TY - THES AB - Algorithms in computational 3-manifold topology typically take a triangulation as an input and return topological information about the underlying 3-manifold. However, extracting the desired information from a triangulation (e.g., evaluating an invariant) is often computationally very expensive. In recent years this complexity barrier has been successfully tackled in some cases by importing ideas from the theory of parameterized algorithms into the realm of 3-manifolds. Various computationally hard problems were shown to be efficiently solvable for input triangulations that are sufficiently “tree-like.” In this thesis we focus on the key combinatorial parameter in the above context: we consider the treewidth of a compact, orientable 3-manifold, i.e., the smallest treewidth of the dual graph of any triangulation thereof. By building on the work of Scharlemann–Thompson and Scharlemann–Schultens–Saito on generalized Heegaard splittings, and on the work of Jaco–Rubinstein on layered triangulations, we establish quantitative relations between the treewidth and classical topological invariants of a 3-manifold. In particular, among other results, we show that the treewidth of a closed, orientable, irreducible, non-Haken 3-manifold is always within a constant factor of its Heegaard genus. AU - Huszár, Kristóf ID - 8032 SN - 2663-337X TI - Combinatorial width parameters for 3-dimensional manifolds ER - TY - THES AB - During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This so-called Z-ring acts as a scaffold recruiting several division-related proteins to mid-cell and plays a key role in distributing proteins at the division site, a feature driven by the treadmilling motion of FtsZ filaments around the septum. What regulates the architecture, dynamics and stability of the Z-ring is still poorly understood, but FtsZ-associated proteins (Zaps) are known to play an important role. Advances in fluorescence microscopy and in vitro reconstitution experiments have helped to shed light into some of the dynamic properties of these complex systems, but methods that allow to collect and analyze large quantitative data sets of the underlying polymer dynamics are still missing. Here, using an in vitro reconstitution approach, we studied how different Zaps affect FtsZ filament dynamics and organization into large-scale patterns, giving special emphasis to the role of the well-conserved protein ZapA. For this purpose, we use high-resolution fluorescence microscopy combined with novel image analysis workfows to study pattern organization and polymerization dynamics of active filaments. We quantified the influence of Zaps on FtsZ on three diferent spatial scales: the large-scale organization of the membrane-bound filament network, the underlying polymerization dynamics and the behavior of single molecules. We found that ZapA cooperatively increases the spatial order of the filament network, binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner, without compromising filament dynamics. Furthermore, we believe that our automated quantitative methods can be used to analyze a large variety of dynamic cytoskeletal systems, using standard time-lapse movies of homogeneously labeled proteins obtained from experiments in vitro or even inside the living cell. AU - Dos Santos Caldas, Paulo R ID - 8358 SN - 2663-337X TI - Organization and dynamics of treadmilling filaments in cytoskeletal networks of FtsZ and its crosslinkers ER - TY - THES AB - Designing and verifying concurrent programs is a notoriously challenging, time consuming, and error prone task, even for experts. This is due to the sheer number of possible interleavings of a concurrent program, all of which have to be tracked and accounted for in a formal proof. Inventing an inductive invariant that captures all interleavings of a low-level implementation is theoretically possible, but practically intractable. We develop a refinement-based verification framework that provides mechanisms to simplify proof construction by decomposing the verification task into smaller subtasks. In a first line of work, we present a foundation for refinement reasoning over structured concurrent programs. We introduce layered concurrent programs as a compact notation to represent multi-layer refinement proofs. A layered concurrent program specifies a sequence of connected concurrent programs, from most concrete to most abstract, such that common parts of different programs are written exactly once. Each program in this sequence is expressed as structured concurrent program, i.e., a program over (potentially recursive) procedures, imperative control flow, gated atomic actions, structured parallelism, and asynchronous concurrency. This is in contrast to existing refinement-based verifiers, which represent concurrent systems as flat transition relations. We present a powerful refinement proof rule that decomposes refinement checking over structured programs into modular verification conditions. Refinement checking is supported by a new form of modular, parameterized invariants, called yield invariants, and a linear permission system to enhance local reasoning. In a second line of work, we present two new reduction-based program transformations that target asynchronous programs. These transformations reduce the number of interleavings that need to be considered, thus reducing the complexity of invariants. Synchronization simplifies the verification of asynchronous programs by introducing the fiction, for proof purposes, that asynchronous operations complete synchronously. Synchronization summarizes an asynchronous computation as immediate atomic effect. Inductive sequentialization establishes sequential reductions that captures every behavior of the original program up to reordering of coarse-grained commutative actions. A sequential reduction of a concurrent program is easy to reason about since it corresponds to a simple execution of the program in an idealized synchronous environment, where processes act in a fixed order and at the same speed. Our approach is implemented the CIVL verifier, which has been successfully used for the verification of several complex concurrent programs. In our methodology, the overall correctness of a program is established piecemeal by focusing on the invariant required for each refinement step separately. While the programmer does the creative work of specifying the chain of programs and the inductive invariant justifying each link in the chain, the tool automatically constructs the verification conditions underlying each refinement step. AU - Kragl, Bernhard ID - 8332 SN - 2663-337X TI - Verifying concurrent programs: Refinement, synchronization, sequentialization ER - TY - THES AB - The oft-quoted dictum by Arthur Schawlow: ``A diatomic molecule has one atom too many'' has been disavowed. Inspired by the possibility to experimentally manipulate and enhance chemical reactivity in helium nanodroplets, we investigate the rotation of coupled cold molecules in the presence of a many-body environment. In this thesis, we introduce new variational approaches to quantum impurities and apply them to the Fröhlich polaron - a quasiparticle formed out of an electron (or other point-like impurity) in a polar medium, and to the angulon - a quasiparticle formed out of a rotating molecule in a bosonic bath. With this theoretical toolbox, we reveal the self-localization transition for the angulon quasiparticle. We show that, unlike for polarons, self-localization of angulons occurs at finite impurity-bath coupling already at the mean-field level. The transition is accompanied by the spherical-symmetry breaking of the angulon ground state and a discontinuity in the first derivative of the ground-state energy. Moreover, the type of symmetry breaking is dictated by the symmetry of the microscopic impurity-bath interaction, which leads to a number of distinct self-localized states. For the system containing multiple impurities, by analogy with the bipolaron, we introduce the biangulon quasiparticle describing two rotating molecules that align with respect to each other due to the effective attractive interaction mediated by the excitations of the bath. We study this system from the strong-coupling regime to the weak molecule-bath interaction regime. We show that the molecules tend to have a strong alignment in the ground state, the biangulon shows shifted angulon instabilities and an additional spectral instability, where resonant angular momentum transfer between the molecules and the bath takes place. Finally, we introduce a diagonalization scheme that allows us to describe the transition from two separated angulons to a biangulon as a function of the distance between the two molecules. AU - Li, Xiang ID - 8958 SN - 2663-337X TI - Rotation of coupled cold molecules in the presence of a many-body environment ER - TY - THES AB - Form versus function is a long-standing debate in various design-related fields, such as architecture as well as graphic and industrial design. A good design that balances form and function often requires considerable human effort and collaboration among experts from different professional fields. Computational design tools provide a new paradigm for designing functional objects. In computational design, form and function are represented as mathematical quantities, with the help of numerical and combinatorial algorithms, they can assist even novice users in designing versatile models that exhibit their desired functionality. This thesis presents three disparate research studies on the computational design of functional objects: The appearance of 3d print—we optimize the volumetric material distribution for faithfully replicating colored surface texture in 3d printing; the dynamic motion of mechanical structures— our design system helps the novice user to retarget various mechanical templates with different functionality to complex 3d shapes; and a more abstract functionality, multistability—our algorithm automatically generates models that exhibit multiple stable target poses. For each of these cases, our computational design tools not only ensure the functionality of the results but also permit the user aesthetic freedom over the form. Moreover, fabrication constraints were taken into account, which allow for the immediate creation of physical realization via 3D printing or laser cutting. AU - Zhang, Ran ID - 8386 SN - 2663-337X TI - Structure-aware computational design and its application to 3D printable volume scattering, mechanism, and multistability ER - TY - THES AB - Quantum computation enables the execution of algorithms that have exponential complexity. This might open the path towards the synthesis of new materials or medical drugs, optimization of transport or financial strategies etc., intractable on even the fastest classical computers. A quantum computer consists of interconnected two level quantum systems, called qubits, that satisfy DiVincezo’s criteria. Worldwide, there are ongoing efforts to find the qubit architecture which will unite quantum error correction compatible single and two qubit fidelities, long distance qubit to qubit coupling and calability. Superconducting qubits have gone the furthest in this race, demonstrating an algorithm running on 53 coupled qubits, but still the fidelities are not even close to those required for realizing a single logical qubit. emiconductor qubits offer extremely good characteristics, but they are currently investigated across different platforms. Uniting those good characteristics into a single platform might be a big step towards the quantum computer realization. Here we describe the implementation of a hole spin qubit hosted in a Ge hut wire double quantum dot. The high and tunable spin-orbit coupling together with a heavy hole state character is expected to allow fast spin manipulation and long coherence times. Furthermore large lever arms, for hut wire devices, should allow good coupling to superconducting resonators enabling efficient long distance spin to spin coupling and a sensitive gate reflectometry spin readout. The developed cryogenic setup (printed circuit board sample holders, filtering, high-frequency wiring) enabled us to perform low temperature spin dynamics experiments. Indeed, we measured the fastest single spin qubit Rabi frequencies reported so far, reaching 140 MHz, while the dephasing times of 130 ns oppose the long decoherence predictions. In order to further investigate this, a double quantum dot gate was connected directly to a lumped element resonator which enabled gate reflectometry readout. The vanishing inter-dot transition signal, for increasing external magnetic field, revealed the spin nature of the measured quantity. AU - Kukucka, Josip ID - 7996 SN - 2663-337X TI - Implementation of a hole spin qubit in Ge hut wires and dispersive spin sensing ER - TY - THES AB - Deep neural networks have established a new standard for data-dependent feature extraction pipelines in the Computer Vision literature. Despite their remarkable performance in the standard supervised learning scenario, i.e. when models are trained with labeled data and tested on samples that follow a similar distribution, neural networks have been shown to struggle with more advanced generalization abilities, such as transferring knowledge across visually different domains, or generalizing to new unseen combinations of known concepts. In this thesis we argue that, in contrast to the usual black-box behavior of neural networks, leveraging more structured internal representations is a promising direction for tackling such problems. In particular, we focus on two forms of structure. First, we tackle modularity: We show that (i) compositional architectures are a natural tool for modeling reasoning tasks, in that they efficiently capture their combinatorial nature, which is key for generalizing beyond the compositions seen during training. We investigate how to to learn such models, both formally and experimentally, for the task of abstract visual reasoning. Then, we show that (ii) in some settings, modularity allows us to efficiently break down complex tasks into smaller, easier, modules, thereby improving computational efficiency; We study this behavior in the context of generative models for colorization, as well as for small objects detection. Secondly, we investigate the inherently layered structure of representations learned by neural networks, and analyze its role in the context of transfer learning and domain adaptation across visually dissimilar domains. AU - Royer, Amélie ID - 8390 SN - 2663-337X TI - Leveraging structure in Computer Vision tasks for flexible Deep Learning models ER - TY - THES AB - In this thesis we study certain mathematical aspects of evolution. The two primary forces that drive an evolutionary process are mutation and selection. Mutation generates new variants in a population. Selection chooses among the variants depending on the reproductive rates of individuals. Evolutionary processes are intrinsically random – a new mutation that is initially present in the population at low frequency can go extinct, even if it confers a reproductive advantage. The overall rate of evolution is largely determined by two quantities: the probability that an invading advantageous mutation spreads through the population (called fixation probability) and the time until it does so (called fixation time). Both those quantities crucially depend not only on the strength of the invading mutation but also on the population structure. In this thesis, we aim to understand how the underlying population structure affects the overall rate of evolution. Specifically, we study population structures that increase the fixation probability of advantageous mutants (called amplifiers of selection). Broadly speaking, our results are of three different types: We present various strong amplifiers, we identify regimes under which only limited amplification is feasible, and we propose population structures that provide different tradeoffs between high fixation probability and short fixation time. AU - Tkadlec, Josef ID - 7196 TI - A role of graphs in evolutionary processes ER - TY - THES AB - We present solutions to several problems originating from geometry and discrete mathematics: existence of equipartitions, maps without Tverberg multiple points, and inscribing quadrilaterals. Equivariant obstruction theory is the natural topological approach to these type of questions. However, for the specific problems we consider it had yielded only partial or no results. We get our results by complementing equivariant obstruction theory with other techniques from topology and geometry. AU - Avvakumov, Sergey ID - 8156 SN - 2663-337X TI - Topological methods in geometry and discrete mathematics ER - TY - THES AB - Fabrication of curved shells plays an important role in modern design, industry, and science. Among their remarkable properties are, for example, aesthetics of organic shapes, ability to evenly distribute loads, or efficient flow separation. They find applications across vast length scales ranging from sky-scraper architecture to microscopic devices. But, at the same time, the design of curved shells and their manufacturing process pose a variety of challenges. In this thesis, they are addressed from several perspectives. In particular, this thesis presents approaches based on the transformation of initially flat sheets into the target curved surfaces. This involves problems of interactive design of shells with nontrivial mechanical constraints, inverse design of complex structural materials, and data-driven modeling of delicate and time-dependent physical properties. At the same time, two newly-developed self-morphing mechanisms targeting flat-to-curved transformation are presented. In architecture, doubly curved surfaces can be realized as cold bent glass panelizations. Originally flat glass panels are bent into frames and remain stressed. This is a cost-efficient fabrication approach compared to hot bending, when glass panels are shaped plastically. However such constructions are prone to breaking during bending, and it is highly nontrivial to navigate the design space, keeping the panels fabricable and aesthetically pleasing at the same time. We introduce an interactive design system for cold bent glass façades, while previously even offline optimization for such scenarios has not been sufficiently developed. Our method is based on a deep learning approach providing quick and high precision estimation of glass panel shape and stress while handling the shape multimodality. Fabrication of smaller objects of scales below 1 m, can also greatly benefit from shaping originally flat sheets. In this respect, we designed new self-morphing shell mechanisms transforming from an initial flat state to a doubly curved state with high precision and detail. Our so-called CurveUps demonstrate the encodement of the geometric information into the shell. Furthermore, we explored the frontiers of programmable materials and showed how temporal information can additionally be encoded into a flat shell. This allows prescribing deformation sequences for doubly curved surfaces and, thus, facilitates self-collision avoidance enabling complex shapes and functionalities otherwise impossible. Both of these methods include inverse design tools keeping the user in the design loop. AU - Guseinov, Ruslan ID - 8366 KW - computer-aided design KW - shape modeling KW - self-morphing KW - mechanical engineering SN - 2663-337X TI - Computational design of curved thin shells: From glass façades to programmable matter ER - TY - THES AB - The medial habenula (MHb) is an evolutionary conserved epithalamic structure important for the modulation of emotional memory. It is involved in regulation of anxiety, compulsive behavior, addiction (nicotinic and opioid), sexual and feeding behavior. MHb receives inputs from septal regions and projects exclusively to the interpeduncular nucleus (IPN). Distinct sub-regions of the septum project to different subnuclei of MHb: the bed nucleus of anterior commissure projects to dorsal MHb and the triangular septum projects to ventral MHb. Furthermore, the dorsal and ventral MHb project to the lateral and rostral/central IPN, respectively. Importantly, these projections have unique features of prominent co-release of different neurotransmitters and requirement of a peculiar type of calcium channel for release. In general, synaptic neurotransmission requires an activity-dependent influx of Ca2+ into the presynaptic terminal through voltage-gated calcium channels. The calcium channel family most commonly involved in neurotransmitter release comprises three members, P/Q-, N- and R-type with Cav2.1, Cav2.2 and Cav2.3 subunits, respectively. In contrast to most CNS synapses that mainly express Cav2.1 and/or Cav2.2, MHb terminals in the IPN exclusively express Cav2.3. In other parts of the brain, such as the hippocampus, Cav2.3 is mostly located to postsynaptic elements. This unusual presynaptic location of Cav2.3 in the MHb-IPN pathway implies unique mechanisms of glutamate release in this pathway. One potential example of such uniqueness is the facilitation of release by GABAB receptor (GBR) activation. Presynaptic GBRs usually inhibit the release of neurotransmitters by inhibiting presynaptic calcium channels. MHb shows the highest expression levels of GBR in the brain. GBRs comprise two subunits, GABAB1 (GB1) and GABAB2 (GB2), and are associated with auxiliary subunits, called potassium channel tetramerization domain containing proteins (KCTD) 8, 12, 12b and 16. Among these four subunits, KCTD12b is exclusively expressed in ventral MHb, and KCTD8 shows the strongest expression in the whole MHb among other brain regions, indicating that KCTD8 and KCTD12b may be involved in the unique mechanisms of neurotransmitter release mediated by Cav2.3 and regulated by GBRs in this pathway. In the present study, we first verified that neurotransmission in both dorsal and ventral MHb-IPN pathways is mainly mediated by Cav2.3 using a selective blocker of R-type channels, SNX-482. We next found that baclofen, a GBR agonist, has facilitatory effects on release from ventral MHb terminal in rostral IPN, whereas it has inhibitory effects on release from dorsal MHb terminals in lateral IPN, indicating that KCTD12b expressed exclusively in ventral MHb may have a role in the facilitatory effects of GBR activation. In a heterologous expression system using HEK cells, we found that KCTD8 and KCTD12b but not KCTD12 directly bind with Cav2.3. Pre-embedding immunogold electron microscopy data show that Cav2.3 and KCTD12b are distributed most densely in presynaptic active zone in IPN with KCTD12b being present only in rostral/central but not lateral IPN, whereas GABAB, KCTD8 and KCTD12 are distributed most densely in perisynaptic sites with KCTD12 present more frequently in postsynaptic elements and only in rostral/central IPN. In freeze-fracture replica labelling, Cav2.3, KCTD8 and KCTD12b are co-localized with each other in the same active zone indicating that they may form complexes regulating vesicle release in rostral IPN. On electrophysiological studies of wild type (WT) mice, we found that paired-pulse ratio in rostral IPN of KCTD12b knock-out (KO) mice is lower than those of WT and KCTD8 KO mice. Consistent with this finding, in mean variance analysis, release probability in rostral IPN of KCTD12b KO mice is higher than that of WT and KCTD8 KO mice. Although paired-pulse ratios are not different between WT and KCTD8 KO mice, the mean variance analysis revealed significantly lower release probability in rostral IPN of KCTD8 KO than WT mice. These results demonstrate bidirectional regulation of Cav2.3-mediated release by KCTD8 and KCTD12b without GBR activation in rostral IPN. Finally, we examined the baclofen effects in rostral IPN of KCTD8 and KCTD12b KO mice, and found the facilitation of release remained in both KO mice, indicating that the peculiar effects of the GBR activation in this pathway do not depend on the selective expression of these KCTD subunits in ventral MHb. However, we found that presynaptic potentiation of evoked EPSC amplitude by baclofen falls to baseline after washout faster in KCTD12b KO mice than WT, KCTD8 KO and KCTD8/12b double KO mice. This result indicates that KCTD12b is involved in sustained potentiation of vesicle release by GBR activation, whereas KCTD8 is involved in its termination in the absence of KCTD12b. Consistent with these functional findings, replica labelling revealed an increase in density of KCTD8, but not Cav2.3 or GBR at active zone in rostral IPN of KCTD12b KO mice compared with that of WT mice, suggesting that increased association of KCTD8 with Cav2.3 facilitates the release probability and termination of the GBR effect in the absence of KCTD12b. In summary, our study provided new insights into the physiological roles of presynaptic Cav2.3, GBRs and their auxiliary subunits KCTDs at an evolutionary conserved neuronal circuit. Future studies will be required to identify the exact molecular mechanism underlying the GBR-mediated presynaptic potentiation on ventral MHb terminals. It remains to be determined whether the prominent presence of presynaptic KCTDs at active zone could exert similar neuromodulatory functions in different pathways of the brain. AU - Bhandari, Pradeep ID - 7525 KW - Cav2.3 KW - medial habenula (MHb) KW - interpeduncular nucleus (IPN) SN - 2663-337X TI - Localization and functional role of Cav2.3 in the medial habenula to interpeduncular nucleus pathway ER - TY - THES AB - Synthesis of proteins – translation – is a fundamental process of life. Quantitative studies anchor translation into the context of bacterial physiology and reveal several mathematical relationships, called “growth laws,” which capture physiological feedbacks between protein synthesis and cell growth. Growth laws describe the dependency of the ribosome abundance as a function of growth rate, which can change depending on the growth conditions. Perturbations of translation reveal that bacteria employ a compensatory strategy in which the reduced translation capability results in increased expression of the translation machinery. Perturbations of translation are achieved in various ways; clinically interesting is the application of translation-targeting antibiotics – translation inhibitors. The antibiotic effects on bacterial physiology are often poorly understood. Bacterial responses to two or more simultaneously applied antibiotics are even more puzzling. The combined antibiotic effect determines the type of drug interaction, which ranges from synergy (the effect is stronger than expected) to antagonism (the effect is weaker) and suppression (one of the drugs loses its potency). In the first part of this work, we systematically measure the pairwise interaction network for translation inhibitors that interfere with different steps in translation. We find that the interactions are surprisingly diverse and tend to be more antagonistic. To explore the underlying mechanisms, we begin with a minimal biophysical model of combined antibiotic action. We base this model on the kinetics of antibiotic uptake and binding together with the physiological response described by the growth laws. The biophysical model explains some drug interactions, but not all; it specifically fails to predict suppression. In the second part of this work, we hypothesize that elusive suppressive drug interactions result from the interplay between ribosomes halted in different stages of translation. To elucidate this putative mechanism of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using in- ducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks partially causes these interactions. We extend this approach by varying two translation bottlenecks simultaneously. This approach reveals the suppression of translocation inhibition by inhibited translation. We rationalize this effect by modeling dense traffic of ribosomes that move on transcripts in a translation factor-mediated manner. This model predicts a dissolution of traffic jams caused by inhibited translocation when the density of ribosome traffic is reduced by lowered initiation. We base this model on the growth laws and quantitative relationships between different translation and growth parameters. In the final part of this work, we describe a set of tools aimed at quantification of physiological and translation parameters. We further develop a simple model that directly connects the abundance of a translation factor with the growth rate, which allows us to extract physiological parameters describing initiation. We demonstrate the development of tools for measuring translation rate. This thesis showcases how a combination of high-throughput growth rate mea- surements, genetics, and modeling can reveal mechanisms of drug interactions. Furthermore, by a gradual transition from combinations of antibiotics to precise genetic interventions, we demonstrated the equivalency between genetic and chemi- cal perturbations of translation. These findings tile the path for quantitative studies of antibiotic combinations and illustrate future approaches towards the quantitative description of translation. AU - Kavcic, Bor ID - 8657 SN - 2663-337X TI - Perturbations of protein synthesis: from antibiotics to genetics and physiology ER - TY - THES AB - Proteins and their complex dynamic interactions regulate cellular mechanisms from sensing and transducing extracellular signals, to mediating genetic responses, and sustaining or changing cell morphology. To manipulate these protein-protein interactions (PPIs) that govern the behavior and fate of cells, synthetically constructed, genetically encoded tools provide the means to precisely target proteins of interest (POIs), and control their subcellular localization and activity in vitro and in vivo. Ideal synthetic tools react to an orthogonal cue, i.e. a trigger that does not activate any other endogenous process, thereby allowing manipulation of the POI alone. In optogenetics, naturally occurring photosensory domain from plants, algae and bacteria are re-purposed and genetically fused to POIs. Illumination with light of a specific wavelength triggers a conformational change that can mediate PPIs, such as dimerization or oligomerization. By using light as a trigger, these tools can be activated with high spatial and temporal precision, on subcellular and millisecond scales. Chemogenetic tools consist of protein domains that recognize and bind small molecules. By genetic fusion to POIs, these domains can mediate PPIs upon addition of their specific ligands, which are often synthetically designed to provide highly specific interactions and exhibit good bioavailability. Most optogenetic tools to mediate PPIs are based on well-studied photoreceptors responding to red, blue or near-UV light, leaving a striking gap in the green band of the visible light spectrum. Among both optogenetic and chemogenetic tools, there is an abundance of methods to induce PPIs, but tools to disrupt them require UV illumination, rely on covalent linkage and subsequent enzymatic cleavage or initially result in protein clustering of unknown stoichiometry. This work describes how the recently structurally and photochemically characterized green-light responsive cobalamin-binding domains (CBDs) from bacterial transcription factors were re-purposed to function as a green-light responsive optogenetic tool. In contrast to previously engineered optogenetic tools, CBDs do not induce PPI, but rather confer a PPI already upon expression, which can be rapidly disrupted by illumination. This was employed to mimic inhibition of constitutive activity of a growth factor receptor, and successfully implement for cell signalling in mammalian cells and in vivo to rescue development in zebrafish. This work further describes the development and application of a chemically induced de-dimerizer (CDD) based on a recently identified and structurally described bacterial oxyreductase. CDD forms a dimer upon expression in absence of its cofactor, the flavin derivative F420. Safety and of domain expression and ligand exposure are demonstrated in vitro and in vivo in zebrafish. The system is further applied to inhibit cell signalling output from a chimeric receptor upon F420 treatment. CBDs and CDD expand the repertoire of synthetic tools by providing novel mechanisms of mediating PPIs, and by recognizing previously not utilized cues. In the future, they can readily be combined with existing synthetic tools to functionally manipulate PPIs in vitro and in vivo. AU - Kainrath, Stephanie ID - 7680 TI - Synthetic tools for optogenetic and chemogenetic inhibition of cellular signals ER - TY - THES AB - The development of the human brain occurs through a tightly regulated series of dynamic and adaptive processes during prenatal and postnatal life. A disruption of this strictly orchestrated series of events can lead to a number of neurodevelopmental conditions, including Autism Spectrum Disorders (ASDs). ASDs are a very common, etiologically and phenotypically heterogeneous group of disorders sharing the core symptoms of social interaction and communication deficits and restrictive and repetitive interests and behaviors. They are estimated to affect one in 59 individuals in the U.S. and, over the last three decades, mutations in more than a hundred genetic loci have been convincingly linked to ASD pathogenesis. Yet, for the vast majority of these ASD-risk genes their role during brain development and precise molecular function still remain elusive. De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin 3 (CUL3) lead to ASD. In the study described here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 heterozygous knockout mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3+/-, Cul3+/fl Emx1-Cre and Cul3fl/fl Emx1-Cre mutant brains display cortical lamination abnormalities due to defective migration of post-mitotic excitatory neurons, as well as reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal cortical organization, Cul3 heterozygous deletion is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level we show that Cul3 regulates cytoskeletal and adhesion protein abundance in the mouse embryonic cortex. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neural cells results in atypical organization of the actin mesh at the cell leading edge. Of note, heterozygous deletion of Cul3 in adult mice does not induce the majority of the behavioral defects observed in constitutive Cul3 haploinsufficient animals, pointing to a critical time-window for Cul3 deficiency. In conclusion, our data indicate that Cul3 plays a critical role in the regulation of cytoskeletal proteins and neuronal migration. ASD-associated defects and behavioral abnormalities are primarily due to dosage sensitive Cul3 functions at early brain developmental stages. AU - Morandell, Jasmin ID - 8620 SN - 2663-337X TI - Illuminating the role of Cul3 in autism spectrum disorder pathogenesis ER - TY - THES AB - Mitochondria are sites of oxidative phosphorylation in eukaryotic cells. Oxidative phosphorylation operates by a chemiosmotic mechanism made possible by redox-driven proton pumping machines which establish a proton motive force across the inner mitochondrial membrane. This electrochemical proton gradient is used to drive ATP synthesis, which powers the majority of cellular processes such as protein synthesis, locomotion and signalling. In this thesis I investigate the structures and molecular mechanisms of two inner mitochondrial proton pumping enzymes, respiratory complex I and transhydrogenase. I present the first high-resolution structure of the full transhydrogenase from any species, and a significantly improved structure of complex I. Improving the resolution from 3.3 Å available previously to up to 2.3 Å in this thesis allowed us to model bound water molecules, crucial in the proton pumping mechanism. For both enzymes, up to five cryo-EM datasets with different substrates and inhibitors bound were solved to delineate the catalytic cycle and understand the proton pumping mechanism. In transhydrogenase, the proton channel is gated by reversible detachment of the NADP(H)-binding domain which opens the proton channel to the opposite sites of the membrane. In complex I, the proton channels are gated by reversible protonation of key glutamate and lysine residues and breaking of the water wire connecting the proton pumps with the quinone reduction site. The tight coupling between the redox and the proton pumping reactions in transhydrogenase is achieved by controlling the NADP(H) exchange which can only happen when the NADP(H)-binding domain interacts with the membrane domain. In complex I, coupling is achieved by cycling of the whole complex between the closed state, in which quinone can get reduced, and the open state, in which NADH can induce quinol ejection from the binding pocket. On the basis of these results I propose detailed mechanisms for catalytic cycles of transhydrogenase and complex I that are consistent with a large amount of previous work. In both enzymes, conformational and electrostatic mechanisms contribute to the overall catalytic process. Results presented here could be used for better understanding of the human pathologies arising from deficiencies of complex I or transhydrogenase and could be used to develop novel therapies. AU - Kampjut, Domen ID - 8340 SN - 2663-337X TI - Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes ER - TY - THES AB - Metabolic adaptation is a critical feature of migrating cells. It tunes the metabolic programs of migrating cells to allow them to efficiently exert their crucial roles in development, inflammatory responses and tumor metastasis. Cell migration through physically challenging contexts requires energy. However, how the metabolic reprogramming that underlies in vivo cell invasion is controlled is still unanswered. In my PhD project, I identify a novel conserved metabolic shift in Drosophila melanogaster immune cells that by modulating their bioenergetic potential controls developmentally programmed tissue invasion. We show that this regulation requires a novel conserved nuclear protein, named Atossa. Atossa enhances the transcription of a set of proteins, including an RNA helicase Porthos and two metabolic enzymes, each of which increases the tissue invasion of leading Drosophila macrophages and can rescue the atossa mutant phenotype. Porthos selectively regulates the translational efficiency of a subset of mRNAs containing a 5’-UTR cis-regulatory TOP-like sequence. These 5’TOPL mRNA targets encode mitochondrial-related proteins, including subunits of mitochondrial oxidative phosphorylation (OXPHOS) components III and V and other metabolic-related proteins. Porthos powers up mitochondrial OXPHOS to engender a sufficient ATP supply, which is required for tissue invasion of leading macrophages. Atossa’s two vertebrate orthologs rescue the invasion defect. In my PhD project, I elucidate that Atossa displays a conserved developmental metabolic control to modulate metabolic capacities and the cellular energy state, through altered transcription and translation, to aid the tissue infiltration of leading cells into energy demanding barriers. AU - Emtenani, Shamsi ID - 8983 SN - 2663-337X TI - Metabolic regulation of Drosophila macrophage tissue invasion ER - TY - THES AB - Many flows encountered in nature and applications are characterized by a chaotic motion known as turbulence. Turbulent flows generate intense friction with pipe walls and are responsible for considerable amounts of energy losses at world scale. The nature of turbulent friction and techniques aimed at reducing it have been subject of extensive research over the last century, but no definite answer has been found yet. In this thesis we show that in pipes at moderate turbulent Reynolds numbers friction is better described by the power law first introduced by Blasius and not by the Prandtl–von Kármán formula. At higher Reynolds numbers, large scale motions gradually become more important in the flow and can be related to the change in scaling of friction. Next, we present a series of new techniques that can relaminarize turbulence by suppressing a key mechanism that regenerates it at walls, the lift–up effect. In addition, we investigate the process of turbulence decay in several experiments and discuss the drag reduction potential. Finally, we examine the behavior of friction under pulsating conditions inspired by the human heart cycle and we show that under such circumstances turbulent friction can be reduced to produce energy savings. AU - Scarselli, Davide ID - 7258 SN - 2663-337X TI - New approaches to reduce friction in turbulent pipe flow ER - TY - THES AB - Mutations are the raw material of evolution and come in many different flavors. Point mutations change a single letter in the DNA sequence, while copy number mutations like duplications or deletions add or remove many letters of the DNA sequence simultaneously. Each type of mutation exhibits specific properties like its rate of formation and reversal. Gene expression is a fundamental phenotype that can be altered by both, point and copy number mutations. The following thesis is concerned with the dynamics of gene expression evolution and how it is affected by the properties exhibited by point and copy number mutations. Specifically, we are considering i) copy number mutations during adaptation to fluctuating environments and ii) the interaction of copy number and point mutations during adaptation to constant environments.   AU - Tomanek, Isabella ID - 8653 KW - duplication KW - amplification KW - promoter KW - CNV KW - AMGET KW - experimental evolution KW - Escherichia coli SN - 2663-337X TI - The evolution of gene expression by copy number and point mutations ER - TY - THES AB - Self-organization is a hallmark of plant development manifested e.g. by intricate leaf vein patterns, flexible formation of vasculature during organogenesis or its regeneration following wounding. Spontaneously arising channels transporting the phytohormone auxin, created by coordinated polar localizations of PIN-FORMED 1 (PIN1) auxin exporter, provide positional cues for these as well as other plant patterning processes. To find regulators acting downstream of auxin and the TIR1/AFB auxin signaling pathway essential for PIN1 coordinated polarization during auxin canalization, we performed microarray experiments. Besides the known components of general PIN polarity maintenance, such as PID and PIP5K kinases, we identified and characterized a new regulator of auxin canalization, the transcription factor WRKY DNA-BINDING PROTEIN 23 (WRKY23). Next, we designed a subsequent microarray experiment to further uncover other molecular players, downstream of auxin-TIR1/AFB-WRKY23 involved in the regulation of auxin-mediated PIN repolarization. We identified a novel and crucial part of the molecular machinery underlying auxin canalization. The auxin-regulated malectin-type receptor-like kinase CAMEL and the associated leucine-rich repeat receptor-like kinase CANAR target and directly phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated repolarization leading to defects in auxin transport, ultimately to leaf venation and vasculature regeneration defects. Our results describe the CAMEL-CANAR receptor complex, which is required for auxin feed-back on its own transport and thus for coordinated tissue polarization during auxin canalization. AU - Hajny, Jakub ID - 8822 SN - 2663-337X TI - Identification and characterization of the molecular machinery of auxin-dependent canalization during vasculature formation and regeneration ER - TY - THES AB - Cytoplasm is a gel-like crowded environment composed of tens of thousands of macromolecules, organelles, cytoskeletal networks and cytosol. The structure of the cytoplasm is thought to be highly organized and heterogeneous due to the crowding of its constituents and their effective compartmentalization. In such an environment, the diffusive dynamics of the molecules is very restricted, an effect that is further amplified by clustering and anchoring of molecules. Despite the jammed nature of the cytoplasm at the microscopic scale, large-scale reorganization of cytoplasm is essential for important cellular functions, such as nuclear positioning and cell division. How such mesoscale reorganization of the cytoplasm is achieved, especially for very large cells such as oocytes or syncytial tissues that can span hundreds of micrometers in size, has only begun to be understood. In this thesis, I focus on the recent advances in elucidating the molecular, cellular and biophysical principles underlying cytoplasmic organization across different scales, structures and species. First, I outline which of these principles have been identified by reductionist approaches, such as in vitro reconstitution assays, where boundary conditions and components can be modulated at ease. I then describe how the theoretical and experimental framework established in these reduced systems have been applied to their more complex in vivo counterparts, in particular oocytes and embryonic syncytial structures, and discuss how such complex biological systems can initiate symmetry breaking and establish patterning. Specifically, I examine an example of large-scale reorganizations taking place in zebrafish embryos, where extensive cytoplasmic streaming leads to the segregation of cytoplasm from yolk granules along the animal-vegetal axis of the embryo. Using biophysical experimentation and theory, I investigate the forces underlying this process, to show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the embryo. This wave functions in segregation by both pulling cytoplasm animally and pushing yolk granules vegetally. Cytoplasm pulling is mediated by bulk actin network flows exerting friction forces on the cytoplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. This study defines a novel role of bulk actin polymerization waves in embryo polarization via cytoplasmic segregation. Lastly, I describe the cytoplasmic reorganizations taking place during zebrafish oocyte maturation, where the initial segregation of the cytoplasm and yolk granules occurs. Here, I demonstrate a previously uncharacterized wave of microtubule aster formation, traveling the oocyte along the animal-vegetal axis. Further research is required to determine the role of such microtubule structures in cytoplasmic reorganizations therein. Collectively, these studies provide further evidence for the coupling between cell cytoskeleton and cell cycle machinery, which can underlie a core self-organizing mechanism for orchestrating large-scale reorganizations in a cell-cycle-tunable manner, where the modulations of the force-generating machinery and cytoplasmic mechanics can be harbored to fulfill cellular functions. AU - Shamipour, Shayan ID - 8350 SN - 2663-337X TI - Bulk actin dynamics drive phase segregation in zebrafish oocytes ER -