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A novel USP11-TCEAL1-mediated mechanism protects transcriptional elongation by RNA Polymerase II
(2024)
Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge.
Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding.
In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS.
Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma.
In this study, we developed an innovative nanoparticle formulation to facilitate the delivery of antitumor antibodies to tumor sites. The study commenced with the utilization of 13 bispecific antibody fusion proteins, which targeted the Fn14 receptor, thereby validating the pivotal role of crosslinking in Fn14 receptor activation. Subsequently, gold nanoparticles were activated using COOH-PEG-SH in combination with EDC/NHS, and subsequently conjugated with two Fn14-targeting antibodies, PDL192 and 5B6. Following this, a pH-sensitive shell was generated on the outer layer of the antibody-coupled gold nanoparticles through the application of chemically modified polylysine. The resultant complexes, termed MPL-antibody-AuNP, demonstrated a release profile reminiscent of the tumor microenvironment (TME). Notably, these complexes released antibody-AuNPs only in slightly acidic conditions while remaining intact in neutral or basic environments. Functionality analysis further affirmed the pH-sensitive property of MPL-antibody-AuNPs, demonstrating that the antibodies only initiated potent Fn14 activation in slightly acidic environments. This formulation holds potential for applicability to antibodies or ligands targeting the 80 TNFRSF family, given that gold nanoparticles successfully served as platforms for antibody crosslinking, thereby transforming these antibodies into potent agonists. Moreover, the TME disintegration profile of MPL mitigates the potential cytotoxic effects of antibodies, thereby circumventing associated adverse side effects. This study not only showcases the potential of nanoparticle formulations in targeted therapy, but also provides a solid foundation for further investigations on their clinical application in the context of targeting category II TNFRSF receptors with antibodies or ligands.
Structure and dynamics of the plasma membrane: a single-molecule study in \(Trypanosoma\) \(brucei\)
(2024)
The unicellular, flagellated parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and nagana in livestock. In the last decades, it has become an established eukaryotic model organism in the field of biology, as well as in the interdisciplinary field of biophysics. For instance, the dense variant surface glycoprotein (VSG) coat offers the possibility to study the dynamics of GPI-anchored proteins in the plasma membrane of living cells. The fluidity of the VSG coat is not only an interesting object of study for its own sake, but is critically important for the survival of the parasite in the mammalian host. In order to maintain the integrity of the coat, the entire VSG coat is recycled within a few minutes. This is surprisingly fast for a purely diffusive process with the flagellar pocket (FP) as the sole site for endo- and exocytosis. Previous studies characterising VSG dynamics using FRAP reported diffusion coefficients that were not sufficient to to enable fast turnover based on passive VSG randomisation on the trypanosome surface.
In this thesis, live-cell single-molecule fluorescence microscopy (SMFM) was employed to elucidate whether VSG diffusion coefficients were priorly underestimated or whether directed forces could be involved to bias VSGs towards the entrance of the FP. Embedding the highly motile trypanosomes in thermo-stable hydrogels facilitated the investigation of VSG dynamics on living trypanosomes at the mammalian host's temperature of 37°C. To allow for a spatial correlation of the VSG dynamics to the FP entrance, a cell line was employed harbouring a fluorescently labelled structure as a reference. Sequential two-colour SMFM was then established to allow for recording and registration of the dynamic and static single-molecule information.
In order to characterise VSG dynamics, an algorithm to obtain reliable information from short trajectories was adapted (shortTrAn). It allowed for the quantification of the local dynamics in two distinct scenarios: diffusion and directed motion. The adaptation of the algorithm to the VSG data sets required the introduction of an additional projection filter. The algorithm was further extended to take into account the localisation errors inherent to single-particle tracking. The results of the quantification of diffusion and directed motion were presented in maps of the trypanosome surface, including an outline generated from a super-resolved static structure as a reference. Information on diffusion was displayed in one map, an ellipse plot. The colour code represented the local diffusion coefficient, while the shape of the ellipses provided an indication of the diffusion behaviour (aniso- or isotropic diffusion). The eccentricity of the ellipses was used to quantify deviations from isotropic diffusion. Information on directed motion was shown in three maps: A velocity map, representing the amplitude of the local velocities in a colour code. A quiver plot, illustrating the orientation of directed motion, and a third map which indicated the relative standard error of the local velocities colour-coded. Finally, a guideline based on random walk simulations was used to identify which of the two motion scenarios dominated locally. Application of the guideline to the VSG dynamics analysed by shortTrAn yielded supermaps that showed the locally dominant motion mode colour-coded.
I found that VSG dynamics are dominated by diffusion, but several times faster than previously determined. The diffusion behaviour was additionally characterised by spatial heterogeneity. Moreover, isolated regions exhibiting the characteristics of round and elongated traps were observed on the cell surface. Additionally, VSG dynamics were studied with respect to the entrance of the FP. VSG dynamics in this region displayed similar characteristics compared to the remainder of the cell surface and forces biasing VSGs into the FP were not found.
Furthermore, I investigated a potential interference of the attachment of the cytoskeleton to the plasma membrane with the dynamics of VSGs which are anchored to the outer leaflet of the membrane. Preliminary experiments were conducted on osmotically swollen trypanosomes and trypanosomes depleted for a microtubule-associated protein anchoring the subpellicular microtubule cytoskeleton to the plasma membrane. The measurements revealed a trend that detachment of the cytoskeleton could be associated with a reduction in the VSG diffusion coefficient and a loss of elongated traps. The latter could be an indication that these isolated regions were caused by underlying structures associated with the cytoskeleton.
The measurements on cells with an intact cytoskeleton were complemented by random walk simulations of VSG dynamics with the newly determined diffusion coefficient on long time scales not accessible in experiments. Simulations showed that passive VSG randomisation is fast enough to allow for a turnover of the full VSG coat within a few minutes. According to an estimate based on the known rate of endocytosis and the newly determined VSG diffusion coefficient, the majority of exocytosed VSGs could escape from the FP to the cell surface without being immediately re-endocytosed.
Human prosociality, encompassing generosity, cooperation, and volunteering, holds a vital role in our daily lives. Over the last decades, the question of whether prosociality undergoes changes over the adult lifespan has gained increased research attention. Earlier studies suggested increased prosociality in older compared to younger individuals. However, recent meta-analyses revealed that this age effect might be heterogeneous and modest. Moreover, the contributing factors and mechanisms behind these age-related variations remain to be identified. To unravel age-related differences in prosociality, the first study of this dissertation employed a meta-analytical approach to summarize existing findings and provide insight into their heterogeneity by exploring linear and quadratic age effects on self-reported and behavioral prosociality. Additionally, two empirical research studies investigated whether these age-related differences in prosociality were observed in real life, assessed through ecological momentary assessment (Study 2), and in a controlled laboratory setting by applying a modified dictator game (Study 3). Throughout these three studies, potential underlying behavioral and computational mechanisms were explored. The outcome of the meta-analysis (Study 1) revealed small linear age effects on prosociality and significant age group differences between younger and older adults, with higher levels of prosociality in older adults. Explorative evidence emerged in favor of a quadratic age effect on behavioral prosociality, indicating the highest levels in midlife. Additionally, heightened prosocial behavior among middle-aged adults was observed compared to younger adults, whereas no significant differences in prosocial behavior were noted between middle-aged and older adults. Situational and contextual features, such as the setting of the study and specific paradigm characteristics, moderated the age-prosociality relationship, highlighting the importance of the (social) context when studying prosociality. For Study 2, no significant age effect on real-life prosocial behavior was observed. However, evidence for a significant linear and quadratic age effect on experiencing empathy in real life emerged, indicating a midlife peak. Additionally, across all age groups, the link between an opportunity to empathize and age significantly predicted real-life prosocial behavior. This effect, indicating higher levels of prosocial behavior when there was a situation possibly evoking empathy, was most pronounced in midlife. Study 3 presented age differences in how older and younger adults integrate values related to monetary gains for self and others to make a potential prosocial decision. Younger individuals effectively combined both values in a multiplicative fashion, enhancing decision-making efficiency. Older adults showed an additive effect of values for self and other and displayed increased decision-making efficiency when considering the values separately. However, among older adults, individuals with better inhibitory control were better able to integrate information about both values in their decisions. Taken together, the findings of this dissertation offer new insights into the multi-faceted nature of prosociality across adulthood and the mechanisms that help explain these age-related disparities. While this dissertation observed increasing prosociality across the adult lifespan, it also questions the assumption that older adults are inherently more prosocial. The studies highlight midlife as a potential peak period in social development but also emphasize the importance of the (social) context and that different operationalizations might capture distinct facets of prosociality. This underpins the need for a comprehensive framework to understand age effects of prosociality better and guide potential interventions.
In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells.
First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models.
The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices.
The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds.
Few topics have been the subject of more controversy than those encapsulated by the terms "sex" and "gender". Social-cultural and biological-evolutionary argumentation patterns frequently clash and especially the public debate appears to be stuck in a stalemate between the two competing parties.
From a psychological perspective both topics appear deeply intertwined and are not easy to be separated. This study pursues an integrative approach to better understand the roots of differences best subsumed under the term sex/gender. It will become apparent that both nature and nurture variables interact and form the complex system of human behavior and experience.
The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc’s structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel–Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field.
In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines.
In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs.
In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs.
Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy.
1,1,2-trifluoroethene (HFO-1123) is intended for use as a refrigerant. Inhalation studies on HFO-1123 in rats suggested a low potential for toxicity, with no-observed-adverse-effect levels greater then 20,000 ppm. However, single inhalation exposure of Goettingen Minipigs and New Zealand White Rabbits resulted in mortality. It was assumed that conjugation of HFO-1123 with glutathione, via glutathione S-transferase, gives rise to S-(1,1,2-trifluoroethyl)-L-glutathione (1123-GSH), which is then transformed to the corresponding cysteine S-conjugate (S-(1,1,2-trifluoroethyl)-L-cysteine, 1123-CYS). Subsequent beta-lyase mediated cleavage of 1123-CYS may result in monofluoroacetic acid, a potent inhibitor of aconitase. Species-differences in 1123-GSH formation and 1123-CYS cleavage to MFA may explain species-differences in HFO-1123 toxicity.
This study was designed to test the hypothesis, that GSH-dependent biotransformation and subsequent beta-lyase mediated formation of monofluoroacetic acid, a potent inhibitor of aconitase in the citric acid cycle, may play a key role in HFO-1123 toxicity and to evaluate if species-differences in the extent of MFA formation may account for the species-differences in HFO-1123 toxicity. The overall objective was to determine species-differences in HFO-1123 biotransformation in susceptible vs. less susceptible species and humans as a basis for human risk assessment.
To this end, in vitro biotransformation of HFO-1123 and 1123-CYS was investigated in renal and hepatic subcellular fractions of mice, rats, humans, Goettingen Minipigs and NZW Rabbits. Furthermore, cytotoxicity and metabolism of 1123-CYS was assessed in cultured renal epithelial cells. Enzyme kinetic parameters for beta-lyase mediated cleavage of 1123-CYS in renal and hepatic cytosolic fractions were determined, and 19F-NMR was used to identify fluorine containing metabolites arising from 1123-CYS cleavage. Quantification of 1123-GSH formation in hepatic S9 fractions after incubation with HFO-1123 was performed by LC-MS/MS and hepatic metabolism of HFO-1123 was monitored by 19F-NMR.
Rates of 1123-GSH formation were increased in rat, mouse and NZW Rabbit compared to human and Goettingen hepatic S9, indicating increased GSH dependent biotransformation in rats, mouse and NZW Rabbits. NZW Rabbit hepatic S9 exhibited increased 1123-GSH formation in the presence compared to the absence of acivicin, a specific gamma-GT inhibitor. This indicates increased gamma-GT mediated cleavage of 1123-GSH in NZW Rabbit hepatic S9 compared to the other species. 19F-NMR confirmed formation of 1123-GSH as the main metabolite of GSH mediated biotransformation of HFO-1123 in hepatic S9 fractions next to F-. Increased F- formation was detected in NZW Rabbit and Goettingen Minipig hepatic S9 in the presence of an NADPH regenerating system, indicating a higher rate of CYP-450 mediated metabolism in these species. Based on these findings, it is possible that CYP-450 mediated metabolism may contribute to HFO-1123 toxicity.
In contrast to the increased formation of 1123-GSH in rat, mouse and NZW Rabbit hepatic S9 (compared to human and Goettingen Minipig), enzyme kinetic studies revealed a significantly higher beta-lyase activity towards 1123-CYS in renal cytosol of Goettingen Minipigs compared to cytosol from rats, mice, humans and NZW Rabbits. However, beta-lyase cleavage in renal NZW Rabbit cytosol was slightly increased compared to rat, mouse and human renal cytosols. 19F-NMR analysis confirmed increased time-dependent formation of MFA in renal Goettingen Minipig cytosol and NZW Rabbit (compared to human and rat cytosolic fractions). Three structurally not defined MFA-derivatives were detected exclusively in NZW Rabbit and Goettingen Minipig cytosols. Also, porcine kidney cells were more sensitive to cytotoxicity of 1123-CYS compared to rat and human kidney cells.
Overall, increased beta-lyase mediate cleavage of 1123-CYS to MFA in Goettingen Minipig and NZW Rabbit kidney (compared to human and rat) may support the hypothesis that enzymatic cleavage by beta-lyases may account for the species-differences in HFO-1123 toxicity. However, the extent of GST mediated biotransformation in the liver as the initial step in HFO-1123 metabolism does not fully agree with this hypothesis, since 1123-GSH formation occurs at higher rates in rat, mouse and NZW Rabbit S9 as compared to the Goettingen Minipig.
Based on the inconsistencies between the extent of GST and beta-lyase mediated biotransformation of HFO-1123 obtained by this study, a decisive statement about an increased biotransformation of HFO-1123 in susceptible species with a direct linkage to the species-specific toxicity cannot be drawn. Resulting from this, a clear and reliable conclusion regarding the risk for human health originating from HFO-1123 cannot be made. However, considering the death of Goettingen Minipigs and NZW Rabbits after inhalation exposure of HFO-1123 at concentrations great than 500 ppm and greater than 1250 ppm, respectively, this indicates a health concern for humans under peak exposure conditions. For a successful registration of HFO-1123 and its use as a refrigerant, further in vitro and in vivo investigations addressing uncertainties in the species-specific toxicity of HFO-1123 are urgently needed.
Idiopathic Pulmonary Fibrosis (IPF) is a progressive parenchymal lung disease with limited therapeutic treatments. Pathologically altered lung fibroblasts, called myofibroblasts, exhibit increased proliferation, migration, and collagen production, and drive IPF development and progression. Fibrogenic factors such as Platelet derived growth factor-BB (PDGF-BB) contribute to these pathological alterations. Endogenous counter-regulating factors are barely known. Published studies have described a protective role of exogenously administered C-type Natriuretic Peptide (CNP) in pathological tissue remodeling, for example in heart and liver fibrosis. CNP and its cyclic GMP producing guanylyl cyclase B (GC-B) receptor are expressed in the lungs, but it is unknown whether CNP can attenuate lung fibrosis by this pathway. To address this question, we performed studies in primary cultured lung fibroblasts.
To examine the effects of the CNP/GC-B pathway on PDGF-BB-induced collagen
production, proliferation, and migration in vitro, lung fibroblasts were cultured from wildtype control and GC-B knockout mice. Human lung fibroblasts from patients with IPF and healthy controls were obtained from the UGMLC Biobank. In RIA experiments, CNP, at 10nM and 100nM, markedly and similarly increased cGMP levels in both the murine and human lung fibroblasts, demonstrating GC-B/cGMP signaling. CNP reduced PDGF-BB induced proliferation and migration of lung fibroblasts in BrdU incorporation and gap closure assays, respectively. CNP strongly decreased PDGF-BB-induced collagen 1/3 expression as measured by immunocytochemistry and immunoblotting. Importantly, the protective actions of CNP were preserved in IPF fibroblasts. It is known that the profibrotic actions of PDGF-BB are partly mediated by phosphorylation and nuclear export of Forkhead Box O3 (FoxO3), a transcription factor downregulated in IPF. CNP prevented PDGF-BB elicited FoxO3 phosphorylation and nuclear exclusion in both murine and human control and IPF fibroblasts. CNP signaling and functions were abolished in GC-B-deficient lung fibroblasts.
Taken together, the results show that CNP moderates the PDGF-BB-induced activation and differentiation of human and murine lung fibroblasts to myofibroblasts. This effect is mediated CNP-dependent by GC-B/cGMP signaling and FoxO3 regulation. To follow up the patho-physiological relevance of these results, we are generating mice with fibroblast-restricted GC-B deletion for studies in the model of bleomycin-induced pulmonary fibrosis.
Humans actively interact with the world through a wide range of body movements. To understand human cognition in its natural state, we need to incorporate ecologically relevant body movement into our account. One fundamental body movement during daily life is natural walking. Despite its ubiquity, the impact of natural walking on brain activity and cognition has remained a realm underexplored.
In electrophysiology, previous studies have shown a robust reduction of ongoing alpha power in the parieto-occipital cortex during body movements. However, what causes the reduction of ongoing alpha, namely whether this is due to body movement or prevalent sensory input changes, was unknown. To clarify this, study 1 was performed to test if the alpha reduction is dependent on visual input. I compared the resting state alpha power during natural walking and standing, in both light and darkness. The results showed that natural walking led to decreased alpha activity over the occipital cortex compared to standing, regardless of the lighting condition. This suggests that the movement-induced modulation of occipital alpha activity is not driven by visual input changes during walking. I argue that the observed alpha power reduction reflects a change in the state of the subject based on disinhibition induced by walking. Accordingly, natural walking might enhance visual processing and other cognitive processes that involve occipital cortical activity.
I first tested this hypothesis in vision. Study 2 was performed to examine the possible effects of natural walking across visual processing stages by assessing various neural markers during different movement states. The findings revealed an amplified early visual response, while a later visual response remain unaffected. A follow-up study 3 replicated the walking-induced enhancement of the early visual evoked potential and showed that the enhancement was dependent on specific stimulus-related parameters (eccentricity, laterality, distractor presence). Importantly, the results provided evidence that the enhanced early visual responses are indeed linked to the modulation of ongoing occipital alpha power. Walking also modulated the stimulus-induced alpha power. Specifically, it showed that when the target appeared in the fovea area without a distractor, walking exhibited a significantly reduced modulation of alpha power, and showed the largest difference to standing condition. This effect of eccentricity indicates that during later visual processing stages, the visual input in the fovea area is less processed than in peripheral areas while walking.
The two visual studies showed that walking leads to an enhancement in temporally early visual processes which can be predicted by the walking-induced change in ongoing alpha oscillation likely marking disinhibition. However, while walking affects neural markers of early sensory processes, it does not necessarily lead to a change in the behavioural outcome of a sensory task. The two visual studies suggested that the behavioural outcome seems to be mainly based on later processing stages.
To test the effects of walking outside the visual domain, I turned to audition in study 4. I investigated the influence of walking in a particular path vs. simply stepping on auditory processing. Specifically, the study tested whether enhanced processing due to natural walking can be found in primary auditory brain activity and whether the processing preferences are dependent on the walking path. In addition, I tested whether the changed spatial processing that was reported in previous visual studies can be seen in the auditory domain. The results showed enhanced sensory processing due to walking in the auditory domain, which was again linked to the modulation of occipital alpha oscillation. The auditory processing was further dependent on the walking path. Additionally, enhanced peripheral sensory processing, as found in vision, was also present in audition.
The findings outside vision supported the idea of natural walking affecting cognition in a rather general way. Therefore in my study 5, I examined the effect of natural walking on higher cognitive processing, namely divergent thinking, and its correlation with the modulation of ongoing alpha oscillation. I analyzed alpha oscillations and behavioural performance during restricted and unrestricted movement conditions while subjects completed a Guilford's alternate uses test. The results showed that natural walking, as well as missing body restriction, reduces the occipital alpha ongoing power independent of the task phase which goes along with higher test scores. The occipital alpha power reduction can therefore be an indicator of a changed state that allows improved higher cognitive processes.
In summary, the research presented in this thesis highlights that natural walking can change different processes in the visual and auditory domain as well as higher cognitive processes. The effect can be attributed to the movement of natural walking itself rather than to changes in sensory input during walking. The results further indicate that the walking-induced modulation of ongoing occipital alpha oscillations drives the cognitive effects. We therefore suggest that walking changes the inhibitory state which can influence awareness and attention. Such a mechanism could facilitate an adaptive enhancement in cognitive processes and thereby optimize movement-related behaviour such as navigation.
The pancreas is the key organ for the maintenance of euglycemia. This is regulated in particular by α-cell-derived glucagon and β-cell-derived insulin, which are released in response to nutrient deficiency and elevated glucose levels, respectively. Although glucose is the main regulator of insulin secretion, it is significantly enhanced by various potentiators.
Platelets are anucleate cell fragments in the bloodstream that are essential for hemostasis to prevent and stop bleeding events. Besides their classical role, platelets were implemented to be crucial for other physiological and pathophysiological processes, such as cancer progression, immune defense, and angiogenesis. Platelets from diabetic patients often present increased reactivity and basal activation. Interestingly, platelets store and release several substances that have been reported to potentiate insulin secretion by β-cells. For these reasons, the impact of platelets on β-cell functioning was investigated in this thesis.
Here it was shown that both glucose and a β-cell-derived substance/s promote platelet activation and binding to collagen. Additionally, platelet adhesion specifically to the microvasculature of pancreatic islets was revealed, supporting the hypothesis of their influence on glucose homeostasis. Genetic or pharmacological ablation of platelet functioning and platelet depletion consistently resulted in reduced insulin secretion and associated glucose intolerance. Further, the platelet-derived lipid fraction was found to enhance glucose-stimulated insulin secretion, with 20-hydroxyeicosatetraenoic acid (20-HETE) and possibly also lyso-precursor of platelet-activating factor (lysoPAF) being identified as crucial factors. However, the acute platelet-stimulated insulin secretion was found to decline with age, as did the levels of platelet-derived 20-HETE. In addition to their direct stimulatory effect on insulin secretion, specific defects in platelet activation have also been shown to affect glucose homeostasis by potentially influencing islet vascular development. Taking together, the results of this thesis suggest a direct and indirect mechanism of platelets in the regulation of insulin secretion that ensures glucose homeostasis, especially in young individuals.
The increase in intensively used areas and climate change are direct and indirect consequences of anthropogenic actions, caused by a growing population and increasing greenhouse gas emissions. The number of research studies, investigating the effects of land use and climate change on ecosystems, including flora, fauna, and ecosystem services, is steadily growing. This thesis contributes to this research area by investigating land-use and climate effects on decomposer communities (arthropods and microbes) and the ecosystem service ‘decomposition of dead material’.
Chapter II deals with consequences of intensified land use and climate change for the ecosystem service ‘decomposition of dead organic material’ (necromass). Considering the severe decline in insects, we experimentally excluded insects from half of the study objects. The decomposition of both dung and carrion was robust to land-use changes. Dung decomposition, moreover, was unaffected by temperature and the presence/ absence of insects. Along the altitudinal gradient, however, highest dung decomposition was observed at medium elevation between 600 and 700 m above sea level (although insignificant). As a consequence, we assume that at this elevation there is an ideal precipitation:temperature ratio for decomposing organisms, such as earthworms or collembolans. Carrion decomposition was accelerated by increasing elevation and by the presence of insects, indicating that increasing variability in climate and an ongoing decline in insects could modify decomposition processes and consequently natural nutrient cycles. Moreover, we show that different types of dead organic material respond differently to environmental factors and should be treated separately in future studies.
In Chapter III, we investigated land-use and climate effects on dung-visiting beetles and their resource specialization. Here, all beetles that are preferentially found on dung, carrion or other rotten material were included. Both α- and γ-diversity were strongly reduced in agricultural and urban areas. High precipitation reduced dung-visiting beetle abundance, whereas γ-diversity was lowest in the warmest regions. Resource specialization decreased with increasing temperatures. The results give evidence that land use as well as climate can alter dung-visiting beetle diversity and resource specialization and may hence influence the natural balance of beetle communities and their contribution to the ecosystem service ‘decomposition of dead material’.
The following chapter, Chapter IV, contributes to the findings in Chapter II. Here, carrion decomposition is not only explained by land-use intensity and climate but also by diversity and community composition of two taxonomic groups found on carrion, beetles and bacteria. The results revealed a strong correlation between bacteria diversity and community composition with temperature. Carrion decomposition was to a great extent directed by bacterial community composition and precipitation. The role of beetles was neglectable in carrion decomposition. With this study, I show that microbes, despite their microscopic size, direct carrion decomposition and may not be neglected in future decomposition studies.
In Chapter V a third necromass type is investigated, namely deadwood. The aim was to assess climate and land-use effects on deadwood-inhabiting fungi and bacteria. Main driver for microbial richness (measured as number of OTUs) was climate, including temperature and precipitation. Warmer climates promoted the diversity of bacteria, whereas fungi richness was unaffected by temperature. In turn, fungi richness was lower in urban landscapes compared to near-natural landscapes and bacteria richness was higher on meadows than on forest sites. Fungi were extremely specialized on their host tree, independent of land use and climate. Bacteria specialization, however, was strongly directed by land use and climate. These results underpin previous studies showing that fungi are highly specialized in contrast to bacteria and add new insights into the robustness of fungi specialization to climate and land use.
I summarize that climate as well as intensive land use influence biodiversity. Temperature and precipitation, however, had positive and negative effects on decomposer diversity, while anthropogenic land use had mostly negative effects on the diversity of decomposers.
Academic education is seen as an important place for the development of professionalism of (future) adult educators. Since adult education academia, research, and practice is closely intertwined with global and international de- velopments, there is a need for adult education programmes to prepare their students for these interconnections. This can be examined in the context of international teaching and learning settings that integrate international, inter- cultural, or global perspectives into teaching and learning and are part of the internationalisation efforts of higher education. The focus of this international and comparative study is on how international teaching and learning settings contribute to the academic professionalisation in adult education in three mas- ter’s programmes with a focus on adult education at the University of Würzburg (Germany), University of Belgrade (Serbia) and University of Florence (Italy). International teaching and learning settings are examined on the structural and individual level of academic professionalisation. The aim is to explore the provision of international teaching and learning settings in the master’s pro- grammes on the one hand, and to analyse the contribution of international teach- ing and learning settings to the development of students’ professionalism on the other. For this purpose, three focus group interviews with programme heads, (academic) staff, and students as well as 22 guided interviews with graduates of the three master’s programmes at the three university locations are collected and analysed in an international and comparative study design. The study reveals similarities and differences in the forms, framework con- ditions, and goals of international teaching and learning settings between the three master’s programmes. Overarching contexts that guide the internationalisation of the master’s programmes become apparent (e.g. education and higher education policy, internationalisation of the university, programme structure). The triangulation of the interview data of the graduates shows that the interna- tional environment, the structural arrangement, and the practical relevance of the international teaching and learning settings support the development of the graduates’ professionalism. The results underline the relevance of international teaching and learning settings for the development of professionalism in adult education and point to the requirement for a systematic and comprehensive in- ternationalisation of adult education programmes.
Wireless communication networks already comprise an integral part of both the private and industrial sectors and are successfully replacing existing wired networks. They enable the development of novel applications and offer greater flexibility and efficiency. Although some efforts are already underway in the aerospace sector to deploy wireless communication networks on board spacecraft, none of these projects have yet succeeded in replacing the hard-wired state-of-the-art architecture for intra-spacecraft communication. The advantages are evident as the reduction of the wiring harness saves time, mass, and costs, and makes the whole integration process more flexible. It also allows for easier scaling when interconnecting different systems.
This dissertation deals with the design and implementation of a wireless network architecture to enhance intra-spacecraft communications by breaking with the state-of-the-art standards that have existed in the space industry for decades. The potential and benefits of this novel wireless network architecture are evaluated, an innovative design using ultra-wideband technology is presented. It is combined with a Medium Access Control (MAC) layer tailored for low-latency and deterministic networks supporting even mission-critical applications. As demonstrated by the Wireless Compose experiment on the International Space Station (ISS), this technology is not limited to communications but also enables novel positioning applications.
To adress the technological challenges, extensive studies have been carried out on electromagnetic compatibility, space radiation, and data robustness. The architecture was evaluated from various perspectives and successfully demonstrated in space.
Overall, this research highlights how a wireless network can improve and potentially replace existing state-of-the-art communication systems on board spacecraft in future missions. And it will help to adapt and ultimately accelerate the implementation of wireless networks in space systems.
The goal of this thesis is to study the topological and algebraic properties of the quasiconformal automorphism groups of simply and multiply connected domains in the complex plain, in which the quasiconformal automorphism groups are endowed with the supremum metric on the underlying domain. More precisely, questions concerning central topological properties such as (local) compactness, (path)-connectedness and separability and their dependence on the boundary of the corresponding domains are studied, as well as completeness with respect to the supremum metric. Moreover, special subsets of the quasiconformal automorphism group of the unit disk are investigated, and concrete quasiconformal automorphisms are constructed. Finally, a possible application of quasiconformal unit disk automorphisms to symmetric cryptography is presented, in which a quasiconformal cryptosystem is defined and studied.
Trust carries the capacity to shift the focus from risks to opportunities of a situation. Scientific studies from the field of trust research point out that besides situation-specific factors (i.e., stimuli of the environment), cross-situationally stable interindividual differences (i.e., personality) are involved in the emergence of trust. Stable interindividual differences are particularly influential to the subjective experience of situational conditions when crucial information is incomplete. The online shopping environment classifies as a prime example of markets with asymmetric information. Research has examined online consumer trust in the light of signaling theory to understand the effects of trust-enhancing signals. Previous research largely neglects interindividual differences in the perception, processing and reaction to these signals. Against this background, this scientific work has two primary objectives: the investigation of (1) interindividual differences in the evaluation of trust-enhancing signals and (2) a personality-based personalization of trust-enhancing signals in its effect on cognition and behavior. For this purpose, an interactive online shop setup was created, which served as realistic environmental framework. First, the results show a trust-enhancing effect of both objective and subjective personalization, with a superiority of subjective over objective personalization. Second, results suggest a particular susceptibility of the beliefs component of trust. Third, the results suggest that personalization exerts a specifically strong effect in what is, by definition, the particularly uncertain environment of credence goods. Fourth, results indicate that while the trust-enhancing effects of personalization operate (largely) independently of personality, the effect of personality on trust seems to depend on the condition of signal presentation. Taken together, the present work makes a contribution to understanding the effect of personality-adapted signaling environments on the emergence of trust and decision making in the specific context of B2C e-commerce.
This work illustrates how the targeted tailoring of supramolecular cavities can not only accomplish high binding due to optimized stereoelectronic shape matches between host and guest but also how molecular engineering of the binding site by a refined substitution periphery of the cavity makes enantiospecific guest recognition and host mediated chirality transfer feasible. Moreover, an enzyme mimic, following the Pauling-Jencks model of enzyme catalysis was realized by the smart design of a PBI host composed of moderately twisted chromophores, which drives the substrate inversion according to the concepts of transition state stabilization and ground state destabilization. The results of this thesis contribute to a better understanding of structure-specific interactions in host-guest complexes as well as the corresponding thermodynamic and kinetic properties and represent an appealing blueprint for the design of new artificial complex structures of high stereoelectronic shape complementarity in order to achieve the goal of sophisticated supramolecular receptors and enzyme mimicry.
Learning accompanies us throughout our lives, from early childhood education through
school, training and university to learning at work. However, much of what we learn is quickly
forgotten. The use of practice tests is a learning strategy that contributes to the acquisition of
sustainable knowledge, i.e. knowledge that is permanently available and can be retrieved when
it is needed. This dissertation first presents findings from previous research on testing in real
educational contexts and discusses theoretically why certain learner or situational
characteristics might influence the effectiveness of the testing effect. Furthermore, a cycle of
three experiments is presented, which were used to investigate whether the positive effect of
practice tests on retention (testing effect) depends on personal or situational characteristics and
also promotes the retention of lecture content that was not directly tested (transfer) in the context
of regular psychology lectures in teacher training courses. In an additional chapter, feedback
from students on the implementation of the study in the classroom context is examined in more
detail. Finally, the results of the three studies are discussed and placed in relation to the theories
presented. The central conclusion from the studies presented is that the testing effect appears to
be a very effective learning strategy that can be used effectively in university teaching and leads
to better learning outcomes regardless of learner characteristics. However, the practice tests
should cover the entire range of relevant content, as transfer effects to non-tested content are
not to be expected.
Anxiety patients overgeneralize fear, also because of an inability to perceptually discriminate threat and safety signals. Therefore, some studies have developed discrimination training that successfully reduced the occurrence of fear generalization. The present work is the first to take a treatment-like approach by using discrimination training after generalization has occurred. Therefore, two studies were conducted with healthy participants using the same fear conditioning and generalization paradigm, with two faces as conditioned stimuli (CSs), and four facial morphs between CSs as generalization stimuli (GSs). Only one face (CS+) was followed by a loud scream (unconditioned stimulus, US). In Study 1, participants underwent either fear-relevant (discriminating faces) or fear-irrelevant discrimination training (discriminating width of lines) or a non-discriminative control training between the two generalization tests, each with or without feedback (n = 20 each). Generalization of US expectancy was reduced more effectively by fear-relevant compared to fear-irrelevant discrimination training. However, neither discrimination training was more effective than non-discriminative control training. Moreover, feedback reduced generalization of US expectancy only in discrimination training. Study 2 was designed to replicate the effects of the discrimination-training conditions in a large sample (N = 244) and examine their benefits in individuals at risk for anxiety disorders. Again, feedback reduced fear generalization particularly well for US expectancy. Fear relevance was not confirmed to be particularly fear-reducing in healthy participants, but may enhance training effects in individuals at risk of anxiety disorder. In summary, this work provides evidence that existing fear generalization can be reduced by discrimination training, likely involving several (higher-level) processes besides perceptual discrimination (e.g., motivational mechanisms in feedback conditions). Its use may be promising as part of individualized therapy for patients with difficulty discriminating similar stimuli.
Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells is highly effective in haematological malignancies. This success, however, has not been achieved in solid tumours so far. In contrast to hematologic malignancies, solid tumours include a hostile tumour microenvironment (TME), that poses additional challenges for curative effects and consistent therapeutic outcome. These challenges manifest in physical and immunological barriers that dampen efficacy of the CAR T cells. Preclinical testing of novel cellular immunotherapies is performed mainly in 2D cell culture and animal experiments. While 2D cell culture is an easy technique for efficacy analysis, animal studies reveal information about toxicity in vivo. However, 2D cell culture cannot fully reflect the complexity observed in vivo, because cells are cultured without anchorage to a matrix and only short-term periods are feasible. Animal studies provide a more complex tissue environment, but xenografts often lack human stroma and tumour inoculation occurs mostly ectopically. This emphasises the need for standardisable and scalable tumour models with incorporated TME-aspects, which enable preclinical testing with enhanced predictive value for the clinical outcome of immunotherapies. Therefore, microphysiologic 3D tumour models based on the biological SISmuc (Small Intestinal mucosa and Submucosa) matrix with preserved basement membrane were engaged and improved in this work to serve as a modular and versatile tumour model for efficacy testing of CAR T cells. In order to reflect a variety of cancer entities, TME-aspects, long-term stability and to enhance the read-out options they were further adapted to achieve scalable and standardisable defined microphysiologic 3D tumour models. In this work, novel culture modalities (semi-static, sandwich-culture) were characterised and established that led to an increased and organised tissue generation and long-term stability. Application of the SISmuc matrix was extended to sarcoma and melanoma models and serial bioluminescence intensity (BLI)-based in vivo imaging analysis was established in the microphysiologic 3D tumour models, which represents a time-efficient read-out method for quality evaluation of the models and treatment efficacy analysis, that is independent of the cell phenotype. Isolation of cancer-associated-fibroblasts (CAFs) from lung (tumour) tissue was demonstrated and CAF-implementation further led to stromal-enriched microphysiologic 3D tumour models with in vivo-comparable tissue-like architecture. Presence of CAFs was confirmed by CAF-associated markers (FAP, α-SMA, MMP-2/-9) and cytokines correlated with CAF phenotype, angiogenesis, invasion and immunomodulation. Additionally, an endothelial cell barrier was implemented for static and dynamic culture in a novel bioreactor set-up, which is of particular interest for the analysis of immune cell diapedesis. Studies in microphysiologic 3D Ewing’s sarcoma models indicated that sarcoma cells could be sensitised for GD2-targeting CAR T cells. After enhancing the scale of assessment of the microphysiologic 3D tumour models and improving them for CAR T cell testing, the tumour models were used to analyse their sensitivity towards differently designed receptor tyrosine kinase-like orphan receptor 1 (ROR1) CAR T cells and to study the effects of the incorporated TME-aspects on the CAR T cell treatment respectively. ROR1 has been described as a suitable target for several malignancies including triple negative breast cancer (TNBC), as well as lung cancer. Therefore, microphysiologic 3D TNBC and lung cancer models were established. Analysis of ROR1 CAR T cells that differed in costimulation, spacer length and targeting domain, revealed, that the microphysiologic 3D tumour models are highly sensitive and can distinguish optimal from sub-optimal CAR design. Here, higher affinity of the targeting domain induced stronger anti-tumour efficacy and anti-tumour function depended on spacer length, respectively. Long-term treatment for 14 days with ROR1 CAR T cells was demonstrated in dynamic microphysiologic 3D lung tumour models, which did not result in complete tumour cell removal, whereas direct injection of CAR T cells into TNBC and lung tumour models represented an alternative route of application in addition to administration via the medium flow, as it induced strong anti-tumour response. Influence of the incorporated TME-aspects on ROR1 CAR T cell therapy represented by CAF-incorporation and/or TGF-β supplementation was analysed. Presence of TGF-β revealed that the specific TGF-β receptor inhibitor SD-208 improves ROR1 CAR T cell function, because it effectively abrogated immunosuppressive effects of TGF-β in TNBC models. Implementation of CAFs should provide a physical and immunological barrier towards ROR1 CAR T cells, which, however, was not confirmed, as ROR1 CAR T cell function was retained in the presence of CAFs in stromal-enriched microphysiologic 3D lung tumour models. The absence of an effect of CAF enrichment on CAR T cell efficacy suggests a missing component for the development of an immunosuppressive TME, even though immunomodulatory cytokines were detected in co-culture models. Finally, improved gene-edited ROR1 CAR T cells lacking exhaustion-associated genes (PD-1, TGF-β-receptor or both) were challenged by the combination of CAF-enrichment and TGF-β in microphysiologic 3D TNBC models. Results indicated that the absence of PD-1 and TGF-β receptor leads to improved CAR T cells, that induce strong tumour cell lysis, and are protected against the hostile TME. Collectively, the microphysiologic 3D tumour models presented in this work reflect aspects of the hostile TME of solid tumours, engage BLI-based analysis and provide long-term tissue homeostasis. Therefore, they present a defined, scalable, reproducible, standardisable and exportable model for translational research with enhanced predictive value for efficacy testing and candidate selection of cellular immunotherapy, as exemplified by ROR1 CAR T cells.