Involvement of TRPV3 mutants in Olmsted syndrome is well-documented, yet the precise mechanisms underlying their pathogenicity remain elusive, particularly concerning trafficking and functionality within the cell membrane with controversial hypotheses in literature. Furthermore, and despite reports suggesting a role in NH4+ uptake in ruminant species, the permeability of the TRPV3 channel to this ion remained uncertain. This study aims to elucidate the NH4+ permeability of human and bovine TRPV3 channels using Xenopus laevis oocytes and HEK-293 cells, with a primary focus on the human channel. Immunoblotting and immunostaining confirm the expression and trafficking of TRPV3 channels in both models as well as in native tissue. Notably, human skin equivalents and bovine rumen both showed distinct staining of the apical membrane of the top layer of keratinocytes with weaker cytosolic staining in the middle layers, suggesting potential roles in epithelial function. Functional assays, including single-channel patch clamp experiments and whole-cell studies with agonists, demonstrate the channels' responsiveness and permeability to NH4+ showing larger conductances in TRPV3 overexpressing cells. Furthermore, pH-sensitive microelectrodes were used to measure acidification by NH4+ in Xenopus laevis oocytes showing endogenous channels capable of NH4+ transport while also demonstrating significantly greater effects in TRPV3 expressing cells. These observations challenge conventional dogmas regarding membrane transport primarily via NH3 diffusion. Expression of the TRPV3 mutant G573S in HEK-293 cells elucidates its impact on cell viability, with significant cell death observed and partial rescue via treatment with the established blocker ruthenium red. Crucially, immunofluorescence validated cytosolic expression, with membrane staining detected in a minority of cells. These observations argue for the hypothesis of a functional expression in the membrane where the gain-of-function mutation triggers the cascade ending in increased cell death. Our study highlights the versatility of TRPV3 channels not only in Ca2+ signaling as extensively described in the literature, but also more generally as a cation transporter including for NH4+, with implications for various biological processes including skin physiology, ion homeostasis, pH regulation, and nitrogen utilization. These insights contribute to our evolving understanding of the biological role of TRPV3 and hold potential for informing future research and clinical interventions.
Weniger anzeigenEsta tesis doctoral realiza un estudio histórico de los sistemas de identificación de delincuentes en Colombia desde la mitad del siglo XIX hasta la década de 1920. En concreto se centra en el proceso de circulación global y apropiación local de la antropometría criminal o judicial, conocida como bertillonage, al interior de los cuerpos de vigilancia del país, específicamente de la Policía Nacional, como parte de su agenda reformista de principios del siglo XX. Periodo en el que este saber se posicionó como una herramienta para el mantenimiento del orden social y, por lo tanto, como una condición para el funcionamiento de los cuerpos policiales dada su metodológica capacidad de individualización de reincidentes. Observaremos cómo la capacidad de lectura del cuerpo humano de este saber persiguió dos utópicas finalidades, identificar a las amenazas sociales y prevenir el crimen consolidando un entramado documental que permitió el incremento de la memoria documental del Estado, y que ató al individuo a sus acciones.
Weniger anzeigenCells are faced with coordinating countless, simultaneous, partly antagonistic biochemical reactions. This is especially true for neurons that must orchestrate the complex task of neurotransmission. One solution to this problem is the formation of specialized compartments. To understand the molecular mechanisms of such compartments this thesis investigates two system in neuronal cells: i. the plasma membrane and its underlying cytoskeleton and ii. synaptic vesicle clusters inside synaptic boutons. Towards this end, a combinatorial approach of computational modeling, single particle tracking and super-resolution microscopy is employed. A periodic array of actin rings in the neuronal axon initial segment has been known to confine membrane protein motion. Still, a local enrichment of ion channels offers an alternative explanation. Using computational modeling this thesis now shows that ion channels, in contrast to actin rings, cannot mediate confinement. Furthermore, by employing single particle tracking and super-resolution microscopy, this work shows that actin rings are close to the plasma membrane and that actin rings confine membrane proteins in several neuronal cell types. Further, it is shown that actin ring disruption leads to a reduction of membrane compartmentalization. Synaptic boutons in the axon of neurons are the location of synaptic vesicle release. Synaptic vesicles form dense clusters inside boutons, that are essential for pre-synaptic function. In vitro experiments have suggested that the soluble phosphoprotein synapsin 1 controls synaptic clustering via liquid liquid phase separation. However, the in vivo mechanism remains elusive. This work now shows via two-color single molecule tracking in live neurons that synapsin 1 drives synaptic vesicle clustering. Furthermore, using a synapsin knock-out model it is shown that synapsin 1 controls the mobility of synaptic vesicles through its intrinsically disordered region, which is responsible for phase separation. By studying the dynamics of compartmentalized systems in neuronal cells this work uncovers two molecular mechanisms: actin rings form membrane diffusion barriers and synapsin 1 controls synaptic vesicle clustering and mobility through liquid liquid phase separation. Thus, this thesis makes important strides towards deepening the understanding of neuronal function by uncovering how compartmentalization operates in both the plasma membrane and the cytosol of neuronal cells.
Weniger anzeigenHeterogeneous-Agent New Keynesian models (HANK)---which replace the representative household by a whole distribution of households---are the about to become the new state-off-the-art model of modern monetary economics because they allow for more realistic consumption patterns of households and a rich description of income and wealth distributions. This PhD thesis contributes to the ongoing advancements in the HANK literature in three ways. First, it employs empirically realistic, medium-sized state-of-the-art HANK models to analyze the macroeconomic and distributional impact of contemporary policy issues such as climate change. This thesis shows that the macroeconomic impact of higher carbon prices is similar than those of carbon shocks: they increase inflation and decreases economic activity. Nevertheless, higher carbon prices can be welfare-beneficial if the government pays back the increased revenue from higher carbon prices as lump-sum transfers to households. Second, it deepens our understanding of the interplay between (systematic) monetary and fiscal policy within this framework. It shows that, compared to independent monetary policies, a monetary union does not redistribute the impact of business cycle shocks vertically, that is, between households of different wealth and income brackets. It does, however, redistribute horizontally across countries, that is, between households within the same wealth and income brackets. For the euro area, this redistribution is strongest at the tails of the distribution. In addition, it shows that by using distortionary taxes and adjusting the government debt level, fiscal policy can be a perfect substitute for monetary policy when the latter is constrained by the effective lower bound, an exchange rate peg, or a monetary union. Third, this thesis connects HANK models with the fast-growing field of behavioral macroeconomics by integrating empirically supported behavioral elements. It develops a HANK model in which households’ expectations underreact to aggregate news as is robustly found in survey data. This behavioral HANK model then matches four recent important empirical findings in the monetary economics literature that previously could not be reconciled within one model. It also shows that incorporating them all into a single model matters greatly for the business cycle implications of monetary policy as, for example, after supply shocks, inflation increases much more strongly. In addition, this thesis shows that allowing for empirically documented heterogeneity in cognitive skills and overconfidence thereon enhances the empirical fit of HANK models to match the wealth distribution and the distribution of marginal propensity to consume. Key to this result is that overconfident households underestimate their precautionary savings motive, thereby oftentimes ending up hand-to-mouth, as they also do in the data.
Weniger anzeigenCommon in vitro cell culture systems are widely used to understand physiological processes and disease development. However, these have a number of limitations such as the lack of the physiological 3D tissue architecture but also regulatory mechanisms, such as the circadian rhythm. For that reason, in vitro methods need to be improved to mimic as closely as possible the in vivo situation found in humans, and the regulatory framework of toxicological relevant pathways should be studied to better understand physiological processes. Even if the circadian rhythm has been linked to various important physiological processes, the molecular mechanism of these circadian regulations remains unravelled. The circadian rhythm is characterized by internal oscillations of physiological processes with a recurring periodicity of approximately 24 h and its synchronization depends mainly on the light-dark periods of the day. On the molecular level, the circadian rhythm is driven by the recurring expression of CLOCK genes, which regulate up to 48% of all human genes and ensures the proper daytime-depended activity. The uniform circadian rhythm of all cells in a tissue is often lost in common 2D in vitro cell culture systems but can be easily restored through artificial synchronization of these cells. Subsequently, such synchronized cell culture systems lead to enhanced quantitative and qualitative cellular response with a higher human relevance. A highly toxicological relevant pathway, which might be under circadian control is the AhR (Aryl hydrocarbon Receptor) signaling pathway. In particular, in synchronized human breast cells the cellular response upon AhR-ligand binding shows a circadian pattern indicating a circadian regulation of the AhR pathway. This study describes for the first time important aspects of the mechanism behind the circadian regulation of the AhR signaling pathway. The circadian expression of AhR target genes, e.g. CYP1A1, is caused by a circadian activation of the CYP1A1 promoter but not a circadian expression of AhR itself. Upon ligand exposure, e.g. TCDD (2,3,7,8-Tetrachlordibenzodioxin), AhR translocates into the nucleus and binds not only with ARNT but also with the circadian rhythm regulator BMAL1. These interactions seem to be circadian phase-dependent, indicating a possible competition in AhR binding. Additionally, the AhR co-factor, p23, negatively modulated the circadian regulation of the AhR signaling and SP1 was identified as a novel AhR co-factor exhibiting a circadian protein expression, which possibly sustains the proper circadian regulation of the AhR pathway. Besides that, preliminary data suggest that GSK3β could be involved in the circadian regulation of the AhR pathway. Summarizing, all the findings of this thesis propose a possible mechanism for the circadian regulation of the AhR pathway and this work contributes to a better understanding of the circadian regulation of an important toxicological pathway, that might lead to the development of improved cell culture-based in vitro systems for toxicological and risk assessment strategies as well as for drug development with a higher human relevance.
Weniger anzeigenDehydration of oceanic lithosphere in subduction zones is a crucial geodynamic factor on Earth and is linked to various geological phenomena. Efficient recycling of fluids liberated at depth back to the surface is essential to maintain the deep volatile cycle in balance over geological timescales. Rock dehydration itself is a multiscale process involving multiple physical processes, each acting on different time and length scales.
Natural examples of exhumed meta-serpentinites comprise a channelized network of olivine-rich veins that formed during dehydration and have served as a pathway for fluid escape at depth during dehydration. These vein networks can provide information about the processes that led to their formation and hence about the fluid release mechanisms. Previous studies have shown the importance of chemical heterogeneities for microscale channelization, and numerical models can successfully reproduce slab-scale fluid flow focusing that is observed using geophysical methods. However, the strikingly high content of metamorphic olivine and the intrinsic rock properties that lead to channelization going towards the slab scale, are not yet well understood.
This dissertation systematically studies rock dehydration as a multi-scale process. By combining a dataset of fully hydrated serpentinite with a numerical model, we aim to reproduce and explain the formation of a channelized, olivine-rich vein network observed in nature. The model thus connects the microscale mineral dehydration reactions to the macroscale fluid escape from the slab into the mantle.
The first scientific chapter presents a multiscale dataset of a serpentinite from the Mirdita ophiolite in Albania. The dataset includes geological, chemical, and structural data from the sub-micrometer up to the outcrop scale. Chemical heterogeneities are demonstrated to be scale invariant over at least four spatial orders of magnitudes. Thermodynamic modeling along the subduction zone P-T path shows porosity to be spatially heterogeneous on each scale during the P-T window of a dehydration reaction.
As a first step towards a dynamic model, the third chapter presents a numerical model for reactive fluid flow in an existing vein network. The model investigates the effects of chemical heterogeneities on the composition of the released fluid. A fluid released in a low-silica environment has a low content of dissolved silica. Fluid flow from a low-silica to a relatively higher-silica environment can trigger the dehydration reaction of antigorite to olivine, even at constant temperature. The model shows that local metasomatism can produce veins of almost pure olivine in a matrix that still contains abundant antigorite.
In the fourth chapter, the reactive fluid flow model is formulated in GENERIC (General Equation for Non-Equilibrium Reversible-Irreversible Coupling) to mathematically analyze the underlying system of equations. This is the first application of GENERIC to a geoscience problem. The analysis shows that the system is well-posed and confirms the existence of a general solution for the system of equations.
The outlook presents preliminary results of a reactive porosity wave model, which is a logical and promising next step in combining the findings of the previous chapters into a single numerical model. Porosity waves are a mechanical fluid flow focusing mechanism and thus one possible mechanism for slab-scale channelized fluid fluxes. The model couples mechanical porosity waves with the effects of silica transport in the fluid and thermal effects. It serves as a large-scale model for fluid escape from the slab. Based on the findings of scale-invariant occurrence of chemical heterogeneities, it uses chemical mappings from the multiscale dataset as initial conditions and as a proxy for kilometer-scale chemical heterogeneities. This is the first time that a reactive porosity wave model has been combined with data from natural rock samples.
Weniger anzeigenMarija Pavlović's dissertation, Cold War Kids in Neoliberal Dystopia: Transgression, Disruption, and Fragmentation in the Work of Chuck Palahniuk and Victor Pelevin, examines the evolution of transgression, disruption, and fragmentation in literature from postmodernism to contemporary forms. Introducing hypertrashrealism, she articulates this new literary movement as both a response to and a development beyond postmodern tendencies. The study significantly draws on Ihab Hassan's theory of postmodernism, providing a comparative framework that underscores key shifts in narrative and thematic approaches in the works of Palahniuk and Pelevin. This analysis emphasizes the critical transformation in literary styles and themes, reflecting contemporary societal and cultural dynamics, aiming to define a contemporary narrative alternative to the exhausted term "postmodernism" and the cumbersome "post-postmodernism."
Weniger anzeigenDank des medizinischen Fortschritts ist die Lebenserwartung in den letzten Jahrzehnten kontinuierlich gestiegen. Chronische Erkrankungen vor allem des kardiovaskulären Systems schmälern jedoch diesen Erfolg, da durch sie die Jahre in Gesundheit nicht in gleichem Maße zugenommen haben. Die Folge ist eine alternde Gesellschaft mit hoher Krankheitslast und zunehmendem Pflegebedarf. Die Stärkung endogener regenerativer Ressourcen könnte eine anhaltende Resistenz gegenüber pathogenen Stimuli bis ins hohe Alter ermöglichen, so dass der Anteil gesunder Lebensjahre zunimmt und zu mehr Lebensqualität bei geringerer Belastung des Gesundheits- und Pflegesektors führt. Die hier dargestellten Arbeiten zeigen, wie eine Modulation des mTOR-Signalnetzwerkes protektive Mechanismen auf zellulärer Ebene aktivieren kann, die vor pathologischen vorzeitigen Alterungsprozessen und altersassoziierten Erkrankungen schützen können. Insbesondere der Hemmung von mTORC1 bei gleichzeitiger Aktivierung von mTORC2 kommt hierbei eine herausragende Bedeutung zu: In humanen MSC, die als Vorläuferzellen für glatte Gefäßmuskelzellen ein relevantes Beispiel für den vaskulären Regenerationsapparat darstellen, wurde durch dieses Aktivierungsmuster des mTOR-Netzwerkes Autophagie gefördert und in der Folge zelluläre Seneszenz und Apoptose verhindert. Insgesamt resultierte diese Aktivierung protektiver Zellschicksale in einer Reduktion osteoblastärer Differenzierung und einem Schutz vor Kalzifizierung. Hieraus lässt sich das Potenzial ableiten, über eine entsprechende Beeinflussung des mTOR-Netzwerkes die endogene Regenerationsfähigkeit des Gefäßsystems durch MSC als vaskulären Progenitorzellen aufrechtzuerhalten. Außerdem stabilisierte der mTOR-Modulator Rapamycin in MSC einen kontraktilen glattmuskulären Phänotyp. Als weiteres Beispiel diente ein Nierentransplantationsmodell der Ratte. Hier zeigte Rapamycin in der unmittelbaren Posttransplantationsphase eine deutliche Reduktion zellulärer Seneszenz in multiplen Organkompartimenten ohne negative Auswirkungen auf die vaskuläre Integrität und die Rückbildung des Konservierungs-Reperfusionsschadens. Hierdurch kann die Regenerationsfähigkeit der transplantierten Niere auch gegenüber künftigen schädigenden Einflüssen erhalten bleiben, so dass sie besser vor interstitieller Fibrose und Tubulusatrophie geschützt ist. Eine gezielte Beeinflussung des mTOR-Netzwerkes durch pharmakologische und nicht-pharmakologische Maßnahmen könnte über eine Aktivierung protektiver Zellschicksale das endogene Regenerationspotenzial körpereigener Vorläuferzellen erhalten und so vor vorzeitiger Alterung in Folge pathogener Stimuli und vor degenerativen Schäden im Alter schützen. Dies könnte eine Verlängerung der Gesundheitserwartung ermöglichen und so die Bedarfssteigerung finanzieller und humaner Ressourcen trotz des demographischen Wandels hin zu einer immer älteren Gesellschaft dämpfen.
Weniger anzeigenThis dissertation comprises four independent chapters contributing to the literature on the Economics of Education and Labor Economics. They shed light on the determinants of high school graduates' wage expectations (Chapter 1) and how these expectations, along with the labor market conditions at graduation (Chapter 2), program rankings (Chapter 3), and the direct costs of studying (Chapter 4), affect postsecondary human capital investment. A comprehensive introduction and conclusion precede and close the individual contributions.
Chapter 1 uses detailed data from a unique survey of high school graduates in Germany, and documents a gender gap in expected full-time earnings of more than 15%. We decompose this early gender gap and find that especially differences in coefficients help explain different expectations. In particular, the effects of having time for family as a career motive and being a first-generation college student are associated with large penalties in female wage expectations exclusively. This is especially true for higher-expected career paths. Resulting expected returns to education are associated with college enrollment of women and could thus entrench subsequent gaps in realized earnings.
Chapter 2 asks if countercyclical college-going, a stylized fact in economics, holds with a dual education system as an outside option. This study uses German register data to show that reduced local labor demand at high school graduation decreases college enrollment and attainment. More students choose vocationally oriented colleges and apprenticeships instead of traditional universities. Conditional on economic preferences, these effects are reversed for high SES graduates. Survey data further reveals that low-achieving young men expect higher relative returns to vocational education during recessions. The findings suggest that investment in firm-specific human capital is perceived as an insurance mechanism.
Chapter 3 asks how public information on the within-field-of-study quality distribution of universities affects college choice. We combine an ordinal tier ranking published by the German newspaper "Die Zeit" and register data on higher education students. Differences-in-differences estimates show that being ranked in the top tier increases the average distance traveled by freshmen within a program by over 7%. The results are larger in dynamic specifications and robust to controlling for the local rent level. Rankings based on recommendations by faculty are overall less effective. We discuss how information provision on program quality can affect educational mismatch and implications for inequality.
Chapter 4 analyzes how tuition fees affect not only university enrollment but also completion rates. Following a landmark court ruling in 2005, more than half of Germany's universities started charging tuition fees, which were later abolished in a staggered manner. We exploit the fact that even students who were already enrolled had to start paying fees. We show that fees increase study effort and degree completion among these students. However, fees also decrease first-time university enrollment among high school graduates. Combining this enrollment impact with the effect on completion, we find that fees around the zero-price margin have only little effect on overall educational attainment. We conclude by discussing policies targeting the separate effect margins of fees and caution against a general abolition.
Weniger anzeigenThe prevalence of extended-spectrum beta-lactamase (ESBL)- and AmpC beta-lactamaseproducing Escherichia (E.) coli in the broiler production chain is high. The finding of these resistant bacteria in high numbers in barns and in the environment of broiler farms raises concerns about the use of potentially contaminated chicken manure as fertilizer on fields. As it is assumed that ESBL-/AmpC-producing E. coli can enter the human food chain by contaminated water, vegetables, and fruit after land application of chicken manure, better insights into the survival and the reduction kinetics of these bacteria during storage and chicken manure treatments are needed. Thus, the present work aimed to determine the reduction kinetics of ESBL-/AmpC-producing E. coli during composting and anaerobic digestion with a special focus on potential inactivation factors, above all temperature, carbon/nitrogen (C/N) ratio, and moisture content (MC). To that aim, three studies were carried out.
In the first study, laboratory-scale anaerobic digestion tests were conducted at 37, 42 and 55 °C to investigate the influence of temperature on the reduction kinetics of ESBL-/AmpC-producing E. coli during anaerobic digestion of chicken manure. One ESBL- (CTX-M15) and one plasmid-mediated AmpC- (CMY-2) producing E. coli strain were added to the substrate to achieve an initial bacterial count of 7 log10 colony forming units (cfu) per mL. Both E. coli strains were below the detection limit of <100 cfu/mL after 35 days, with decimal reduction times (Dvalues) of 3-6 days at 37 °C, 1.5 days at 42 °C and 48 min at 55 °C. However, at 37 and 42 °C, ESBL-/AmpC-producing E. coli remained partially detectable by enrichment until the end of the trials after 35 days. Hence, the results showed a strong temperature dependency, whereas no direct correlation could be observed between pH, volatile fatty acids (VFAs) or ammonia (NH3) and E. coli reduction.
The second study aimed to determine the effects of the C/N ratio and MC on the survival of ESBL-producing E. coli during laboratory-scale chicken manure composting in bioreactors. To that aim, a ESBL- (CTX-M15) producing E. coli strain was added to nine compost mixtures with different combinations of MC (20, 40, 60%) and C/N ratios (10:1, 20:1, 40:1) to obtain an initial bacterial count of 7 log10 cfu/g. The fastest decrease in E. coli was observed in all mixtures with a C/N ratio of 10:1 with D-values of 0.27 days to 0.60 days. Additionally, in dry mixtures with an MC of 20% and a C/N ratio of either 10:1 or 40:1, ESBL-producing E. coli were reduced faster than in the moist mixtures despite lower maximum temperatures within the bioreactors. The highest D-value of 4.82 days was determined for a mixture with a C/N ratio of 40:1 and 40% MC. While temperature was the main inactivation factor, in dry mixtures, desiccation also played a significant role. Significant effects of the C/N ratio and MC on the number of ESBL-producing E. coli as well as on temperature development were shown. In addition, it was demonstrated that even suboptimal composting mixtures – typical for chicken manure – led to a rapid reduction in ESBL-producing E. coli.
To validate the results of the laboratory composting trials in a more practical environment, in a third study, pilot-scale composting of chicken manure was performed at the test grounds in Potsdam. Here, the influences of three different management treatments for chicken manure, composting in (i) static piles, (ii) static piles covered with a compost fleece, and (iii) periodically turned piles, were determined with respect to the survival of an artificially added, nonresistant E. coli strain (DSM 1116) during different environmental conditions (summer and winter). The results showed an even faster reduction in E. coli and higher temperatures within the compost mass than in the laboratory-scale composting trials. Within 24 hours, E. coli were no longer detectable by direct count in any piles in either center or subsurface locations. By day 28, E. coli were also no longer qualitatively detectable in any samples. Within these 24 hours, temperatures exceeded 50 °C in all piles, which was most likely responsible for the fast inactivation of E. coli. The statistical analysis revealed the significant influences of sample location (center, subsurface) and the total hours at temperatures ≥ 50 °C and ≥ 55 °C in the piles on the survival of E. coli in the chicken manure compost. Although no influences of season or manure treatment method were shown, periodical turning is recommended, especially in winter, to increase the likelihood of exposure of all parts of the compost, including possible ESBL-/AmpC-producing E. coli, to high temperatures.
Taken together, the results of the three studies showed the effectiveness of both composting and anaerobic digestion in terms of ESBL-/AmpC-producing E. coli reduction. The reasons for the reduction in ESBL-/AmpC-producing-E. coli were multifactorial, with temperature being the main inactivation factor. Most likely due to the chicken manure characteristics of a low MC and a low C/N ratio, even in stored, nonmanaged or only minimally managed chicken manure, ESBL-producing-E. coli levels were reduced below the detection limit after one day. Therefore, concluding the results of this work, the risk of spreading ESBL-/AmpC-producing E. coli to land by using chicken manure, chicken manure compost, or digestate from biogas plants running on chicken manure is considered low. Composting of chicken manure with periodic turning or anaerobic digestion at thermophilic temperatures are the safest methods in terms of ESBL-/AmpC-producing E. coli reduction to treat chicken manure before application to land and are therefore highly recommended.
Weniger anzeigenThe infiltration of T cells into the tumor tissue is in many cases insufficient to prevent tumor progression. In addition, tumor-infiltrating lymphocytes (TILs) in the tumor are chronically exposed to high levels of tumor antigen, which can lead to their elimination. Therefore, it is unclear how well T cells with therapeutically effective T cell receptors (TCRs) are represented in progressively growing tumors. For the successful treatment of cancer by adoptive transfer of TCR-engineered T cells (TCR- Ts), it is crucial to use TCRs of sufficiently high affinity. To determine whether the quality of tumor antigen-specific TCRs from TILs is generally inferior to that of T cells generated from antigen-negative donors, I used TCRs recognizing either of two well- characterized MHC-I-presented neoantigens. First, I analyzed a panel of TCRs directed against a neoantigen from the mutated gene cyclin-dependent kinase 4 (CDK4). These TCRs were derived from TILs, healthy donors, or from mice with human TCR gene loci. On the other hand, I isolated TCRs specific for a neoantigen derived from the mutated gene p68 (mp68) from tumor-bearing and tumor-free, immunized mice. To induce the expansion of mp68-specific T cell clones in tumor-bearing mice, I developed a cancer model in which antigen expression could be induced 3 weeks after tumor cell transplantation, thus preventing priming of mp68-specific T cells during transplantation-associated inflammation. I transferred all TCRs into donor T cells to evaluate their ability to secrete cytokines after co-culture with target cells as well as their potential to kill tumor cells in vitro. To assess the therapeutic efficacy of the TCRs, I performed adoptive T cell transfer on tumor-bearing mice. I found that the in vitro experiments that best predicted in vivo tumor control were long-term cytotoxicity assays. Furthermore, I observed that both, TIL- and immunization-derived TCRs were of variable quality which underscores the importance of testing TCRs experimentally before therapy. Most importantly, I could show that the majority of TIL-derived TCRs were therapeutically effective, and that their quality was not inferior to that of healthy donor-derived TCRs. Therefore, my results support the use of TIL-derived TCRs for adoptive transfer of TCR-Ts directed against neoantigens.
Weniger anzeigenPreclinical research using animal models is still imperative in biomedical sciences to address the unmet clinical needs in many areas such as fracture healing disorders. General ethical and legal requirements must be carefully considered to provide scientific quality and integrity in animal-based studies. Sufficient reporting, adequate pain assessment and adapted pain management in animal-based research are crucial to generate reliable, reproducible, and comparable data, in line with the humane use of animals in research.
However, ongoing discussions in the scientific community and various systematic literature analyses have indicated that the overall reporting quality in many areas of animal-based biomedical research is still inadequate. Therefore, the first aim of this thesis was to evaluate the status quo of the reporting accuracy of different general and model-specific Parameters and the respective analgesia regimes in studies using adequately stabilized mouse femoral fracture models. High reporting accuracies were identified for the used fixation methods and fracturing procedures as well as the used strain, age, and sex of the mice, while insufficient reporting was found regarding the substrain and genetic background of the mice, body weight, hygiene monitoring, anesthesia, and analgesia. The systematic review also showed marked differences in the route, timepoint and duration of application as well as analgesic dosages, further indicating a need for more evidence-based data on model-adapted pain Management protocols.
The adequate pain assessment and an adapted stress-reduced pain management in laboratory rodents are ongoing challenges, yet essential to improve animal welfare and increase scientific validity and reproducibility. To reduce handling-associated stress while covering the period of greatest pain, the application of analgesics via the drinking water or the injection of sustained-release preparations can be used as an addition or an alternative to repeated injections. However, no sustained-release preparation for pain management in mice is currently available on the European market. The introduction of such a sustained-release buprenorphine would, therefore, be of great benefit for extended pain relief in a variety of different mouse models. Thus, the second part of the thesis focused on the evaluation of the analgesic efficacy of a newly developed depot formulation of buprenorphine (BUP-Depot) for prolonged post-operative pain relief in two mouse osteotomy models. Following a single injection, the analyses of various general and model-specific parameters yielded promising pain-relieving properties of the BUP-Depot for up to 72h post-surgical in female and male mice in both models. The availability of such a sustained-release formulation of buprenorphine would greatly help to further broaden the field of analgesic options in Europe and could have the potential to further refine analgesia in animal-based studies in Europe.
In the third part of the thesis, rearing behavior was evaluated to serve as an additional and meaningful model-specific indicator for pain in the first 72h after femoral osteotomy in mice. The analyzed data indicated significantly reduced rearing behavior after osteotomy, independent of sex or fixation, while anesthesia and analgesia alone did not impact rearing behavior. However, there was no clear evidence that the analysis of rearing behavior could serve as a meaningful sole indicator for residual model-specific skeletal pain in the analyzed models. Thus, further studies are needed to evaluate the value and predictive capacity of rearing behavior to assess pain in skeletal-impaired mouse models.
Overall, this thesis demonstrates that the accuracy of reporting in mouse fracture models needs to be further improved and that guidelines such as the ARRIVE guidelines should be more actively addressed in future studies. Furthermore, the data demonstrated safe use and efficient analgesia of a newly developed sustained-release buprenorphine for 72h after femoral osteotomy and underline the need and relevance of the availability of sustained-release analgesics for refined analgesia in laboratory mice in Europe. Lastly, this thesis indicates an ongoing need for further in-depth analysis and evaluation of easy-to-use and reliable indicators for pain in laboratory mice to ensure animal welfare.
Weniger anzeigenDas Interesse an der Verwendung von Nutzhanf (legal anbaubarer Hanf mit maximal 0,3 % Tetrahydrocannabinol-Gehalt) wächst und in den letzten Jahren wurden viele neuartige Hanfprodukte auf den Markt gebracht – darunter auch Tierfutter. Nutzhanf enthält Cannabinoide. Einige dieser Stoffe wirken beim Menschen und beim Tier psychoaktiv (z. B. Δ9-Tetrahydrocannabinol, Δ9-THC) oder haben anderweitige pharmakologische Wirkungen (z. B. Cannabidiol, CBD). Unklar war bislang, in welchem Umfang diese Cannabinoide aus nutzhanfhaltigem Futter in Produkte tierischen Ursprungs übergehen und ob dies ein Risiko für den Verbraucher darstellt.
In der vorliegenden Studie „Transfer von Δ9-Tetrahydrocannabinol (Δ9-THC) und anderen Cannabinoiden aus nutzhanfhaltigen Futtermitteln in die Milch von Kühen“ werden die Ergebnisse eines Fütterungsversuchs mit Nutzhanfsilage bei zehn laktierenden Milchkühen vorgestellt. In der Futterration der Tiere wurde zunächst für sieben Tage ein Teil der Maissilage durch Nutzhanfsilage aus der gesamten Hanfpflanze (GP-Silage, sehr niedrige Cannabinoid-Konzentration) ersetzt. Anschließend erfolgte für sechs Tage der teilweise Ersatz der Maissilage durch Nutzhanfsilage, die nur aus Blättern, Blütenständen und Samen der Pflanze bestand (BBS-Silage, höhere Cannabinoid-Konzentration). Die Verfütterung erfolgte in zwei Dosierungen (Gruppe 1: niedriger Nutzhanfgehalt; Gruppe 2: hoher Nutzhanfgehalt). Im Laufe des Versuchs wurden Milch- und Blutproben gewonnen und die Vitalparameter sowie das Verhalten der Kühe erfasst. Die Analytik erfolgte mittels Flüssigchromatographie-Tandem-Massenspektrometrie-basierter Methode (HPLC-MS/MS), welche die Unterscheidung zwischen psychoaktivem Δ9-THC und seiner nicht-psychoaktiven Vorstufe Δ9-Tetrahydrocannabinolsäure (Δ9-THCA) ermöglichte. Für Δ9-THC und CBD wurden Transferraten von 0,24 ± 0,04 % bzw. 0,15 ± 0,05 % aus dem Futter in die Milch berechnet. In verschiedenen Expositionsszenarien, welche mittels EFSA-RACE-Tool berechnet wurden, überschritten mehrere Verbrauchergruppen bei Verzehr von Milch und Milchprodukten die akute Referenzdosis (ARfD) für Δ9-THC, wenn Nutzhanfsilage zur Fütterung der Milchkühe verwendet wurde. Besonders ausgeprägt war die Überschreitung bei Kindern bei Verwendung der BBS-Silage. Im Hinblick auf die Tiergesundheit führte die Verfütterung der cannabinoidarmen GP-Silage zu keinen signifikanten Effekten bei den Kühen, wogegen die Verfütterung der cannabinoidreichen BBS-Silage zur signifikanten Reduktion der Herz- und Atemfrequenz, gestörter Bewegungskoordination, geröteten Augen, Nasensekretion, Hypersalivation und ausgeprägtem Zungenspiel führte.
Weniger anzeigen1 Summary 1.1 Overview of Presented Topics In this cumulative dissertation the following topics are presented: 1) Interfacial modulation of MoS2 with oxo-functionalized graphene and its derivatives a. Oxo-functionalized graphene/MoS2 and reduced oxo-functionalized graphene/MoS2 heterostructures b. Porous oxo-functionalized graphene/MoS2 2) Interfacial modulation of laser-induced functional graphene/MoS2 heterostructures Oligophenyl-functionalized graphene/MoS2 The research was conducted in collaboration with the groups of 1) Dr. Patryk Kusch from the Department of Physics at Freie Universität Berlin; 2) Prof. Dr. Hyeon S. Shin from the Department of Chemistry at Ulsan National Institute of Science and Technology. The complete results and experimental details are included in the attached publications in Chapter 5 and 6. To keep the explanation of heterostructures concise, we define the vertical stacking order of the heterostructure layers as (substrate/)bottom layer/upper layer. 1.2 Summary of Results 1.2.1 Interfacial modulation of MoS2 with oxo-functionalized graphene and its derivatives a. Oxo-functionalized graphene/MoS2 and reduced oxo-functionalized graphene/MoS2 heterostructures Tuning the electronic and optical properties of monolayer MoS2 and gaining profound insights into the fundamental mechanisms that govern these properties is of utmost significance for the development of efficient optoelectronic devices, such as photodetectors, photodiodes etc. Intrinsic structural defects of monolayer MoS2, such as S vacancies, induce electrons gathering in neighboring Mo atoms, which function as nonradiative traps, thereby impairing the photoluminescence (PL) efficiency. Stacking MoS2 layers on different two-dimensional (2D) materials, such as hexagonal boron nitride (h-BN) and graphene, provides a way to modulate the PL performance. Oxygen-functionalized graphene (oxo-G), a graphene derivative, has a defective graphene network with oxygen species decorating the edges and the plane. Electron-withdrawing groups, such as hydroxyl, epoxy, and organosulfates, make oxo-G a p-doping material. By reducing oxo-G (r-oxo-G), sp2-hybridized graphene domains with a lateral size of up to 10 nm are recovered with the removal of most oxygen groups. During the reduction process, in-plane defects such as vacancies, holes, and non-six-membered carbon rings with sp3 hybridization are formed, which act as structural motifs or active sites and significantly change the electronic and surface properties of roxo-G. Therefore, the use of oxo-G and r-oxo-G is suggested for tuning the carrier concentration of MoS2. Herein, heterostructures of monolayer MoS2 with three types of monolayer graphene are fabricated: mechanically exfoliated pristine graphene, oxo-G (a high amount of oxygen of 60%), and r-oxo-G (a defect density of 0.5%). Raman and PL spectroscopy combined with Kelvin probe force microscopy (KPFM, collaboration with the Shin group, Ulsan National Institute of Science and Technology) measurements are carried out to study optoelectronic properties and mechanism of interface interaction. Oxo-G with a work function (WF) of 5.67 eV and r-oxo-G with a WF of 5.85 eV serving as hole injection layers significantly enhance the PL intensity of MoS2, whereas pristine graphene with a WF of 5.02 eV resulted in PL quenching of MoS2. The electron-withdrawing functional groups of oxo-G and the defects in r-oxo-G layers facilitate the recombination of neutral exciton and result in PL enhancement. Furthermore, the r-oxo-G/MoS2 heterostructure exhibits a higher increase (5-fold) in the overall PL intensity than the oxo-G/MoS2 (3-fold) heterostructure. Our research demonstrates the PL modulation of monolayer MoS2 by monolayer graphene with a varying ability in extracting electrons. The enhancement of PL plays a vital role in high performance optoelectronic devices by improving photovoltaic efficiency, sensitivity, and photoresponse etc. b. Porous oxo-functionalized graphene/MoS2 The in-plane lattice defects in r-oxo-G can affect the electron transfer between graphene and MoS2, leading to an enhancement in the PL of MoS2. To investigate interfacial charge transfer and PL performance, porous graphene with large lattice defects is prepared and stacked with MoS2. Oxo-G with a low density of initial vacancy defects (0.8%) is used as a precursor to etch pores assisted by a Mn-species at 400 °C in Ar atmosphere. By controlling the reaction conditions, it is possible to gain a certain control over the size of pores on porous oxo-G (Pr-oxo-G) with diameters between 100–200 nm. The PL of MoS2 on SiO2, oxo-G, Pr-oxo-G6h (etching time of 6 h), and Pr-oxoG12h (etching time of 12 h) are studied. The amplitudes of the PL are increased for oxo-G/MoS2 (4 times), Pr-oxo-G6h/MoS2 (3 times), and Pr-oxo-G12h/MoS2 (10 times), compared to the PL of the pristine MoS2 monolayer. Overall, Pr-oxoG12h reflects a p-doped material, as indicated by Raman shifts, achieving the highest PL enhancement. The Pr-oxoG12h/MoS2 PL intensity map measured by scanning nearfield optical microscopy (s-SNOM) with nano-scale resolution shows a constant PL intensity over the MoS2 flake, exhibiting no sign of a spatial PL modulation that may arise from free-standing MoS2. (collaboration with Dr. Patryk Kusch, FU Berlin) Furthermore, the Pr-oxo-G6h/MoS2 showed slightly lower PL intensity than oxo-G/MoS2. The Mn-impurities in Pr-oxo-G6h/MoS2 are supposed to limit the increase of the PL of MoS2, and the interaction of Mnspecies with carbonyl groups may be responsible. 1.2.2 Interfacial modulation of laser-induced functional graphene/MoS2 heterostructures Oligophenyl-functionalized graphene/MoS2 The electron-withdrawing effects of the functional groups and lattice defects in graphene have been demonstrated to enhance the PL intensity of monolayer MoS2. In addition, Interlayer van der Waals interactions and interlayer distance are very important factors in studying the PL of graphene/MoS2 heterostructures (G/MoS2) as they are only a few atomic thin. Functionalization of graphene with specific functional groups is of great significance for the further development of covalent modification of graphene and the interface construction in G/MoS2 heterostructures, thereby facilitating the study of interlayer coupling of G/MoS2 heterostructures. Monotopic covalently modified graphene, oligophenyl-functionalized graphene (F-G), are prepared by a laser-induced reaction and stacked with a monolayer MoS2. The functionalization of graphene is regioselective with the assistance of the mapping function of the scanning Raman spectrometer. Through Raman, PL, KPFM and scanning near-field optical microscopy (collaboration with Dr. Patryk Kusch, FU Berlin) measurements, the boundaries and the distinct characteristics of the functionalized and the non-functionalized areas are identified on the heterostructure. More importantly, the layer stacking sequence of F-G and MoS2 brings different interface structures in perpendicular orientation. MoS2 supported by F-G (F-G/MoS2) results in a sandwiched structure consisting of graphene/oligophenyl-groups/MoS2 with an enlarged interlayer distance of 8 nm between the graphene basal plane and MoS2. In the case of MoS2 stacked underneath F-G (MoS2/F-G) a direct interface is formed between the graphene basal plane and MoS2, with the oligophenyl-groups located on the top surface of the heterostructure. The different interfaces in the heterostructures result in a significant difference in the PL enhancement of MoS2. F-G/MoS2 shows a 5-fold PL enhancement, while MoS2/F-G only shows a 1.8-fold PL enhancement compared to pristine G/MoS2. Accordingly, the results indicate that the oligophenyl-groups in F-G/MoS2 not only have a pdoping effect on MoS2 but also largely prevent electron donation from the graphene basal plane with the enlarged interlayer distance. Consequently, the PL enhancement is restored with the thermal de-functionalization of F-G. Thus, we conclude that the functional groups can be considered as separate molecular component with the vertical arrangement in the functionalized heterostructure system. The photoactive graphene acts as a template for perpendicular molecular alignment in the heterointerface construction, thus opening more possibilities for the fabrication of heterointerfaces. In this thesis, the interfaces of G/MoS2 are engineered through the introduction of oxo-functional groups, structural defects, and laser-induced perpendicular functional-groups on graphene. The interfacial modulation via interlayer charge transfers and interlayer distances results in significant changes in the PL properties of G/MoS2 heterostructures. These findings offer novel insights into the design and exploration of optoelectronic devices. Furthermore, the PL enhancement of G/MoS2 opens up numerous possibilities for optoelectronic applications, for instance, wavelength-tunable phototransistors, broadband photodetectors, single-photon emission sites for quantum information science, improved electrical performance for high-speed optoelectronics, and signal enhancement in photodetectors and sensors. Based on the findings in this thesis, there are still ample opportunities for future research to continue this study. First, the development of diverse interlayer functional groups with diverse electron transfer capabilities and spatial effects can be achieved through laser-induced functionalization methods to realize a more versatile interface modulation. Second, further investigation into the interface modulation of different 2D materials beyond graphene and MoS2 is expected. Third, a thorough fundamental understanding of hetero-interfaces is required. A deeper insight into the effects of interface modulation on the electronic structure, band alignment, and van der Waals interactions of heterostructures can be achieved through theoretical studies and techniques such as charge transport measurements, ultrafast optical spectroscopy, and magnetic characterization.
Weniger anzeigenAnimals need to switch behavioral strategies to adapt to changing environmental conditions. The ability to choose the most advantageous response to a situation is crucial for survival. These decisions are influenced by environmental factors and internal signals such as physiological needs. During foraging behavior, larval zebrafish approach potential preys and avoid potential predators. The choice is influenced by external factors, such as the size of the object they are interacting with: small visual stimuli are perceived as preys while large objects are considered predators. Moreover, it was previously shown that behavioral choice during feeding behavior is modulated by metabolic state: food-deprived larvae are more likely to take risks during hunting and approach small, prey-like objects compared to fed fish. It was also demonstrated that this modulation is mediated by a differential processing of visual stimuli. In zebrafish the visual information is relayed from the retina to a midbrain structure, the optic tectum, where sensory inputs are integrated. Distinct downstream motor centers are subsequently activated to perform either an approach or an escape. This makes the optic tectum a major center for decision-making. In food-deprived larvae, tectal neurons respond preferentially to small visual stimuli compared to fed siblings, showing a shift of the tuning towards cues important for survival. These results suggest feeding induces a change in the excitability of tectal neurons to modulate behavioral choice, however the molecular mechanisms underlying this phenomenon are still unclear. To fill the gap by investigating the molecular pathways mediating the influence of metabolic state on behavioral choice, a proteomic study was performed to identify proteins differentially abundant in fed versus food-deprived larvae. Among all the hits, an especially interesting candidate was selected: a small peptide known to modulate neuronal excitability, Pcp4a, which was less abundant in fed larvae compared to food-deprived fish. pcp4a mRNA levels were also found to be lower in brain samples from fed larvae, suggesting that feeding modulates its expression through a transcriptional mechanism. PCP4, the mammalian ortholog of Pcp4a is known to bind to calmodulin, a molecule involved in several processes during neuronal activation, and to inhibit its target enzymes such as CaMKII. This results in modulation of neuron excitability in vivo. Since pcp4a is expressed in the optic tectum in zebrafish, we hypothesized that it may play a role in the regulation of tectal neurons excitability by feeding state. To test this hypothesis, I first looked at the role of pcp4a in the modulation of behavioral choice by feeding state using a loss-of-function model. Food-deprived larvae lacking Pcp4a show increased avoidance of small objects in a size discrimination assay, thus phenocopying behavioral choice of fed larvae. To understand if the effect was due to modulation of tectal neurons excitability, I investigated the response to visual stimuli of different size in tectal neurons expressing pcp4a (pcp4a+). Tectal pcp4a+ neurons in fed larvae responded preferentially to large objects compared to starved siblings; the same response profile was observed in larvae lacking Pcp4a, suggesting that Pcp4a mediates the effect of feeding on tectal neurons excitability. I then looked into the neuromodulatory mechanisms mediating the influence of metabolic signals on pcp4a expression. I found that feeding increased activity of dopaminergic neurons in the pretectum and hypothalamus, which could exert an effect on tectal pcp4a+ neurons through their direct and indirect projections to the optic tectum. Indeed, pharmacological activation of dopaminergic signaling through D2 receptors decreased pcp4a expression in food-deprived larvae, mimicking the effect of feeding. Dopamine controls pcp4a transcription through the D2 receptor - cAMP signaling cascade. Pharmacological activation of dopaminergic signaling induced a shift of the response profile of pcp4a+ tectal neurons towards large stimuli by altering the tuning properties of individual neurons. In this study we elucidate a novel molecular mechanism mediating the effect of metabolic state on behavioral choice in zebrafish. In our model, feeding activates dopaminergic neurons in the pretectum and hypothalamus, which project to the optic tectum. Dopaminergic signaling through D2 receptors induces a decrease of pcp4a expression through a transcriptional mechanism, which results in a shift of the response profile of pcp4a+ tectal neurons towards large visual stimuli through a cell-autonomous mechanism. This ultimately leads to increased avoidance of small stimuli during foraging behavior. This study advances our knowledge of the molecular mechanisms mediating neuromodulation of decision-making behavior.
Weniger anzeigenThis dissertation examines the construction and instrumentalisation of Generation Y as a discourse in the US and its translation to Turkey. It is guided by the inquiry to understand the enduring emphasis and popularity of generational thinking, despite the lack of empirical evidence and its theoretical shortcomings.
The analysis examines, in sequential order, influential actors shaping the Gen Y discourse in the US, uncovering implicit and explicit claims associated with it. The focus then shifts to Turkey, where the discourse gained prominence during the Gezi Park Protests, exploring local influencers and delving into the evolution and translation of Generation Y discourse within this unique context. The investigation extends to companies in Turkey, examining how employees and companies relate to and instrumentalise the Gen Y discourse in their management practices.
To address these inquiries, the dissertation adopts a constructivist approach, combining thematic analysis of texts on Gen Y from mainstream newspapers, popular business magazines, consultancy reports, and bestselling books in Turkish and English, with the in-depth interviews conducted with white-collar workers in Turkey.
The dissertation highlights the role of management and consultancy practitioners in constructing and disseminating the Generation Y discourse, suggesting that they employ strategies similar to those used for popular management fads and fashions, including the use of an ambiguous and polysemous language. The analysis reveals that, despite theoretical controversies, Generation Y is portrayed as a scientific concept based on birth cohorts while simultaneously drawing strategically on historical connotations of generational identity, aligning itself with alternative understandings of generations (such as kinship structures and youth as a driving force in shaping history). This research suggests that the Gen Y discourse is deeply embedded in the idea of a “new” era marked by digitalisation and widespread internet use. In this context, "generational conflicts" and "becoming outdated" are presented as a single problem, forming a central message within the discourse. Consequently, Gen Y discourse extends beyond addressing generational conflicts or explaining youth movements; it is presented as a panacea offering solutions to all current and foreseeable future problems.
Weniger anzeigenThe development of metal-nitrogen-carbons (M-N-Cs) has gained significant momentum in the 21st century, driven by their potential as sustainable alternatives to precious metals for electrocatalytic applications. However, as the pyrolytic synthesis often produces highly disordered carbonized materials, it can be challenging to accurately characterize the active sites and their involvement in the catalytic reactions. In this regard, quantum chemical methodologies like density functional theory (DFT) could provide crucial atomic-level information to guide the characterization and optimization of M-N-Cs. In this thesis, I present computational studies related to the synthesis of M-N-Cs in the first part, followed by their electrocatalytic applications in the second part. In parallel to these studies, potential factors for the material degradation during the synthesis and applications are also identified, especially for the generally active but unstable Fe-N-Cs.
Inspired by the recent success of the templating-transmetallation strategy in producing M-N-Cs with dense active sites, the first part of the thesis features a computational investigation on the chemical properties of metals at the MN4 sites. By constructing and optimizing structural models for the incorporation of MN4 motifs in conductive carbon matrices, we determined several advantages of using molecular DFT methods for modeling the active sites, especially the structurally complex pyrrolic MN4 motifs. Using these models, we further examined the trends among the first-row transition metals pertaining their binding within the MN4 site as well as their relative affinity to the pyrrolic or pyridinic motifs. It could be shown that metals with more significant ionic binding character, such as Zn and Mg, are more likely to form the pyrrolic MN4 sites than the catalytically active metals like Fe and Co, leading to the successful characterization of the pyrrolic MN4 sites from synthesis strategies which utilized Zn2+ or Mg2+ as the sacrificial templating ions. In addition, the binding of Zn was determined to be the weakest among the transition metals, making it an optimal choice for the subsequent transmetallation.
As the transmetallation step generally retains the original structure of the active site, in addition to its morphological properties and active site densities, it became a crucial starting point for intrinsic activity comparisons among metals in the M-N-Cs. In the second part of this thesis, the electrocatalytic mechanisms for the oxygen reduction reaction (ORR) and the carbon dioxide reduction reaction (CO2RR) on the previously identified pyrrolic MN4 sites are presented along with corresponding side reactions including the Fenton reaction and the hydrogen evolution reaction (HER). The combined efforts of theory and experiment led to the identification of inconspicuous species at the interface, such as hydroxides for alkaline ORR, water for the Fenton reaction, and cations for the CO2RR, which in fact play an active role in the mechanisms of the respective reactions. For the ORR, we determined the involvement of the outer-sphere electron transfer in the reaction mechanism, especially in the alkaline medium, leading to the formation of H2O2 whose subsequent reactions could depend on specific interactions with the MN4 site. For the CO2RR, a theory-experiment contradiction in the activity and selectivity of Ni-N-Cs could be resolved with the inclusion of alkali metal ions in the mechanism, whose adsorption was made favorable by the pyrrolic N atoms at the MN4 site. Overall, these insights derived with DFT provide strong theoretical support for the knowledge-based optimization of the activity, selectivity and stability of M-N-Cs for sustainable energy conversion applications.
Weniger anzeigenIn dieser Habilitationsarbeit wurden hochspezifische pathophysiologische Konzepte im Kontext verschiedener klinischer Präsentationen einiger ausgewählter neuromuskulärer Erkrankungen untersucht. Dabei kam ein Spektrum von experimentellen, morphologischen und klinischen Techniken zum Einsatz, um verschiedene Aspekte der jeweiligen Erkrankung zu untersuchen. Im Kontext inflammatorischer (IBM, SSc) und hereditärer (MADD, Danon-Erkrankung) Myopathien konnten morphologische und molekulare Muster herausgearbeitet werden, die mit der klinischen Präsentation korrelieren. Darüber hinaus wurde das bisherige pathophysiologische Verständnis der IBM erweitert und das darauf basierende Konzept der IBM-Spektrumserkrankung eingeführt. Im Rahmen dieser Arbeiten wurden auch mögliche Kandidaten für einen neuen IBM-Biomarker identifiziert. Zudem wurden sowohl im Kontext hereditärer (Danon-Erkrankung), wie auch erworbener Myopathien (IBM/PM-Mito) relevante mitochondriale Veränderungen identifiziert, denen ein bislang nicht ausreichend verstandener Anteil an der Pathophysiologie und dem Phänotyp der Erkrankungen zukommt. Schließlich wurde das klinische Spektrum der TTR-Amyloidose erweitert und die ATTRwt neben der ATTRv als behandelbare Ursache für Polyneuropathien in höherem Lebensalter identifiziert. Zusammengefasst stellen die hier präsentierten klinischen und experimentellen Arbeiten die Grundlage für weitere Forschungsprojekte dar, die, gleichberechtigt zum wissenschaftlichen Erkenntnisgewinn, die Verbesserung der Versorgung von Patient*innen mit neuromuskulären Erkrankungen zum Ziel haben werden.
Weniger anzeigenDuring lactation, the gut mucosa undergoes expansion to accommodate increased food consumption and absorption, in order to meet the increased energy demands associated with nurturing neonates. Previous studies have investigated the impact of litter size, hormones, and diet on lactation-induced gut hyperplasia, yet the role of immune cells remained unexplored. The recently discovered innate lymphocytes type 3 (ILC3s) have emerged as key players not only in organizing lymphoid tissues but also in protecting the intestinal epithelium against tissue damage and genotoxic stress. Thus, we explored the contribution of ILC3s to lactation-induced gut hyperplasia. Our immunofluorescence analysis revealed that mice lacking RORγt, the transcription factor crucial for ILC3 development and function, exhibited shorter villi and reduced proliferation in the crypts, where the intestinal stem cells reside. Subsequent analysis using IL-22-deficient mice, a cytokine primarily expressed by ILC3s, suggests that ILC3s promote lactation-induced gut hyperplasia in an IL-22-dependent manner. Furthermore, our findings demonstrated that STAT3 in the intestinal epithelium, known to be stimulated by IL-22 and EGF, also contributes to increased proliferation and villus growth in lactating mice.
Flow cytometry experiments and histological analyses confirmed increased ILC3 proliferation during lactation and enlargement of the clusters ILC3s form proximal to the crypts, namely cryptopatches and isolated lymphoid follicles. Bulk RNA sequencing and leukocyte stimulation experiments between non-pregnant (NP) and lactating mice, confirmed the upregulation of IL-22 in ILC3s during lactation, a finding further validated through the analysis of IL-22 tdTomato reporter mice. Single-cell RNA sequencing of intestinal epithelial cells from both ILC3-sufficient and deficient mice unveiled downregulation of the EGF signaling in the gut, which was validated by quantification of EGFR phosphorylation in epithelial cells. The phosphorylation of EGFR, linked to ligand binding, was confirmed to be associated with EGF upregulation during lactation, originating from mesenchymal PDPN+ cells. Notably, RORγt-dependent cells were found to regulate EGF production in mesenchymal cells in an IL-22-dependent manner upon lactation.
Moreover, our study demonstrated that IL-22 modulates genes associated with nutrient absorption in gut epithelium in vitro, differentially to the IL-20 and IL-24 cytokines, which also bind to the IL-10RB receptor. Lastly, we observed that lactating RORγt and IL22-deficient mice consumed more food than the WT control mice, presumably to compensate for reduced food absorption. However, due to their reduced absorptive epithelial surface, the body weight of IL-22-sufficient offspring nurtured from ILC3- and IL-22-deficient mice was reduced.
Weniger anzeigenOpioid receptor ligands (opioids) represent the gold standard for severe pain treatment. However, approved opioids possess many serious side effects even if they are used according to medical instructions. As a result, opioid use leads to thousands of hospitalizations and deaths each year. Four different opioid receptor subtypes exist but approved opioids mainly activate the μ-opioid receptor (MOR). Activation of the κ-opioid receptor (KOR) emerged as a promising strategy to sufficiently alleviate pain while improving the side effect profile of opioids. However, KOR-related side effects and the high structural similarity of the opioid receptor subtypes renders rational drug design challenging. The opioid receptor system is not only involved in pain modulation, but also in the maintenance of mood homeostasis. Antagonism of the KOR recently emerged as a promising strategy for the development of novel antidepressants with fast-onset antidepressant effects that are also effective against suicidal intent. However, the chemical space of KOR selective antagonists is limited. No short-acting and selective KOR antagonists have been approved so far. This dissertation encompasses retrospective and prospective studies that focus on in silico techniques to address current questions in opioid research. The first three articles, conducted retrospective in silico analyses of HS-731, an opioid agonist, Compound A, a KOR/MOR dual antagonist, and Salvinorin A (SalA), a non-basic, KOR-selective agonist. These analyses aimed to improve our limited understanding of the factors that determine opioid receptor activity and selectivity. The investigated compounds are of high pharmacological interest. HS-731 represents an analgesic with a potentially improved safety profile due to its exclusion from the central nervous system (CNS). Compound A has a unique scaffold that may overcome the unfavorable kinetic profile of current antidepressant opioids. SalA represents a non-basic agonist with exceptional KOR selectivity that potentially binds dissimilar to basic opioids at the KOR. Protein-ligand binding modes of the compounds at the opioid receptor subtypes were predicted and selectivity determinants rationalized. Important protein-ligand interactions responsible for the opioid receptor activity of these ligands were highlighted. A new binding site for SalA above the typical morphinan binding site that is highly non-conserved was discovered. The SalA binding mode is consistent with structure-activity relationship (SAR) data and rationalizes the exceptional receptor subtype selectivity of SalA at the KOR. In addition, a partial agonism-inducing mechanism for the KOR was hypothesized involving an interaction between the extracellular oriented parts of the transmembrane helices 5 and 6. Typical opioids possess a basic amine moiety interacting with a conserved aspartate residue inside the binding site. Since the discover of non-basic and highly KOR-selective SalA, the design of non-basic opioids became a new and promising strategy for the selective activation of the KOR, circumventing MOR-associated severe side effects. However, the number of known non-basic opioids is strongly limited and rational drug design of new non-basic opioids is the hindered by the lack of understanding about the binding mode of SalA. In the fourth article included in this dissertation, a prospective 3D pharmacophore-based virtual screening campaign was conducted. The campaign was based on the SalA binding mode postulated in the third article. The aim was to determine new non-basic and selective KOR ligands. Pharmacological in vitro experiments confirmed two of the suggested compounds to activate the KOR with nanomolar potency and good subtype selectivity. One hit compound possesses a full agonistic and one compound shows a partial agonistic profile at the KOR. Both hit compounds share a novel spiro-moiety-containing scaffold. Overall, the findings of this dissertation provide new mechanistic insights into opioid receptor activation in the context of current progress in structural biology, and lay the basis for improved, rational drug design of safer analgesics and novel antidepressants with KOR activity.
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