@misc{23183, abstract = {Cooperative verification is gaining momentum in recent years. The usual setup in cooperative verification is that verifier A is run with some pre-defined resources, and if it is not able to verify the program, the verification task is passed to verifier B together with information learned about the program by the verifier A, then the chain can continue to verifier C, and so on. This scheme, which is employed by most of cooperative verification tools, is static and sequential: tools run one after another in a fixed pre-defined order and fixed parameters and resource limits. Bubaak is a portfolio-based program analysis tool for verification of C programs that run multiple program verifiers in parallel and allows to exchange verification artifacts between them on run-time. Moreover, the new version of Bubaak allows to combine sequential and parallel portfolios into workflows. In brief, one or more verification tasks are initially started and each of the tasks, when it is done (e.g., because of hitting a time limit or finishing its job), can rewrite itself into a new task or generate a set of new verification tasks. This all happens dynamically based on the result of the task that finished. This artifact contains Bubaak and Bubaak-SpLit (which is a specific configuration for Bubaak) that competed in SV-COMP 2024.}, author = {Chalupa, Marek}, publisher = {Zenodo}, title = {{Bubaak at SV-COMP 2024}}, doi = {10.5281/zenodo.10065866}, year = {2023}, } @phdthesis{12491, abstract = {The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions.}, author = {Zens, Bettina}, isbn = {978-3-99078-027-5}, issn = {2663-337X}, keywords = {cryo-EM, cryo-ET, FIB milling, method development, FIBSEM, extracellular matrix, ECM, cell-derived matrices, CDMs, cell culture, high pressure freezing, HPF, structural biology, tomography, collagen}, pages = {187}, publisher = {Institute of Science and Technology Austria}, title = {{Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography}}, doi = {10.15479/at:ista:12491}, year = {2023}, } @phdthesis{13984, abstract = {Social insects fight disease using their individual immune systems and the cooperative sanitary behaviors of colony members. These social defenses are well explored against externally-infecting pathogens, but little is known about defense strategies against internally-infecting pathogens, such as viruses. Viruses are ubiquitous and in the last decades it has become evident that also many ant species harbor viruses. We present one of the first studies addressing transmission dynamics and collective disease defenses against viruses in ants on a mechanistic level. I successfully established an experimental ant host – viral pathogen system as a model for the defense strategies used by social insects against internal pathogen infections, as outlined in the third chapter. In particular, we studied how garden ants (Lasius neglectus) defend themselves and their colonies against the generalist insect virus CrPV (cricket paralysis virus). We chose microinjections of virus directly into the ants’ hemolymph because it allowed us to use a defined exposure dose. Here we show that this is a good model system, as the virus is replicating and thus infecting the host. The ants mount a clear individual immune response against the viral infection, which is characterized by a specific siRNA pattern, namely siRNAs mapping against the viral genome with a peak of 21 and 22 bp long fragments. The onset of this immune response is consistent with the timeline of viral replication that starts already within two days post injection. The disease manifests in decreased survival over a course of two to three weeks. Regarding group living, we find that infected ants show a strong individual immune response, but that their course of disease is little affected by nestmate presence, as described in chapter four. Hence, we do not find social immunity in the context of viral infections in ants. Nestmates, however, can contract the virus. Using Drosophila S2R+ cells in culture, we showed that 94 % of the nestmates contract active virus within four days of social contact to an infected individual. Virus is transmitted in low doses, thus not causing disease transmission within the colony. While virus can be transmitted during short direct contacts, we also assume transmission from deceased ants and show that the nestmates’ immune system gets activated after contracting a low viral dose. We find considerable potential for indirect transmission via the nest space. Virus is shed to the nest, where it stays viable for one week and is also picked up by other ants. Apart from that, we want to underline the potential of ant poison as antiviral agent. We determined that ant poison successfully inactivates CrPV in vitro. However, we found no evidence for effective poison use to sanitize the nest space. On the other hand, local application of ant poison by oral poison uptake, which is part of the ants prophylactic behavioral repertoire, probably contributes to keeping the gut of each individual sanitized. We hypothesize that oral poison uptake might be the reason why we did not find viable virus in the trophallactic fluid. The fifth chapter encompasses preliminary data on potential social immunization. However, our experiments do not confirm an actual survival benefit for the nestmates upon pathogen challenge under the given experimental settings. Nevertheless, we do not want to rule out the possibility for nestmate immunization, but rather emphasize that considering different experimental timelines and viral doses would provide a multitude of options for follow-up experiments. In conclusion, we find that prophylactic individual behaviors, such as oral poison uptake, might play a role in preventing viral disease transmission. Compared to colony defense against external pathogens, internal pathogen infections require a stronger component of individual physiological immunity than behavioral social immunity, yet could still lead to collective protection.}, author = {Franschitz, Anna}, isbn = {978-3-99078-034-3}, issn = {2663-337X}, pages = {89}, publisher = {Institute of Science and Technology Austria}, title = {{Individual and social immunity against viral infections in ants}}, doi = {10.15479/at:ista:13984}, year = {2023}, } @phdthesis{12897, abstract = {Inverse design problems in fabrication-aware shape optimization are typically solved on discrete representations such as polygonal meshes. This thesis argues that there are benefits to treating these problems in the same domain as human designers, namely, the parametric one. One reason is that discretizing a parametric model usually removes the capability of making further manual changes to the design, because the human intent is captured by the shape parameters. Beyond this, knowledge about a design problem can sometimes reveal a structure that is present in a smooth representation, but is fundamentally altered by discretizing. In this case, working in the parametric domain may even simplify the optimization task. We present two lines of research that explore both of these aspects of fabrication-aware shape optimization on parametric representations. The first project studies the design of plane elastic curves and Kirchhoff rods, which are common mathematical models for describing the deformation of thin elastic rods such as beams, ribbons, cables, and hair. Our main contribution is a characterization of all curved shapes that can be attained by bending and twisting elastic rods having a stiffness that is allowed to vary across the length. Elements like these can be manufactured using digital fabrication devices such as 3d printers and digital cutters, and have applications in free-form architecture and soft robotics. We show that the family of curved shapes that can be produced this way admits geometric description that is concise and computationally convenient. In the case of plane curves, the geometric description is intuitive enough to allow a designer to determine whether a curved shape is physically achievable by visual inspection alone. We also present shape optimization algorithms that convert a user-defined curve in the plane or in three dimensions into the geometry of an elastic rod that will naturally deform to follow this curve when its endpoints are attached to a support structure. Implemented in an interactive software design tool, the rod geometry is generated in real time as the user edits a curve and enables fast prototyping. The second project tackles the problem of general-purpose shape optimization on CAD models using a novel variant of the extended finite element method (XFEM). Our goal is the decoupling between the simulation mesh and the CAD model, so no geometry-dependent meshing or remeshing needs to be performed when the CAD parameters change during optimization. This is achieved by discretizing the embedding space of the CAD model, and using a new high-accuracy numerical integration method to enable XFEM on free-form elements bounded by the parametric surface patches of the model. Our simulation is differentiable from the CAD parameters to the simulation output, which enables us to use off-the-shelf gradient-based optimization procedures. The result is a method that fits seamlessly into the CAD workflow because it works on the same representation as the designer, enabling the alternation of manual editing and fabrication-aware optimization at will.}, author = {Hafner, Christian}, isbn = {978-3-99078-031-2}, issn = {2663-337X}, pages = {180}, publisher = {Institute of Science and Technology Austria}, title = {{Inverse shape design with parametric representations: Kirchhoff Rods and parametric surface models}}, doi = {10.15479/at:ista:12897}, year = {2023}, } @article{13188, abstract = {The Kirchhoff rod model describes the bending and twisting of slender elastic rods in three dimensions, and has been widely studied to enable the prediction of how a rod will deform, given its geometry and boundary conditions. In this work, we study a number of inverse problems with the goal of computing the geometry of a straight rod that will automatically deform to match a curved target shape after attaching its endpoints to a support structure. Our solution lets us finely control the static equilibrium state of a rod by varying the cross-sectional profiles along its length. We also show that the set of physically realizable equilibrium states admits a concise geometric description in terms of linear line complexes, which leads to very efficient computational design algorithms. Implemented in an interactive software tool, they allow us to convert three-dimensional hand-drawn spline curves to elastic rods, and give feedback about the feasibility and practicality of a design in real time. We demonstrate the efficacy of our method by designing and manufacturing several physical prototypes with applications to interior design and soft robotics.}, author = {Hafner, Christian and Bickel, Bernd}, issn = {1557-7368}, journal = {ACM Transactions on Graphics}, keywords = {Computer Graphics, Computational Design, Computational Geometry, Shape Modeling}, number = {5}, publisher = {Association for Computing Machinery}, title = {{The design space of Kirchhoff rods}}, doi = {10.1145/3606033}, volume = {42}, year = {2023}, } @phdthesis{14280, abstract = {Cell division in Escherichia coli is performed by the divisome, a multi-protein complex composed of more than 30 proteins. The divisome spans from the cytoplasm through the inner membrane to the cell wall and the outer membrane. Divisome assembly is initiated by a cytoskeletal structure, the so-called Z-ring, which localizes at the center of the E. coli cell and determines the position of the future cell septum. The Z-ring is composed of the highly conserved bacterial tubulin homologue FtsZ, which forms treadmilling filaments. These filaments are recruited to the inner membrane by FtsA, a highly conserved bacterial actin homologue. FtsA interacts with other proteins in the periplasm and thus connects the cytoplasmic and periplasmic components of the divisome. A previous model postulated that FtsA regulates maturation of the divisome by switching from an oligomeric, inactive state to a monomeric and active state. This model was based mostly on in vivo studies, as a biochemical characterization of FtsA has been hampered by difficulties in purifying the protein. Here, we studied FtsA using an in vitro reconstitution approach and aimed to answer two questions: (i) How are dynamics from cytoplasmic, treadmilling FtsZ filaments coupled to proteins acting in the periplasmic space and (ii) How does FtsA regulate the maturation of the divisome? We found that the cytoplasmic peptides of the transmembrane proteins FtsN and FtsQ interact directly with FtsA and can follow the spatiotemporal signal of FtsA/Z filaments. When we investigated the underlying mechanism by imaging single molecules of FtsNcyto, we found the peptide to interact transiently with FtsA. An in depth analysis of the single molecule trajectories helped to postulate a model where PG synthases follow the dynamics of FtsZ by a diffusion and capture mechanism. Following up on these findings we were interested in how the self-interaction of FtsA changes when it encounters FtsNcyto and if we can confirm the proposed oligomer-monomer switch. For this, we compared the behavior of the previously identified, hyperactive mutant FtsA R286W with wildtype FtsA. The mutant outperforms WT in mirroring and transmitting the spatiotemporal signal of treadmilling FtsZ filaments. Surprisingly however, we found that this was not due to a difference in the self-interaction strength of the two variants, but a difference in their membrane residence time. Furthermore, in contrast to our expectations, upon binding of FtsNcyto the measured self-interaction of FtsA actually increased. We propose that FtsNcyto induces a rearrangement of the oligomeric architecture of FtsA. In further consequence this change leads to more persistent FtsZ filaments which results in a defined signalling zone, allowing formation of the mature divisome. The observed difference between FtsA WT and R286W is due to the vastly different membrane turnover of the proteins. R286W cycles 5-10x faster compared to WT which allows to sample FtsZ filaments at faster frequencies. These findings can explain the observed differences in toxicity for overexpression of FtsA WT and R286W and help to understand how FtsA regulates divisome maturation.}, author = {Radler, Philipp}, isbn = {978-3-99078-033-6}, issn = {2663-337X}, keywords = {Cell Division, Reconstitution, FtsZ, FtsA, Divisome, E.coli}, pages = {156}, publisher = {Institute of Science and Technology Austria}, title = {{Spatiotemporal signaling during assembly of the bacterial divisome}}, doi = {10.15479/at:ista:14280}, year = {2023}, } @phdthesis{12964, abstract = {Pattern formation is of great importance for its contribution across different biological behaviours. During developmental processes for example, patterns of chemical gradients are established to determine cell fate and complex tissue patterns emerge to define structures such as limbs and vascular networks. Patterns are also seen in collectively migrating groups, for instance traveling waves of density emerging in moving animal flocks as well as collectively migrating cells and tissues. To what extent these biological patterns arise spontaneously through the local interaction of individual constituents or are dictated by higher level instructions is still an open question however there is evidence for the involvement of both types of process. Where patterns arise spontaneously there is a long standing interest in how far the interplay of mechanics, e.g. force generation and deformation, and chemistry, e.g. gene regulation and signaling, contributes to the behaviour. This is because many systems are able to both chemically regulate mechanical force production and chemically sense mechanical deformation, forming mechano-chemical feedback loops which can potentially become unstable towards spatio and/or temporal patterning. We work with experimental collaborators to investigate the possibility that this type of interaction drives pattern formation in biological systems at different scales. We focus first on tissue-level ERK-density waves observed during the wound healing response across different systems where many previous studies have proposed that patterns depend on polarized cell migration and arise from a mechanical flocking-like mechanism. By combining theory with mechanical and optogenetic perturbation experiments on in vitro monolayers we instead find evidence for mechanochemical pattern formation involving only scalar bilateral feedbacks between ERK signaling and cell contraction. We perform further modeling and experiment to study how this instability couples with polar cell migration in order to produce a robust and efficient wound healing response. In a following chapter we implement ERK-density coupling and cell migration in a 2D active vertex model to investigate the interaction of ERK-density patterning with different tissue rheologies and find that the spatio-temporal dynamics are able to both locally and globally fluidize a tissue across the solid-fluid glass transition. In a last chapter we move towards lower spatial scales in the context of subcellular patterning of the cell cytoskeleton where we investigate the transition between phases of spatially homogeneous temporal oscillations and chaotic spatio-temporal patterning in the dynamics of myosin and ROCK activities (a motor component of the actomyosin cytoskeleton and its activator). Experimental evidence supports an intrinsic chemical oscillator which we encode in a reaction model and couple to a contractile active gel description of the cell cortex. The model exhibits phases of chemical oscillations and contractile spatial patterning which reproduce many features of the dynamics seen in Drosophila oocyte epithelia in vivo. However, additional pharmacological perturbations to inhibit myosin contractility leaves the role of contractile instability unclear. We discuss alternative hypotheses and investigate the possibility of reaction-diffusion instability.}, author = {Boocock, Daniel R}, isbn = {978-3-99078-032-9}, issn = {2663-337X}, pages = {146}, publisher = {Institute of Science and Technology Austria}, title = {{Mechanochemical pattern formation across biological scales}}, doi = {10.15479/at:ista:12964}, year = {2023}, } @article{12802, abstract = {Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.}, author = {Knaus, Lisa and Basilico, Bernadette and Malzl, Daniel and Gerykova Bujalkova, Maria and Smogavec, Mateja and Schwarz, Lena A. and Gorkiewicz, Sarah and Amberg, Nicole and Pauler, Florian and Knittl-Frank, Christian and Tassinari, Marianna and Maulide, Nuno and Rülicke, Thomas and Menche, Jörg and Hippenmeyer, Simon and Novarino, Gaia}, issn = {0092-8674}, journal = {Cell}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {9}, pages = {1950--1967.e25}, publisher = {Elsevier}, title = {{Large neutral amino acid levels tune perinatal neuronal excitability and survival}}, doi = {10.1016/j.cell.2023.02.037}, volume = {186}, year = {2023}, } @phdthesis{12809, abstract = {Understanding the mechanisms of learning and memory formation has always been one of the main goals in neuroscience. Already Pavlov (1927) in his early days has used his classic conditioning experiments to study the neural mechanisms governing behavioral adaptation. What was not known back then was that the part of the brain that is largely responsible for this type of associative learning is the cerebellum. Since then, plenty of theories on cerebellar learning have emerged. Despite their differences, one thing they all have in common is that learning relies on synaptic and intrinsic plasticity. The goal of my PhD project was to unravel the molecular mechanisms underlying synaptic plasticity in two synapses that have been shown to be implicated in motor learning, in an effort to understand how learning and memory formation are processed in the cerebellum. One of the earliest and most well-known cerebellar theories postulates that motor learning largely depends on long-term depression at the parallel fiber-Purkinje cell (PC-PC) synapse. However, the discovery of other types of plasticity in the cerebellar circuitry, like long-term potentiation (LTP) at the PC-PC synapse, potentiation of molecular layer interneurons (MLIs), and plasticity transfer from the cortex to the cerebellar/ vestibular nuclei has increased the popularity of the idea that multiple sites of plasticity might be involved in learning. Still a lot remains unknown about the molecular mechanisms responsible for these types of plasticity and whether they occur during physiological learning. In the first part of this thesis we have analyzed the variation and nanodistribution of voltagegated calcium channels (VGCCs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type glutamate receptors (AMPARs) on the parallel fiber-Purkinje cell synapse after vestibuloocular reflex phase reversal adaptation, a behavior that has been suggested to rely on PF-PC LTP. We have found that on the last day of adaptation there is no learning trace in form of VGCCs nor AMPARs variation at the PF-PC synapse, but instead a decrease in the number of PF-PC synapses. These data seem to support the view that learning is only stored in the cerebellar cortex in an initial learning phase, being transferred later to the vestibular nuclei. Next, we have studied the role of MLIs in motor learning using a relatively simple and well characterized behavioral paradigm – horizontal optokinetic reflex (HOKR) adaptation. We have found behavior-induced MLI potentiation in form of release probability increase that could be explained by the increase of VGCCs at the presynaptic side. Our results strengthen the idea of distributed cerebellar plasticity contributing to learning and provide a novel mechanism for release probability increase. }, author = {Alcarva, Catarina}, issn = {2663-337X}, pages = {115}, publisher = {Institute of Science and Technology Austria}, title = {{Plasticity in the cerebellum: What molecular mechanisms are behind physiological learning}}, doi = {10.15479/at:ista:12809}, year = {2023}, } @phdthesis{12781, abstract = {Most energy in humans is produced in form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). Complex I is the first and the largest enzyme of the respiratory chain which is essential for energy production. It couples the transfer of two electrons from NADH to ubiquinone with proton translocation across bacterial or inner mitochondrial membrane. The coupling mechanism between electron transfer and proton translocation is one of the biggest enigma in bioenergetics and structural biology. Even though the enzyme has been studied for decades, only recent technological advances in cryo-EM allowed its extensive structural investigation. Complex I from E.coli appears to be of special importance because it is a perfect model system with a rich mutant library, however the structure of the entire complex was unknown. In this thesis I have resolved structures of the minimal complex I version from E. coli in different states including reduced, inhibited, under reaction turnover and several others. Extensive structural analyses of these structures and comparison to structures from other species allowed to derive general features of conformational dynamics and propose a universal coupling mechanism. The mechanism is straightforward, robust and consistent with decades of experimental data available for complex I from different species. Cyanobacterial NDH (cyanobacterial complex I) is a part of broad complex I superfamily and was studied as well in this thesis. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Here, I solved structure of NDH and revealed additional state, which was not observed before. The novel “resting” state allowed to propose the mechanism of CET regulation. Moreover, conformational dynamics of NDH resembles one in complex I which suggest more broad universality of the proposed coupling mechanism. In summary, results presented here helped to interpret decades of experimental data for complex I and contributed to fundamental mechanistic understanding of protein function. }, author = {Kravchuk, Vladyslav}, isbn = {978-3-99078-029-9}, issn = {2663-337X}, pages = {127}, publisher = {Institute of Science and Technology Austria}, title = {{Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog}}, doi = {10.15479/at:ista:12781}, year = {2023}, } @phdthesis{12891, abstract = {The tight spatiotemporal coordination of signaling activity determining embryo patterning and the physical processes driving embryo morphogenesis renders embryonic development robust, such that key developmental processes can unfold relatively normally even outside of the full embryonic context. For instance, embryonic stem cell cultures can recapitulate the hallmarks of gastrulation, i.e. break symmetry leading to germ layer formation and morphogenesis, in a very reduced environment. This leads to questions on specific contributions of embryo-specific features, such as the presence of extraembryonic tissues, which are inherently involved in gastrulation in the full embryonic context. To address this, we established zebrafish embryonic explants without the extraembryonic yolk cell, an important player as a signaling source and for morphogenesis during gastrulation, as a model of ex vivo development. We found that dorsal-marginal determinants are required and sufficient in these explants to form and pattern all three germ layers. However, formation of tissues, which require the highest Nodal-signaling levels, is variable, demonstrating a contribution of extraembryonic tissues for reaching peak Nodal signaling levels. Blastoderm explants also undergo gastrulation-like axis elongation. We found that this elongation movement shows hallmarks of oriented mesendoderm cell intercalations typically associated with dorsal tissues in the intact embryo. These are disrupted by uniform upregulation of BMP signaling activity and concomitant explant ventralization, suggesting that tight spatial control of BMP signaling is a prerequisite for explant morphogenesis. This control is achieved by Nodal signaling, which is critical for effectively downregulating BMP signaling in the mesendoderm, highlighting that Nodal signaling is not only directly required for mesendoderm cell fate specification and morphogenesis, but also by maintaining low levels of BMP signaling at the dorsal side. Collectively, we provide insights into the capacity and organization of signaling and morphogenetic domains to recapitulate features of zebrafish gastrulation outside of the full embryonic context.}, author = {Schauer, Alexandra}, issn = {2663-337X}, pages = {190}, publisher = {Institute of Science and Technology Austria}, title = {{Mesendoderm formation in zebrafish gastrulation: The role of extraembryonic tissues}}, doi = {10.15479/at:ista:12891}, year = {2023}, } @article{13201, abstract = {As a crucial nitrogen source, nitrate (NO3−) is a key nutrient for plants. Accordingly, root systems adapt to maximize NO3− availability, a developmental regulation also involving the phytohormone auxin. Nonetheless, the molecular mechanisms underlying this regulation remain poorly understood. Here, we identify low-nitrate-resistant mutant (lonr) in Arabidopsis (Arabidopsis thaliana), whose root growth fails to adapt to low-NO3− conditions. lonr2 is defective in the high-affinity NO3− transporter NRT2.1. lonr2 (nrt2.1) mutants exhibit defects in polar auxin transport, and their low-NO3−-induced root phenotype depends on the PIN7 auxin exporter activity. NRT2.1 directly associates with PIN7 and antagonizes PIN7-mediated auxin efflux depending on NO3− levels. These results reveal a mechanism by which NRT2.1 in response to NO3− limitation directly regulates auxin transport activity and, thus, root growth. This adaptive mechanism contributes to the root developmental plasticity to help plants cope with changes in NO3− availability.}, author = {Wang, Yalu and Yuan, Zhi and Wang, Jinyi and Xiao, Huixin and Wan, Lu and Li, Lanxin and Guo, Yan and Gong, Zhizhong and Friml, Jiří and Zhang, Jing}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {25}, publisher = {National Academy of Sciences}, title = {{The nitrate transporter NRT2.1 directly antagonizes PIN7-mediated auxin transport for root growth adaptation}}, doi = {10.1073/pnas.2221313120}, volume = {120}, year = {2023}, } @phdthesis{12531, abstract = {All visual experiences of the vertebrates begin with light being converted into electrical signals by the eye retina. Retinal ganglion cells (RGCs) are the neurons of the innermost layer of the mammal retina, and they transmit visual information to the rest of the brain. It has been shown that RGCs vary in their morphology and genetic profiles, moreover they can be unambiguously grouped into subtypes that share the same morphological and/or molecular properties. However, in terms of RGCs function, it remains unclear how many distinct types there are and what response properties their typology relies on. Even given the recent studies that successfully classified RGCs in a patch of the retina [1] and in scotopic conditions [2], the question remains whether the found subtypes persist across the entire retina. In this work, using a novel imaging method, we show that, when sampled from a large portion of the retina, RGCs can not be clearly divided into functional subtypes. We found that in photopic conditions, which implies more prominent natural scene statistic differences across the visual field, response properties can be exhibited by cells differently depending on their location in the retina, which leads to formation of a gradient of features rather than distinct classes. This finding suggests that RGCs follow a global organization across the visual field of the animal, adapting each RGC subtype to the requirements imposed by the natural scene statistics.}, author = {Kirillova, Kseniia}, issn = {2791-4585}, pages = {46}, publisher = {Institute of Science and Technology Austria}, title = {{Panoramic functional gradients across the mouse retina}}, doi = {10.15479/at:ista:12531}, year = {2023}, } @phdthesis{14697, abstract = {During my Ph.D. research, I managed a series of projects, each focused on the mechanisms underlying cell migration. My work involved an in-depth examination of the complex strategies employed by neutrophils, with a specific focus on their ability to synchronize spatial-temporal cues and optimize their gradient perception. However, it is essential to acknowledge that not all projects yielded successful results, as some ideas were discontinued and are archived for future reference within this thesis. My main project investigated how neutrophils decode spatial cues for precise navigation. Human neutrophils showcased distinct movement patterns based on source type – linear or point-like. By combining single-cell tracking in 3D environments with proxy dyes, this project linked cell behaviors to gradient changes, revealing a stronger response to semi-exponential gradients from point sources. In addition, neutrophils exhibited oscillating migration speeds, using speed minima to adjust trajectories toward sources. Experiencing continuous concentration changes, they accelerated over time and employed a "Run and Fumble" strategy, alternating between consistent runs and strategic "tumbles" for efficient navigation. The project extended to the possibility of cells amplifying perceived gradients by enclosing their immediate surroundings, pushing attractants forward for enrichment while depleting it at the cell rear. Microfluidic devices were employed, and various experimental parameters configurations were optimized. Although significant differences in migratory efficacy were detected across pore sizes and device heights, quantifying gradient manipulation effects proved challenging. The "Laser-Assisted Protein Adsorption by Photobleaching" (LAPAP) project was promising, as it allowed the printing of gradients. Initially successful with dendritic cells, we aimed to adapt it for neutrophils. Through extensive experimentation with multiple parameters, we attempted to trigger responses from neutrophils. Despite these efforts and collaboration, the project failed due to practical challenges and limitations. Facing a lack of neutrophil-like cells at IST, we initially established the SCF-HoxB8 primary murine cell line. Despite their existence, their migratory behavior was largely unexplored due to potential limitations. Through differentiation protocol refinements we enhanced their migratory capabilities, though their capacity still lagged behind human neutrophils. Despite this, the improved migration potential of these cells pointed toward their utility for in vitro murine neutrophil migration studies.}, author = {Stopp, Julian A}, isbn = {978-3-99078-038-1}, issn = {2663-337X}, pages = {226}, publisher = {Institute of Science and Technology Austria}, title = {{Neutrophils on the hunt : Migratory strategies employed by neutrophils to fulfill their effector function}}, doi = {10.15479/at:ista:14697}, year = {2023}, } @article{14360, abstract = {To navigate through diverse tissues, migrating cells must balance persistent self-propelled motion with adaptive behaviors to circumvent obstacles. We identify a curvature-sensing mechanism underlying obstacle evasion in immune-like cells. Specifically, we propose that actin polymerization at the advancing edge of migrating cells is inhibited by the curvature-sensitive BAR domain protein Snx33 in regions with inward plasma membrane curvature. The genetic perturbation of this machinery reduces the cells’ capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. Our results show how cells can read out their surface topography and utilize actin and plasma membrane biophysics to interpret their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR domain proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.}, author = {Sitarska, Ewa and Almeida, Silvia Dias and Beckwith, Marianne Sandvold and Stopp, Julian A and Czuchnowski, Jakub and Siggel, Marc and Roessner, Rita and Tschanz, Aline and Ejsing, Christer and Schwab, Yannick and Kosinski, Jan and Sixt, Michael K and Kreshuk, Anna and Erzberger, Anna and Diz-Muñoz, Alba}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Sensing their plasma membrane curvature allows migrating cells to circumvent obstacles}}, doi = {10.1038/s41467-023-41173-1}, volume = {14}, year = {2023}, } @phdthesis{14323, abstract = {Morphogens are signaling molecules that are known for their prominent role in pattern formation within developing tissues. In addition to patterning, morphogens also control tissue growth. However, the underlying mechanisms are poorly understood. We studied the role of morphogens in regulating tissue growth in the developing vertebrate neural tube. In this system, opposing morphogen gradients of Shh and BMP establish the dorsoventral pattern of neural progenitor domains. Perturbations in these morphogen pathways result in alterations in tissue growth and cell cycle progression, however, it has been unclear what cellular process is affected. To address this, we analysed the rates of cell proliferation and cell death in mouse mutants in which signaling is perturbed, as well as in chick neural plate explants exposed to defined concentrations of signaling activators or inhibitors. Our results indicated that the rate of cell proliferation was not altered in these assays. By contrast, both the Shh and BMP signaling pathways had profound effects on neural progenitor survival. Our results indicate that these pathways synergise to promote cell survival within neural progenitors. Consistent with this, we found that progenitors within the intermediate region of the neural tube, where the combined levels of Shh and BMP are the lowest, are most prone to cell death when signaling activity is inhibited. In addition, we found that downregulation of Shh results in increased apoptosis within the roof plate, which is the dorsal source of BMP ligand production. This revealed a cross-interaction between the Shh and BMP morphogen signaling pathways that may be relevant for understanding how gradients scale in neural tubes with different overall sizes. We further studied the mechanism acting downstream of Shh in cell survival regulation using genetic and genomic approaches. We propose that Shh transcriptionally regulates a non-canonical apoptotic pathway. Altogether, our study points to a novel role of opposing morphogen gradients in tissue size regulation and provides new insights into complex interactions between Shh and BMP signaling gradients in the neural tube.}, author = {Kuzmicz-Kowalska, Katarzyna}, issn = {2663-337X}, pages = {151}, publisher = {Institute of Science and Technology Austria}, title = {{Regulation of neural progenitor survival by Shh and BMP in the developing spinal cord}}, doi = {10.15479/at:ista:14323}, year = {2023}, } @article{12837, abstract = {As developing tissues grow in size and undergo morphogenetic changes, their material properties may be altered. Such changes result from tension dynamics at cell contacts or cellular jamming. Yet, in many cases, the cellular mechanisms controlling the physical state of growing tissues are unclear. We found that at early developmental stages, the epithelium in the developing mouse spinal cord maintains both high junctional tension and high fluidity. This is achieved via a mechanism in which interkinetic nuclear movements generate cell area dynamics that drive extensive cell rearrangements. Over time, the cell proliferation rate declines, effectively solidifying the tissue. Thus, unlike well-studied jamming transitions, the solidification uncovered here resembles a glass transition that depends on the dynamical stresses generated by proliferation and differentiation. Our finding that the fluidity of developing epithelia is linked to interkinetic nuclear movements and the dynamics of growth is likely to be relevant to multiple developing tissues.}, author = {Bocanegra, Laura and Singh, Amrita and Hannezo, Edouard B and Zagórski, Marcin P and Kicheva, Anna}, issn = {1745-2481}, journal = {Nature Physics}, pages = {1050--1058}, publisher = {Springer Nature}, title = {{Cell cycle dynamics control fluidity of the developing mouse neuroepithelium}}, doi = {10.1038/s41567-023-01977-w}, volume = {19}, year = {2023}, } @phdthesis{13081, abstract = {During development, tissues undergo changes in size and shape to form functional organs. Distinct cellular processes such as cell division and cell rearrangements underlie tissue morphogenesis. Yet how the distinct processes are controlled and coordinated, and how they contribute to morphogenesis is poorly understood. In our study, we addressed these questions using the developing mouse neural tube. This epithelial organ transforms from a flat epithelial sheet to an epithelial tube while increasing in size and undergoing morpho-gen-mediated patterning. The extent and mechanism of neural progenitor rearrangement within the developing mouse neuroepithelium is unknown. To investigate this, we per-formed high resolution lineage tracing analysis to quantify the extent of epithelial rear-rangement at different stages of neural tube development. We quantitatively described the relationship between apical cell size with cell cycle dependent interkinetic nuclear migra-tions (IKNM) and performed high cellular resolution live imaging of the neuroepithelium to study the dynamics of junctional remodeling. Furthermore, developed a vertex model of the neuroepithelium to investigate the quantitative contribution of cell proliferation, cell differentiation and mechanical properties to the epithelial rearrangement dynamics and validated the model predictions through functional experiments. Our analysis revealed that at early developmental stages, the apical cell area kinetics driven by IKNM induce high lev-els of cell rearrangements in a regime of high junctional tension and contractility. After E9.5, there is a sharp decline in the extent of cell rearrangements, suggesting that the epi-thelium transitions from a fluid-like to a solid-like state. We found that this transition is regulated by the growth rate of the tissue, rather than by changes in cell-cell adhesion and contractile forces. Overall, our study provides a quantitative description of the relationship between tissue growth, cell cycle dynamics, epithelia rearrangements and the emergent tissue material properties, and novel insights on how epithelial cell dynamics influences tissue morphogenesis.}, author = {Bocanegra, Laura}, issn = {2663-337X}, pages = {93}, publisher = {Institute of Science and Technology Austria}, title = {{Epithelial dynamics during mouse neural tube development}}, doi = {10.15479/at:ista:13081}, year = {2023}, } @phdthesis{12800, abstract = {The evolutionary processes that brought about today’s plethora of living species and the many billions more ancient ones all underlie biology. Evolutionary pathways are neither directed nor deterministic, but rather an interplay between selection, migration, mutation, genetic drift and other environmental factors. Hybrid zones, as natural crossing experiments, offer a great opportunity to use cline analysis to deduce different evolutionary processes - for example, selection strength. Theoretical cline models, largely assuming uniform distribution of individuals, often lack the capability of incorporating population structure. Since in reality organisms mostly live in patchy distributions and their dispersal is hardly ever Gaussian, it is necessary to unravel the effect of these different elements of population structure on cline parameters and shape. In this thesis, I develop a simulation inspired by the A. majus hybrid zone of a single selected locus under frequency dependent selection. This simulation enables us to untangle the effects of different elements of population structure as for example a low-density center and long-range dispersal. This thesis is therefore a first step towards theoretically untangling the effects of different elements of population structure on cline parameters and shape. }, author = {Julseth, Mara}, issn = {2791-4585}, pages = {21}, publisher = {Institute of Science and Technology Austria}, title = {{The effect of local population structure on genetic variation at selected loci in the A. majus hybrid zone}}, doi = {10.15479/at:ista:12800}, year = {2023}, } @phdthesis{14422, abstract = {Animals exhibit a remarkable ability to learn and remember new behaviors, skills, and associations throughout their lifetime. These capabilities are made possible thanks to a variety of changes in the brain throughout adulthood, regrouped under the term "plasticity". Some cells in the brain —neurons— and specifically changes in the connections between neurons, the synapses, were shown to be crucial for the formation, selection, and consolidation of memories from past experiences. These ongoing changes of synapses across time are called synaptic plasticity. Understanding how a myriad of biochemical processes operating at individual synapses can somehow work in concert to give rise to meaningful changes in behavior is a fascinating problem and an active area of research. However, the experimental search for the precise plasticity mechanisms at play in the brain is daunting, as it is difficult to control and observe synapses during learning. Theoretical approaches have thus been the default method to probe the plasticity-behavior connection. Such studies attempt to extract unifying principles across synapses and model all observed synaptic changes using plasticity rules: equations that govern the evolution of synaptic strengths across time in neuronal network models. These rules can use many relevant quantities to determine the magnitude of synaptic changes, such as the precise timings of pre- and postsynaptic action potentials, the recent neuronal activity levels, the state of neighboring synapses, etc. However, analytical studies rely heavily on human intuition and are forced to make simplifying assumptions about plasticity rules. In this thesis, we aim to assist and augment human intuition in this search for plasticity rules. We explore whether a numerical approach could automatically discover the plasticity rules that elicit desired behaviors in large networks of interconnected neurons. This approach is dubbed meta-learning synaptic plasticity: learning plasticity rules which themselves will make neuronal networks learn how to solve a desired task. We first write all the potential plasticity mechanisms to consider using a single expression with adjustable parameters. We then optimize these plasticity parameters using evolutionary strategies or Bayesian inference on tasks known to involve synaptic plasticity, such as familiarity detection and network stabilization. We show that these automated approaches are powerful tools, able to complement established analytical methods. By comprehensively screening plasticity rules at all synapse types in realistic, spiking neuronal network models, we discover entire sets of degenerate plausible plasticity rules that reliably elicit memory-related behaviors. Our approaches allow for more robust experimental predictions, by abstracting out the idiosyncrasies of individual plasticity rules, and provide fresh insights on synaptic plasticity in spiking network models. }, author = {Confavreux, Basile J}, issn = {2663-337X}, pages = {148}, publisher = {Institute of Science and Technology Austria}, title = {{Synapseek: Meta-learning synaptic plasticity rules}}, doi = {10.15479/at:ista:14422}, year = {2023}, }