TY - THES AB - Accumulation of interstitial fluid (IF) between embryonic cells is a common phenomenon in vertebrate embryogenesis. Unlike other model systems, where these accumulations coalesce into a large central cavity – the blastocoel, in zebrafish, IF is more uniformly distributed between the deep cells (DC) before the onset of gastrulation. This is likely due to the presence of a large extraembryonic structure – the yolk cell (YC) at the position where the blastocoel typically forms in other model organisms. IF has long been speculated to play a role in tissue morphogenesis during embryogenesis, but direct evidence supporting such function is still sparse. Here we show that the relocalization of IF to the interface between the YC and DC/epiblast is critical for axial mesendoderm (ME) cell protrusion formation and migration along this interface, a key process in embryonic axis formation. We further demonstrate that axial ME cell migration and IF relocalization engage in a positive feedback loop, where axial ME migration triggers IF accumulation ahead of the advancing axial ME tissue by mechanically compressing the overlying epiblast cell layer. Upon compression, locally induced flow relocalizes the IF through the porous epiblast tissue resulting in an IF accumulation ahead of the leading axial ME. This IF accumulation, in turn, promotes cell protrusion formation and migration of the leading axial ME cells, thereby facilitating axial ME extension. Our findings reveal a central role of dynamic IF relocalization in orchestrating germ layer morphogenesis during gastrulation. AU - Huljev, Karla ID - 9397 SN - 2663-337X TI - Coordinated spatiotemporal reorganization of interstitial fluid is required for axial mesendoderm migration in zebrafish gastrulation ER - TY - THES AB - Left-right asymmetries can be considered a fundamental organizational principle of the vertebrate central nervous system. The hippocampal CA3-CA1 pyramidal cell synaptic connection shows an input-side dependent asymmetry where the hemispheric location of the presynaptic CA3 neuron determines the synaptic properties. Left-input synapses terminating on apical dendrites in stratum radiatum have a higher density of NMDA receptor subunit GluN2B, a lower density of AMPA receptor subunit GluA1 and smaller areas with less often perforated PSDs. On the other hand, left-input synapses terminating on basal dendrites in stratum oriens have lower GluN2B densities than right-input ones. Apical and basal synapses further employ different signaling pathways involved in LTP. SDS-digested freeze-fracture replica labeling can visualize synaptic membrane proteins with high sensitivity and resolution, and has been used to reveal the asymmetry at the electron microscopic level. However, it requires time-consuming manual demarcation of the synaptic surface for quantitative measurements. To facilitate the analysis of replica labeling, I first developed a software named Darea, which utilizes deep-learning to automatize this demarcation. With Darea I characterized the synaptic distribution of NMDA and AMPA receptors as well as the voltage-gated Ca2+ channels in CA1 stratum radiatum and oriens. Second, I explored the role of GluN2B and its carboxy-terminus in the establishment of input-side dependent hippocampal asymmetry. In conditional knock-out mice lacking GluN2B expression in CA1 and GluN2B-2A swap mice, where GluN2B carboxy-terminus was exchanged to that of GluN2A, no significant asymmetries of GluN2B, GluA1 and PSD area were detected. We further discovered a previously unknown functional asymmetry of GluN2A, which was also lost in the swap mouse. These results demonstrate that GluN2B carboxy-terminus plays a critical role in normal formation of input-side dependent asymmetry. AU - Kleindienst, David ID - 9562 SN - 2663-337X TI - 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning ER - TY - THES AB - In this thesis, we consider several of the most classical and fundamental problems in static analysis and formal verification, including invariant generation, reachability analysis, termination analysis of probabilistic programs, data-flow analysis, quantitative analysis of Markov chains and Markov decision processes, and the problem of data packing in cache management. We use techniques from parameterized complexity theory, polyhedral geometry, and real algebraic geometry to significantly improve the state-of-the-art, in terms of both scalability and completeness guarantees, for the mentioned problems. In some cases, our results are the first theoretical improvements for the respective problems in two or three decades. AU - Goharshady, Amir Kafshdar ID - 8934 SN - 2663-337X TI - Parameterized and algebro-geometric advances in static program analysis ER - TY - THES AB - Bacteria-host interactions represent a continuous trade-off between benefit and risk. Thus, the host immune response is faced with a non-trivial problem – accommodate beneficial commensals and remove harmful pathogens. This is especially difficult as molecular patterns, such as lipopolysaccharide or specific surface organelles such as pili, are conserved in both, commensal and pathogenic bacteria. Type 1 pili, tightly regulated by phase variation, are considered an important virulence factor of pathogenic bacteria as they facilitate invasion into host cells. While invasion represents a de facto passive mechanism for pathogens to escape the host immune response, we demonstrate a fundamental role of type 1 pili as active modulators of the innate and adaptive immune response. AU - Tomasek, Kathrin ID - 10307 SN - 2663-337X TI - Pathogenic Escherichia coli hijack the host immune response ER - TY - THES AB - Nitrogen is an essential macronutrient determining plant growth, development and affecting agricultural productivity. Root, as a hub that perceives and integrates local and systemic signals on the plant’s external and endogenous nitrogen resources, communicates with other plant organs to consolidate their physiology and development in accordance with actual nitrogen balance. Over the last years, numerous studies demonstrated that these comprehensive developmental adaptations rely on the interaction between pathways controlling nitrogen homeostasis and hormonal networks acting globally in the plant body. However, molecular insights into how the information about the nitrogen status is translated through hormonal pathways into specific developmental output are lacking. In my work, I addressed so far poorly understood mechanisms underlying root-to-shoot communication that lead to a rapid re-adjustment of shoot growth and development after nitrate provision. Applying a combination of molecular, cell, and developmental biology approaches, genetics and grafting experiments as well as hormonal analytics, I identified and characterized an unknown molecular framework orchestrating shoot development with a root nitrate sensory system. AU - Abualia, Rashed ID - 10303 SN - 2663-337X TI - Role of hormones in nitrate regulated growth ER - TY - THES AB - The brain is one of the largest and most complex organs and it is composed of billions of neurons that communicate together enabling e.g. consciousness. The cerebral cortex is the largest site of neural integration in the central nervous system. Concerted radial migration of newly born cortical projection neurons, from their birthplace to their final position, is a key step in the assembly of the cerebral cortex. The cellular and molecular mechanisms regulating radial neuronal migration in vivo are however still unclear. Recent evidence suggests that distinct signaling cues act cell-autonomously but differentially at certain steps during the overall migration process. Moreover, functional analysis of genetic mosaics (mutant neurons present in wild-type/heterozygote environment) using the MADM (Mosaic Analysis with Double Markers) analyses in comparison to global knockout also indicate a significant degree of non-cell-autonomous and/or community effects in the control of cortical neuron migration. The interactions of cell-intrinsic (cell-autonomous) and cell-extrinsic (non-cell-autonomous) components are largely unknown. In part of this thesis work we established a MADM-based experimental strategy for the quantitative analysis of cell-autonomous gene function versus non-cell-autonomous and/or community effects. The direct comparison of mutant neurons from the genetic mosaic (cell-autonomous) to mutant neurons in the conditional and/or global knockout (cell-autonomous + non-cell-autonomous) allows to quantitatively analyze non-cell-autonomous effects. Such analysis enable the high-resolution analysis of projection neuron migration dynamics in distinct environments with concomitant isolation of genomic and proteomic profiles. Using these experimental paradigms and in combination with computational modeling we show and characterize the nature of non-cell-autonomous effects to coordinate radial neuron migration. Furthermore, this thesis discusses recent developments in neurodevelopment with focus on neuronal polarization and non-cell-autonomous mechanisms in neuronal migration. AU - Hansen, Andi H ID - 9962 KW - Neuronal migration KW - Non-cell-autonomous KW - Cell-autonomous KW - Neurodevelopmental disease SN - 2663-337X TI - Cell-autonomous gene function and non-cell-autonomous effects in radial projection neuron migration ER - TY - THES AB - Plant motions occur across a wide spectrum of timescales, ranging from seed dispersal through bursting (milliseconds) and stomatal opening (minutes) to long-term adaptation of gross architecture. Relatively fast motions include water-driven growth as exemplified by root cell expansion under abiotic/biotic stresses or during gravitropism. A showcase is a root growth inhibition in 30 seconds triggered by the phytohormone auxin. However, the cellular and molecular mechanisms are still largely unknown. This thesis covers the studies about this topic as follows. By taking advantage of microfluidics combined with live imaging, pharmaceutical tools, and transgenic lines, we examined the kinetics of and causal relationship among various auxininduced rapid cellular changes in root growth, apoplastic pH, cytosolic Ca2+, cortical microtubule (CMT) orientation, and vacuolar morphology. We revealed that CMT reorientation and vacuolar constriction are the consequence of growth itself instead of responding directly to auxin. In contrast, auxin induces apoplast alkalinization to rapidly inhibit root growth in 30 seconds. This auxin-triggered apoplast alkalinization results from rapid H+- influx that is contributed by Ca2+ inward channel CYCLIC NUCLEOTIDE-GATED CHANNEL 14 (CNGC14)-dependent Ca2+ signaling. To dissect which auxin signaling mediates the rapid apoplast alkalinization, we combined microfluidics and genetic engineering to verify that TIR1/AFB receptors conduct a non-transcriptional regulation on Ca2+ and H+ -influx. This non-canonical pathway is mostly mediated by the cytosolic portion of TIR1/AFB. On the other hand, we uncovered, using biochemical and phospho-proteomic analysis, that auxin cell surface signaling component TRANSMEMBRANE KINASE 1 (TMK1) plays a negative role during auxin-trigger apoplast alkalinization and root growth inhibition through directly activating PM H+ -ATPases. Therefore, we discovered that PM H+ -ATPases counteract instead of mediate the auxintriggered rapid H+ -influx, and that TIR1/AFB and TMK1 regulate root growth antagonistically. This opposite effect of TIR1/AFB and TMK1 is consistent during auxin-induced hypocotyl elongation, leading us to explore the relation of two signaling pathways. Assisted with biochemistry and fluorescent imaging, we verified for the first time that TIR1/AFB and TMK1 can interact with each other. The ability of TIR1/AFB binding to membrane lipid provides a basis for the interaction of plasma membrane- and cytosol-localized proteins. Besides, transgenic analysis combined with genetic engineering and biochemistry showed that vi they do function in the same pathway. Particularly, auxin-induced TMK1 increase is TIR1/AFB dependent, suggesting TIR1/AFB regulation on TMK1. Conversely, TMK1 also regulates TIR1/AFB protein levels and thus auxin canonical signaling. To follow the study of rapid growth regulation, we analyzed another rapid growth regulator, signaling peptide RALF1. We showed that RALF1 also triggers a rapid and reversible growth inhibition caused by H + influx, highly resembling but not dependent on auxin. Besides, RALF1 promotes auxin biosynthesis by increasing expression of auxin biosynthesis enzyme YUCCAs and thus induces auxin signaling in ca. 1 hour, contributing to the sustained RALF1-triggered growth inhibition. These studies collectively contribute to understanding rapid regulation on plant cell growth, novel auxin signaling pathway as well as auxin-peptide crosstalk. AU - Li, Lanxin ID - 10083 SN - 2663-337X TI - Rapid cell growth regulation in Arabidopsis ER - TY - THES AB - Indirect reciprocity in evolutionary game theory is a prominent mechanism for explaining the evolution of cooperation among unrelated individuals. In contrast to direct reciprocity, which is based on individuals meeting repeatedly, and conditionally cooperating by using their own experiences, indirect reciprocity is based on individuals’ reputations. If a player helps another, this increases the helper’s public standing, benefitting them in the future. This lets cooperation in the population emerge without individuals having to meet more than once. While the two modes of reciprocity are intertwined, they are difficult to compare. Thus, they are usually studied in isolation. Direct reciprocity can maintain cooperation with simple strategies, and is robust against noise even when players do not remember more than their partner’s last action. Meanwhile, indirect reciprocity requires its successful strategies, or social norms, to be more complex. Exhaustive search previously identified eight such norms, called the “leading eight”, which excel at maintaining cooperation. However, as the first result of this thesis, we show that the leading eight break down once we remove the fundamental assumption that information is synchronized and public, such that everyone agrees on reputations. Once we consider a more realistic scenario of imperfect information, where reputations are private, and individuals occasionally misinterpret or miss observations, the leading eight do not promote cooperation anymore. Instead, minor initial disagreements can proliferate, fragmenting populations into subgroups. In a next step, we consider ways to mitigate this issue. We first explore whether introducing “generosity” can stabilize cooperation when players use the leading eight strategies in noisy environments. This approach of modifying strategies to include probabilistic elements for coping with errors is known to work well in direct reciprocity. However, as we show here, it fails for the more complex norms of indirect reciprocity. Imperfect information still prevents cooperation from evolving. On the other hand, we succeeded to show in this thesis that modifying the leading eight to use “quantitative assessment”, i.e. tracking reputation scores on a scale beyond good and bad, and making overall judgments of others based on a threshold, is highly successful, even when noise increases in the environment. Cooperation can flourish when reputations are more nuanced, and players have a broader understanding what it means to be “good.” Finally, we present a single theoretical framework that unites the two modes of reciprocity despite their differences. Within this framework, we identify a novel simple and successful strategy for indirect reciprocity, which can cope with noisy environments and has an analogue in direct reciprocity. We can also analyze decision making when different sources of information are available. Our results help highlight that for sustaining cooperation, already the most simple rules of reciprocity can be sufficient. AU - Schmid, Laura ID - 10293 SN - 2663-337X TI - Evolution of cooperation via (in)direct reciprocity under imperfect information ER - TY - THES AB - Plants maintain the capacity to develop new organs e.g. lateral roots post-embryonically throughout their whole life and thereby flexibly adapt to ever-changing environmental conditions. Plant hormones auxin and cytokinin are the main regulators of the lateral root organogenesis. Additionally to their solo activities, the interaction between auxin and cytokinin plays crucial role in fine-tuning of lateral root development and growth. In particular, cytokinin modulates auxin distribution within the developing lateral root by affecting the endomembrane trafficking of auxin transporter PIN1 and promoting its vacuolar degradation (Marhavý et al., 2011, 2014). This effect is independent of transcription and translation. Therefore, it suggests novel, non-canonical cytokinin activity occuring possibly on the posttranslational level. Impact of cytokinin and other plant hormones on auxin transporters (including PIN1) on the posttranslational level is described in detail in the introduction part of this thesis in a form of a review (Semeradova et al., 2020). To gain insights into the molecular machinery underlying cytokinin effect on the endomembrane trafficking in the plant cell, in particular on the PIN1 degradation, we conducted two large proteomic screens: 1) Identification of cytokinin binding proteins using chemical proteomics. 2) Monitoring of proteomic and phosphoproteomic changes upon cytokinin treatment. In the first screen, we identified DYNAMIN RELATED PROTEIN 2A (DRP2A). We found that DRP2A plays a role in cytokinin regulated processes during the plant growth and that cytokinin treatment promotes destabilization of DRP2A protein. However, the role of DRP2A in the PIN1 degradation remains to be elucidated. In the second screen, we found VACUOLAR PROTEIN SORTING 9A (VPS9A). VPS9a plays crucial role in plant’s response to cytokin and in cytokinin mediated PIN1 degradation. Altogether, we identified proteins, which bind to cytokinin and proteins that in response to cytokinin exhibit significantly changed abundance or phosphorylation pattern. By combining information from these two screens, we can pave our way towards understanding of noncanonical cytokinin effects. AU - Semerádová, Hana ID - 10135 SN - 2663-337X TI - Molecular mechanisms of the cytokinin-regulated endomembrane trafficking to coordinate plant organogenesis ER - TY - THES AB - Most real-world flows are multiphase, yet we know little about them compared to their single-phase counterparts. Multiphase flows are more difficult to investigate as their dynamics occur in large parameter space and involve complex phenomena such as preferential concentration, turbulence modulation, non-Newtonian rheology, etc. Over the last few decades, experiments in particle-laden flows have taken a back seat in favour of ever-improving computational resources. However, computers are still not powerful enough to simulate a real-world fluid with millions of finite-size particles. Experiments are essential not only because they offer a reliable way to investigate real-world multiphase flows but also because they serve to validate numerical studies and steer the research in a relevant direction. In this work, we have experimentally investigated particle-laden flows in pipes, and in particular, examined the effect of particles on the laminar-turbulent transition and the drag scaling in turbulent flows. For particle-laden pipe flows, an earlier study [Matas et al., 2003] reported how the sub-critical (i.e., hysteretic) transition that occurs via localised turbulent structures called puffs is affected by the addition of particles. In this study, in addition to this known transition, we found a super-critical transition to a globally fluctuating state with increasing particle concentration. At the same time, the Newtonian-type transition via puffs is delayed to larger Reynolds numbers. At an even higher concentration, only the globally fluctuating state is found. The dynamics of particle-laden flows are hence determined by two competing instabilities that give rise to three flow regimes: Newtonian-type turbulence at low, a particle-induced globally fluctuating state at high, and a coexistence state at intermediate concentrations. The effect of particles on turbulent drag is ambiguous, with studies reporting drag reduction, no net change, and even drag increase. The ambiguity arises because, in addition to particle concentration, particle shape, size, and density also affect the net drag. Even similar particles might affect the flow dissimilarly in different Reynolds number and concentration ranges. In the present study, we explored a wide range of both Reynolds number and concentration, using spherical as well as cylindrical particles. We found that the spherical particles do not reduce drag while the cylindrical particles are drag-reducing within a specific Reynolds number interval. The interval strongly depends on the particle concentration and the relative size of the pipe and particles. Within this interval, the magnitude of drag reduction reaches a maximum. These drag reduction maxima appear to fall onto a distinct power-law curve irrespective of the pipe diameter and particle concentration, and this curve can be considered as the maximum drag reduction asymptote for a given fibre shape. Such an asymptote is well known for polymeric flows but had not been identified for particle-laden flows prior to this work. AU - Agrawal, Nishchal ID - 9728 KW - Drag Reduction KW - Transition to Turbulence KW - Multiphase Flows KW - particle Laden Flows KW - Complex Flows KW - Experiments KW - Fluid Dynamics SN - 2663-337X TI - Transition to turbulence and drag reduction in particle-laden pipe flows ER -