as putative Grsf1 binding partners? 2. Does systemic inactivation of the Grsf1 gene alter the phenotype of mice? To answer the first question, we employed the yeast 2-hybrid system and identified three different proteins as putative Grsf1 binding proteins: I) tetratricopeptide 7B (TTC7B), II) selenocysteine-binding protein 2 (SBP2) and III) SNF8 subunit of ESCRT-II (SNF8)]. Co-immunoprecipitation experiments using co-transfected human embryonic kidney cells (HEK) confirmed the postulated protein-protein interaction. To address the second question, we first generated floxed Grsf1 transgenic mice, in which exons 4 and 5 of the Grsf1 gene were flanked by recognition sequences for Cre-recombinase. These floxed Grsf1 mice were crossed with deleter mice expressing the Cre-recombinase under the control of the CMV promoter [B6.C-Tg(CMV-cre)1Cgn/J]. Offsprings expressed heterozygously a truncated version of the Grsf1 gene and the resulting dysfunctional Grsf1 mRNA was targeted for nonsense mediated decay. Intercrossing the resulting Grsf1+/- mice we obtain homozygous Grsf1-/- mice, in which the Grsf1 gene was inactivated in all tissues. Inactivation of the Grsf1 gene was demonstrated by quantification of the Grsf1 mRNA in a number of organs and by Western blot analysis in testis. We found that Grsf1-/- mice developed normally and did not show major phenotypic abnormalities. Although we observed subtle growth retardation in male Grsf1-/- mice, female individuals developed normally. The reproduction behavior of Grsf1-/- mice was normal and we did not detect restricted mobility or life expectancy. In the future, these Grsf1-/- mice can be tested in different animal disease models to explore the biological role of Grsf1 protein in mouse models of human diseases.
Weniger anzeigenHintergrund: Die Anwendung von Fast-Track (FT) Anästhesieverfahren zeigte positive Auswirkungen auf Morbidität und Mortalität mit Reduktion der Beatmungs- und Intensivstationsliegedauer, sodass FT-Prozeduren in der Herzchirurgie zunehmend an Bedeutung gewinnen. Hinsichtlich der sicheren Anwendbarkeit in der minimalinvasiven Mitralklappenchirurgie (MIMVS) liefert die Studienlage noch keine umfassenden Daten. Zu diesem Zwecke analysierten wir 491 MIMVS-Patienten und -Patientinnen unter FT-Anästhesieverfahren, um Ausmaß, Charakteristika und assoziierte Risikofaktoren des Fast-Track-Failures (FTF) zu evaluieren. Methoden: Im Deutschen Herzzentrum der Charité (DHZC) wurden retrospektiv 491 Patienten und Patientinnen, die sich zwischen 2014 und 2020 einer MIMVS mit FT bei Mitralklappeninsuffizienz Carpentier Typ I und/oder II unterzogen, in die Studie eingeschlossen. Es wurden uni- und multivariable Analysen durchgeführt, um unabhängige prä- und perioperative Assoziationsfaktoren für das Scheitern des FT-Protokolls zu identifizieren. Unsere Definition des FTF beinhaltet vier Elemente: (1) >10 Stunden Beatmung und/oder (2) >24 Stunden Aufenthalt auf der Intensivpflegestation (IPS) und/oder (3) Reintubation nach erfolgter Extubation und/oder (4) IPS-Wiederaufnahme. Ergebnisse: Ein FTF lag bei 237 (48.3%) unserer Gesamtpopulation aus 491 Patienten und Patientinnen vor. Die häufigsten Ursachen waren ein IPS-Aufenthalt über 24 Stunden in 155 Fällen (65.4%) und eine Beatmungsdauer von über 10 Stunden (142, 59.9%). Die mittlere Operationsdauer beträgt 150 Minuten und die des kardiopulmonalen Bypasses (CPB) 101 Minuten. Eine Notwendigkeit für Erythrozytentransfusionen bestand in 69 Fällen (14.1%) und die einer operativen Revision aufgrund einer Blutung in 29 Fällen (5.9%). Die multivariable logistische Regressione zeigten eine unabhängige Assoziation zum Fast-Track-Failure für folgende Faktoren: Revision aufgrund einer Blutung (OR 8.36, KI [2.81 – 36.01]; p = <0.001), chronische Nierenerkrankung (GFR <85 ml/min/1,73m2) (OR 2.03, KI [1.14 – 3.70]; p = 0.018), koronare Herzkrankheit (OR 1.90, KI [1.13 – 3.23]; p = 0.016), präoperativer NYHA III oder IV (OR 2.05, KI [1.38 – 3.08]; p = <0.001) und die Operationsdauer in Minuten (OR 1.01, KI [1.01 – 1.01]; p = <0.001). Zusammenfassung: Die Inzidenz des FTF ist in der von uns untersuchten Population von Patienten und Patientinnen mit Carpentier Typ I und/oder II Mitralklappeninsuffizienz und minimalinvasiver Mitralklappenreparatur nicht zu vernachlässigen. Als nicht-modifizierbare, mit dem FTF assoziierte Faktoren, identifizierten wir einen NYHA-Status III und IV, eine vorbestehende chronische Nierenerkrankung und eine begleitende koronare Herzkrankheit. Hinsichtlich potenziell modifizierbarer Faktoren identifizierten wir postoperative Blutungen, die eine chirurgische Revision bedürfen sowie die Dauer des Eingriffs als Assoziationen für einen FTF bei minimalinvasiver Mitralklappenreparatur.
Weniger anzeigenThe high variability in remission rates highlights the importance of optimizing treatment response in depression. Interventional psychiatry treatments are an option for patients who have been unable to achieve remission with established therapies. Their efficacy could be further improved by identifying subgroups of individuals more likely to respond based on specific features. Moreover, interventional therapies could boost classical behavioral interventions. The aim of the present dissertation was to investigate antidepressant-response predictors to interventional psychiatry techniques and the potential of neuromodulation to enhance the effect of psychotherapy while providing a better understanding of the neural mechanisms and treatment- related effects on brain structure. In the first study we used voxel-based morphometry to investigate structural predictors of response in 33 depressed patients treated with a single sub-anesthetic ketamine infusion. We found that greater baseline gray matter volume (GMV) of the bilateral rostral anterior cingulate cortex (rACC) significantly predicts symptom reduction after ketamine. It could be speculated that ketamine acts by enhancing a well-preserved rACC, thus promoting adaptive self-referential processing and cognitive control. In the second study, we applied multivariate pattern analysis techniques to baseline data of 71 patients treated with ECT. sMRI-based classification of ECT responders and non-responders reached an accuracy of 69 %, with a cluster in the right anterior parahippocampal gyrus (aPHCr) contributing the most to the classification. A post-hoc regression- based prediction analysis on continuous symptom improvements showed a significant relationship between smaller GMV on the aPHCr and treatment response. These results are in line with previous reports that ECT acts by promoting neurogenesis in temporal structures. Finally, we conducted a double-blind, placebo-controlled randomized clinical trial to investigate whether the efficacy of cognitive behavioral therapy (CBT) can be enhanced by concurrent transcranial direct current stimulation (tDCS). We set three groups: 53 patients to CBT alone, 48 to CBT+tDCS, and 47 to CBT+sham-tDCS. The interventions showed to be safe and reduced depressive symptoms with a significant effect over time. However, we found no significant interactions between group and time. The findings suggest structural predictors may be specific to treatment mechanisms (such as enhancing well-preserved structures or facilitating neurogenesis). A better understanding of these would enable the design of further synergistic interventions that target specific neurobiological processes. Using a combination of clinical variables, functional brain imaging, laboratory data, and genetic data, it may be possible to gain a systematic understanding of treatment effects.
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