Vessel calcifications are a main risk factor for the development of cardiovascular diseases, a leading cause of morbidity and mortality in western societies. Even though vascular calcifications have been researched for centuries, patients still suffer from a huge therapeutic gap, because neither an effective prophylaxis, nor an effective treatment is available. Currently, consent exists that vascular calcification is an active cellular process that resembles bone formation. Vascular Smooth Muscle Cells (VSMCs) and their transdifferentiation into osteoblast like cells are considered decisive in the formation of vascular calcifications. Several stimuli can induce osteoblastic transdifferentiation, including cellular aging processes, oxidative stress and inflammation. These factors can be linked into a concept termed inflammaging via the formation of damage associated molecular patterns, persistent DNA damage and induction of a senescence associated secretory phenotype. The objective of this dissertation was to analyse the effects of the cellular stressors Azathioprine and Doxorubicin and the pro-inflammatory cytokine interleukin 1ß on the induction of osteoblastic transdifferentiation of VSMCs and vascular calcification in different experimental settings. The cellular stressors induce the formation of reactive oxygen species, cellular senescence, secretion of pro-inflammatory cytokines such as interleukin 1ß and interleukin 6, upregulation of NLRP3 inflammasome components as well as osteoblastic transdifferentiation and calcification of VSMCs. Stimulation with interleukin 1ß induced a pro-inflammatory auto-loop, osteoblastic transdifferentiation and calcification of VSMCs, but did not induce markers of senescence in VSMCs apart from components of the cellular senescence associated secretory phenotype. For both, cellular stressors and interleukin 1ß, the NLRP3 inflammasome is decisively involved in the calcification process, as inhibition of NLRP3 significantly reduces the calcification induced by cellular stressors and interleukin 1ß. This dissertation is in line with other recent research that emphasizes the involvement of inflammaging in the pathology of vascular calcification. Several therapeutic targets arise from the insight that oxidative stress, inflammation and senescence are crucially involved in the pathophysiology of VSMC calcification. These new therapeutic approaches can help to meet the high unmet clinical need of patients suffering from vascular calcifications.Weniger anzeigen
Extreme precipitation events can have serious adverse consequences for the population. Their severity and possible impacts are mainly determined by their intensity and duration. Therefore, when planning and operating water management systems or protecting infrastructure from flooding, it is important to consider the frequency at which particularly intense or long-lasting heavy rainfall events can be expected. This information is commonly summarized in intensity-duration-frequency (IDF) curves. The methods used to estimate IDF curves are based on extreme value statistics, where the challenge is modeling events of rarely occurring magnitude or those not even observed yet. A further obstacle is the availability of data, since the time series of precipitation measurements with a high temporal resolution are usually relatively short and the spatial coverage of precipitation gauges is sparse. Therefore, the estimation of IDF curves is often associated with considerable uncertainties. This is problematic, because overestimation leads to major additional construction costs, while underestimation implies an unreasonable residual risk of failure and might result in severe consequences.
Therefore, the objective of this work is to develop IDF models that allow for estimation with reduced uncertainties. We achieve this by more efficient usage of the available data. Thus, as a first step, we model the annual precipitation intensity maxima over a range of durations simultaneously within one model. To this end, we use a duration-dependent Generalized Extreme Value (d-GEV) distribution. We demonstrate that this results in a significant reduction of uncertainties, especially in the estimation of the shape parameter. This parameter determines the upper tail of the distribution and thus comprises information on very rare, severe events.
Within our first study, we extend the d-GEV model by incorporating spatial variations (in the form of covariates). This provides two advantages: on the one hand, we can combine information from different stations, which leads to a further reduction of uncertainties. On the other hand, we are able to spatially interpolate the IDF relationship, which allows us to obtain estimates at any location within the study area, including locations without observations. We analyze the performance of the model in a case study in detail. Our results show that including spatial variations into the model improves the estimation of rare events and yields reliable estimates at locations with little or no data. Using two examples of heavy rainfall events in Berlin, we further demonstrate how the spatial d-GEV model can be applied to analyze the extremeness of events on different timescales and in space. However, another result of our first study indicates a disadvantage of the d-GEV model: it is not flexible enough to describe a wide range of durations, as it does not improve the estimates for every duration. In our second study, we therefore investigate possible extensions of the model and discuss for which applications certain enhancements might be beneficial.
Since the d-GEV distribution is commonly applied to model annual maxima, only a small fraction of the observations is used to estimate IDF curves. In our third study, we implement seasonal variations into the model, allowing us to use monthly maxima, instead. This again results in a significant reduction in uncertainties. Moreover, we can investigate the influence of seasonal variations on the IDF curves. Our results suggest that especially at stations with large differences in the seasonality of short and long-lasting extreme events, the initial d-GEV model does not provide a good approximation. At these stations, the flexible d-GEV model is clearly able to better describe the IDF relationship. Additionally, modeling monthly instead of annual maxima allows for more reliable estimation of the shape parameter, therefore, we are able to perform a more careful analysis of the dependence of the shape parameter on duration.
The methods presented in this thesis not only serve to reduce uncertainties in the estimation of IDF curves but also provide a consistent and parameter parsimonious approach to model variations of extreme rainfall. Future studies can benefit from our insights to investigate the influence of climate change on the intensity and frequency of extreme events on different timescales with greater accuracy.Weniger anzeigen
Strain accumulation and stress release along multiscale geological fault networks are fundamental mechanisms for earthquake and rupture processes in the lithosphere. Due to long periods of seismic quiescence, the scarcity of large earthquakes and incompleteness of paleoseismic, historical and instrumental record, there is a fundamental lack of insight into the multiscale, spatio-temporal nature of earthquake dynamics in fault networks. This thesis constitutes another step towards reliable earthquake prediction and quantitative hazard analysis. Its focus lies on developing a mathematical model for prototypical, layered fault networks on short time scales as well as their efficient numerical simulation.
This exposition begins by establishing a fault system consisting of layered bodies with viscoelastic Kelvin-Voigt rheology and non-intersecting faults featuring rate-and-state friction as proposed by Dieterich and Ruina. The individual bodies are assumed to experience small viscoelastic deformations, but possibly large relative tangential displacements. Thereafter, semi-discretization in time with the classical Newmark scheme of the variational formulation yields a sequence of continuous, nonsmooth, coupled, spatial minimization problems for the velocities and states in each time step, that are decoupled by means of a fixed point iteration. Subsequently, spatial discretization is based on linear and piecewise constant finite elements for the rate and state problems, respectively. A dual mortar discretization of the non-penetration constraints entails a hierarchical decomposition of the discrete solution space, that enables the localization of the non-penetration condition. Exploiting the resulting structure, an algebraic representation of the parametrized rate problem can be solved efficiently using a variant of the Truncated Nonsmooth Newton Multigrid (TNNMG) method. It is globally convergent due to nonlinear, block Gauß–Seidel type smoothing and employs nonsmooth Newton and multigrid ideas to enhance robustness and efficiency of the overall method. A key step in the TNNMG algorithm is the efficient computation of a correction obtained from a linearized, inexact Newton step.
The second part addresses the numerical homogenization of elliptic variational problems featuring fractal interface networks, that are structurally similar to the ones arising in the linearized correction step of the TNNMG method. Contrary to the previous setting, this model incorporates the full spatial complexity of geological fault networks in terms of truly multiscale fractal interface geometries. Here, the construction of projections from a fractal function space to finite element spaces with suitable approximation and stability properties constitutes the main contribution of this thesis. The existence of these projections enables the application of well-known approaches to numerical homogenization, such as localized orthogonal decomposition (LOD) for the construction of multiscale discretizations with optimal a priori error estimates or subspace correction methods, that lead to algebraic solvers with mesh- and scale-independent convergence rates.
Finally, numerical experiments with a single fault and the layered multiscale fault system illustrate the properties of the mathematical model as well as the efficiency, reliability and scale-independence of the suggested algebraic solver.Weniger anzeigen
The development of our society relying on utilization of raw materials from Earth has left unprecedented marks on our planet’s environment. A key issue is the climate change phenomenon caused by the continuous increase in the atmospheric concentration of the greenhouse gas CO2 due to combustion of fossil fuels as main energy source. The mitigation of the CO2 emissions via its capture and conversion, increase in the utilization of renewable energy and recycling technologies, and eliminating the dependence from fossil fuels is a strategy for building sustainable society. A promising concept for tackling the CO2 emission via its conversion into valuable products (hydrocarbons and alcohols etc.) is the electrochemical reduction of CO2 (CO2ER), that has many advantages over the other conversion concepts. Cu is unique in terms of material that can intrinsically catalyze CO2 reduction into hydrocarbons and alcohols. However, there are many Cu catalyst/experimental conditions/engineering - related challenges and other issues of various nature that affect the product selectivity and therefore still hinder the large-scale application of the CO2ER. Regarding the catalyst and experimental conditions challenges, possible alternative for overcoming the selectivity issues is step- wise CO2ER i.e., two-electron electrochemical reduction of CO2 into CO and subsequent conversion of CO into hydrocarbons, alcohols and other valuable products. Furthermore, another two-electron product, that is formic acid (HCOOH) or formate (HCOO–) that find various industrial applications and are also promising alternative as fuel in fuel cells, together with CO can be produced with high selectivity on various cheap and abundant electrocatalysts. Namely, the Cu rich Cu-Sn materials appear to be promising catalysts for CO2ER into CO, while Sn rich Cu-Sn and Cu-S for production of HCOO–, and therefore they are worth and inspiring to be more thoroughly studied in terms of their composition- structure relations with the catalytic activity for electrochemical conversion of CO2. Hence, the first main goal of this thesis is dedicated to study of the composition-structure-CO2ER activity relations in the Cu-Sn and Cu-S based electrocatalyst materials. On the other hand, the second main goal encompasses providing simple, cheap and fast synthesis methods for both Cu-Sn and Cu-S based materials, and moreover, including a successful proof-of-concept for recycling/repurposing waste for deriving CO selective Cu-Sn electrocatalyst, which are prerequisites toward possible application of these materials for large-scale conversion of CO2 and building a sustainable society based on recycling in order to mitigate and finally cease the extraction of natural resources. The thesis is divided into three studies, from which the first study represents determination of the composition and speciation of Cu and Sn in Cu-Sn electrocatalysts under CO2 electrolysis in order to reveal the relationship between these parameters and the CO2ER selectivity alteration between CO and HCOO– at various applied potentials. For the purpose of this study, SnO2 functionalized CuO nanowires with varying thickness of surface SnO2 layers (low and high Sn), were synthesized. The CO2ER product quantification was performed using chromatography, while the material characterization methods comprised of mainly spectroscopy-based techniques including ex-situ soft x-ray XAS, in-situ hard x-ray XAS and quasi in-situ XPS, supported by microscopy/electron diffraction (EF-TEM, HR-TEM and SAED) and computational modeling (DFT). The results show that thin layer of SnO2 (low Sn) functionalized CuO nanowires electrocatalysts that are selective for CO2ER into CO, reaching maximal FE of ~80% at –0.7 V, undergo surface transformation generating Cu0 and SnOx (Snd+) species under all examined potentials. The presence of Snd+ is supporting the Sn to Cu charge redistribution mechanism and therefore promoting desorption of the Cu bound *CO intermediate, leading to significantly higher CO evolution, compared to the activity of pristine Cu. On the other hand, the results show that the increase in the surface Sn content is beneficial for CO2ER into HCOO–, achieving the highest FE (80%) at –0.9 V for the catalyst with highest Sn content. Altering the potential toward more negative values is leading to increase in the surface fraction of metallic Sn specie that readily bind the *OCHO* intermediate following the HCOO– pathway, accompanied with significant suppression of the competitive hydrogen evolution reaction (HER) due to weak binding of the *H intermediate. Even though these Cu-Sn materials can reach very high selectivity for both CO and HCOO– in dependence of the surface Sn content, sophisticated, expensive and time-consuming approach, that includes atomic layer deposition (ALD) of SnO2, was used for their synthesis. An important requirement for future practical application of the CO2ER catalysts is definitely simple, cheap and fast synthesis. Therefore, in the scope of the second study, facile one-step electrochemical method was developed for deriving Cu-Sn foam with low Sn content from waste bronze. The bronze derived Cu-Sn foam reached 80% FE for CO at –0.8 V, competing with the best catalysts for this purpose, which makes it promising for future large-scale application. This study is showing that recycling/repurposing waste material for CO2ER catalyst synthesis is achievable, which is an important step towards sustainable supply of materials for this purpose. The third study is based on investigation of the composition-structure relations in Cu-S catalysts selective for CO2ER into HCOO–, and moreover presenting a facile method for synthesis of these materials based on direct reaction between elemental Cu and S dissolved in toluene, hence avoiding usage of expensive and extremely toxic precursors. The most important finding in this study, based on examination of the Cu-S catalysts with quasi in-situ XPS, reveals that under CO2 electrolysis the materials do not undergo complete reduction and Cu+ surface species persist at all examined potentials (–0.5 to –0.9 V), compared to pristine Cu which is completely reduced to metallic under identical conditions. The presence of residual surface sulfur species is most probably stabilizing the Cu+ with oxophilic nature on which the *OCHO* intermediate favorably binds and further converts into HCOO–. However, the HCOO– selectivity that can reach up to 70-75% is dependent on activation of the electrocatalyst that is related to the Cu:S surface composition and various electrode-electrolyte interface effects. Namely, besides the S2–, presence of unexpected SO42– specie is found on the surface of the electrocatalysts that are subjected to applied potential of –0.9 V, most probably due to local pH increase effects. These local effects are not fully understood from this study which is inspiring for further research that involve probing the electrode-electrolyte interface with other surface sensitive methods under in- situ conditions such as Raman and infrared spectroscopy. Finally, the future challenges include an adaptation of the facile synthesis methods developed in this work to prepare gas-diffusion electrodes loaded with Cu-Sn and Cu-S catalysts. Examining their CO2ER activity in gas-diffusion electrolyzers is important to achieve high current densities and, hence, industrial relevant conversion rates that are required for future large-scale applications.Weniger anzeigen
Die vorliegende Arbeit setzt sich mit den Briefen Papst Alexanders IV. auseinander, die während seines Pontifikats zwischen 1254 und 1261 entstanden sind. Knapp 3260 dieser Schreiben haben sich im Hauptregister des Papstes erhalten, zahlreiche Weitere zudem in Empfängerüberlieferung. Diese Litterae dienen der Wissenschaft bis heute, um einen Einblick in die päpstliche Politik um die Mitte des 13. Jahrhunderts zu erlangen. Dabei wurde gerade der Pontifikat Alexanders IV. von früheren Historikern häufig als ein „Bild hilfloser Schwäche“ abgetan. Dagegen stellt sich diese Arbeit, die von der Hypothese ausgeht, dass diese Litterae nicht als schlichte Tatsachenberichte zu deuten sind, sondern in einem hohen Maß durch Rhetorik ausgeschmückt wurden. Es ist anzunehmen, dass durch die Verwendung gewisser Formulierungen und Darstellungsweisen seitens der päpstlichen Kurie ein spezifisches Bild vom Papsttum konstruiert wurde. Um diese These zu überprüfen, wurden die Briefe Alexanders IV. innerhalb zweier Querschnittuntersuchungen eingehend epistolographisch untersucht. Zum einen wurden die Verhandlungen um das Königreich Sizilien, zum anderen die Verbindung zwischen Alexander IV. und den Bettelorden betrachtet. So konnten die Grundlagen der sprachlichen Gestaltung der päpstlichen Kanzlei erkannt und die Frage nach der darstellerischen Inszenierung Alexanders IV. beantworten werden. Es wurde nicht nur der kuriale Briefaufbau aufgeschlüsselt, sondern auch spezielle Allegorien aufgezeigt, die der päpstlichen Inszenierung dienten. Für die Analyse der einzelnen Briefe wurde dabei der Ansatz Tanja Brosers genutzt, den sie für ihre eigene Untersuchung der Biefe Papst Clemensʼ IV. auf den Grundlagen der Briefanalyse von Carl Erdmann entwickelt hat. Anhand dieser epistolographischen Untersuchung konnte tatsächlich ein „Bild hilfloser Schwäche“ ausgemacht werden. Hierbei handelt es sich jedoch nachweislich um einen Teil der päpstlichen Inszenierung, die seitens der Kanzlei als Argumentationsmuster genutzt wurde. Ähnliches ließ sich in vergleichenden Analysen auch für die Vorgänger Alexanders IV. Gregor IX. und Innocenz IV. nachweisen. Auf der Grundlage dieser Ergebnisse konnte damit auch das bisherige Urteil der Wissenschaft über Alexander IV. relativiert werden.Weniger anzeigen
Freshwater systems have always been under the most variate disturbance regimes. However, the number of multi-stressors and the frequency/intensity of stochastic pulse perturbations they will face in the future is predicted to increase. Stochastic pulse perturbations such as storms and floods are well known to temporarily disrupt ecosystem services that are fundamental for global existence within short time scales - as drinking water supply, irrigation, and food provision. Eutrophication - one of the main freshwater press perturbations deteriorating water quality - may enhance harmful cyanobacterial blooms, modify the structure of food webs, and alter long-term ecological stability, resulting in substantial ecological, social, and economic losses. Despite all our knowledge on the effects of stochastic events and eutrophication operating in isolation, we know little about how their interactions can shape the resilience of aquatic ecosystems.
The aim of this thesis is to identify general processes of recovery and response pathways taking place after mortality pulse perturbations and assess how resource availability can modify them. For that, the existing literature was reviewed, and experiments were executed using aquatic systems of different ecological complexity. Chapter 2 starts with a global review of how climate change may modify in-lake processes that are prone to escalate into feedback loops of relevance to freshwater biogeochemistry (i.e., mixing regimes modifying nutrient upwelling). Chapter 3 explored if Early- Warning Signals of Regime Shift (EWS) could produce sufficient mechanistic empirical evidence to be used as a reliable methodological framework for quantifying Ecological Resilience. For that, we reviewed the literature for aquatic experiments that have designed interactions between pulse and press perturbations. Using the lessons learned from both reviews, the experiment of Chapter 4 used an eco-evolutionary microcosm with complex intraspecific interactions to focus on methodological constraints of using recovery rate and time for addressing engineering and ecological resilience. Also, this experiment investigated the role of resource availability on the processes ruling the recovery from pulse perturbations at a community-alike level. Last, in chapter 5, ecological complexity was scaled up using mesocosms filled with natural lake water to identify key processes involving the effect of nutrient availability on the microbial community response to pulse perturbations. Here the focus was on how eutrophication changes the phytoplankton resistance and resilience to mortality events that cause internal nutrient turnover. Together, these four chapters provided the core of the thesis.
Overall, we identified that climate change can indeed create feedback loops capable of altering the nutrient dynamics in lakes, as well as the direction of change depending on the lake characteristics. We scrutinized processes that can lead to an increase of total nutrients in the epilimnion in the long run while creating longer periods of dissolved nutrient limitation within a season. Those processes are related to In-lake mixing regimes, which are susceptible to atmospheric conditions and responsible for the internal mixing of nutrients in lakes. Therefore, the presence of feedback loops in lake dynamics might become increasingly relevant under ongoing climate change.
When reviewing the reliability of EWS to address the loss of ecological resilience in aquatic systems, we observed that the field is still at the stage of proof-of-concept. Complex experiments designed to assess the interaction between pulse and press perturbations were virtually absent. Most experiments considered either a pulse or a press perturbation affecting the system, and often without a mechanistic understanding of why an EWS was observed or not. Amongst all EWS, recovery rate or time were the most capable of bringing together how long-term changes in the system affect the response to stochastic pulse perturbations. Recovery rates showed reliable results when predicting population collapses; however, the translation of individual populational-level recovery rates to more complex community-based ecosystem dynamics is far from consolidated.
Moving on into process-based approaches, our experiments highlighted that the effects of resource availability can be decomposed into two distinguished processes of recovery. One is the capability of the system to fully recover from disturbance. Another is the recovery pattern that the system will embody after the pulse perturbation. In both experiments we observed the importance of biotic interaction for determining how the system responds to perturbations and the effect of resource availability on shaping the direction of those interactions. Competition, facilitation, intrinsic growth rates, population turnover, the complexity of ecological interactions, and rates of energy transfer, all played a role in the processes underlying 'resilience' (latu sensu).
The eco-evolutionary microcosm taught us that resource availability modulates the speed of the recovery process (rate and time) but not necessarily the level of recovery. Systems with low resource availability showed weaker recovery rates and longer recovery times for both compositional and functional dimensions compared to when resources were high. However, all tested systems showed substantial plasticity in absorbing and recovering from a pulse perturbation (complete recovery). Therefore, the slower recovery process caused by low resource availability did not change “the measure of the persistence of the system” or “their ability to absorb change and disturbance and still maintain the same relationships between populations or state variables" – the formal definition of ecological resilience (Holling, 1973). When dealing with systems of different resource availability, ecological (sensu Holling) and engineering resilience (sensu Pimm) may potentially decouple in time. Therefore, we must be especially careful when using recovery from perturbation as a proxy of ecological resilience if resource availability is the main driver of ecosystem change.
When scaling up the trophic complexity to a mesocosms level, we observed that pulse perturbations increased the amount of autochthonous nutrients in the system, selecting for 'small and fast-growers' at first. As more resources were allocated in the system before the pulse perturbation, more nutrients were released during the mortality event; and with more dissolved nutrients available for uptake, higher peaks of phytoplankton concentration were observed as a response to the pulse perturbation. However, the level of resource availability in the mesocosms did not change the resistance or the recovery from a mortality pulse perturbation. Together, the chlorophyll-a displacement declined after each sequential pulse perturbation despite increases in resource availability. The more perturbed the system was, the less responsive it became, and this can be interpreted as a possible community pathway towards stability. The mesocosms experiment indicated the capability of the system to adapt to sequential pulse perturbations in a way to reduce its impacts and that resource availability and sequential pulse perturbations are antagonistic forces shaping the response of the system to mortality events.
Both experiments executed in this thesis presented a convergent story. Resource availability did not change the capability of the systems to recover from pulse perturbations, despite changing their compositional structure and process rates. However, the evidence that resource availability is unlikely to influence the capability of systems to recover should not lead to underestimations of the risks related to changing process rates in ecological systems. Systems under low resource availability might become more vulnerable to changes in the frequency of pulse perturbations, while systems with high resource availability may present stronger responses immediately after the pulse perturbation (i.e., peaks of Chl-a concentrations). This is an important consideration when managing waterbodies with strict regulations (i.e., drinking water provision) since systems with a surplus of resource availability may more easily trespass regulatory guideline values (e.g., maximum Chl-a threshold levels for safe water supply) following a pulse perturbation, while oligotrophic systems may become especially vulnerable to changes in the frequency of sequential weather-related pulse perturbations.Weniger anzeigen
Despite intense research efforts, epithelial ovarian cancer (EOC) invariably remains the most lethal gynecological malignancy in women. In the absence of biomarkers capable of detecting EOC at an early, still curable stage, altered glycosylation of proteins attracted attention as a potential source of complementary screening markers. While changes of total serum N-glycome in EOC patients have been broadly examined, there is so far only limited data available on glycosylation alterations occurring in individual serum-derived glycoproteins and ovarian cancer tissue. Therefore, to fill this gap of knowledge, EOC-related glycosylation changes were investigated in this thesis at the level of immunoglobulin G (IgG), the most abundant serum glycoprotein, and at the level of EOC tissue, implementing cutting-edge technology, MALDI mass spectrometry imaging (MALDI-MSI). In humans, IgG occurs as four subclasses, IgG1, IgG2, IgG3, and IgG4, which differ sharply in respect to their biological characteristics and effector functions despite high homology at the amino acid level. Although IgG glycosylation in EOC patients has already been investigated previously, EOC-related glycosylation alterations were determined at the level of enzymatically released N-glycans. In consequence, currently available data give only global information and is neither subclass- nor site (Fc/Fab)-specific. Therefore, in order to provide a deeper insight into glycomic changes accompanying the development and progression of EOC, in this work, glycosylation alterations were investigated in a subclass-specific manner, performing the analysis at the glycopeptide level. To this end, IgG was isolated from blood sera of 87 EOC patients (FIGO stage I-IV) and 74 age- and sex-matched healthy controls. In order to separate IgG2 and IgG3, whose peptide backbones encompassing the Asn297-glycosylation are frequently identical, a two-step affinity purification employing Protein A and Protein G Sepharose was performed. Isolated IgG fractions containing IgG1, IgG2, IgG4, and IgG3, respectively, were digested with trypsin, and the resulting glycopeptides were enriched using cotton-HILIC columns. Finally, measurements were performed by MALDITOF-MS in negative ionization mode. By summing up relative intensities of respective glycopeptide structures, six (or five in the case of IgG2) glycosylation traits, namely a-, mono-, digalactosylation, sialylation, bisection, and fucosylation, were calculated separately for IgG1-3 subclasses (analysis of IgG4 was omitted due to its very low serum concentration and overlapping glycopeptide signals). In all investigated IgG subclasses, mono- and digalactosylation were found to decrease in patients suffering from early- and late-stage EOC. Notably, as glycosylation profiles of all investigated subclasses were observed to alter with increasing age, the EOC-related alterations were more pronounced in younger patients. Additionally, the detected subclass-specific glycosylation features indicate that each IgG subclass responds slightly differently to the outbreak of the disease. Above all, IgG1 showed the most pronounced EOC-related glycosylation alterations, and its agalactosylation showed the strongest association with CA 125 routine diagnostic marker. Additionally, as compared to IgG2 and IgG3, the combination of IgG1 glycosylation with the CA 125 marker showed the best performance in distinguishing healthy controls from EOC patients, irrespective of patients’ age and EOC stage. Furthermore, the results of this work imply that a common analytical practice of simultaneous analysis of IgG2 and IgG3 subclasses should be avoided due to their noticeably different glycosylation phenotypes. The aim of the second part of this work was to determine glycosylation alterations occurring directly on glycoproteins expressed in ovarian cancer cells and their surroundings. Analyses were performed using MALDI-MSI technique that enables the visualization of the spatial distribution of numerous analytes in a single imaging experiment. The investigated material consisted of formalin-fixed paraffin-embedded tissue specimens of the most common and aggressive EOC type, high-grade serous ovarian cancer, less aggressive low-grade serous, clear cell, and endometrioid ovarian cancer as well as non-malignant borderline ovarian tumor. All tissue specimens were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval. Negatively charged sialic acids were chemically derivatized in a linkage-specific manner, after which all tissue-contained N-glycans were released in situ with PNGase F. Ultimately, released N-glycans were measured using a rapifleX MALDI Tissuetyper. The applied protocol allowed the detection of over 60 m/z species corresponding to high-mannose-, hybrid-, and complex-type neutral and sialylated N-glycans. Importantly, observed N-glycans were diversely distributed within distinct tissue regions, defined by a pathologist prior to the analysis. High-mannose N-glycans were predominantly detected in tumor regions, while neutral complex-type N-glycans were more abundant in non-tumor tissue areas. Due to the mass shift introduced via a chemical derivatization reaction, the sialylated structures carrying α2,3- and α2,6-linked sialic acids could be distinguished. Notably, this is the first study, in which in situ sialylation of EOC tissue was investigated in its stabilized form and a linkage-specific manner. Importantly, the distribution of differently sialylated N-glycan structures within the tissue was observed to vary. While α2,6-sialylated N-glycans were enriched in tumor and tumor-related necrotic regions, α2,3-sialylated N-glycans were predominantly present in non-tumor tissue. Ultimately, statistical analyses revealed tissue type-specific N-glycan structures, which support EOC diagnosis and pave the way to serve as biomarkers to discriminate between cancerous and noncancerousovarian tissue.Weniger anzeigen
The Cassini spacecraft orbited Saturn from 2004 to 2017. During this period, Cassini’s onboard dust instrument – the Cosmic Dust Analyzer (CDA) – recorded numerous in situ time of flight mass spectra, generated from μm to sub-μm sized dust particles. Most of these spectra originate from ice particles sampled in Saturn’s large, diffuse E-ring. This ring is mainly sustained by material ejected from the cryo-volcanic plumes at the south polar terrain of Saturn’s icy moon Enceladus. Previous analyses of CDA spectra revealed the existence of three different compositional main types of ice particles: salt- and organic-poor (Type 1), salt-poor but enriched in organics (Type 2) and salt-rich (Type 3). A new compositional type was discovered during the course of this work, called Type 5. Its CDA spectra indicate a very high salt concentration; far above the level found for Type 3 particles of about 1 % by mass. So this work represents the first, large-scale investigation of the spatial distribution of these four types to study the compositional structure of the E-ring. For this purpose, more than 10000 CDA spectra of ice grains were analyzed for their composition and particle size with respect to the spacecraft position and trajectory.
The results of this investigation not only revealed distinct spatial correlations and trends but are also in good agreement with previously published E-ring results, obtained during close Enceladus flybys. Moreover, they show that, although the absolute number density of all E-ring grains drops when moving radially outwards from Enceladus or vertically away from Saturn’s equatorial ring plane, the relative frequencies of the compositional types vary significantly. Type 1 particles generally represent the majority of all ice particles in the E-ring and become relatively more frequent at larger radial and vertical distances. Type 2 particles exhibit the opposite trend with the relative frequency declining at larger distances. Thus, it is proposed that Type 2 particles are converted into secondary Type 1 particles, through photochemical degradation of the embedded organic molecules. For Type 3 particles the radial and vertical trends differ. Radially, the relative frequency first rises to a maximum and then decreases, while vertically it remains almost constant, with only a minor indication of a North-South asymmetry. The newly discovered particle type (Type 5) is most frequently detected in a relatively narrow region of the E-ring, between the orbital distances of Dione and Rhea, confirming existing theories about the dynamical evolution of E-ring grains. This argues against a direct Enceladian source, instead favoring a formation within the E-ring itself. Hence, it is proposed that Type 5 represents secondary particles, formed from Type 3 grains via plasma sputtering of the more volatile water component.
To understand the impact, that particle composition has on the determination of the particle size, particularly the organic substances in the ice particles, laboratory experiments were undertaken using a Laser-Induced Liquid Beam Ion Desorption (LILBID) approach. By irradiating a μm-sized liquid water beam with a pulsed, infrared laser, LILBID simulates the impact ionization process of ice grains in space. The experiments revealed a distinct difference in ion yields between salt-rich and organic-rich compositions. While the latter increases the ion yields just slightly, compared to pure water, the addition of salts increases them significantly. Considering these results, it is inferred that Type 2 particles are, on average, the largest particles. The average sizes of the other three types overlap within the errors, depending on the particular radial distance to Saturn. The overall shape of the radial size distribution seems to be intimately related to the particles’ salinities. The average sizes of the salt-poor particle types (Type 1 and 2) decrease linearly in the inner E-ring up to 10 RSaturn (Saturn radius RSaturn = 60268 km) from Saturn, then remain more or less constant. In comparison, salt-rich particles (Type 3 and 5) exhibit a similar initial decrease but reach their minima sooner, at around 8 RSaturn. These minima are followed by a distinct increase in the populations’ average grain sizes in the outer E-ring. Although it is currently unclear why the average grain sizes of salty ice grains increase again in the outer E-ring, the two different distribution shapes – those of the salt-poor types on one side and the salt-rich types on the other – again indicate that two different transformation processes, responsible for the transformation of Type 2 to Type 1 and Type 3 to Type 5, are occurring, as also inferred from the compositional data.Weniger anzeigen
Optimization of nanosuspensions produced by wet bead milling is a complex process involving many factors that affect milling efficiency. This thesis focused on the identification of key stabilizer properties that have a direct outcome in the formation of a stable nanosuspension. Furthermore, critical process parameters in the preparation of fixed dose combination (FDC) nanosuspensions were described. Finally, formulation parameters determining the long-term physical stability of FDC nanosuspensions were described. A comprehensive stabilizer screening was initially performed on the individual drugs. Loteprednol was milled in the presence of five surfactants at two concentrations. Higher concentration generally resulted in smaller particle sizes. Surfactant hydrophobicity (r = 0.68) and surface activity (r = 0.87) were observed to positively affect milling efficiency. For PEO PPO-PEP block polymers (Poloxamer) a sufficient PPO fraction was crucial to interact with the drug surface as indicated in decreasing contact angles at increasing milling efficiency. The best candidate for milling loteprednol was Poloxamer 407. Two factors could however be further optimized: milling to smaller particle sizes and a more monomodal particle size distribution. Poloxamer 407 milling efficiency was further tuned from a d90 of 550 to about 200 nm by introducing glycerol or NaCl to the milling media. Poloxamer 407 conformation as a function of osmotic agent concentration suggested a stretching of the polymer chains thus increasing the steric stabilization. Chain collapse due to high medium ionic strength was then also accompanied by a loss in milling efficiency. The success rate of high molecular weight polymers on the other hand was generally limited by insufficient wetting and increased media viscosity. In contrast to surfactants, milling with high molecular weight polymers left a significant µm fraction of about 50% of total volume, ending far below the milling efficiency of surfactants. Under accelerated storage at 40 °C/75%RH, surfactants however only insufficiently stabilized the loteprednol nanosuspensions, with a growth rate of at least 2.5 µm/month while particle size was completely maintained for non ionic polymers. It became apparent that a combination of 0.5% Poloxamer 407 and 0.125% HPMC E4M could significantly increase milling efficiency to a d90 of 200 nm and result in superior storage stability with a negligible growth rate of 0.005 µm/month. This was explained by a synergistic co-adsorption of both stabilizers.
Combination therapy with two drugs being classified as poorly soluble, could be formulated as an FDC nanosuspension. Several difficulties arise during formulation development of such products, especially regarding their production and characterization. This thesis presented the approach of selective dissolution, providing the in situ particle size of the individual compound within the FDC. The approach was based on the difference in drug solubilities, facilitating the in situ dissolution of nepafenac during the laser diffraction measurement and thus facilitating the particle size determination of loteprednol within the FDC. Two approaches were considered for the preparation of the FDC nanosuspensions, separate or combined milling of the two individual drugs. Using selective dissolution, precipitation of loteprednol was detected in the presence of the second drug nepafenac after dilution. This was attributed to critical changes in solubility after combining the two single nanosuspensions, making the approach of separate milling unfeasible for this FDC. It became apparent that milling nepafenac and loteprednol together increased milling efficiency of the more challenging compound loteprednol by 50% for surfactants and by 10 30% for non-ionic polymers compared to the single nanosuspension. The increase in loteprednol milling efficiency was attributed to an increase in total active milling volume due the presence of nepafenac. The same effect was seen in a combination of dexamethasone and nepafenac. Nepafenac reached nm-sizes very early in the process, which seemed an important factor as a simple increase in e.g. volume fraction did not alter loteprednol or dexamethasone milling efficiency. Drug brittleness was indicated as a key parameter. Brittleness of three drug powders was determined and brittleness was the highest for nepafenac, followed by loteprednol and dexamethasone. As particle size determines dissolution rate, dissolution studies were subsequently performed for the nepafenac-dexamethasone FDC and the beneficial effect of combined milling on the dissolution of dexamethasone and nepafenac FDC nanosuspensions could be shown. Nanosuspensions are thermodynamically unstable systems and tend to agglomerate or exhibit crystal growth upon storage. When preparing an FDC nanosuspension, both milling efficiency and storage stability were considered. Poloxamer 407 was the best candidate to obtain a loteprednol and nepafenac FDC nanosuspension with a d90 of 240 nm. It however failed to maintain the nanosuspension physically stable upon storage at 40 °C/75%RH. In contrast, high molecular weight polymers left a clear µm-fraction in the FDC nanosuspension after milling. Kinetic adsorption seemed to however be relevant over storage time, where minimum particle sizes with a d90 around 400 nm were reached within 2-month storage. This suggested, that the increased breakage rate during combined milling resulted in loteprednol breakage, with the fresh surfaces however not readily being covered with polymer. A relaxation of the system by either free polymer adsorption or deagglomeration due to repulsive effects explained the decrease in particle size over time. Aside stabilizer choice, other formulation parameters were investigated to increase the physical stability of the inherently unstable FDC nanosuspension. Increased viscosity and the introduction of a stabilizer combination both were successful in preventing physical instabilities and decreased crystal growth rate from 1.5 to 0.01 µm/month and below. Viscosity of the system was increased from 5 to 25 mPas which also was accompanied with a decrease in sedimentation velocity from 11 and 3 µm/s. Increasing the formulation’s viscosity however worsened redispersibility after 6-month storage. As crystals could not be maintained in a discrete suspended state, flocculation was induced by a stabilizer combination of 0.5% Poloxamer 407 and 0.125% HPMC E4M. Although flocs settled more rapidly than crystals at increased viscosity, flocculated crystals formed a lattice that resisted complete settling and thus was less prone to cake formation. The stabilizer combination was therefore the formulation approach with the highest milling efficiency and best characteristics upon storage for the FDC nanosuspension.Weniger anzeigen
Sowohl theoretisch als auch empirisch konnte bereits die Schlüsselrolle von Familien im Entwicklungsprozess ihrer Kinder herausgestellt werden. Aus diesem Grund wird in neueren Modellen frühpädagogischer Qualität die Zusammenarbeit mit Familien auch als eigenständige Qualitätskomponente angenommen (vgl. Kluczniok & Roßbach, 2014). Hierbei liegt die Annahme zugrunde, dass eine familienergänzende Kindertagesbetreuung sich besonders positiv auf die Entwicklung der Kinder auswirken kann, wenn Fachkräfte neben den Kindern auch deren Familien in ihr pädagogisches Handeln miteinbeziehen. Im Fachdiskurs werden für die Zusammenarbeitsthematik unterschiedliche Begrifflichkeiten genutzt, die auch mit einem veränderten Verständnis von der Zusammenarbeit zwischen Kita und Familie einhergehen (vgl. Stange, 2012). In dieser Arbeit wird in Anlehnung an Betz (2015) unter der Qualitätsdimension Zusammenarbeit mit Familien jegliche Form der organisierten Kommunikation und Kooperation zwischen frühpädagogischen Fachkräften und Eltern verstanden; folglich gelten frühpädagogische Fachkräfte als verantwortlich für die Gestaltung der Zusammenarbeit und es ist Bestandteil ihres professionellen Aufgabenrepertoires, eine qualitativ hochwertige Zusammenarbeit umzusetzen (vgl. Roth, 2014). Betrachtet man bisherige Forschung im Bereich der Zusammenarbeitsthematik so wird deutlich, dass oftmals nur Belege für die Bedeutung der Elternbeteiligung sowie einem partnerschaftlichen Verhältnis zwischen Kita und Familie geliefert wurden (z. B. Fantuzzo et al., 2013; Mautone et al., 2015; Serpell & Mashburn, 2012). Unbeantwortet bleibt meist, welche Voraussetzungen seitens der Kita für eine erfolgreiche Zusammenarbeit geschaffen werden müssen. Das vorliegende Dissertationsvorhaben beabsichtigt daher, innerhalb vier Teilstudien zu neuen Erkenntnissen hinsichtlich der Zusammenarbeit mit Familien als Qualitätsdimension beizutragen. Die erste Teilstudie untersuchte die Struktur und Beschaffenheit der Qualität der Fachkraft-Eltern-Kommunikation im Rahmen der morgendlichen Bringsituation sowie Qualitätsunterschiede nach strukturellen Rahmenbedingungen der Einrichtungen anhand eines neu entwickelten Beobachtungsinstruments. Die Ergebnisse konfirmatorischer Faktorenanalysen zeigten, dass sich die Qualität der Fachkraft-Eltern-Kommunikation in die Dimensionen Gesprächs- und Interaktionsbereitschaft, Kommunikationsstrategien und Verabschiedung differenzieren ließ und sich somit die Prinzipien der personenzentrierten Gesprächsführung auf den frühpädagogischen Kontext übertragen lassen. Einrichtungen mit einem höheren Anteil an akademisch ausgebildetem Personal, mit günstigeren Fachkraft-Kind-Verhältnissen sowie mit einer Organisation der pädagogischen Arbeit in Stammgruppen wiesen eine höhere Qualität der Fachkraft-Eltern-Kommunikation auf. Unterschiede hinsichtlich des Anteils an Kindern mit Migrationshintergrund sowie der Einrichtungsgröße konnten nicht festgestellt werden. Die zweite Teilstudie nutzte das Beobachtungsinstrument aus Teilstudie 1 und untersuchte, inwieweit die beobachtete Qualität der Fachkraft-Eltern-Kommunikation, aber auch fragebogenbasierte Qualitätsmerkmale, die seitens der Familien eingeschätzt wurden, mit dem Vertrauen von Eltern zusammenhingen. Entsprechend gängiger Vertrauenstheorien wurde zudem getestet, inwiefern das Vertrauen mit familiären Hintergrundmerkmalen assoziiert ist. Die Ergebnisse zeigten, dass Familien in Einrichtungen mit einer höheren beobachteten Qualität ein höheres Vertrauen in das Personal aufwiesen. Mit Blick auf die Qualitätseinschätzungen der Familien sowie deren Zufriedenheit mit der Kommunikation im Rahmen der Tür- und Angelgespräche ließen sich ebenfalls signifikant positive Zusammenhänge feststellen. Die Analysen zeigten des Weiteren, dass Eltern mit einer anderen Familiensprache als Deutsch und Eltern mit verhaltensauffälligen Kindern ein signifikant niedrigeres Vertrauen in die Kita ihres Kindes besaßen. Ein statistisch bedeutsamer Zusammenhang zwischen dem Bildungsniveau der Eltern und ihrem Vertrauen konnte nicht nachgewiesen werden. Teilstudie 3 fokussierte sich auf die Angebotsstruktur der Einrichtungen zur Zusammenarbeit mit Familien sowie deren Bedeutung für das Vertrauen von Eltern. Des Weiteren wurde untersucht, inwieweit strukturelle Voraussetzungen der Einrichtungen und professionelle Kompetenzen frühpädagogischer Fachkräfte die Angebotsform und -intensität vorhersagen. Die Ergebnisse zeigten, dass die Anzahl an Aktivitäten zur Elternbildung sowie zur Partizipations- und Entscheidungsfindung positiv mit dem Vertrauen von Eltern zusammenhingen. Die anderen beiden Angebotsformen zur sozialen Vernetzung der Eltern sowie zur Einbeziehung der Familienkulturen standen in keinem statistisch bedeutsamen Zusammenhang zum elterlichen Vertrauen. Ferner zeigte sich, dass Kitas, die von einem höheren prozentualen Anteil an Kindern mit Anspruch auf das Bildungs- und Teilhabepaket besucht wurden, in den letzten zwölf Monaten mehr Aktivitäten zur Elternbildung angeboten haben. Kitas mit einem höheren prozentualen Anteil an Kindern mit Migrationshintergrund boten mehr Aktivitäten zur Einbeziehung der Familienkulturen an. Die vertraglich vereinbarte Vorbereitungszeit der Fachkräfte hingegen stand in einem signifikant positiven Zusammenhang zur Intensität der Angebote zur Partizipation und Entscheidungsfindung. Der Fachkraft-Kind-Schlüssel stand zu keiner der vier Angebotsformen in einem statistisch bedeutsamen Zusammenhang. Im Hinblick auf die professionellen Kompetenzen der Fachkräfte zeigte sich, dass in Kitas, in denen das Personal über ein umfangreiches Wissen zum Handlungsfeld Zusammenarbeit mit Familien verfügte, mehr Aktivitäten zur sozialen Vernetzung und zur Elternbildung angeboten wurden. Darüber hinaus wurde ein signifikant positiver Zusammenhang zwischen den Überzeugungen der Fachkräfte und der Anzahl der Angebote für soziale Vernetzung gefunden. Vor dem Hintergrund steigender Anforderungen im Handlungsfeld Zusammenarbeit mit Familien untersuchte Teilstudie 4, inwieweit sich pädagogische Fachberatung als Qualifizierungsressource für Einrichtungen und ihre pädagogischen Fachkräfte eignet. Hierzu wurde das fachdidaktische Wissen von Fachberatungen als bedeutsame Kompetenzfacette näher beleuchtet. Die Befunde zeigten, dass Fachberatungen zwar insgesamt viele Methoden zur Zusammenarbeit mit Familien kennen, jedoch deutliche Unterschiede zwischen den einzelnen Fachberatungen existieren. Im Rahmen einer Clusteranalyse konnten die folgenden vier Wissenstypen von Fachberatungen generiert werden: Novizen, Generalisten, Spezialisten und Experten. Fast ein Drittel der befragten Fachberatungen lässt sich dem Cluster Novizen zuordnen. Diese Fachberatungen nennen im Durchschnitt nicht einmal eine Methode. Einzelne Zusammenhänge zu den fachlichen Voraussetzungen der Fachberatungen können als Indiz für eine Vermittlung von Methodenwissen in Ausbildung, Praxis sowie Fort- und Weiterbildung gewertet werden. Insbesondere die Fort- und Weiterbildungsaktivität von Fachberatungen scheint ein geeignetes Instrument zur Qualifizierung von Fachberatungen im Bereich Zusammenarbeit mit Familien zu sein. Insgesamt lassen sich aus den Ergebnissen wichtige Erkenntnisse (1) über die Struktur der Qualität der Fachkraft-Eltern-Kommunikation sowie (2) über deren Beschaffenheit ziehen. Die Ergebnisse verdeutlichen ferner (2) die Bedeutsamkeit der Qualität der Fachkraft-Eltern-Kommunikation und der Angebotsform und -intensität für das Vertrauen von Eltern und zeigen, dass sowohl strukturelle Rahmenbedingungen der Einrichtungen als auch Kompetenzfacetten der frühpädagogischen Fachkräfte bedeutsame Voraussetzungen für die Qualität in der Zusammenarbeit darstellen. Die Bedeutung weiterer Professionalisierungsbestrebungen wird unterstrichen. Implikationen für die Forschung sowie die pädagogische Praxis werden diskutiert.Weniger anzeigen
Die pulmonalarterielle Hypertonie ist eine seltene, zum Tode führende Erkrankung, welche durch eine endotheliale Dysfunktion, pulmonale Vasokonstriktion, pulmonalvaskuläre Hyperreagibilität und pulmonalarterielles Remodeling gekennzeichnet ist. Das Endothelinsystem spielt in der Pathogenese der PAH eine zentrale Rolle. Die Ziele dieser Arbeit lagen zum einen in der Untersuchung des Endothelin-Systems in der PAH-assoziierten pulmonalen TH2-Inflammation und zum anderen in der Untersuchung altersabhängiger Endothelin-1-vermittelter Veränderungen von Herz und pulmonaler Zirkulation in der Maus. Die Induktion der pulmonalen TH2-Inflammation erfolgte mittels systemischer Ovalbumin-Sensibilisierung und nachfolgender Ovalbumin-Atemwegsexposition (OVA/OVA). Mit Hilfe transgener Mäuse konnten unter anderem im ex vivo Modell der isoliert perfundierten und ventilierten Mauslunge die Effekte der ETB-Defizienz (ETB-/-) und der humanen Prepro-Endothelin-1-Überexpression (ETtg) auf pulmonalarteriellen Mitteldruck und pulmonalvaskuläre (Hyper-) Reagibilität untersucht werden. Zudem erfolgten vergleichende histologische Analysen in Bezug auf pulmonale Kollagendeposition und perivaskuläre Inflammation. Mittels Fulton-Index erfolgte die Untersuchung rechtsventrikulärer Hypertrophie. Zytokin-Analysen aus der bronchoalveolären Lavage (BAL) und mRNS-Analysen aus dem Lungenhomogenat erlaubten eine genauere Charakterisierung der Inflammation und der Regulationsmechanismen in den einzelnen untersuchten Gruppen. ETB-defiziente Mäuse wiesen einen pulmonalen Hypertonus inklusive pulmonalvaskulärer Hyperreagibilität und rechtsventrikulärer Hypertrophie auf. Infolge der pulmonalen TH2-Inflammation aggravierten sowohl die pulmonalvaskuläre Hyperreagibilität als auch die rechtsventrikuläre Hypertrophie. Die perivaskulären Leukozyteninfiltrate, die pulmonale Kollagendeposition, die pulmonale Endothelin-1-Expression und die IL-12p40-Spiegel der BAL zeigten sich in den OVA/OVA-behandelten ETB-/--Mäusen im Vergleich zu den WT-Tieren deutlich verstärkt. Zudem konnte in den ETB-/--Mäusen eine deutliche Zunahme der Endothelin-1-vermittelten pulmonalvaskulären Thromboxan-Freisetzung nachgewiesen werden. Während die Prepro-Endothelin-1-Überexpression in den jungen ETtg-Tieren mit einer pulmonalvaskulären Hyperreagibilität einherging, kam es in den stark gealterten ETtg-Mäusen zu einem fixierten pulmonalen Hypertonus inklusive rechtsventrikulärer Hypertrophie. Zusammenfassend führte die ETB-Defizienz infolge der pulmonalen TH2-Inflammation zu einem vermehrten pulmonalen Leukozyteninflux mit deutlich erhöhten IL-12p40-Spiegeln und zu einer verstärkten Expression von Endothelin-1. Diese Veränderungen begünstigten möglicherweise die nach OVA/OVA-Behandlung detektierte Zunahme der rechtsventrikulären Hypertrophie und pulmonalvaskulären Hyperreagibilität, an der auch eine vermehrte pulmonalvaskuläre Thromboxanfreisetzung beteiligt zu sein scheint. Die hier erhobenen Daten unterstützen die Hypothese der protektiven Rolle des ETB-Rezeptors im pulmonalen Gefäßsystem. Zudem zeigen sie, dass Endothelin-1 als alleiniger Stimulus ausreicht, PAH-assoziierte Veränderungen an Herz und pulmonaler Zirkulation zu induzieren. Um zu klären, ob in der Behandlung der pulmonalarteriellen Hypertonie eine separate ETA-Inhibierung der dualen Endothelinrezeptor-Blockade überlegen sein könnte, sind jedoch weiterführende Analysen, inklusive separater und simultaner ETA- und/ oder ETB-Inhibierung, erforderlich.Weniger anzeigen
An abdominal aortic aneurysm (AAA) is defined as a permanent local dilatation of the abdominal aorta, usually accompanied by thrombus formation. Once ruptured, an AAA is associated with an overall mortality rate of over 90%. Even though AAAs represent one of the leading causes of sudden death in developed countries, the exact etiology and pathophysiology have not yet been fully elucidated. Nowadays, AAAs are clinically diagnosed by either computed tomography, ultrasound or magnetic resonance imaging (MRI), yet those modalities only deliver information on the aneurysm anatomy, size and form. It is generally accepted that AAA size correlates with the probability for rupture and related clinical events. However, a growing body of literature argues that the pathophysiology of AAA expansion is more multifaceted, with inflammation and degradation of the extracellular matrix (ECM) playing a pivotal role. Visualizing the complex nature of aneurysms requires the development of novel imaging modalities that facilitate more detailed depiction of AAAs. Magnetic resonance elastography (MRE) is an imaging technology that combines lowfrequency vibrations and MRI to create stiffness maps of body tissues. Remodeling of the ECM during AAA progression leads to stiffness changes, providing a potential imaging marker. Therefore, we assessed murine aneurysms by ex vivo microscopic multifrequency magnetic resonance elastography (μMMRE). By examining the aneurysmal thrombus, we discovered that regional variations in stiffness were strongly correlated to local histology-quantified ECM accumulation. With this proof-of-concept study, we demonstrated that MRE represents a suitable method for detecting shear wave speed changes reflected in varying stiffness values in the aneurysmal thrombus that in turn are representative of ECM remodeling. With successful clinical translation, this imaging modality could help detect potential fatal changes in the biomechanical structure of the AAA mimicked by ECM changes. Magnetic particle imaging (MPI) is an innovative imaging modality, enabling a highly sensitive detection of magnetic nanoparticles (MNPs). Since MNPs are a suitable surrogate marker for molecular targeting of macrophages and the aneurysmal development involves inflammation as a fundamental process, we tested the feasibility of imaging AAA inflammation with MPI. We demonstrated that the MNP accumulation in ex vivo murine aneurysms can be visualized and quantified with MPI. In addition, the colocalization of macrophages and MNPs was visible in histology. The ability to detect the spatial distribution and local concentration of MNPs establishes MPI as a promising tool for monitoring inflammatory progression in AAAs.Weniger anzeigen
The latest events of establishing Social Credit Systems (SCSs) in China have received much attention, not only locally, but also on the international level. A lot has been written about the SCSs, putting the focus on the construction of big data platforms, surveillance control mechanisms and the reputational system etc. However, its social impact and its functions in the dating market in today’s China is an understudied topic. Its operation of selecting people by means of ‘value’ predicts major changes in the realm of the social fabric, such as the mate selection process of Chinese women. Today, women are still torn between traditional family patterns and their own choice concerning mate selection. From now on, women might have to take a third component into account, namely the integrity of their future life partner in terms of the SCSs. Using qualitative data of surveys and interviews, this dissertation investigates the influences of the SCS pilots in regard to the mate selection process of urban Chinese women to find out to what extent the SCSs have an impact on the partner selection in China.Weniger anzeigen
Located in the western part of the Cuvelai Basin, the Iishana system is a transboundary region covering parts of southern Angola and northern Namibia. Hydrologically, this region is characterized by a network of episodically water-bearing channels in which numerous pans are embedded. These pans, which fill up during the rainy season, form an important water resource for the rural population, especially for agricultural and domestic use. The Iishana system is one of the most densely populated areas in southwestern Africa, and this high population trend is increasing (NamStat 2013). To date, the majority of the population (80–90%) currently lives in rural areas. However, (small) cities are experiencing steady growth. The semi-arid climate in this area has distinct rainy and dry seasons and is characterized by high interannual variability, resulting both in intense droughts and in strong flood events. As a result, water is sometimes a scarce resource in this region. The strong population growth and the temperature increase predicted as a result of global climate change will put further pressure on available water resources. However, as this region is also subject to volatile rainfall dynamics, in addition to droughts, the Iishana system also experiences repeated, severe flood events. Most recently, flood events occurred in 2008 to 2011, 2013, and 2017, resulting in the loss of life, the loss of crop yields and consequent loss of livelihood for many people, and the destruction of key infrastructure elements. To date, there has been no complex 2D-hydrodynamic model for the Iishana system and no transferable modeling approach to identify potential locations for water storage and facilitate the planning and development of flood retention measures. In this study, various methods have been developed and applied to address these issues. This has allowed for the validation of existing findings as well as the discovery of new insights, which are briefly summarized below. First, an investigation was performed to test the influence of topography on hydrology, with a special emphasis on infrastructure elements. The focus here was on improving the raw DEM for subsequent calculations. For this purpose, filter corrections were performed on the TanDEM X raw data, and road dams, culverts, and bridges were recorded by means of kinematic surveys. As a result, the definition of the flow paths was improved. It became clear that northern roads, especially those running from east to west, have a strong influence on the runoff behavior in the study area due to their height and their orientation orthogonal to the water flow of the Iishana. Based on the corrected DEM and the application of a modified Blue Spot Analysis, further new findings emerged. Approximately 190,000 pans with a total storage volume of about 1.9 km³ and a total area of 4,021 km² were identified. The part of the study area located in Angola accounts for two thirds of the potential storage volume while only one third of the storage volume is in Namibia. Furthermore, about one third of all pans are located in the episodically water-bearing channels. Based on previous results in other regions, a calculation of the surface-volume relationship (SA/V rate) was performed for the first time for the Iishana system. This enabled the identification of about 2,000 pans that are primarily suitable for an expansion of storage volume. Using continuous and spatio-temporally varying TRMM precipitation data, a 2D-hydrodynamic modeling and reconstruction of the 2008/2009 flood event was performed using the FloodArea model. Although the results represent a snapshot, they nevertheless contribute to an improved understanding of the interconnected runoff system and highlight potential flood hazards. Depending on the weighting of evapotranspiration in the calculation of the model, the potential storage volume can be quantified between 0.116 km³ and 0.547 km³. The total inundation area was calculated at 1.860 km². In addition, three main runoff paths were identified, of which the central and the eastern runoff paths pose a particular threat to the regional capital of Oshakati. Furthermore, with the help of the model, for the first time it was possible to identify areas where, after the end of the rainy season, water availability is naturally shortest (Namibia) or longest (Angola). Based on these numerous, new results, scenario calculations for neighboring catchments as well as calculations for other precipitation periods can be performed in the future. Thus, the duration of water availability after the end of a rainy season can be determined and possible locations for retention measures can be identified for various locations.Weniger anzeigen
Das primäre Ziel dieser Arbeit war es, die molekularbiologischen Ursachen des nicht-allergischen Angioödems zu untersuchen. Es ist bekannt, dass das nicht-allergische Angioödem durch einen erhöhten Bradykininspiegel verursacht wird, jedoch ist der genaue Signalweg noch unklar. Leider gibt es nur wenige Studien über Veränderungen der Bradykinin B2-Rezeptor-Expression im kardiovaskulären System. Über die Wirkung von Bradykinin auf die eNOS-Expression wurde bereits berichtet, aber bis zum Beginn meiner Arbeit hat keine Studie über die Wirkung von eNOS auf die B2-Rezeptorexpression berichtet. In dieser Arbeit wurde der Einfluss des NO/cGMP-Signalweges auf Bradykinin B2-Rezeptoren untersucht. Eine Langzeit-Wirkung des NO/cGMP-Signalwegs auf die B2-Expression wurde in vivo in genetisch veränderten Mäusen untersucht. Der Mangel an eNOS-abgeleitetem NO im kardiovaskulären System wurde in eNOS-defizienten Mäusen untersucht, die Auswirkungen der erhöhten Aktivität der vaskulären eNOS wurden in Mäusen mit endothelspezifischer Überexpression von eNOS untersucht. Die Ergebnisse zeigten keine signifikanten Effekte von NO auf die Bradykinin B2-Rezeptorexpression. Um die akuten Effekte der Abwesenheit oder Aktivierung des NO/cGMP-Signalweges zu untersuchen, wurden Wildtyp (WT) Mäuse mit NOS-Inhibitoren oder NO-Donatoren entsprechend behandelt. Für In-vitro-Studien wurden porcine Aortenendothelzellen (PAEC) und porcine aortale glatte Muskelzellen mit NO-Donatoren oder PAEC mit NOS-Inhibitor behandelt. Auch in dieser Untersuchung wurden keine signifikanten Veränderungen der Bradykinin B2-Rezeptorexpression festgestellt. Bradykinin-inkubierte Endothelzellen zeigten ebenfalls keine signifikante Veränderung der Bradykinin B2-Expression. Zusätzlich wurde die Wirkung von Fibrinogen auf Bradykinin B2-Rezeptoren untersucht und es wurden keine signifikanten Veränderungen festgestellt. Diese Ergebnisse zeigten eine überraschende Stabilität der Bradykinin B2-Rezeptoren und keinen Hinweis auf die molekularbiologischen Ursachen, die das nicht-allergische Angioödem auslösen. Funktionstests des Bradykinin B2-Rezeptors in eNOS-defizienten und Wildtyp-Mäusen zeigten, dass Bradykinin eine Vasokonstriktion in der Aorta der eNOS-defizienten und Wildtyp-Maus induziert. Die Hemmung der Prostaglandinbildung zeigte, dass im Wildtyp diese Vasokonstriktion durch gleichzeitige Bildung von NO abgeschwächt wurde. Diese NO-abhängigen Veränderungen der Aortenverengung von Mäusen legen nahe, dass NO zwar wichtig für die B2-Rezeptorfunktion ist, aber keinen Einfluss auf die B2-Rezeptor Proteinexpression hat. Diese Mechanismen könnten Relevanz für die Bradykinin-vermittelten molekularbiologischen Ursachen des nicht-allergischen Angioödems haben.Weniger anzeigen
Embryonic development and organogenesis depend on the precise spatiotemporal expression of specific sets of genes. Precisely controlled gene expression ensures cell state transitions, especially in the early stages of development, as gastrulation. These complex multi-layered cellular processes are orchestrated by the interfacing of the epigenome, 3-dimensional (3D) nuclear organization, cis-regulatory elements (CREs) with transcription factors (TF), and long non-coding RNAs (lncRNAs). In the gastrulating embryo, definitive endoderm is specified from the pluripotent epiblast following a series of regulatory events, including the activation of SOX17, a key TF of that particular germ layer. Although SOX17 has been extensively studied in early embryonic development, the precise control of its activation, the locus, and the epigenetic rules governing its genetic regulatory network (GRN) remains poorly investigated. In my thesis, I in-depth characterized the human SOX17 locus, exploring the relevance and regulatory impact of 3D nuclear organization, its distal CREs, and their activity. I applied a series of loss of function (LOF) and transgenic experiments to dissect the locus at a satisfactory resolution. In particular, I showed SOX17 among a subset of developmental regulators topologically isolated within CTCF-CTCF loop domains and highlighted the importance of gene control in 3D within this type of domain. I pinpointed the relevance of SOX17’s distal CREs and their definitive endoderm-specific interaction and showed this interaction to be highly dependent on CTCF-CTCF loop-formation to guarantee proper gene control. I found CRE-dependent SOX17 gene deregulation associated with poor definitive endoderm differentiation outcome and a stalled “mesendodermal-like” phenotype. Assessing the genetic identity of different CREs, I divulged the presence of a novel lncRNA within the locus, namely LNCSOX17. I fully characterized LNCSOX17 and established its identity as a bona fide lncRNA through a series of genetic perturbations. I demonstrated the importance of LNCSOX17 for forming definitive endoderm and the lack of participation in SOX17 cis-acting gene control. I associated the loss of LNCSOX17 RNA but not its active transcription at the locus with an aberrant endodermal transcriptome, a lack of epithelial-to-mesenchymal transition (EMT), and the hyperactivity of the detrimental definitive endoderm JNK/JUN/AP1 signaling pathway. I found definitive endoderm lacking LNCSOX17 to be functionally impeded in the generation of pancreatic progenitor populations. The studies within this thesis serve as valuable examples to support the functional relevance of 3D nuclear organization and its importance for developmental gene control in cis via CTCF-CTCF loop domain-mediated CRE-promoter contact facilitation. They associate developmental gene expression levels with various phenotypes, identify a so far unknown developmental lncRNA molecule, and imply its relevance for the formation of definitive endoderm. The outlined results advance our knowledge of developmental TF gene-control and its importance for the development of human definitive endoderm.Weniger anzeigen
For efficacious adoptive T cell therapy (ATT), the appropriate selection of tumor-specific antigens (TSAs) is crucial. Recurrent somatic driver mutations yield ideal TSAs for ATT to achieve complete tumor elimination, possibly without relapse. In silico algorithms predict epitopes as TSAs for ATT, however, with low probability. Thus, the accuracy of prediction algorithms still needs validation in describing processed neoepitopes. Simultaneous validation of proteasomal processing of predicted TSAs and identification of neoantigen-specific T cell receptors (TCRs) is possible from transgenic mice. One such model developed by Li et al. in 2010 was the ABabDII mice having the complete human TCR gene loci, knocked out for murine TCR and MHC class I gene loci, and it is transgenic for human HLA class I allele, HLA-A*02:01 (HLA-A2). Though isolation of human TCRs is possible from ABabDII mice, the model is limited as a source of obtaining only HLA-A2-restricted TCRs, thereby restricting epitope identification to HLA-A2 antigens. In this doctoral study, a novel mouse model with six human HLA alleles as a single haplotype, named ABab.I, was developed on ABabDII background to diversify HLA genes in the existing ABabDII mice for epitope discovery and broaden TCR repertoire for non-HLA-A2 restricted TCR isolation. In silico screening for putative neoepitopes predicted to bind high-ranked HLA alleles selected the six alleles in ABab.I mice; novel class I haplotype like in humans as every individual bears six HLA class I alleles. Initially, the in silico screening predicted 23 and 152 high-affinity TSAs (IC50 < 50 nM) from recurrent point mutations (n=266) that bound HLA-A2, and other frequent class I alleles (n=18), respectively. In the first phase of this doctoral thesis, epitope immunogenicity was tested in 4 out of 23 HLA- A2-binders in ABabDII mice. Only 1 out of 4 immunized epitopes elicited CD8+ T cell (CTL) responses. However, this was confirmed to be not endogenously processed when tested with TCRs raised from ABabDII mice. This form of reverse immunology is laborious and still a concern with open questions. A previous study on identifying epitopes from the vaccinia virus by Assarsson et al. in 2007 aligns with the in silico screen predicted data examined in this thesis. Thus, prediction algorithms offer a low probability to select immunogenic epitopes. In the second phase, the ABab.I mouse model with a novel set of chimeric class I fusion alleles, HLA-A*03:01, A*11:01, B*07:02, B*15:01, C*04:01, and C*07:02, was developed using PiggyBac transposon strategy. The introduction of six HLA alleles as a single genotype resembling a natural human situation broadened the TCR repertoire four-fold by enriching the peripheral pool with five times more unique and rarer V(D)J-TCRß clonotypes than ABabDII mice. Ultimately, ABab.I mice would serve as a versatile in vivo model system for epitope discovery, besides being a valuable tool to identify novel TCRs against high HLA-affinity tumor-specific antigens (IC50 < 50 nM) derived from recurrent somatic point mutations.Weniger anzeigen
This dissertation comprises four empirical chapters which contribute to the fields of labor economics, inequality research, and health economics.
The first chapter studies the relationship between the spatial distribution of labor market inspections and non-compliance with Germany’s Minimum Wage Law. By combining novel administrative data on labor market inspections with the German Socio-economic Panel (SOEP), we document that the inspection probability is higher in regions with higher non-compliance. This implies a risk-based allocation of inspection efforts and, hence, their endogeneity. Using fixed-effects and an instrumental variable approach, we show that higher inspection efforts have a limited effect on compliance. Based on a theoretical framework and international evidence, we discuss challenges for law enforcement, the political importance of compliance, and potential improvement measures.
The second chapter focuses on inequality in monthly earnings in Germany and the role of desired and actual working hours. We document a significant rise in monthly earnings inequality between 1993 and 2018. The main contributors are inter-temporal increases in working hours inequality and increases in the covariance between working hours and hourly wages, while changes in the distribution of hourly wages play a minor role. We develop a novel double decomposition technique which reveals that these results are particularly pronounced in the growing groups of female employees and service sector employees. If employees had been able to realize their desired optimal working hours, the increase in inequality would have been more moderate. This is mainly because employees with low hourly wages work less than desired, a finding that is reinforced over time—even after controlling for various covariates.
The third chapter investigates the labor market effects of transitory and persistent health shocks. Using machine learning based on sick days and hospitalizations, we derive two novel health shock indicators: one for transitory and one for persistent shocks. By using these new indicators, we overcome issues in the measurement of health, such as heterogeneity and measurement error. In an event study framework, we analyze the respective effects of either shock type on employment, yearly working hours, and labor earnings, but also partner earnings and household net income. Persistent shocks induce large negative employment effects that end up impacting household net incomes. In contrast, transitory shocks induce only minor employment effects that leave household net incomes unaffected. We also investigate effect heterogeneity and find that individuals over 50 years of age are particularly affected by health shocks. Accordingly, persistent health shocks reduce employment of individuals above 50 by 25 percentage points.
The fourth chapter analyzes the effect of occupational routine task intensity on workers’ mental and physical health in the context of technological progress and automation driving the deroutinization of job tasks. By combining individual-level health information of German employees with data on occupational task profiles and applying an instrumental variable strategy, I find that male and female workers are oppositely affected by occupational routine task intensity. For women, routine tasks are more likely cognitive routine tasks that negatively affect mental health. For men, routine tasks are more likely manual routine tasks that negatively affect physical health, but have a positive effect on mental health. When considering the overall workforce, the effects on mental health balance out, but a significant negative effect of routine task intensity on physical health remains.Weniger anzeigen
Panksepp (1998) was the first researcher to propose a theory on the evolutionary development of emotions that accounted for the role of language. In his theory, Panksepp assumed that emotions arise in subcortical areas of the brain, are linked to learning experiences in the limbic system, and interact with higher cognitive functions including language, in the cortical areas. Thus, these three levels should play a role when looking at the affective component of words here called affective semantics. The representation of affective semantics can be examined by testing explicit valence decisions, that is, whether a word is evaluated as positive or negative. Furthermore, the relationship of language and emotions can also implicitly be tested, for example, when participants make lexical decisions on stimuli that are systematically manipulated valence. Recently, the explicit and implicit modes of valence processing have been extensively examined in adults. Consequently, the corresponding behavioral and neuronal correlates of valence are already well-established. However, it remains unclear how these explicit and implicit influences develop during childhood on both a behavioral and neural level. This dissertation seeks to address this open issue and attempts to provide answers regarding how children compared to adults process the valence of words. Three empirical studies were conducted to approach the central question of whether children between the ages of six and twelve show the same behavioral phenomena and neuronal correlates of valence processing as adults.
As a basis for the studies, a word database was designed to specifically target the vocabulary of children called the Berlin Affective Word List for Children. The words were selected based on the Berlin Affective Word List, which is a German word database for adults. The adult version of the affective word list was used as a template in order to ensure comparability between child and adult ratings. Study I (Sylvester et al., 2016) examines the question of whether children between the ages of six and twelve evaluate words in the same way as adults when they rate words in terms of their valence, arousal, and imageability. The results point to similar affective semantic representations in children and adults, including a U-shaped function relating valence and arousal ratings and an inverse U-shaped function relating valence and reaction times encompassing the positivity superiority effect (Lüdtke & Jacobs, 2015), indicating that positive words lead to shortest reaction times.
Subsequently, the neuronal correlates of explicit valence processing were examined in Study II (Sylvester et al., 2021a). According to the framework of Panksepp (1998; 2005a; 2005b), neural activation was expected in the affective semantic network which includes the amygdala, striatum, thalamus, insula, orbitofrontal, supplementary and cingulate cortex, inferior and middle frontal, superior and middle temporal gyrus. In order to test the affective semantic network, children between the ages of six and nine and adults between ages 19 and 30 performed a valence decision task. Based on the behavioral results of Study I, similar rating behavior was observed in children and adults. Furthermore, similar neural activation patterns were expected across age groups.
The neural response patterns of the children and adults of Study II are very similar to the results previously reported in adults. Specifically, we observed extensive activation within the presumed affective semantic neural network over all three valence categories (positive, negative, and neutral). However, there were differences regarding the regions predominantly recruited for valence decisions, for example there was an increased activation in amygdala in children. Moreover, activation beyond the assumed affective semantic network was observed, for example, in occipito-parietal regions, especially among adults. The different results between children and adults suggest that differences in the size of the mental lexicon between children and adults and/or the experiences from which affective semantics are derived may lead to different neural activation. It is suggested that children primarily recruit information from regions primarily associated with affective processing, whereas adults recruit regions devoted to the integration of information from various resources related to affective semantics, which includes regions in the supplementary motor cortex, middle temporal and precentral gyrus. Thus, adults seem to rely their valence decisions more strongly on multimodal affective semantic hubs since they already have integrated affective semantic information, whereas children tend to recruit valence-related information directly from the ‘source’.
In Study III (Sylvester et al., 2021b), the implicit influences of valence were assessed by using lexical decisions. The question arose as to whether the similarities found for the explicit valence decisions in Study II could also be found for implicit ones. Consequently, a similar neural pattern with activations in the large-scale affective semantic network was assumed. Similar to the results in Study II, both age cohorts showed greatly overlapping activation for processing (positive, negative and neutral) words. However, when looking more precisely at differences between the valence categories, children and adults show different processing streams. Although the adults showed similar neuronal activation as adults in previous studies, i.e., activation in orbitofrontal and anterior cingulate cortex activation (e.g., Kuchinke et al., 2005), a different picture emerged in children. The children mainly showed activation in regions associated with lexico-semantic processing, i.e., supramarginal and superior parietal gyrus activation (e.g., Price, 2012). In contrast, we observed less activation in regions directly linked to valence processing. However, positive words compare more favorably between children and adults, possibly due to that positive words are learned earlier in life (Ponari et al., 2018). These results are consistent with the interpretation of Study II in that children primarily display task-specific activation, meaning the recruitment of primarily valence-specific region during explicit valence decisions occurs whereas valence plays a subordinate role during lexical decisions. These results indicate that less connected affective semantic information is processed in children than in adults. In contrast, the adults demonstrated greater activation of valence-related areas, suggesting that valence is an important implicit source of information in lexical decisions, facilitating the decision process.
In summary, the three empirical studies of the current work are among the first to show that children and adults have similar affective semantic representations. It is, however, noteworthy that some aspects of affective semantic processing are still not adult-like in children up to the age of twelve, specifically they show more task-specific activation. Furthermore, the differences in neural activation in the affective semantic network, especially the IFG (Hofmann & Jacobs, 2014) indicate deviations between the mental lexica in children and adults. As such, the neural results in the present dissertation are interpreted in the light of differences between the children’s and adults’ mental lexicons, resulting from children’s limited experience with experiential and distributional data and/or their smaller vocabulary.
Based on the central results of this dissertation, a developmental hypothesis for affective semantics is proposed. The developmental hypothesis assumes that the amount of the apperceptive mass (Kintsch, 1980), as defined here as the size of the vocabulary and the amount of available experienced and distributed data, moderates the neural processing of affective semantics. The results could contribute to the developmental computational and cognitive models to model the influence of vocabulary on and for learning affective semantics.Weniger anzeigen
Zebrafish is an excellent model to study cardiac regeneration. This freshwater teleost fish can fully regenerate its heart after myocardial infarction (MI) within one to two months after the injury. Cryoinjury method is widely used to induce the injury in the zebrafish heart since it mimics the closest human MI. Current protocols used in adult zebrafish, including cryoinjury method, do not involve any analgesia treatment and they do not take into consideration the adverse effects that can arise out of these techniques. Nociception is a basic response of the animal to harmful external or internal stimuli, which is essential for survival and adaptability to the environment. Fish can react to noxious stimuli, similarly to the higher vertebrates, which should be considered in the design of scientific experiments. In this study, I showed that cryoinjury is a noxious stimulus for zebrafish by assessing zebrafish behaviour Moreover, I showed that morphine treatment for 6h after the cryoinjury procedure, but not lidocaine, significantly improved zebrafish behaviour using swimming speed as a readout. Importantly, the morphine treatment for 6h did not impair regeneration based on histological analysis and did not affect regenerative capacities based on the transcriptome analysis using scRNA-seq and qPCR. We advise using morphine as the analgesic of choice to reduce adverse effects associated with the cryoinjury procedure in adult zebrafish. The refinement of the current cryoinjury protocol allowed us to investigate cellular drivers of zebrafish heart repair with consideration of zebrafish welfare with the improved protocol. Several pro-regenerative factors and signalling pathways are known to be crucial for zebrafish heart regeneration, however, the cellular composition of the zebrafish regenerating heart and cell types involved in the process are still incompletely understood. To systematically investigate the diversity of activated cell states in the zebrafish regenerating heart, we used single cell transcriptomics and spatiotemporal analysis. We showed that upon the injury there is a significant induction of several cell states with fibroblasts characteristics and we confirmed the pro-regenerative role of transient col12a1a-expressing fibroblasts. To determine the origin of transient cell states, we performed Cre/lox lineage tracing, in order to understand the events leading to heart regeneration. We reported that transient fibroblasts can originate both from epicardium and endocardium, similarly to the development process. We also identified Wnt signalling as a key regulator of endocardial-derived fibroblasts, which was at least partially mediated by the impaired revascularization during regeneration. In this study, we identified the great diversity of specialized activated fibroblasts as one of the factors crucial for zebrafish heart regeneration, which gives us new possibilities towards improving mammalian heart repair.Weniger anzeigen