TY - THES AB - We describe arrangements of three-dimensional spheres from a geometrical and topological point of view. Real data (fitting this setup) often consist of soft spheres which show certain degree of deformation while strongly packing against each other. In this context, we answer the following questions: If we model a soft packing of spheres by hard spheres that are allowed to overlap, can we measure the volume in the overlapped areas? Can we be more specific about the overlap volume, i.e. quantify how much volume is there covered exactly twice, three times, or k times? What would be a good optimization criteria that rule the arrangement of soft spheres while making a good use of the available space? Fixing a particular criterion, what would be the optimal sphere configuration? The first result of this thesis are short formulas for the computation of volumes covered by at least k of the balls. The formulas exploit information contained in the order-k Voronoi diagrams and its closely related Level-k complex. The used complexes lead to a natural generalization into poset diagrams, a theoretical formalism that contains the order-k and degree-k diagrams as special cases. In parallel, we define different criteria to determine what could be considered an optimal arrangement from a geometrical point of view. Fixing a criterion, we find optimal soft packing configurations in 2D and 3D where the ball centers lie on a lattice. As a last step, we use tools from computational topology on real physical data, to show the potentials of higher-order diagrams in the description of melting crystals. The results of the experiments leaves us with an open window to apply the theories developed in this thesis in real applications. AU - Iglesias Ham, Mabel ID - 201 SN - 2663-337X TI - Multiple covers with balls ER - TY - THES AB - The most common assumption made in statistical learning theory is the assumption of the independent and identically distributed (i.i.d.) data. While being very convenient mathematically, it is often very clearly violated in practice. This disparity between the machine learning theory and applications underlies a growing demand in the development of algorithms that learn from dependent data and theory that can provide generalization guarantees similar to the independent situations. This thesis is dedicated to two variants of dependencies that can arise in practice. One is a dependence on the level of samples in a single learning task. Another dependency type arises in the multi-task setting when the tasks are dependent on each other even though the data for them can be i.i.d. In both cases we model the data (samples or tasks) as stochastic processes and introduce new algorithms for both settings that take into account and exploit the resulting dependencies. We prove the theoretical guarantees on the performance of the introduced algorithms under different evaluation criteria and, in addition, we compliment the theoretical study by the empirical one, where we evaluate some of the algorithms on two real world datasets to highlight their practical applicability. AU - Zimin, Alexander ID - 68 SN - 2663-337X TI - Learning from dependent data ER - TY - THES AB - A proof system is a protocol between a prover and a verifier over a common input in which an honest prover convinces the verifier of the validity of true statements. Motivated by the success of decentralized cryptocurrencies, exemplified by Bitcoin, the focus of this thesis will be on proof systems which found applications in some sustainable alternatives to Bitcoin, such as the Spacemint and Chia cryptocurrencies. In particular, we focus on proofs of space and proofs of sequential work. Proofs of space (PoSpace) were suggested as more ecological, economical, and egalitarian alternative to the energy-wasteful proof-of-work mining of Bitcoin. However, the state-of-the-art constructions of PoSpace are based on sophisticated graph pebbling lower bounds, and are therefore complex. Moreover, when these PoSpace are used in cryptocurrencies like Spacemint, miners can only start mining after ensuring that a commitment to their space is already added in a special transaction to the blockchain. Proofs of sequential work (PoSW) are proof systems in which a prover, upon receiving a statement x and a time parameter T, computes a proof which convinces the verifier that T time units had passed since x was received. Whereas Spacemint assumes synchrony to retain some interesting Bitcoin dynamics, Chia requires PoSW with unique proofs, i.e., PoSW in which it is hard to come up with more than one accepting proof for any true statement. In this thesis we construct simple and practically-efficient PoSpace and PoSW. When using our PoSpace in cryptocurrencies, miners can start mining on the fly, like in Bitcoin, and unlike current constructions of PoSW, which either achieve efficient verification of sequential work, or faster-than-recomputing verification of correctness of proofs, but not both at the same time, ours achieve the best of these two worlds. AU - Abusalah, Hamza M ID - 83 SN - 2663-337X TI - Proof systems for sustainable decentralized cryptocurrencies ER - TY - THES AB - Modern computer vision systems heavily rely on statistical machine learning models, which typically require large amounts of labeled data to be learned reliably. Moreover, very recently computer vision research widely adopted techniques for representation learning, which further increase the demand for labeled data. However, for many important practical problems there is relatively small amount of labeled data available, so it is problematic to leverage full potential of the representation learning methods. One way to overcome this obstacle is to invest substantial resources into producing large labelled datasets. Unfortunately, this can be prohibitively expensive in practice. In this thesis we focus on the alternative way of tackling the aforementioned issue. We concentrate on methods, which make use of weakly-labeled or even unlabeled data. Specifically, the first half of the thesis is dedicated to the semantic image segmentation task. We develop a technique, which achieves competitive segmentation performance and only requires annotations in a form of global image-level labels instead of dense segmentation masks. Subsequently, we present a new methodology, which further improves segmentation performance by leveraging tiny additional feedback from a human annotator. By using our methods practitioners can greatly reduce the amount of data annotation effort, which is required to learn modern image segmentation models. In the second half of the thesis we focus on methods for learning from unlabeled visual data. We study a family of autoregressive models for modeling structure of natural images and discuss potential applications of these models. Moreover, we conduct in-depth study of one of these applications, where we develop the state-of-the-art model for the probabilistic image colorization task. AU - Kolesnikov, Alexander ID - 197 SN - 2663-337X TI - Weakly-Supervised Segmentation and Unsupervised Modeling of Natural Images ER - TY - THES AB - This thesis is concerned with the inference of current population structure based on geo-referenced genetic data. The underlying idea is that population structure affects its spatial genetic structure. Therefore, genotype information can be utilized to estimate important demographic parameters such as migration rates. These indirect estimates of population structure have become very attractive, as genotype data is now widely available. However, there also has been much concern about these approaches. Importantly, genetic structure can be influenced by many complex patterns, which often cannot be disentangled. Moreover, many methods merely fit heuristic patterns of genetic structure, and do not build upon population genetics theory. Here, I describe two novel inference methods that address these shortcomings. In Chapter 2, I introduce an inference scheme based on a new type of signal, identity by descent (IBD) blocks. Recently, it has become feasible to detect such long blocks of genome shared between pairs of samples. These blocks are direct traces of recent coalescence events. As such, they contain ample signal for inferring recent demography. I examine sharing of IBD blocks in two-dimensional populations with local migration. Using a diffusion approximation, I derive formulas for an isolation by distance pattern of long IBD blocks and show that sharing of long IBD blocks approaches rapid exponential decay for growing sample distance. I describe an inference scheme based on these results. It can robustly estimate the dispersal rate and population density, which is demonstrated on simulated data. I also show an application to estimate mean migration and the rate of recent population growth within Eastern Europe. Chapter 3 is about a novel method to estimate barriers to gene flow in a two dimensional population. This inference scheme utilizes geographically localized allele frequency fluctuations - a classical isolation by distance signal. The strength of these local fluctuations increases on average next to a barrier, and there is less correlation across it. I again use a framework of diffusion of ancestral lineages to model this effect, and provide an efficient numerical implementation to fit the results to geo-referenced biallelic SNP data. This inference scheme is able to robustly estimate strong barriers to gene flow, as tests on simulated data confirm. AU - Ringbauer, Harald ID - 200 SN - 2663-337X TI - Inferring recent demography from spatial genetic structure ER - TY - THES AB - The aim of this thesis was the development of new strategies for optical and optogenetic control of proliferative and pro-survival signaling, and characterizing them from the molecular mechanism up to cellular effects. These new light-based methods have unique features, such as red light as an activator, or the avoidance of gene delivery, which enable to overcome current limitations, such as light delivery to target tissues and feasibility as therapeutic approach. A special focus was placed on implementing these new light-based approaches in pancreatic β-cells, as β-cells are the key players in diabetes and especially their loss in number negatively affects disease progression. Currently no treatment options are available to compensate the lack of functional β-cells in diabetic patients. In a first approach, red-light-activated growth factor receptors, in particular receptor tyrosine kinases were engineered and characterized. Receptor activation with light allows spatio-temporal control compared to ligand-based activation, and especially red light exhibits deeper tissue penetration than other wavelengths of the visible spectrum. Red-light-activated receptor tyrosine kinases robustly activated major growth factor related signaling pathways with a high temporal resolution. Moreover, the remote activation of the proliferative MAPK/Erk pathway by red-light-activated receptor tyrosine kinases in a pancreatic β-cell line was also achieved, through one centimeter thick mouse tissue. Although red-light-activated receptor tyrosine kinases are particularly attractive for applications in animal models due to the deep tissue penetration of red light, a drawback, especially with regard to translation into humans, is the requirement of gene therapy. In a second approach an endogenous light-sensitive mechanism was identified and its potential to promote proliferative and pro-survival signals was explored, towards light-based tissue regeneration without the need for gene transfer. Blue-green light illumination was found to be sufficient for the activation of proliferation and survival promoting signaling pathways in primary pancreatic murine and human islets. Blue-green light also led to an increase in proliferation of primary islet cells, an effect which was shown to be mostly β-cell specific in human islets. Moreover, it was demonstrated that this approach of pancreatic β-cell expansion did not have any negative effect on the β-cell function, in particular on their insulin secretion capacity. In contrast, a trend for enhanced insulin secretion under high glucose conditions after illumination was detected. In order to unravel the detailed characteristics of this endogenous light-sensitive mechanism, the precise light requirements were determined. In addition, the expression of light sensing proteins, OPN3 and rhodopsin, was detected. The observed effects were found to be independent of handling effects such as temperature differences and cytochrome c oxidase dependent ATP increase, but they were found to be enhanced through the knockout of OPN3. The exact mechanism of how islets cells sense light and the identity of the photoreceptor remains unknown. Summarized two new light-based systems with unique features were established that enable the activation of proliferative and pro-survival signaling pathways. While red-light-activated receptor tyrosine kinases open a new avenue for optogenetics research, by allowing non-invasive control of signaling in vivo, the identified endogenous light-sensitive mechanism has the potential to be the basis of a gene therapy-free therapeutical approach for light-based β-cell expansion. AU - Gschaider-Reichhart, Eva ID - 418 SN - 2663-337X TI - Optical and optogenetic control of proliferation and survival ER - TY - THES AB - In this thesis we will discuss systems of point interacting fermions, their stability and other spectral properties. Whereas for bosons a point interacting system is always unstable this ques- tion is more subtle for a gas of two species of fermions. In particular the answer depends on the mass ratio between these two species. Most of this work will be focused on the N + M model which consists of two species of fermions with N, M particles respectively which interact via point interactions. We will introduce this model using a formal limit and discuss the N + 1 system in more detail. In particular, we will show that for mass ratios above a critical one, which does not depend on the particle number, the N + 1 system is stable. In the context of this model we will prove rigorous versions of Tan relations which relate various quantities of the point-interacting model. By restricting the N + 1 system to a box we define a finite density model with point in- teractions. In the context of this system we will discuss the energy change when introducing a point-interacting impurity into a system of non-interacting fermions. We will see that this change in energy is bounded independently of the particle number and in particular the bound only depends on the density and the scattering length. As another special case of the N + M model we will show stability of the 2 + 2 model for mass ratios in an interval around one. Further we will investigate a different model of point interactions which was discussed before in the literature and which is, contrary to the N + M model, not given by a limiting procedure but is based on a Dirichlet form. We will show that this system behaves trivially in the thermodynamic limit, i.e. the free energy per particle is the same as the one of the non-interacting system. AU - Moser, Thomas ID - 52 SN - 2663-337X TI - Point interactions in systems of fermions ER - TY - THES AB - A qubit, a unit of quantum information, is essentially any quantum mechanical two-level system which can be coherently controlled. Still, to be used for computation, it has to fulfill criteria. Qubits, regardless of the system in which they are realized, suffer from decoherence. This leads to loss of the information stored in the qubit. The upper bound of the time scale on which decoherence happens is set by the spin relaxation time. In this thesis I studied a two-level system consisting of a Zeeman-split hole spin confined in a quantum dot formed in a Ge hut wire. Such Ge hut wires have emerged as a promising material system for the realization of spin qubits, due to the combination of two significant properties: long spin coherence time as expected for group IV semiconductors due to the low hyperfine interaction and a strong valence band spin-orbit coupling. Here, I present how to fabricate quantum dot devices suitable for electrical transport measurements. Coupled quantum dot devices allowed the realization of a charge sensor, which is electrostatically and tunnel coupled to a quantum dot. By integrating the charge sensor into a radio-frequency reflectometry setup, I performed for the first time single-shot readout measurements of hole spins and extracted the hole spin relaxation times in Ge hut wires. AU - Vukušić, Lada ID - 69 SN - 2663-337X TI - Charge sensing and spin relaxation times of holes in Ge hut wires ER - TY - THES AB - Neuronal networks in the brain consist of two main types of neuron, glutamatergic principal neurons and GABAergic interneurons. Although these interneurons only represent 10–20% of the whole population, they mediate feedback and feedforward inhibition and are involved in the generation of high-frequency network oscillations. A hallmark functional property of GABAergic interneurons, especially of the parvalbumin‑expressing (PV+) subtypes, is the speed of signaling at their output synapse across species and brain regions. Several molecular and subcellular factors may underlie the submillisecond signaling at GABAergic synapses. Such as the selective use of P/Q type Ca2+ channels and the tight coupling between Ca2+ channels and Ca2+ sensors of exocytosis. However, whether the molecular identity of the release sensor contributes to these signaling properties remains unclear. Besides, these interneurons are mainly show depression in response to train of stimuli. How could they keep sufficient release to control the activity of postsynaptic principal neurons during high network activity, is largely elusive. For my Ph.D. work, we firstly examined the Ca2+ sensor of exocytosis at the GABAergic basket cell (BC) to Purkinje cell (PC) synapse in the cerebellum. Immunolabeling suggested that BC terminals selectively expressed synaptotagmin 2 (Syt2), whereas synaptotagmin 1 (Syt1) was enriched in excitatory terminals. Genetic elimination of Syt2 reduced action potential-evoked release to ~10% compared to the wild-type control, identifying Syt2 as the major Ca2+ sensor at BC‑PC synapses. Differential adenovirus-mediated rescue revealed Syt2 triggered release with shorter latency and higher temporal precision, and mediated faster vesicle pool replenishment than Syt1. Furthermore, deletion of Syt2 severely reduced and delayed disynaptic inhibition following parallel fiber stimulation. Thus, the selective use of Syt2 as the release sensor at BC–PC synapse ensures fast feedforward inhibition in cerebellar microcircuits. Additionally, we tested the function of another synaptotagmin member, Syt7, for inhibitory synaptic transmission at the BC–PC synapse. Syt7 is thought to be a Ca2+ sensor that mediates asynchronous transmitter release and facilitation at synapses. However, it is strongly expressed in fast-spiking, PV+ GABAergic interneurons and the output synapses of these neurons produce only minimal asynchronous release and show depression rather than facilitation. How could Syt7, a facilitation sensor, contribute to the depressed inhibitory synaptic transmission needs to be further investigated and understood. Our results indicated that at the BC–PC synapse, Syt7 contributes to asynchronous release, pool replenishment and facilitation. In combination, these three effects ensure efficient transmitter release during high‑frequency activity and guarantee frequency independence of inhibition. Taken together, our results confirmed that Syt2, which has the fastest kinetic properties among all synaptotagmin members, is mainly used by the inhibitory BC‑PC synapse for synaptic transmission, contributing to the speed and temporal precision of transmitter release. Furthermore, we showed that Syt7, another highly expressed synaptotagmin member in the output synapses of cerebellar BCs, is used for ensuring efficient inhibitor synaptic transmission during high activity. AU - Chen, Chong ID - 324 SN - 2663-337X TI - Synaptotagmins ensure speed and efficiency of inhibitory neurotransmitter release ER - TY - THES AB - Consortial subscription contracts regulate the digital access to publications between publishers and scientific libraries. However, since a couple of years the tendency towards a freely accessible publishing (Open Access) intensifies. As a consequence of this trend the contractual relationship between licensor and licensee is gradually changing as well: More and more contracts exercise influence on open access publishing. The present study attempts to compare Austrian examples of consortial licence contracts, which include components of open access. It describes the difference between pure subscription contracts and differing innovative deals including open access components. Thereby it becomes obvious that for the evaluation of this licence contracts new methods are needed. An essential new element of such analyses is the evaluation of the open access publication numbers. So this study tries to carry out such publication analyses for Austrian open access deals focusing on quantitative questions: How does the number of publications evolve? How does the open access share change? Publications reports of the publishers and database queries from Scopus form the data basis. The analysis of the data points out that differing approaches of contracts result in highly divergent results: Particular deals can prioritize a saving in costs or else the increase of the open access rate. It is to be assumed that within the following years further numerous open access deals will be negotiated. The finding of this study shall provide guidance. AU - Villányi, Márton ID - 278 TI - Lizenzverträge mit Open-Access-Komponenten an österreichischen Bibliotheken ER - TY - THES AB - The eigenvalue density of many large random matrices is well approximated by a deterministic measure, the self-consistent density of states. In the present work, we show this behaviour for several classes of random matrices. In fact, we establish that, in each of these classes, the self-consistent density of states approximates the eigenvalue density of the random matrix on all scales slightly above the typical eigenvalue spacing. For large classes of random matrices, the self-consistent density of states exhibits several universal features. We prove that, under suitable assumptions, random Gram matrices and Hermitian random matrices with decaying correlations have a 1/3-Hölder continuous self-consistent density of states ρ on R, which is analytic, where it is positive, and has either a square root edge or a cubic root cusp, where it vanishes. We, thus, extend the validity of the corresponding result for Wigner-type matrices from [4, 5, 7]. We show that ρ is determined as the inverse Stieltjes transform of the normalized trace of the unique solution m(z) to the Dyson equation −m(z) −1 = z − a + S[m(z)] on C N×N with the constraint Im m(z) ≥ 0. Here, z lies in the complex upper half-plane, a is a self-adjoint element of C N×N and S is a positivity-preserving operator on C N×N encoding the first two moments of the random matrix. In order to analyze a possible limit of ρ for N → ∞ and address some applications in free probability theory, we also consider the Dyson equation on infinite dimensional von Neumann algebras. We present two applications to random matrices. We first establish that, under certain assumptions, large random matrices with independent entries have a rotationally symmetric self-consistent density of states which is supported on a centered disk in C. Moreover, it is infinitely often differentiable apart from a jump on the boundary of this disk. Second, we show edge universality at all regular (not necessarily extreme) spectral edges for Hermitian random matrices with decaying correlations. AU - Alt, Johannes ID - 149 SN - 2663-337X TI - Dyson equation and eigenvalue statistics of random matrices ER - TY - THES AB - Autism spectrum disorders (ASD) are a group of genetic disorders often overlapping with other neurological conditions. Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g. autism spectrum disorder, intellectual disability, epilepsy) remains a great challenge. Recent advancements in geno mics, like whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that were discovered, the etiological variability and the heterogeneous phenotypic outcomes, the need for genotype -along with phenotype- based diagnosis of individual patients becomes a requisite. Driven by this rationale, in a previous study our group described mutations, identified via whole - exome sequencing, in the gene BCKDK – encoding for a key regulator of branched chain amin o acid (BCAA) catabolism - as a cause of ASD. Following up on the role of BCAAs, in the study described here we show that the solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized mainly at the blood brain barrier (BBB), has an essential role in maintaining normal levels of brain BCAAs. In mice, deletion of Slc7a5 from the endothelial cells of the BBB leads to atypical brain amino acid profile, abnormal mRNA translation and severe neurolo gical abnormalities. Additionally, deletion of Slc7a5 from the neural progenitor cell population leads to microcephaly. Interestingly, we demonstrate that BCAA intracerebroventricular administration ameliorates abnormal behaviors in adult mutant mice. Furthermore, whole - exome sequencing of patients diagnosed with neurological dis o r ders helped us identify several patients with autistic traits, microcephaly and motor delay carrying deleterious homozygous mutations in the SLC7A5 gene. In conclusion, our data elucidate a neurological syndrome defined by SLC7A5 mutations and support an essential role for t he BCAA s in human bra in function. Together with r ecent studies (described in chapter two) that have successfully made the transition into clinical practice, our findings on the role of B CAAs might have a crucial impact on the development of novel individualized therapeutic strategies for ASD. AU - Tarlungeanu, Dora-Clara ID - 395 SN - 2663-337X TI - The branched chain amino acids in autism spectrum disorders ER - TY - THES AB - Asymmetries have long been known about in the central nervous system. From gross anatomical differences, such as the presence of the parapineal organ in only one hemisphere of the developing zebrafish, to more subtle differences in activity between both hemispheres, as seen in freely roaming animals or human participants under PET and fMRI imaging analysis. The presence of asymmetries has been demonstrated to have huge behavioural implications, with their disruption often leading to the generation of neurological disorders, memory problems, changes in personality, and in an organism's health and well-being. For my Ph.D. work I aimed to tackle two important avenues of research. The first being the process of input-side dependency in the hippocampus, with the goal of finding a key gene responsible for its development (Gene X). The second project was to do with experience-induced laterality formation in the hippocampus. Specifically, how laterality in the synapse density of the CA1 stratum radiatum (s.r.) could be induced purely through environmental enrichment. Through unilateral tracer injections into the CA3, I was able to selectively measure the properties of synapses within the CA1 and investigate how they differed based upon which hemisphere the presynaptic neurone originated. Having found the existence of a previously unreported reversed (left-isomerism) i.v. mutant, through morpholocal examination of labelled terminals in the CA1 s.r., I aimed to elucidate a key gene responsible for the process of left or right determination of inputs to the CA1 s.r.. This work relates to the previous finding of input-side dependent asymmetry in the wild-type rodent, where the origin of the projecting neurone to the CA1 will determine the morphology of a synapse, to a greater degree than the hemisphere in which the projection terminates. Using left- and right-isomerism i.v. mice, in combination with whole genome sequence analysis, I highlight Ena/VASP-like (Evl) as a potential target for Gene X. In relation to this topic, I also highlight my work in the recently published paper of how knockout of PirB can lead to a lack of input-side dependency in the murine hippocampus. For the second question, I show that the environmental enrichment paradigm will lead to an asymmetry in the synapse densities in the hippocampus of mice. I also highlight that the nature of the enrichment is of less consequence than the process of enrichment itself. I demonstrate that the CA3 region will dramatically alter its projection targets, in relation to environmental stimulation, with the asymmetry in synaptic density, caused by enrichment, relying heavily on commissural fibres. I also highlight the vital importance of input-side dependent asymmetry, as a necessary component of experience-dependent laterality formation in the CA1 s.r.. However, my results suggest that it isn't the only cause, as there appears to be a CA1 dependent mechanism also at play. Upon further investigation, I highlight the significant, and highly important, finding that the changes seen in the CA1 s.r. were predominantly caused through projections from the left-CA3, with the right-CA3 having less involvement in this mechanism. AU - Case, Matthew J ID - 51 SN - 2663-337X TI - From the left to the right: A tale of asymmetries, environments, and hippocampal development ER - TY - THES AB - Genomic imprinting is an epigenetic process that leads to parent of origin-specific gene expression in a subset of genes. Imprinted genes are essential for brain development, and deregulation of imprinting is associated with neurodevelopmental diseases and the pathogenesis of psychiatric disorders. However, the cell-type specificity of imprinting at single cell resolution, and how imprinting and thus gene dosage regulates neuronal circuit assembly is still largely unknown. Here, MADM (Mosaic Analysis with Double Markers) technology was employed to assess genomic imprinting at single cell level. By visualizing MADM-induced uniparental disomies (UPDs) in distinct colors at single cell level in genetic mosaic animals, this experimental paradigm provides a unique quantitative platform to systematically assay the UPD-mediated imbalances in imprinted gene expression at unprecedented resolution. An experimental pipeline based on FACS, RNA-seq and bioinformatics analysis was established and applied to systematically map cell-type-specific ‘imprintomes’ in the mouse brain. The results revealed that parental-specific expression of imprinted genes per se is rarely cell-type-specific even at the individual cell level. Conversely, when we extended the comparison to downstream responses resulting from imbalanced imprinted gene expression, we discovered an unexpectedly high degree of cell-type specificity. Furthermore, we determined a novel function of genomic imprinting in cortical astrocyte production and in olfactory bulb (OB) granule cell generation. These results suggest important functional implication of genomic imprinting for generating cell-type diversity in the brain. In addition, MADM provides a powerful tool to study candidate genes by concomitant genetic manipulation and fluorescent labelling of single cells. MADM-based candidate gene approach was utilized to identify potential imprinted genes involved in the generation of cortical astrocytes and OB granule cells. We investigated p57Kip2, a maternally expressed gene and known cell cycle regulator. Although we found that p57Kip2 does not play a role in these processes, we detected an unexpected function of the paternal allele previously thought to be silent. Finally, we took advantage of a key property of MADM which is to allow unambiguous investigation of environmental impact on single cells. The experimental pipeline based on FACS and RNA-seq analysis of MADM-labeled cells was established to probe the functional differences of single cell loss of gene function compared to global loss of function on a transcriptional level. With this method, both common and distinct responses were isolated due to cell-autonomous and non-autonomous effects acting on genotypically identical cells. As a result, transcriptional changes were identified which result solely from the surrounding environment. Using the MADM technology to study genomic imprinting at single cell resolution, we have identified cell-type-specific gene expression, novel gene function and the impact of environment on single cell transcriptomes. Together, these provide important insights to the understanding of mechanisms regulating cell-type specificity and thus diversity in the brain. AU - Laukoter, Susanne ID - 10 SN - 2663-337X TI - Role of genomic imprinting in cerebral cortex development ER - TY - THES AB - In the here presented thesis, we explore the role of branched actin networks in cell migration and antigen presentation, the two most relevant processes in dendritic cell biology. Branched actin networks construct lamellipodial protrusions at the leading edge of migrating cells. These are typically seen as adhesive structures, which mediate force transduction to the extracellular matrix that leads to forward locomotion. We ablated Arp2/3 nucleation promoting factor WAVE in DCs and found that the resulting cells lack lamellipodial protrusions. Instead, depending on the maturation state, one or multiple filopodia were formed. By challenging these cells in a variety of migration assays we found that lamellipodial protrusions are dispensable for the locomotion of leukocytes and actually dampen the speed of migration. However, lamellipodia are critically required to negotiate complex environments that DCs experience while they travel to the next draining lymph node. Taken together our results suggest that leukocyte lamellipodia have rather a sensory- than a force transducing function. Furthermore, we show for the first time structure and dynamics of dendritic cell F-actin at the immunological synapse with naïve T cells. Dendritic cell F-actin appears as dynamic foci that are nucleated by the Arp2/3 complex. WAVE ablated dendritic cells show increased membrane tension, leading to an altered ultrastructure of the immunological synapse and severe T cell priming defects. These results point towards a previously unappreciated role of the cellular mechanics of dendritic cells in T cell activation. Additionally, we present a novel cell culture based system for the differentiation of dendritic cells from conditionally immortalized hematopoietic precursors. These precursor cells are genetically tractable via the CRISPR/Cas9 system while they retain their ability to differentiate into highly migratory dendritic cells and other immune cells. This will foster the study of all aspects of dendritic cell biology and beyond. AU - Leithner, Alexander F ID - 323 SN - 2663-337X TI - Branched actin networks in dendritic cell biology ER - TY - THES AB - The whole life cycle of plants as well as their responses to environmental stimuli is governed by a complex network of hormonal regulations. A number of studies have demonstrated an essential role of both auxin and cytokinin in the regulation of many aspects of plant growth and development including embryogenesis, postembryonic organogenic processes such as root, and shoot branching, root and shoot apical meristem activity and phyllotaxis. Over the last decades essential knowledge on the key molecular factors and pathways that spatio-temporally define auxin and cytokinin activities in the plant body has accumulated. However, how both hormonal pathways are interconnected by a complex network of interactions and feedback circuits that determines the final outcome of the individual hormone actions is still largely unknown. Root system architecture establishment and in particular formation of lateral organs is prime example of developmental process at whose regulation both auxin and cytokinin pathways converge. To dissect convergence points and pathways that tightly balance auxin - cytokinin antagonistic activities that determine the root branching pattern transcriptome profiling was applied. Genome wide expression analyses of the xylem pole pericycle, a tissue giving rise to lateral roots, led to identification of genes that are highly responsive to combinatorial auxin and cytokinin treatments and play an essential function in the auxin-cytokinin regulated root branching. SYNERGISTIC AUXIN CYTOKININ 1 (SYAC1) gene, which encodes for a protein of unknown function, was detected among the top candidate genes of which expression was synergistically up-regulated by simultaneous hormonal treatment. Plants with modulated SYAC1 activity exhibit severe defects in the root system establishment and attenuate developmental responses to both auxin and cytokinin. To explore the biological function of the SYAC1, we employed different strategies including expression pattern analysis, subcellular localization and phenotypic analyses of the syac1 loss-of-function and gain-of-function transgenic lines along with the identification of the SYAC1 interaction partners. Detailed functional characterization revealed that SYAC1 acts as a developmentally specific regulator of the secretory pathway to control deposition of cell wall components and thereby rapidly fine tune elongation growth. AU - Hurny, Andrej ID - 539 SN - 2663-337X TI - Identification and characterization of novel auxin-cytokinin cross-talk components ER - TY - THES AB - The hippocampus is a key brain region for spatial memory and navigation and is needed at all stages of memory, including encoding, consolidation, and recall. Hippocampal place cells selectively discharge at specific locations of the environment to form a cognitive map of the space. During the rest period and sleep following spatial navigation and/or learning, the waking activity of the place cells is reactivated within high synchrony events. This reactivation is thought to be important for memory consolidation and stabilization of the spatial representations. The aim of my thesis was to directly test whether the reactivation content encoded in firing patterns of place cells is important for consolidation of spatial memories. In particular, I aimed to test whether, in cases when multiple spatial memory traces are acquired during learning, the specific disruption of the reactivation of a subset of these memories leads to the selective disruption of the corresponding memory traces or through memory interference the other learned memories are disrupted as well. In this thesis, using a modified cheeseboard paradigm and a closed-loop recording setup with feedback optogenetic stimulation, I examined how the disruption of the reactivation of specific spiking patterns affects consolidation of the corresponding memory traces. To obtain multiple distinctive memories, animals had to perform a spatial task in two distinct cheeseboard environments and the reactivation of spiking patterns associated with one of the environments (target) was disrupted after learning during four hours rest period using a real-time decoding method. This real-time decoding method was capable of selectively affecting the firing rates and cofiring correlations of the target environment-encoding cells. The selective disruption led to behavioural impairment in the memory tests after the rest periods in the target environment but not in the other undisrupted control environment. In addition, the map of the target environment was less stable in the impaired memory tests compared to the learning session before than the map of the control environment. However, when the animal relearned the task, the same map recurred in the target environment that was present during learning before the disruption. Altogether my work demonstrated that the reactivation content is important: assembly-related disruption of reactivation can lead to a selective memory impairment and deficiency in map stability. These findings indeed suggest that reactivated assembly patterns reflect processes associated with the consolidation of memory traces. AU - Gridchyn, Igor ID - 48 SN - 2663-337X TI - Reactivation content is important for consolidation of spatial memory ER - TY - THES AB - Immune cells migrating to the sites of infection navigate through diverse tissue architectures and switch their migratory mechanisms upon demand. However, little is known about systemic regulators that could allow the acquisition of these mechanisms. We performed a genetic screen in Drosophila melanogaster to identify regulators of germband invasion by embryonic macrophages into the confined space between the ectoderm and mesoderm. We have found that bZIP circadian transcription factors (TFs) Kayak (dFos) and Vrille (dNFIL3) have opposite effects on macrophage germband infiltration: Kayak facilitated and Vrille inhibited it. These TFs are enriched in the macrophages during migration and genetically interact to control it. Kayak sets a less coordinated mode of migration of the macrophage group and increases the probability and length of Levy walks. Intriguingly, the motility of kayak mutant macrophages was also strongly affected during initial germband invasion but not along another less confined route. Inhibiting Rho1 signaling within the tail ectoderm partially rescued the Kayak mutant phenotype, strongly suggesting that migrating macrophages have to overcome a barrier imposed by the stiffness of the ectoderm. Also, Kayak appeared to be important for the maintenance of the round cell shape and the rear edge translocation of the macrophages invading the germband. Complementary to this, the cortical actin cytoskeleton of Kayak- deficient macrophages was strongly affected. RNA sequencing revealed the filamin Cheerio and tetraspanin TM4SF to be downstream of Kayak. Chromatin immunoprecipitation and immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Indeed, Cheerio, TM4SF and Diaphanous are required within macrophages for germband invasion, and expression of constitutively active Diaphanous in macrophages was able to rescue the kayak mutant phenotype. Moreover, Cher and Diaphanous are also reduced in the macrophages overexpressing Vrille. We hypothesize that Kayak, through its targets, increases actin polymerization and cortical tension in macrophages and thus allows extra force generation necessary for macrophage dissemination and migration through confined stiff tissues, while Vrille counterbalances it. AU - Belyaeva, Vera ID - 9 SN - 2663-337X TI - Transcriptional regulation of macrophage migration in the Drosophila melanogaster embryo ER - TY - THES AB - A major challenge in neuroscience research is to dissect the circuits that orchestrate behavior in health and disease. Proteins from a wide range of non-mammalian species, such as microbial opsins, have been successfully transplanted to specific neuronal targets to override their natural communication patterns. The goal of our work is to manipulate synaptic communication in a manner that closely incorporates the functional intricacies of synapses by preserving temporal encoding (i.e. the firing pattern of the presynaptic neuron) and connectivity (i.e. target specific synapses rather than specific neurons). Our strategy to achieve this goal builds on the use of non-mammalian transplants to create a synthetic synapse. The mode of modulation comes from pre-synaptic uptake of a synthetic neurotransmitter (SN) into synaptic vesicles by means of a genetically targeted transporter selective for the SN. Upon natural vesicular release, exposure of the SN to the synaptic cleft will modify the post-synaptic potential through an orthogonal ligand gated ion channel. To achieve this goal we have functionally characterized a mixed cationic methionine-gated ion channel from Arabidopsis thaliana, designed a method to functionally characterize a synthetic transporter in isolated synaptic vesicles without the need for transgenic animals, identified and extracted multiple prokaryotic uptake systems that are substrate specific for methionine (Met), and established a primary/cell line co-culture system that would allow future combinatorial testing of this orthogonal transmitter-transporter-channel trifecta. Synthetic synapses will provide a unique opportunity to manipulate synaptic communication while maintaining the electrophysiological integrity of the pre-synaptic cell. In this way, information may be preserved that was generated in upstream circuits and that could be essential for concerted function and information processing. AU - Mckenzie, Catherine ID - 6266 SN - 2663-337X TI - Design and characterization of methods and biological components to realize synthetic neurotransmission ER - TY - THES AB - The Wnt/planar cell polarity (Wnt/PCP) pathway determines planar polarity of epithelial cells in both vertebrates and invertebrates. The role that Wnt/PCP signaling plays in mesenchymal contexts, however, is only poorly understood. While previous studies have demonstrated the capacity of Wnt/PCP signaling to polarize and guide directed migration of mesenchymal cells, it remains unclear whether endogenous Wnt/PCP signaling performs these functions instructively, as it does in epithelial cells. Here we developed a light-switchable version of the Wnt/PCP receptor Frizzled 7 (Fz7) to unambiguously distinguish between an instructive and a permissive role of Wnt/PCP signaling for the directional collective migration of mesendoderm progenitor cells during zebrafish gastrulation. We show that prechordal plate (ppl) cell migration is defective in maternal-zygotic fz7a and fz7b (MZ fz7a,b) double mutant embryos, and that Fz7 functions cell-autonomously in this process by promoting ppl cell protrusion formation and directed migration. We further show that local activation of Fz7 can direct ppl cell migration both in vitro and in vivo. Surprisingly, however, uniform Fz7 activation is sufficient to fully rescue the ppl cell migration defect in MZ fz7a,b mutant embryos, indicating that Wnt/PCP signaling functions permissively rather than instructively in directed mesendoderm cell migration during zebrafish gastrulation. AU - Capek, Daniel ID - 50 SN - 2663-337X TI - Optogenetic Frizzled 7 reveals a permissive function of Wnt/PCP signaling in directed mesenchymal cell migration ER - TY - THES AB - Expression of genes is a fundamental molecular phenotype that is subject to evolution by different types of mutations. Both the rate and the effect of mutations may depend on the DNA sequence context of a particular gene or a particular promoter sequence. In this thesis I investigate the nature of this dependence using simple genetic systems in Escherichia coli. With these systems I explore the evolution of constitutive gene expression from random starting sequences at different loci on the chromosome and at different locations in sequence space. First, I dissect chromosomal neighborhood effects that underlie locus-dependent differences in the potential of a gene under selection to become more highly expressed. Next, I find that the effects of point mutations in promoter sequences are dependent on sequence context, and that an existing energy matrix model performs poorly in predicting relative expression of unrelated sequences. Finally, I show that a substantial fraction of random sequences contain functional promoters and I present an extended thermodynamic model that predicts promoter strength in full sequence space. Taken together, these results provide new insights and guides on how to integrate information on sequence context to improve our qualitative and quantitative understanding of bacterial gene expression, with implications for rapid evolution of drug resistance, de novo evolution of genes, and horizontal gene transfer. AU - Steinrück, Magdalena ID - 26 SN - 2663-337X TI - The influence of sequence context on the evolution of bacterial gene expression ER - TY - THES AB - Antibiotic resistance can emerge spontaneously through genomic mutation and render treatment ineffective. To counteract this process, in addition to the discovery and description of resistance mechanisms,a deeper understanding of resistanceevolvabilityand its determinantsis needed. To address this challenge, this thesisuncoversnew genetic determinants of resistance evolvability using a customized robotic setup, exploressystematic ways in which resistance evolution is perturbed due to dose-responsecharacteristics of drugs and mutation rate differences,and mathematically investigates the evolutionary fate of one specific type of evolvability modifier -a stress-induced mutagenesis allele.We find severalgenes which strongly inhibit or potentiate resistance evolution. In order to identify them, we first developedan automated high-throughput feedback-controlled protocol whichkeeps the population size and selection pressure approximately constant for hundreds of cultures by dynamically re-diluting the cultures and adjusting the antibiotic concentration. We implementedthis protocol on a customized liquid handling robot and propagated 100 different gene deletion strains of Escherichia coliin triplicate for over 100 generations in tetracycline and in chloramphenicol, and comparedtheir adaptation rates.We find a diminishing returns pattern, where initially sensitive strains adapted more compared to less sensitive ones. Our data uncover that deletions of certain genes which do not affect mutation rate,including efflux pump components, a chaperone and severalstructural and regulatory genes can strongly and reproducibly alterresistance evolution. Sequencing analysis of evolved populations indicates that epistasis with resistance mutations is the most likelyexplanation. This work could inspire treatment strategies in which targeted inhibitors of evolvability mechanisms will be given alongside antibiotics to slow down resistance evolution and extend theefficacy of antibiotics.We implemented astochasticpopulation genetics model, toverifyways in which general properties, namely, dose-response characteristics of drugs and mutation rates, influence evolutionary dynamics. In particular, under the exposure to antibiotics with shallow dose-response curves,bacteria have narrower distributions of fitness effects of new mutations. We show that in silicothis also leads to slower resistance evolution. We see and confirm with experiments that increased mutation rates, apart from speeding up evolution, also leadto high reproducibility of phenotypic adaptation in a context of continually strong selection pressure.Knowledge of these patterns can aid in predicting the dynamics of antibiotic resistance evolutionand adapting treatment schemes accordingly.Focusing on a previously described type of evolvability modifier –a stress-induced mutagenesis allele –we find conditions under which it can persist in a population under periodic selectionakin to clinical treatment. We set up a deterministic infinite populationcontinuous time model tracking the frequencies of a mutator and resistance allele and evaluate various treatment schemes in how well they maintain a stress-induced mutator allele. In particular,a high diversity of stresses is crucial for the persistence of the mutator allele. This leads to a general trade-off where exactly those diversifying treatment schemes which are likely to decrease levels of resistance could lead to stronger selection of highly evolvable genotypes.In the long run, this work will lead to a deeper understanding of the genetic and cellular mechanisms involved in antibiotic resistance evolution and could inspire new strategies for slowing down its rate. AU - Lukacisinova, Marta ID - 6263 SN - 2663-337X TI - Genetic determinants of antibiotic resistance evolution ER - TY - THES AB - This dissertation concerns the automatic verification of probabilistic systems and programs with arrays by statistical and logical methods. Although statistical and logical methods are different in nature, we show that they can be successfully combined for system analysis. In the first part of the dissertation we present a new statistical algorithm for the verification of probabilistic systems with respect to unbounded properties, including linear temporal logic. Our algorithm often performs faster than the previous approaches, and at the same time requires less information about the system. In addition, our method can be generalized to unbounded quantitative properties such as mean-payoff bounds. In the second part, we introduce two techniques for comparing probabilistic systems. Probabilistic systems are typically compared using the notion of equivalence, which requires the systems to have the equal probability of all behaviors. However, this notion is often too strict, since probabilities are typically only empirically estimated, and any imprecision may break the relation between processes. On the one hand, we propose to replace the Boolean notion of equivalence by a quantitative distance of similarity. For this purpose, we introduce a statistical framework for estimating distances between Markov chains based on their simulation runs, and we investigate which distances can be approximated in our framework. On the other hand, we propose to compare systems with respect to a new qualitative logic, which expresses that behaviors occur with probability one or a positive probability. This qualitative analysis is robust with respect to modeling errors and applicable to many domains. In the last part, we present a new quantifier-free logic for integer arrays, which allows us to express counting. Counting properties are prevalent in array-manipulating programs, however they cannot be expressed in the quantified fragments of the theory of arrays. We present a decision procedure for our logic, and provide several complexity results. AU - Daca, Przemyslaw ID - 1155 SN - 2663-337X TI - Statistical and logical methods for property checking ER - TY - THES AB - Bacteria and their pathogens – phages – are the most abundant living entities on Earth. Throughout their coevolution, bacteria have evolved multiple immune systems to overcome the ubiquitous threat from the phages. Although the molecu- lar details of these immune systems’ functions are relatively well understood, their epidemiological consequences for the phage-bacterial communities have been largely neglected. In this thesis we employed both experimental and theoretical methods to explore whether herd and social immunity may arise in bacterial popu- lations. Using our experimental system consisting of Escherichia coli strains with a CRISPR based immunity to the T7 phage we show that herd immunity arises in phage-bacterial communities and that it is accentuated when the populations are spatially structured. By fitting a mathematical model, we inferred expressions for the herd immunity threshold and the velocity of spread of a phage epidemic in partially resistant bacterial populations, which both depend on the bacterial growth rate, phage burst size and phage latent period. We also investigated the poten- tial for social immunity in Streptococcus thermophilus and its phage 2972 using a bioinformatic analysis of potentially coding short open reading frames with a signalling signature, encoded within the CRISPR associated genes. Subsequently, we tested one identified potentially signalling peptide and found that its addition to a phage-challenged culture increases probability of survival of bacteria two fold, although the results were only marginally significant. Together, these results demonstrate that the ubiquitous arms races between bacteria and phages have further consequences at the level of the population. AU - Payne, Pavel ID - 6291 SN - 2663-337X TI - Bacterial herd and social immunity to phages ER - TY - THES AB - Antibiotics have diverse effects on bacteria, including massive changes in bacterial gene expression. Whereas the gene expression changes under many antibiotics have been measured, the temporal organization of these responses and their dependence on the bacterial growth rate are unclear. As described in Chapter 1, we quantified the temporal gene expression changes in the bacterium Escherichia coli in response to the sudden exposure to antibiotics using a fluorescent reporter library and a robotic system. Our data show temporally structured gene expression responses, with response times for individual genes ranging from tens of minutes to several hours. We observed that many stress response genes were activated in response to antibiotics. As certain stress responses cross-protect bacteria from other stressors, we then asked whether cellular responses to antibiotics have a similar protective role in Chapter 2. Indeed, we found that the trimethoprim-induced acid stress response protects bacteria from subsequent acid stress. We combined microfluidics with time-lapse imaging to monitor survival, intracellular pH, and acid stress response in single cells. This approach revealed that the variable expression of the acid resistance operon gadBC strongly correlates with single-cell survival time. Cells with higher gadBC expression following trimethoprim maintain higher intracellular pH and survive the acid stress longer. Overall, we provide a way to identify single-cell cross-protection between antibiotics and environmental stressors from temporal gene expression data, and show how antibiotics can increase bacterial fitness in changing environments. While gene expression changes to antibiotics show a clear temporal structure at the population-level, it is unclear whether this clear temporal order is followed by every single cell. Using dual-reporter strains described in Chapter 3, we measured gene expression dynamics of promoter pairs in the same cells using microfluidics and microscopy. Chapter 4 shows that the oxidative stress response and the DNA stress response showed little timing variability and a clear temporal order under the antibiotic nitrofurantoin. In contrast, the acid stress response under trimethoprim ran independently from all other activated response programs including the DNA stress response, which showed particularly high timing variability in this stress condition. In summary, this approach provides insight into the temporal organization of gene expression programs at the single-cell level and suggests dependencies between response programs and the underlying variability-introducing mechanisms. Altogether, this work advances our understanding of the diverse effects that antibiotics have on bacteria. These results were obtained by taking into account gene expression dynamics, which allowed us to identify general principles, molecular mechanisms, and dependencies between genes. Our findings may have implications for infectious disease treatments, and microbial communities in the human body and in nature. AU - Mitosch, Karin ID - 818 SN - 2663-337X TI - Timing, variability and cross-protection in bacteria – insights from dynamic gene expression responses to antibiotics ER - TY - THES AB - This dissertation focuses on algorithmic aspects of program verification, and presents modeling and complexity advances on several problems related to the static analysis of programs, the stateless model checking of concurrent programs, and the competitive analysis of real-time scheduling algorithms. Our contributions can be broadly grouped into five categories. Our first contribution is a set of new algorithms and data structures for the quantitative and data-flow analysis of programs, based on the graph-theoretic notion of treewidth. It has been observed that the control-flow graphs of typical programs have special structure, and are characterized as graphs of small treewidth. We utilize this structural property to provide faster algorithms for the quantitative and data-flow analysis of recursive and concurrent programs. In most cases we make an algebraic treatment of the considered problem, where several interesting analyses, such as the reachability, shortest path, and certain kind of data-flow analysis problems follow as special cases. We exploit the constant-treewidth property to obtain algorithmic improvements for on-demand versions of the problems, and provide data structures with various tradeoffs between the resources spent in the preprocessing and querying phase. We also improve on the algorithmic complexity of quantitative problems outside the algebraic path framework, namely of the minimum mean-payoff, minimum ratio, and minimum initial credit for energy problems. Our second contribution is a set of algorithms for Dyck reachability with applications to data-dependence analysis and alias analysis. In particular, we develop an optimal algorithm for Dyck reachability on bidirected graphs, which are ubiquitous in context-insensitive, field-sensitive points-to analysis. Additionally, we develop an efficient algorithm for context-sensitive data-dependence analysis via Dyck reachability, where the task is to obtain analysis summaries of library code in the presence of callbacks. Our algorithm preprocesses libraries in almost linear time, after which the contribution of the library in the complexity of the client analysis is (i)~linear in the number of call sites and (ii)~only logarithmic in the size of the whole library, as opposed to linear in the size of the whole library. Finally, we prove that Dyck reachability is Boolean Matrix Multiplication-hard in general, and the hardness also holds for graphs of constant treewidth. This hardness result strongly indicates that there exist no combinatorial algorithms for Dyck reachability with truly subcubic complexity. Our third contribution is the formalization and algorithmic treatment of the Quantitative Interprocedural Analysis framework. In this framework, the transitions of a recursive program are annotated as good, bad or neutral, and receive a weight which measures the magnitude of their respective effect. The Quantitative Interprocedural Analysis problem asks to determine whether there exists an infinite run of the program where the long-run ratio of the bad weights over the good weights is above a given threshold. We illustrate how several quantitative problems related to static analysis of recursive programs can be instantiated in this framework, and present some case studies to this direction. Our fourth contribution is a new dynamic partial-order reduction for the stateless model checking of concurrent programs. Traditional approaches rely on the standard Mazurkiewicz equivalence between traces, by means of partitioning the trace space into equivalence classes, and attempting to explore a few representatives from each class. We present a new dynamic partial-order reduction method called the Data-centric Partial Order Reduction (DC-DPOR). Our algorithm is based on a new equivalence between traces, called the observation equivalence. DC-DPOR explores a coarser partitioning of the trace space than any exploration method based on the standard Mazurkiewicz equivalence. Depending on the program, the new partitioning can be even exponentially coarser. Additionally, DC-DPOR spends only polynomial time in each explored class. Our fifth contribution is the use of automata and game-theoretic verification techniques in the competitive analysis and synthesis of real-time scheduling algorithms for firm-deadline tasks. On the analysis side, we leverage automata on infinite words to compute the competitive ratio of real-time schedulers subject to various environmental constraints. On the synthesis side, we introduce a new instance of two-player mean-payoff partial-information games, and show how the synthesis of an optimal real-time scheduler can be reduced to computing winning strategies in this new type of games. AU - Pavlogiannis, Andreas ID - 821 SN - 2663-337X TI - Algorithmic advances in program analysis and their applications ER - TY - THES AB - The lac operon is a classic model system for bacterial gene regulation, and has been studied extensively in E. coli, a classic model organism. However, not much is known about E. coli’s ecology and life outside the laboratory, in particular in soil and water environments. The natural diversity of the lac operon outside the laboratory, its role in the ecology of E. coli and the selection pressures it is exposed to, are similarly unknown. In Chapter Two of this thesis, I explore the genetic diversity, phylogenetic history and signatures of selection of the lac operon across 20 natural isolates of E. coli and divergent clades of Escherichia. I found that complete lac operons were present in all isolates examined, which in all but one case were functional. The lac operon phylogeny conformed to the whole-genome phylogeny of the divergent Escherichia clades, which excludes horizontal gene transfer as an explanation for the presence of functional lac operons in these clades. All lac operon genes showed a signature of purifying selection; this signature was strongest for the lacY gene. Lac operon genes of human and environmental isolates showed similar signatures of selection, except the lacZ gene, which showed a stronger signature of selection in environmental isolates. In Chapter Three, I try to identify the natural genetic variation relevant for phenotype and fitness in the lac operon, comparing growth rate on lactose and LacZ activity of the lac operons of these wild isolates in a common genetic background. Sequence variation in the lac promoter region, upstream of the -10 and -35 RNA polymerase binding motif, predicted variation in LacZ activity at full induction, using a thermodynamic model of polymerase binding (Tugrul, 2016). However, neither variation in LacZ activity, nor RNA polymerase binding predicted by the model correlated with variation in growth rate. Lac operons of human and environmental isolates did not differ systematically in either growth rate on lactose or LacZ protein activity, suggesting that these lac operons have been exposed to similar selection pressures. We thus have no evidence that the phenotypic variation we measured is relevant for fitness. To start assessing the effect of genomic background on the growth phenotype conferred by the lac operon, I compared growth on minimal medium with lactose between lac operon constructs and the corresponding original isolates, I found that maximal growth rate was determined by genomic background, with almost all backgrounds conferring higher growth rates than lab strain K12 MG1655. However, I found no evidence that the lactose concentration at which growth was half maximal depended on genomic background. AU - Jesse, Fabienne ID - 820 SN - 2663-337X TI - The lac operon in the wild ER - TY - THES AB - In this thesis we discuss the exact security of message authentications codes HMAC , NMAC , and PMAC . NMAC is a mode of operation which turns a fixed input-length keyed hash function f into a variable input-length function. A practical single-key variant of NMAC called HMAC is a very popular and widely deployed message authentication code (MAC). PMAC is a block-cipher based mode of operation, which also happens to be the most famous fully parallel MAC. NMAC was introduced by Bellare, Canetti and Krawczyk Crypto’96, who proved it to be a secure pseudorandom function (PRF), and thus also a MAC, under two assumptions. Unfortunately, for many instantiations of HMAC one of them has been found to be wrong. To restore the provable guarantees for NMAC , Bellare [Crypto’06] showed its security without this assumption. PMAC was introduced by Black and Rogaway at Eurocrypt 2002. If instantiated with a pseudorandom permutation over n -bit strings, PMAC constitutes a provably secure variable input-length PRF. For adversaries making q queries, each of length at most ` (in n -bit blocks), and of total length σ ≤ q` , the original paper proves an upper bound on the distinguishing advantage of O ( σ 2 / 2 n ), while the currently best bound is O ( qσ/ 2 n ). In this work we show that this bound is tight by giving an attack with advantage Ω( q 2 `/ 2 n ). In the PMAC construction one initially XORs a mask to every message block, where the mask for the i th block is computed as τ i := γ i · L , where L is a (secret) random value, and γ i is the i -th codeword of the Gray code. Our attack applies more generally to any sequence of γ i ’s which contains a large coset of a subgroup of GF (2 n ). As for NMAC , our first contribution is a simpler and uniform proof: If f is an ε -secure PRF (against q queries) and a δ - non-adaptively secure PRF (against q queries), then NMAC f is an ( ε + `qδ )-secure PRF against q queries of length at most ` blocks each. We also show that this ε + `qδ bound is basically tight by constructing an f for which an attack with advantage `qδ exists. Moreover, we analyze the PRF-security of a modification of NMAC called NI by An and Bellare that avoids the constant rekeying on multi-block messages in NMAC and allows for an information-theoretic analysis. We carry out such an analysis, obtaining a tight `q 2 / 2 c bound for this step, improving over the trivial bound of ` 2 q 2 / 2 c . Finally, we investigate, if the security of PMAC can be further improved by using τ i ’s that are k -wise independent, for k > 1 (the original has k = 1). We observe that the security of PMAC will not increase in general if k = 2, and then prove that the security increases to O ( q 2 / 2 n ), if the k = 4. Due to simple extension attacks, this is the best bound one can hope for, using any distribution on the masks. Whether k = 3 is already sufficient to get this level of security is left as an open problem. Keywords: Message authentication codes, Pseudorandom functions, HMAC, PMAC. AU - Rybar, Michal ID - 838 SN - 2663-337X TI - (The exact security of) Message authentication codes ER - TY - THES AB - The hippocampus is a key brain region for memory and notably for spatial memory, and is needed for both spatial working and reference memories. Hippocampal place cells selectively discharge in specific locations of the environment to form mnemonic represen tations of space. Several behavioral protocols have been designed to test spatial memory which requires the experimental subject to utilize working memory and reference memory. However, less is known about how these memory traces are presented in the hippo campus, especially considering tasks that require both spatial working and long -term reference memory demand. The aim of my thesis was to elucidate how spatial working memory, reference memory, and the combination of both are represented in the hippocampus. In this thesis, using a radial eight -arm maze, I examined how the combined demand on these memories influenced place cell assemblies while reference memories were partially updated by changing some of the reward- arms. This was contrasted with task varian ts requiring working or reference memories only. Reference memory update led to gradual place field shifts towards the rewards on the switched arms. Cells developed enhanced firing in passes between newly -rewarded arms as compared to those containing an unchanged reward. The working memory task did not show such gradual changes. Place assemblies on occasions replayed trajectories of the maze; at decision points the next arm choice was preferentially replayed in tasks needing reference memory while in the pure working memory task the previously visited arm was replayed. Hence trajectory replay only reflected the decision of the animal in tasks needing reference memory update. At the reward locations, in all three tasks outbound trajectories of the current arm were preferentially replayed, showing the animals’ next path to the center. At reward locations trajectories were replayed preferentially in reverse temporal order. Moreover, in the center reverse replay was seen in the working memory task but in the other tasks forward replay was seen. Hence, the direction of reactivation was determined by the goal locations so that part of the trajectory which was closer to the goal was reactivated later in an HSE while places further away from the goal were reactivated earlier. Altogether my work demonstrated that reference memory update triggers several levels of reorganization of the hippocampal cognitive map which are not seen in simpler working memory demand s. Moreover, hippocampus is likely to be involved in spatial decisions through reactivating planned trajectories when reference memory recall is required for such a decision. AU - Xu, Haibing ID - 837 SN - 2663-337X TI - Reactivation of the hippocampal cognitive map in goal-directed spatial tasks ER - TY - THES AB - The thesis encompasses several topics of plant cell biology which were studied in the model plant Arabidopsis thaliana. Chapter 1 concerns the plant hormone auxin and its polar transport through cells and tissues. The highly controlled, directional transport of auxin is facilitated by plasma membrane-localized transporters. Transporters from the PIN family direct auxin transport due to their polarized localizations at cell membranes. Substantial effort has been put into research on cellular trafficking of PIN proteins, which is thought to underlie their polar distribution. I participated in a forward genetic screen aimed at identifying novel regulators of PIN polarity. The screen yielded several genes which may be involved in PIN polarity regulation or participate in polar auxin transport by other means. Chapter 2 focuses on the endomembrane system, with particular attention to clathrin-mediated endocytosis. The project started with identification of several proteins that interact with clathrin light chains. Among them, I focused on two putative homologues of auxilin, which in non-plant systems is an endocytotic factor known for uncoating clathrin-coated vesicles in the final step of endocytosis. The body of my work consisted of an in-depth characterization of transgenic A. thaliana lines overexpressing these putative auxilins in an inducible manner. Overexpression of these proteins leads to an inhibition of endocytosis, as documented by imaging of cargoes and clathrin-related endocytic machinery. An extension of this work is an investigation into a concept of homeostatic regulation acting between distinct transport processes in the endomembrane system. With auxilin overexpressing lines, where endocytosis is blocked specifically, I made observations on the mutual relationship between two opposite trafficking processes of secretion and endocytosis. In Chapter 3, I analyze cortical microtubule arrays and their relationship to auxin signaling and polarized growth in elongating cells. In plants, microtubules are organized into arrays just below the plasma membrane, and it is thought that their function is to guide membrane-docked cellulose synthase complexes. These, in turn, influence cell wall structure and cell shape by directed deposition of cellulose fibres. In elongating cells, cortical microtubule arrays are able to reorient in relation to long cell axis, and these reorientations have been linked to cell growth and to signaling of growth-regulating factors such as auxin or light. In this chapter, I am addressing the causal relationship between microtubule array reorientation, growth, and auxin signaling. I arrive at a model where array reorientation is not guided by auxin directly, but instead is only controlled by growth, which, in turn, is regulated by auxin. AU - Adamowski, Maciek ID - 938 SN - 2663-337X TI - Investigations into cell polarity and trafficking in the plant model Arabidopsis thaliana ER - TY - THES AB - An instance of the Constraint Satisfaction Problem (CSP) is given by a finite set of variables, a finite domain of labels, and a set of constraints, each constraint acting on a subset of the variables. The goal is to find an assignment of labels to its variables that satisfies all constraints (or decide whether one exists). If we allow more general “soft” constraints, which come with (possibly infinite) costs of particular assignments, we obtain instances from a richer class called Valued Constraint Satisfaction Problem (VCSP). There the goal is to find an assignment with minimum total cost. In this thesis, we focus (assuming that P 6 = NP) on classifying computational com- plexity of CSPs and VCSPs under certain restricting conditions. Two results are the core content of the work. In one of them, we consider VCSPs parametrized by a constraint language, that is the set of “soft” constraints allowed to form the instances, and finish the complexity classification modulo (missing pieces of) complexity classification for analogously parametrized CSP. The other result is a generalization of Edmonds’ perfect matching algorithm. This generalization contributes to complexity classfications in two ways. First, it gives a new (largest known) polynomial-time solvable class of Boolean CSPs in which every variable may appear in at most two constraints and second, it settles full classification of Boolean CSPs with planar drawing (again parametrized by a constraint language). AU - Rolinek, Michal ID - 992 SN - 2663-337X TI - Complexity of constraint satisfaction ER - TY - THES AB - Restriction-modification (RM) represents the simplest and possibly the most widespread mechanism of self/non-self discrimination in nature. In order to provide bacteria with immunity against bacteriophages and other parasitic genetic elements, RM systems rely on a balance between two enzymes: the restriction enzyme, which cleaves non-self DNA at specific restriction sites, and the modification enzyme, which tags the host’s DNA as self and thus protects it from cleavage. In this thesis, I use population and single-cell level experiments in combination with mathematical modeling to study different aspects of the interplay between RM systems, bacteria and bacteriophages. First, I analyze how mutations in phage restriction sites affect the probability of phage escape – an inherently stochastic process, during which phages accidently get modified instead of restricted. Next, I use single-cell experiments to show that RM systems can, with a low probability, attack the genome of their bacterial host and that this primitive form of autoimmunity leads to a tradeoff between the evolutionary cost and benefit of RM systems. Finally, I investigate the nature of interactions between bacteria, RM systems and temperate bacteriophages to find that, as a consequence of phage escape and its impact on population dynamics, RM systems can promote acquisition of symbiotic bacteriophages, rather than limit it. The results presented here uncover new fundamental biological properties of RM systems and highlight their importance in the ecology and evolution of bacteria, bacteriophages and their interactions. AU - Pleska, Maros ID - 202 SN - 2663-337X TI - Biology of restriction-modification systems at the single-cell and population level ER - TY - THES AB - The main objects considered in the present work are simplicial and CW-complexes with vertices forming a random point cloud. In particular, we consider a Poisson point process in R^n and study Delaunay and Voronoi complexes of the first and higher orders and weighted Delaunay complexes obtained as sections of Delaunay complexes, as well as the Čech complex. Further, we examine theDelaunay complex of a Poisson point process on the sphere S^n, as well as of a uniform point cloud, which is equivalent to the convex hull, providing a connection to the theory of random polytopes. Each of the complexes in question can be endowed with a radius function, which maps its cells to the radii of appropriately chosen circumspheres, called the radius of the cell. Applying and developing discrete Morse theory for these functions, joining it together with probabilistic and sometimes analytic machinery, and developing several integral geometric tools, we aim at getting the distributions of circumradii of typical cells. For all considered complexes, we are able to generalize and obtain up to constants the distribution of radii of typical intervals of all types. In low dimensions the constants can be computed explicitly, thus providing the explicit expressions for the expected numbers of cells. In particular, it allows to find the expected density of simplices of every dimension for a Poisson point process in R^4, whereas the result for R^3 was known already in 1970's. AU - Nikitenko, Anton ID - 6287 SN - 2663-337X TI - Discrete Morse theory for random complexes ER - TY - THES AB - Plant hormone auxin and its transport between cells belong to the most important mechanisms controlling plant development. Auxin itself could change localization of PINs and thereby control direction of its own flow. We performed an expression profiling experiment in Arabidopsis roots to identify potential regulators of PIN polarity which are transcriptionally regulated by auxin signalling. We identified several novel regulators and performed a detailed characterization of the transcription factor WRKY23 (At2g47260) and its role in auxin feedback on PIN polarity. Gain-of-function and dominant-negative mutants revealed that WRKY23 plays a crucial role in mediating the auxin effect on PIN polarity. In concordance, typical polar auxin transport processes such as gravitropism and leaf vascular pattern formation were disturbed by interfering with WRKY23 function. In order to identify direct targets of WRKY23, we performed consequential expression profiling experiments using a WRKY23 inducible gain-of-function line and dominant-negative WRKY23 line that is defunct in PIN re-arrangement. Among several genes mostly related to the groups of cell wall and defense process regulators, we identified LYSINE-HISTIDINE TRANSPORTER 1 (LHT1; At5g40780), a small amino acid permease gene from the amino acid/auxin permease family (AAAP), we present its detailed characterisation in auxin feedback on PIN repolarization, identified its transcriptional regulation, we propose a potential mechanism of its action. Moreover, we identified also a member of receptor-like protein kinase LRR-RLK (LEUCINE-RICH REPEAT TRANSMEMBRANE PROTEIN KINASE PROTEIN 1; LRRK1; At1g05700), which also affects auxin-dependent PIN re-arrangement. We described its transcriptional behaviour, subcellular localization. Based on global expression data, we tried to identify ligand responsible for mechanism of signalling and suggest signalling partner and interactors. Additionally, we described role of novel phytohormone group, strigolactone, in auxin-dependent PIN re-arrangement, that could be a fundament for future studies in this field. Our results provide first insights into an auxin transcriptional network targeting PIN localization and thus regulating plant development. We highlighted WRKY23 transcriptional network and characterised its mediatory role in plant development. We identified direct effectors of this network, LHT1 and LRRK1, and describe their roles in PIN re-arrangement and PIN-dependent auxin transport processes. AU - Prat, Tomas ID - 1127 SN - 2663-337X TI - Identification of novel regulators of PIN polarity and development of novel auxin sensor ER - TY - THES AB - Cell-cell contact formation constitutes the first step in the emergence of multicellularity in evolution, thereby allowing the differentiation of specialized cell types. In metazoan development, cell-cell contact formation is thought to influence cell fate specification, and cell fate specification has been implicated in cell-cell contact formation. However, remarkably little is yet known about whether and how the interaction and feedback between cell-cell contact formation and cell fate specification affect development. Here we identify a positive feedback loop between cell-cell contact duration, morphogen signaling and mesendoderm cell fate specification during zebrafish gastrulation. We show that long lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for proper ppl cell fate specification. We further show that Nodal signalling romotes ppl cell-cell contact duration, thereby generating an effective positive feedback loop between ppl cell-cell contact duration and cell fate specification. Finally, by using a combination of theoretical modeling and experimentation, we show that this feedback loop determines whether anterior axial mesendoderm cells become ppl progenitors or, instead, turn into endoderm progenitors. Our findings reveal that the gene regulatory networks leading to cell fate diversification within the developing embryo are controlled by the interdependent activities of cell-cell signaling and contact formation. AU - Barone, Vanessa ID - 961 SN - 2663-337X TI - Cell adhesion and cell fate: An effective feedback loop during zebrafish gastrulation ER - TY - THES AB - Contagious diseases must transmit from infectious to susceptible hosts in order to reproduce. Whilst vectored pathogens can rely on intermediaries to find new hosts for them, many infectious pathogens require close contact or direct interaction between hosts for transmission. Hence, this means that conspecifics are often the main source of infection for most animals and so, in theory, animals should avoid conspecifics to reduce their risk of infection. Of course, in reality animals must interact with one another, as a bare minimum, to mate. However, being social provides many additional benefits and group living has become a taxonomically diverse and widespread trait. How then do social animals overcome the issue of increased disease? Over the last few decades, the social insects (ants, termites and some bees and wasps) have become a model system for studying disease in social animals. On paper, a social insect colony should be particularly susceptible to disease, given that they often contain thousands of potential hosts that are closely related and frequently interact, as well as exhibiting stable environmental conditions that encourage microbial growth. Yet, disease outbreaks appear to be rare and attempts to eradicate pest species using pathogens have failed time and again. Evolutionary biologists investigating this observation have discovered that the reduced disease susceptibility in social insects is, in part, due to collectively performed disease defences of the workers. These defences act like a “social immune system” for the colony, resulting in a per capita decrease in disease, termed social immunity. Our understanding of social immunity, and its importance in relation to the immunological defences of each insect, continues to grow, but there remain many open questions. In this thesis I have studied disease defence in garden ants. In the first data chapter, I use the invasive garden ant, Lasius neglectus, to investigate how colonies mitigate lethal infections and prevent them from spreading systemically. I find that ants have evolved ‘destructive disinfection’ – a behaviour that uses endogenously produced acidic poison to kill diseased brood and to prevent the pathogen from replicating. In the second experimental chapter, I continue to study the use of poison in invasive garden ant colonies, finding that it is sprayed prophylactically within the nest. However, this spraying has negative effects on developing pupae when they have had their cocoons artificially removed. Hence, I suggest that acidic nest sanitation may be maintaining larval cocoon spinning in this species. In the next experimental chapter, I investigated how colony founding black garden ant queens (Lasius niger) prevent disease when a co-foundress dies. I show that ant queens prophylactically perform undertaking behaviours, similar to those performed by the workers in mature nests. When a co-foundress was infected, these undertaking behaviours improved the survival of the healthy queen. In the final data chapter, I explored how immunocompetence (measured as antifungal activity) changes as incipient black garden ant colonies grow and mature, from the solitary queen phase to colonies with several hundred workers. Queen and worker antifungal activity varied throughout this time period, but despite social immunity, did not decrease as colonies matured. In addition to the above data chapters, this thesis includes two co-authored reviews. In the first, we examine the state of the art in the field of social immunity and how it might develop in the future. In the second, we identify several challenges and open questions in the study of disease defence in animals. We highlight how social insects offer a unique model to tackle some of these problems, as disease defence can be studied from the cell to the society. AU - Pull, Christopher ID - 819 SN - 2663-337X TI - Disease defence in garden ants ER - TY - THES AB - This thesis describes a brittle fracture simulation method for visual effects applications. Building upon a symmetric Galerkin boundary element method, we first compute stress intensity factors following the theory of linear elastic fracture mechanics. We then use these stress intensities to simulate the motion of a propagating crack front at a significantly higher resolution than the overall deformation of the breaking object. Allowing for spatial variations of the material's toughness during crack propagation produces visually realistic, highly-detailed fracture surfaces. Furthermore, we introduce approximations for stress intensities and crack opening displacements, resulting in both practical speed-up and theoretically superior runtime complexity compared to previous methods. While we choose a quasi-static approach to fracture mechanics, ignoring dynamic deformations, we also couple our fracture simulation framework to a standard rigid-body dynamics solver, enabling visual effects artists to simulate both large scale motion, as well as fracturing due to collision forces in a combined system. As fractures inside of an object grow, their geometry must be represented both in the coarse boundary element mesh, as well as at the desired fine output resolution. Using a boundary element method, we avoid complicated volumetric meshing operations. Instead we describe a simple set of surface meshing operations that allow us to progressively add cracks to the mesh of an object and still re-use all previously computed entries of the linear boundary element system matrix. On the high resolution level, we opt for an implicit surface representation. We then describe how to capture fracture surfaces during crack propagation, as well as separate the individual fragments resulting from the fracture process, based on this implicit representation. We show results obtained with our method, either solving the full boundary element system in every time step, or alternatively using our fast approximations. These results demonstrate that both of these methods perform well in basic test cases and produce realistic fracture surfaces. Furthermore we show that our fast approximations substantially out-perform the standard approach in more demanding scenarios. Finally, these two methods naturally combine, using the full solution while the problem size is manageably small and switching to the fast approximations later on. The resulting hybrid method gives the user a direct way to choose between speed and accuracy of the simulation. AU - Hahn, David ID - 839 SN - 2663-337X TI - Brittle fracture simulation with boundary elements for computer graphics ER - TY - THES AB - Horizontal gene transfer (HGT), the lateral acquisition of genes across existing species boundaries, is a major evolutionary force shaping microbial genomes that facilitates adaptation to new environments as well as resistance to antimicrobial drugs. As such, understanding the mechanisms and constraints that determine the outcomes of HGT events is crucial to understand the dynamics of HGT and to design better strategies to overcome the challenges that originate from it. Following the insertion and expression of a newly transferred gene, the success of an HGT event will depend on the fitness effect it has on the recipient (host) cell. Therefore, predicting the impact of HGT on the genetic composition of a population critically depends on the distribution of fitness effects (DFE) of horizontally transferred genes. However, to date, we have little knowledge of the DFE of newly transferred genes, and hence little is known about the shape and scale of this distribution. It is particularly important to better understand the selective barriers that determine the fitness effects of newly transferred genes. In spite of substantial bioinformatics efforts to identify horizontally transferred genes and selective barriers, a systematic experimental approach to elucidate the roles of different selective barriers in defining the fate of a transfer event has largely been absent. Similarly, although the fact that environment might alter the fitness effect of a horizontally transferred gene may seem obvious, little attention has been given to it in a systematic experimental manner. In this study, we developed a systematic experimental approach that consists of transferring 44 arbitrarily selected Salmonella typhimurium orthologous genes into an Escherichia coli host, and estimating the fitness effects of these transferred genes at a constant expression level by performing competition assays against the wild type. In chapter 2, we performed one-to-one competition assays between a mutant strain carrying a transferred gene and the wild type strain. By using flow cytometry we estimated selection coefficients for the transferred genes with a precision level of 10-3,and obtained the DFE of horizontally transferred genes. We then investigated if these fitness effects could be predicted by any of the intrinsic properties of the genes, namely, functional category, degree of complexity (protein-protein interactions), GC content, codon usage and length. Our analyses revealed that the functional category and length of the genes act as potential selective barriers. Finally, using the same procedure with the endogenous E. coli orthologs of these 44 genes, we demonstrated that gene dosage is the most prominent selective barrier to HGT. In chapter 3, using the same set of genes we investigated the role of environment on the success of HGT events. Under six different environments with different levels of stress we performed more complex competition assays, where we mixed all 44 mutant strains carrying transferred genes with the wild type strain. To estimate the fitness effects of genes relative to wild type we used next generation sequencing. We found that the DFEs of horizontally transferred genes are highly dependent on the environment, with abundant gene–by-environment interactions. Furthermore, we demonstrated a relationship between average fitness effect of a gene across all environments and its environmental variance, and thus its predictability. Finally, in spite of the fitness effects of genes being highly environment-dependent, we still observed a common shape of DFEs across all tested environments. AU - Acar, Hande ID - 1121 SN - 2663-337X TI - Selective barriers to horizontal gene transfer ER - TY - THES AB - The process of gene expression is central to the modern understanding of how cellular systems function. In this process, a special kind of regulatory proteins, called transcription factors, are important to determine how much protein is produced from a given gene. As biological information is transmitted from transcription factor concentration to mRNA levels to amounts of protein, various sources of noise arise and pose limits to the fidelity of intracellular signaling. This thesis concerns itself with several aspects of stochastic gene expression: (i) the mathematical description of complex promoters responsible for the stochastic production of biomolecules, (ii) fundamental limits to information processing the cell faces due to the interference from multiple fluctuating signals, (iii) how the presence of gene expression noise influences the evolution of regulatory sequences, (iv) and tools for the experimental study of origins and consequences of cell-cell heterogeneity, including an application to bacterial stress response systems. AU - Rieckh, Georg ID - 1128 SN - 2663-337X TI - Studying the complexities of transcriptional regulation ER - TY - THES AU - Morri, Maurizio ID - 1124 SN - 2663-337X TI - Optical functionalization of human class A orphan G-protein coupled receptors ER - TY - THES AB - Directed cell migration is a hallmark feature, present in almost all multi-cellular organisms. Despite its importance, basic questions regarding force transduction or directional sensing are still heavily investigated. Directed migration of cells guided by immobilized guidance cues - haptotaxis - occurs in key-processes, such as embryonic development and immunity (Middleton et al., 1997; Nguyen et al., 2000; Thiery, 1984; Weber et al., 2013). Immobilized guidance cues comprise adhesive ligands, such as collagen and fibronectin (Barczyk et al., 2009), or chemokines - the main guidance cues for migratory leukocytes (Middleton et al., 1997; Weber et al., 2013). While adhesive ligands serve as attachment sites guiding cell migration (Carter, 1965), chemokines instruct haptotactic migration by inducing adhesion to adhesive ligands and directional guidance (Rot and Andrian, 2004; Schumann et al., 2010). Quantitative analysis of the cellular response to immobilized guidance cues requires in vitro assays that foster cell migration, offer accurate control of the immobilized cues on a subcellular scale and in the ideal case closely reproduce in vivo conditions. The exploration of haptotactic cell migration through design and employment of such assays represents the main focus of this work. Dendritic cells (DCs) are leukocytes, which after encountering danger signals such as pathogens in peripheral organs instruct naïve T-cells and consequently the adaptive immune response in the lymph node (Mellman and Steinman, 2001). To reach the lymph node from the periphery, DCs follow haptotactic gradients of the chemokine CCL21 towards lymphatic vessels (Weber et al., 2013). Questions about how DCs interpret haptotactic CCL21 gradients have not yet been addressed. The main reason for this is the lack of an assay that offers diverse haptotactic environments, hence allowing the study of DC migration as a response to different signals of immobilized guidance cue. In this work, we developed an in vitro assay that enables us to quantitatively assess DC haptotaxis, by combining precisely controllable chemokine photo-patterning with physically confining migration conditions. With this tool at hand, we studied the influence of CCL21 gradient properties and concentration on DC haptotaxis. We found that haptotactic gradient sensing depends on the absolute CCL21 concentration in combination with the local steepness of the gradient. Our analysis suggests that the directionality of migrating DCs is governed by the signal-to-noise ratio of CCL21 binding to its receptor CCR7. Moreover, the haptotactic CCL21 gradient formed in vivo provides an optimal shape for DCs to recognize haptotactic guidance cue. By reconstitution of the CCL21 gradient in vitro we were also able to study the influence of CCR7 signal termination on DC haptotaxis. To this end, we used DCs lacking the G-protein coupled receptor kinase GRK6, which is responsible for CCL21 induced CCR7 receptor phosphorylation and desensitization (Zidar et al., 2009). We found that CCR7 desensitization by GRK6 is crucial for maintenance of haptotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. In the context of the organism, immobilized haptotactic guidance cues often coincide and compete with soluble chemotactic guidance cues. During wound healing, fibroblasts are exposed and influenced by adhesive cues and soluble factors at the same time (Wu et al., 2012; Wynn, 2008). Similarly, migrating DCs are exposed to both, soluble chemokines (CCL19 and truncated CCL21) inducing chemotactic behavior as well as the immobilized CCL21. To quantitatively assess these complex coinciding immobilized and soluble guidance cues, we implemented our chemokine photo-patterning technique in a microfluidic system allowing for chemotactic gradient generation. To validate the assay, we observed DC migration in competing CCL19/CCL21 environments. Adhesiveness guided haptotaxis has been studied intensively over the last century. However, quantitative studies leading to conceptual models are largely missing, again due to the lack of a precisely controllable in vitro assay. A requirement for such an in vitro assay is that it must prevent any uncontrolled cell adhesion. This can be accomplished by stable passivation of the surface. In addition, controlled adhesion must be sustainable, quantifiable and dose dependent in order to create homogenous gradients. Therefore, we developed a novel covalent photo-patterning technique satisfying all these needs. In combination with a sustainable poly-vinyl alcohol (PVA) surface coating we were able to generate gradients of adhesive cue to direct cell migration. This approach allowed us to characterize the haptotactic migratory behavior of zebrafish keratocytes in vitro. Furthermore, defined patterns of adhesive cue allowed us to control for cell shape and growth on a subcellular scale. AU - Schwarz, Jan ID - 1129 SN - 2663-337X TI - Quantitative analysis of haptotactic cell migration ER - TY - THES AB - Traditionally machine learning has been focusing on the problem of solving a single task in isolation. While being quite well understood, this approach disregards an important aspect of human learning: when facing a new problem, humans are able to exploit knowledge acquired from previously learned tasks. Intuitively, access to several problems simultaneously or sequentially could also be advantageous for a machine learning system, especially if these tasks are closely related. Indeed, results of many empirical studies have provided justification for this intuition. However, theoretical justifications of this idea are rather limited. The focus of this thesis is to expand the understanding of potential benefits of information transfer between several related learning problems. We provide theoretical analysis for three scenarios of multi-task learning - multiple kernel learning, sequential learning and active task selection. We also provide a PAC-Bayesian perspective on lifelong learning and investigate how the task generation process influences the generalization guarantees in this scenario. In addition, we show how some of the obtained theoretical results can be used to derive principled multi-task and lifelong learning algorithms and illustrate their performance on various synthetic and real-world datasets. AU - Pentina, Anastasia ID - 1126 SN - 2663-337X TI - Theoretical foundations of multi-task lifelong learning ER - TY - THES AB - We study partially observable Markov decision processes (POMDPs) with objectives used in verification and artificial intelligence. The qualitative analysis problem given a POMDP and an objective asks whether there is a strategy (policy) to ensure that the objective is satisfied almost surely (with probability 1), resp. with positive probability (with probability greater than 0). For POMDPs with limit-average payoff, where a reward value in the interval [0,1] is associated to every transition, and the payoff of an infinite path is the long-run average of the rewards, we consider two types of path constraints: (i) a quantitative limit-average constraint defines the set of paths where the payoff is at least a given threshold L1 = 1. Our main results for qualitative limit-average constraint under almost-sure winning are as follows: (i) the problem of deciding the existence of a finite-memory controller is EXPTIME-complete; and (ii) the problem of deciding the existence of an infinite-memory controller is undecidable. For quantitative limit-average constraints we show that the problem of deciding the existence of a finite-memory controller is undecidable. We present a prototype implementation of our EXPTIME algorithm. For POMDPs with w-regular conditions specified as parity objectives, while the qualitative analysis problems are known to be undecidable even for very special case of parity objectives, we establish decidability (with optimal complexity) of the qualitative analysis problems for POMDPs with parity objectives under finite-memory strategies. We establish optimal (exponential) memory bounds and EXPTIME-completeness of the qualitative analysis problems under finite-memory strategies for POMDPs with parity objectives. Based on our theoretical algorithms we also present a practical approach, where we design heuristics to deal with the exponential complexity, and have applied our implementation on a number of well-known POMDP examples for robotics applications. For POMDPs with a set of target states and an integer cost associated with every transition, we study the optimization objective that asks to minimize the expected total cost of reaching a state in the target set, while ensuring that the target set is reached almost surely. We show that for general integer costs approximating the optimal cost is undecidable. For positive costs, our results are as follows: (i) we establish matching lower and upper bounds for the optimal cost, both double and exponential in the POMDP state space size; (ii) we show that the problem of approximating the optimal cost is decidable and present approximation algorithms that extend existing algorithms for POMDPs with finite-horizon objectives. We show experimentally that it performs well in many examples of interest. We study more deeply the problem of almost-sure reachability, where given a set of target states, the question is to decide whether there is a strategy to ensure that the target set is reached almost surely. While in general the problem EXPTIME-complete, in many practical cases strategies with a small amount of memory suffice. Moreover, the existing solution to the problem is explicit, which first requires to construct explicitly an exponential reduction to a belief-support MDP. We first study the existence of observation-stationary strategies, which is NP-complete, and then small-memory strategies. We present a symbolic algorithm by an efficient encoding to SAT and using a SAT solver for the problem. We report experimental results demonstrating the scalability of our symbolic (SAT-based) approach. Decentralized POMDPs (DEC-POMDPs) extend POMDPs to a multi-agent setting, where several agents operate in an uncertain environment independently to achieve a joint objective. In this work we consider Goal DEC-POMDPs, where given a set of target states, the objective is to ensure that the target set is reached with minimal cost. We consider the indefinite-horizon (infinite-horizon with either discounted-sum, or undiscounted-sum, where absorbing goal states have zero-cost) problem. We present a new and novel method to solve the problem that extends methods for finite-horizon DEC-POMDPs and the real-time dynamic programming approach for POMDPs. We present experimental results on several examples, and show that our approach presents promising results. In the end we present a short summary of a few other results related to verification of MDPs and POMDPs. AU - Chmelik, Martin ID - 1397 SN - 2663-337X TI - Algorithms for partially observable markov decision processes ER - TY - THES AB - Motivated by topological Tverberg-type problems in topological combinatorics and by classical results about embeddings (maps without double points), we study the question whether a finite simplicial complex K can be mapped into Rd without triple, quadruple, or, more generally, r-fold points (image points with at least r distinct preimages), for a given multiplicity r ≤ 2. In particular, we are interested in maps f : K → Rd that have no global r -fold intersection points, i.e., no r -fold points with preimages in r pairwise disjoint simplices of K , and we seek necessary and sufficient conditions for the existence of such maps. We present higher-multiplicity analogues of several classical results for embeddings, in particular of the completeness of the Van Kampen obstruction for embeddability of k -dimensional complexes into R2k , k ≥ 3. Speciffically, we show that under suitable restrictions on the dimensions(viz., if dimK = (r ≥ 1)k and d = rk \ for some k ≥ 3), a well-known deleted product criterion (DPC ) is not only necessary but also sufficient for the existence of maps without global r -fold points. Our main technical tool is a higher-multiplicity version of the classical Whitney trick , by which pairs of isolated r -fold points of opposite sign can be eliminated by local modiffications of the map, assuming codimension d – dimK ≥ 3. An important guiding idea for our work was that suffciency of the DPC, together with an old result of Özaydin's on the existence of equivariant maps, might yield an approach to disproving the remaining open cases of the the long-standing topological Tverberg conjecture , i.e., to construct maps from the N -simplex σN to Rd without r-Tverberg points when r not a prime power and N = (d + 1)(r – 1). Unfortunately, our proof of the sufficiency of the DPC requires codimension d – dimK ≥ 3, which is not satisfied for K = σN . In 2015, Frick [16] found a very elegant way to overcome this \codimension 3 obstacle" and to construct the first counterexamples to the topological Tverberg conjecture for all parameters(d; r ) with d ≥ 3r + 1 and r not a prime power, by a reduction1 to a suitable lower-dimensional skeleton, for which the codimension 3 restriction is satisfied and maps without r -Tverberg points exist by Özaydin's result and sufficiency of the DPC. In this thesis, we present a different construction (which does not use the constraint method) that yields counterexamples for d ≥ 3r , r not a prime power. AU - Mabillard, Isaac ID - 1123 SN - 2663-337X TI - Eliminating higher-multiplicity intersections: an r-fold Whitney trick for the topological Tverberg conjecture ER - TY - THES AB - CA3 pyramidal neurons are thought to pay a key role in memory storage and pattern completion by activity-dependent synaptic plasticity between CA3-CA3 recurrent excitatory synapses. To examine the induction rules of synaptic plasticity at CA3-CA3 synapses, we performed whole-cell patch-clamp recordings in acute hippocampal slices from rats (postnatal 21-24 days) at room temperature. Compound excitatory postsynaptic potentials (ESPSs) were recorded by tract stimulation in stratum oriens in the presence of 10 µM gabazine. High-frequency stimulation (HFS) induced N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Although LTP by HFS did not requier postsynaptic spikes, it was blocked by Na+-channel blockers suggesting that local active processes (e.g.) dendritic spikes) may contribute to LTP induction without requirement of a somatic action potential (AP). We next examined the properties of spike timing-dependent plasticity (STDP) at CA3-CA3 synapses. Unexpectedly, low-frequency pairing of EPSPs and backpropagated action potentialy (bAPs) induced LTP, independent of temporal order. The STDP curve was symmetric and broad, with a half-width of ~150 ms. Consistent with these specific STDP induction properties, post-presynaptic sequences led to a supralinear summation of spine [Ca2+] transients. Furthermore, in autoassociative network models, storage and recall was substantially more robust with symmetric than with asymmetric STDP rules. In conclusion, we found associative forms of LTP at CA3-CA3 recurrent collateral synapses with distinct induction rules. LTP induced by HFS may be associated with dendritic spikes. In contrast, low frequency pairing of pre- and postsynaptic activity induced LTP only if EPSP-AP were temporally very close. Together, these induction mechanisms of synaptiic plasticity may contribute to memory storage in the CA3-CA3 microcircuit at different ranges of activity. AU - Mishra, Rajiv Kumar ID - 1396 SN - 2663-337X TI - Synaptic plasticity rules at CA3-CA3 recurrent synapses in hippocampus ER - TY - THES AB - Natural environments are never constant but subject to spatial and temporal change on all scales, increasingly so due to human activity. Hence, it is crucial to understand the impact of environmental variation on evolutionary processes. In this thesis, I present three topics that share the common theme of environmental variation, yet illustrate its effect from different perspectives. First, I show how a temporally fluctuating environment gives rise to second-order selection on a modifier for stress-induced mutagenesis. Without fluctuations, when populations are adapted to their environment, mutation rates are minimized. I argue that a stress-induced mutator mechanism may only be maintained if the population is repeatedly subjected to diverse environmental challenges, and I outline implications of the presented results to antibiotic treatment strategies. Second, I discuss my work on the evolution of dispersal. Besides reproducing known results about the effect of heterogeneous habitats on dispersal, it identifies spatial changes in dispersal type frequencies as a source for selection for increased propensities to disperse. This concept contains effects of relatedness that are known to promote dispersal, and I explain how it identifies other forces selecting for dispersal and puts them on a common scale. Third, I analyse genetic variances of phenotypic traits under multivariate stabilizing selection. For the case of constant environments, I generalize known formulae of equilibrium variances to multiple traits and discuss how the genetic variance of a focal trait is influenced by selection on background traits. I conclude by presenting ideas and preliminary work aiming at including environmental fluctuations in the form of moving trait optima into the model. AU - Novak, Sebastian ID - 1125 SN - 2663-337X TI - Evolutionary proccesses in variable emvironments ER - TY - THES AB - In this thesis we present a computer-aided programming approach to concurrency. Our approach helps the programmer by automatically fixing concurrency-related bugs, i.e. bugs that occur when the program is executed using an aggressive preemptive scheduler, but not when using a non-preemptive (cooperative) scheduler. Bugs are program behaviours that are incorrect w.r.t. a specification. We consider both user-provided explicit specifications in the form of assertion statements in the code as well as an implicit specification. The implicit specification is inferred from the non-preemptive behaviour. Let us consider sequences of calls that the program makes to an external interface. The implicit specification requires that any such sequence produced under a preemptive scheduler should be included in the set of sequences produced under a non-preemptive scheduler. We consider several semantics-preserving fixes that go beyond atomic sections typically explored in the synchronisation synthesis literature. Our synthesis is able to place locks, barriers and wait-signal statements and last, but not least reorder independent statements. The latter may be useful if a thread is released to early, e.g., before some initialisation is completed. We guarantee that our synthesis does not introduce deadlocks and that the synchronisation inserted is optimal w.r.t. a given objective function. We dub our solution trace-based synchronisation synthesis and it is loosely based on counterexample-guided inductive synthesis (CEGIS). The synthesis works by discovering a trace that is incorrect w.r.t. the specification and identifying ordering constraints crucial to trigger the specification violation. Synchronisation may be placed immediately (greedy approach) or delayed until all incorrect traces are found (non-greedy approach). For the non-greedy approach we construct a set of global constraints over synchronisation placements. Each model of the global constraints set corresponds to a correctness-ensuring synchronisation placement. The placement that is optimal w.r.t. the given objective function is chosen as the synchronisation solution. We evaluate our approach on a number of realistic (albeit simplified) Linux device-driver benchmarks. The benchmarks are versions of the drivers with known concurrency-related bugs. For the experiments with an explicit specification we added assertions that would detect the bugs in the experiments. Device drivers lend themselves to implicit specification, where the device and the operating system are the external interfaces. Our experiments demonstrate that our synthesis method is precise and efficient. We implemented objective functions for coarse-grained and fine-grained locking and observed that different synchronisation placements are produced for our experiments, favouring e.g. a minimal number of synchronisation operations or maximum concurrency. AU - Tarrach, Thorsten ID - 1130 SN - 2663-337X TI - Automatic synthesis of synchronisation primitives for concurrent programs ER - TY - THES AB - Computer graphics is an extremely exciting field for two reasons. On the one hand, there is a healthy injection of pragmatism coming from the visual effects industry that want robust algorithms that work so they can produce results at an increasingly frantic pace. On the other hand, they must always try to push the envelope and achieve the impossible to wow their audiences in the next blockbuster, which means that the industry has not succumb to conservatism, and there is plenty of room to try out new and crazy ideas if there is a chance that it will pan into something useful. Water simulation has been in visual effects for decades, however it still remains extremely challenging because of its high computational cost and difficult artdirectability. The work in this thesis tries to address some of these difficulties. Specifically, we make the following three novel contributions to the state-of-the-art in water simulation for visual effects. First, we develop the first algorithm that can convert any sequence of closed surfaces in time into a moving triangle mesh. State-of-the-art methods at the time could only handle surfaces with fixed connectivity, but we are the first to be able to handle surfaces that merge and split apart. This is important for water simulation practitioners, because it allows them to convert splashy water surfaces extracted from particles or simulated using grid-based level sets into triangle meshes that can be either textured and enhanced with extra surface dynamics as a post-process. We also apply our algorithm to other phenomena that merge and split apart, such as morphs and noisy reconstructions of human performances. Second, we formulate a surface-based energy that measures the deviation of a water surface froma physically valid state. Such discrepancies arise when there is a mismatch in the degrees of freedom between the water surface and the underlying physics solver. This commonly happens when practitioners use a moving triangle mesh with a grid-based physics solver, or when high-resolution grid-based surfaces are combined with low-resolution physics. Following the direction of steepest descent on our surface-based energy, we can either smooth these artifacts or turn them into high-resolution waves by interpreting the energy as a physical potential. Third, we extend state-of-the-art techniques in non-reflecting boundaries to handle spatially and time-varying background flows. This allows a novel new workflow where practitioners can re-simulate part of an existing simulation, such as removing a solid obstacle, adding a new splash or locally changing the resolution. Such changes can easily lead to new waves in the re-simulated region that would reflect off of the new simulation boundary, effectively ruining the illusion of a seamless simulation boundary between the existing and new simulations. Our non-reflecting boundaries makes sure that such waves are absorbed. AU - Bojsen-Hansen, Morten ID - 1122 SN - 2663-337X TI - Tracking, correcting and absorbing water surface waves ER - TY - THES AB - Hybrid zones represent evolutionary laboratories, where recombination brings together alleles in combinations which have not previously been tested by selection. This provides an excellent opportunity to test the effect of molecular variation on fitness, and how this variation is able to spread through populations in a natural context. The snapdragon Antirrhinum majus is polymorphic in the wild for two loci controlling the distribution of yellow and magenta floral pigments. Where the yellow A. m. striatum and the magenta A. m. pseudomajus meet along a valley in the Spanish Pyrenees they form a stable hybrid zone Alleles at these loci recombine to give striking transgressive variation for flower colour. The sharp transition in phenotype over ~1km implies strong selection maintaining the hybrid zone. An indirect assay of pollinator visitation in the field found that pollinators forage in a positive-frequency dependent manner on Antirrhinum, matching previous data on fruit set. Experimental arrays and paternity analysis of wild-pollinated seeds demonstrated assortative mating for pigmentation alleles, and that pollinator behaviour alone is sufficient to explain this pattern. Selection by pollinators should be sufficiently strong to maintain the hybrid zone, although other mechanisms may be at work. At a broader scale I examined evolutionary transitions between yellow and anthocyanin pigmentation in the tribe Antirrhinae, and found that selection has acted strate that pollinators are a major determinant of reproductive success and mating patterns in wild Antirrhinum. AU - Ellis, Thomas ID - 1398 SN - 2663-337X TI - The role of pollinator-mediated selection in the maintenance of a flower color polymorphism in an Antirrhinum majus hybrid zone ER - TY - THES AB - Evolution of gene regulation is important for phenotypic evolution and diversity. Sequence-specific binding of regulatory proteins is one of the key regulatory mechanisms determining gene expression. Although there has been intense interest in evolution of regulatory binding sites in the last decades, a theoretical understanding is far from being complete. In this thesis, I aim at a better understanding of the evolution of transcriptional regulatory binding sequences by using biophysical and population genetic models. In the first part of the thesis, I discuss how to formulate the evolutionary dynamics of binding se- quences in a single isolated binding site and in promoter/enhancer regions. I develop a theoretical framework bridging between a thermodynamical model for transcription and a mutation-selection-drift model for monomorphic populations. I mainly address the typical evolutionary rates, and how they de- pend on biophysical parameters (e.g. binding length and specificity) and population genetic parameters (e.g. population size and selection strength). In the second part of the thesis, I analyse empirical data for a better evolutionary and biophysical understanding of sequence-specific binding of bacterial RNA polymerase. First, I infer selection on regulatory and non-regulatory binding sites of RNA polymerase in the E. coli K12 genome. Second, I infer the chemical potential of RNA polymerase, an important but unknown physical parameter defining the threshold energy for strong binding. Furthermore, I try to understand the relation between the lac promoter sequence diversity and the LacZ activity variation among 20 bacterial isolates by constructing a simple but biophysically motivated gene expression model. Lastly, I lay out a statistical framework to predict adaptive point mutations in de novo promoter evolution in a selection experiment. AU - Tugrul, Murat ID - 1131 SN - 2663-337X TI - Evolution of transcriptional regulatory sequences ER -