TY - THES AB - Hybrid automata combine finite automata and dynamical systems, and model the interaction of digital with physical systems. Formal analysis that can guarantee the safety of all behaviors or rigorously witness failures, while unsolvable in general, has been tackled algorithmically using, e.g., abstraction, bounded model-checking, assisted theorem proving. Nevertheless, very few methods have addressed the time-unbounded reachability analysis of hybrid automata and, for current sound and automatic tools, scalability remains critical. We develop methods for the polyhedral abstraction of hybrid automata, which construct coarse overapproximations and tightens them incrementally, in a CEGAR fashion. We use template polyhedra, i.e., polyhedra whose facets are normal to a given set of directions. While, previously, directions were given by the user, we introduce (1) the first method for computing template directions from spurious counterexamples, so as to generalize and eliminate them. The method applies naturally to convex hybrid automata, i.e., hybrid automata with (possibly non-linear) convex constraints on derivatives only, while for linear ODE requires further abstraction. Specifically, we introduce (2) the conic abstractions, which, partitioning the state space into appropriate (possibly non-uniform) cones, divide curvy trajectories into relatively straight sections, suitable for polyhedral abstractions. Finally, we introduce (3) space-time interpolation, which, combining interval arithmetic and template refinement, computes appropriate (possibly non-uniform) time partitioning and template directions along spurious trajectories, so as to eliminate them. We obtain sound and automatic methods for the reachability analysis over dense and unbounded time of convex hybrid automata and hybrid automata with linear ODE. We build prototype tools and compare—favorably—our methods against the respective state-of-the-art tools, on several benchmarks. AU - Giacobbe, Mirco ID - 6894 TI - Automatic time-unbounded reachability analysis of hybrid systems ER - TY - THES AB - The development and growth of Arabidopsis thaliana is regulated by a combination of genetic programing and also by the environmental influences. An important role in these processes play the phytohormones and among them, auxin is crucial as it controls many important functions. It is transported through the whole plant body by creating local and temporal concentration maxima and minima, which have an impact on the cell status, tissue and organ identity. Auxin has the property to undergo a directional and finely regulated cell-to-cell transport, which is enabled by the transport proteins, localized on the plasma membrane. An important role in this process have the PIN auxin efflux proteins, which have an asymmetric/polar subcellular localization and determine the directionality of the auxin transport. During the last years, there were significant advances in understanding how the trafficking molecular machineries function, including studies on molecular interactions, function, subcellular localization and intracellular distribution. However, there is still a lack of detailed characterization on the steps of endocytosis, exocytosis, endocytic recycling and degradation. Due to this fact, I focused on the identification of novel trafficking factors and better characterization of the intracellular trafficking pathways. My PhD thesis consists of an introductory chapter, three experimental chapters, a chapter containing general discussion, conclusions and perspectives and also an appendix chapter with published collaborative papers. The first chapter is separated in two different parts: I start by a general introduction to auxin biology and then I introduce the trafficking pathways in the model plant Arabidopsis thaliana. Then, I explain also the phosphorylation-signals for polar targeting and also the roles of the phytohormone strigolactone. The second chapter includes the characterization of bar1/sacsin mutant, which was identified in a forward genetic screen for novel trafficking components in Arabidopsis thaliana, where by the implementation of an EMS-treated pPIN1::PIN1-GFP marker line and by using the established inhibitor of ARF-GEFs, Brefeldin A (BFA) as a tool to study trafficking processes, we identified a novel factor, which is mediating the adaptation of the plant cell to ARF-GEF inhibition. The mutation is in a previously uncharacterized gene, encoding a very big protein that we, based on its homologies, called SACSIN with domains suggesting roles as a molecular chaperon or as a component of the ubiquitin-proteasome system. Our physiology and imaging studies revealed that SACSIN is a crucial plant cell component of the adaptation to the ARF-GEF inhibition. The third chapter includes six subchapters, where I focus on the role of the phytohormone strigolactone, which interferes with auxin feedback on PIN internalization. Strigolactone moderates the polar auxin transport by increasing the internalization of the PIN auxin efflux carriers, which reduces the canalization related growth responses. In addition, I also studied the role of phosphorylation in the strigolactone regulation of auxin feedback on PIN internalization. In this chapter I also present my results on the MAX2-dependence of strigolactone-mediated root growth inhibition and I also share my results on the auxin metabolomics profiling after application of GR24. In the fourth chapter I studied the effect of two small molecules ES-9 and ES9-17, which were identified from a collection of small molecules with the property to impair the clathrin-mediated endocytosis. In the fifth chapter, I discuss all my observations and experimental findings and suggest alternative hypothesis to interpret my results. In the appendix there are three collaborative published projects. In the first, I participated in the characterization of the role of ES9 as a small molecule, which is inhibitor of clathrin- mediated endocytosis in different model organisms. In the second paper, I contributed to the characterization of another small molecule ES9-17, which is a non-protonophoric analog of ES9 and also impairs the clathrin-mediated endocytosis not only in plant cells, but also in mammalian HeLa cells. Last but not least, I also attach another paper, where I tried to establish the grafting method as a technique in our lab to study canalization related processes. AU - Vasileva, Mina K ID - 7172 TI - Molecular mechanisms of endomembrane trafficking in Arabidopsis thaliana ER - TY - THES AB - Single cells are constantly interacting with their environment and each other, more importantly, the accurate perception of environmental cues is crucial for growth, survival, and reproduction. This communication between cells and their environment can be formalized in mathematical terms and be quantified as the information flow between them, as prescribed by information theory. The recent availability of real–time dynamical patterns of signaling molecules in single cells has allowed us to identify encoding about the identity of the environment in the time–series. However, efficient estimation of the information transmitted by these signals has been a data–analysis challenge due to the high dimensionality of the trajectories and the limited number of samples. In the first part of this thesis, we develop and evaluate decoding–based estimation methods to lower bound the mutual information and derive model–based precise information estimates for biological reaction networks governed by the chemical master equation. This is followed by applying the decoding-based methods to study the intracellular representation of extracellular changes in budding yeast, by observing the transient dynamics of nuclear translocation of 10 transcription factors in response to 3 stress conditions. Additionally, we apply these estimators to previously published data on ERK and Ca2+ signaling and yeast stress response. We argue that this single cell decoding-based measure of information provides an unbiased, quantitative and interpretable measure for the fidelity of biological signaling processes. Finally, in the last section, we deal with gene regulation which is primarily controlled by transcription factors (TFs) that bind to the DNA to activate gene expression. The possibility that non-cognate TFs activate transcription diminishes the accuracy of regulation with potentially disastrous effects for the cell. This ’crosstalk’ acts as a previously unexplored source of noise in biochemical networks and puts a strong constraint on their performance. To mitigate erroneous initiation we propose an out of equilibrium scheme that implements kinetic proofreading. We show that such architectures are favored over their equilibrium counterparts for complex organisms despite introducing noise in gene expression. AU - Cepeda Humerez, Sarah A ID - 6473 KW - Information estimation KW - Time-series KW - data analysis SN - 2663-337X TI - Estimating information flow in single cells ER - TY - THES AB - Transcription factors, by binding to specific sequences on the DNA, control the precise spatio-temporal expression of genes inside a cell. However, this specificity is limited, leading to frequent incorrect binding of transcription factors that might have deleterious consequences on the cell. By constructing a biophysical model of TF-DNA binding in the context of gene regulation, I will first explore how regulatory constraints can strongly shape the distribution of a population in sequence space. Then, by directly linking this to a picture of multiple types of transcription factors performing their functions simultaneously inside the cell, I will explore the extent of regulatory crosstalk -- incorrect binding interactions between transcription factors and binding sites that lead to erroneous regulatory states -- and understand the constraints this places on the design of regulatory systems. I will then develop a generic theoretical framework to investigate the coevolution of multiple transcription factors and multiple binding sites, in the context of a gene regulatory network that performs a certain function. As a particular tractable version of this problem, I will consider the evolution of two transcription factors when they transmit upstream signals to downstream target genes. Specifically, I will describe the evolutionary steady states and the evolutionary pathways involved, along with their timescales, of a system that initially undergoes a transcription factor duplication event. To connect this important theoretical model to the prominent biological event of transcription factor duplication giving rise to paralogous families, I will then describe a bioinformatics analysis of C2H2 Zn-finger transcription factors, a major family in humans, and focus on the patterns of evolution that paralogs have undergone in their various protein domains in the recent past. AU - Prizak, Roshan ID - 6071 SN - 2663-337X TI - Coevolution of transcription factors and their binding sites in sequence space ER - TY - THES AB - In the first part of this thesis we consider large random matrices with arbitrary expectation and a general slowly decaying correlation among its entries. We prove universality of the local eigenvalue statistics and optimal local laws for the resolvent in the bulk and edge regime. The main novel tool is a systematic diagrammatic control of a multivariate cumulant expansion. In the second part we consider Wigner-type matrices and show that at any cusp singularity of the limiting eigenvalue distribution the local eigenvalue statistics are uni- versal and form a Pearcey process. Since the density of states typically exhibits only square root or cubic root cusp singularities, our work complements previous results on the bulk and edge universality and it thus completes the resolution of the Wigner- Dyson-Mehta universality conjecture for the last remaining universality type. Our analysis holds not only for exact cusps, but approximate cusps as well, where an ex- tended Pearcey process emerges. As a main technical ingredient we prove an optimal local law at the cusp, and extend the fast relaxation to equilibrium of the Dyson Brow- nian motion to the cusp regime. In the third and final part we explore the entrywise linear statistics of Wigner ma- trices and identify the fluctuations for a large class of test functions with little regularity. This enables us to study the rectangular Young diagram obtained from the interlacing eigenvalues of the random matrix and its minor, and we find that, despite having the same limit, the fluctuations differ from those of the algebraic Young tableaux equipped with the Plancharel measure. AU - Schröder, Dominik J ID - 6179 SN - 2663-337X TI - From Dyson to Pearcey: Universal statistics in random matrix theory ER - TY - THES AB - The regulation of gene expression is one of the most fundamental processes in living systems. In recent years, thanks to advances in sequencing technology and automation, it has become possible to study gene expression quantitatively, genome-wide and in high-throughput. This leads to the possibility of exploring changes in gene expression in the context of many external perturbations and their combinations, and thus of characterising the basic principles governing gene regulation. In this thesis, I present quantitative experimental approaches to studying transcriptional and protein level changes in response to combinatorial drug treatment, as well as a theoretical data-driven approach to analysing thermodynamic principles guiding transcription of protein coding genes. In the first part of this work, I present a novel methodological framework for quantifying gene expression changes in drug combinations, termed isogrowth profiling. External perturbations through small molecule drugs influence the growth rate of the cell, leading to wide-ranging changes in cellular physiology and gene expression. This confounds the gene expression changes specifically elicited by the particular drug. Combinatorial perturbations, owing to the increased stress they exert, influence the growth rate even more strongly and hence suffer the convolution problem to a greater extent when measuring gene expression changes. Isogrowth profiling is a way to experimentally abstract non-specific, growth rate related changes, by performing the measurement using varying ratios of two drugs at such concentrations that the overall inhibition rate is constant. Using a robotic setup for automated high-throughput re-dilution culture of Saccharomyces cerevisiae, the budding yeast, I investigate all pairwise interactions of four small molecule drugs through sequencing RNA along a growth isobole. Through principal component analysis, I demonstrate here that isogrowth profiling can uncover drug-specific as well as drug-interaction-specific gene expression changes. I show that drug-interaction-specific gene expression changes can be used for prediction of higher-order drug interactions. I propose a simplified generalised framework of isogrowth profiling, with few measurements needed for each drug pair, enabling the broad application of isogrowth profiling to high-throughput screening of inhibitors of cellular growth and beyond. Such high-throughput screenings of gene expression changes specific to pairwise drug interactions will be instrumental for predicting the higher-order interactions of the drugs. In the second part of this work, I extend isogrowth profiling to single-cell measurements of gene expression, characterising population heterogeneity in the budding yeast in response to combinatorial drug perturbation while controlling for non-specific growth rate effects. Through flow cytometry of strains with protein products fused to green fluorescent protein, I discover multiple proteins with bi-modally distributed expression levels in the population in response to drug treatment. I characterize more closely the effect of an ionic stressor, lithium chloride, and find that it inhibits the splicing of mRNA, most strongly affecting ribosomal protein transcripts and leading to a bi-stable behaviour of a small ribosomal subunit protein Rps22B. Time-lapse microscopy of a microfluidic culture system revealed that the induced Rps22B heterogeneity leads to preferential survival of Rps22B-low cells after long starvation, but to preferential proliferation of Rps22B-high cells after short starvation. Overall, this suggests that yeast cells might use splicing of ribosomal genes for bet-hedging in fluctuating environments. I give specific examples of how further exploration of cellular heterogeneity in yeast in response to external perturbation has the potential to reveal yet-undiscovered gene regulation circuitry. In the last part of this thesis, a re-analysis of a published sequencing dataset of nascent elongating transcripts is used to characterise the thermodynamic constraints for RNA polymerase II (RNAP) elongation. Population-level data on RNAP position throughout the transcribed genome with single nucleotide resolution are used to infer the sequence specific thermodynamic determinants of RNAP pausing and backtracking. This analysis reveals that the basepairing strength of the eight nucleotide-long RNA:DNA duplex relative to the basepairing strength of the same sequence when in DNA:DNA duplex, and the change in this quantity during RNA polymerase movement, is the key determinant of RNAP pausing. This is true for RNAP pausing while elongating, but also of RNAP pausing while backtracking and of the backtracking length. The quantitative dependence of RNAP pausing on basepairing energetics is used to infer the increase in pausing due to transcriptional mismatches, leading to a hypothesis that pervasive RNA polymerase II pausing is due to basepairing energetics, as an evolutionary cost for increased RNA polymerase II fidelity. This work advances our understanding of the general principles governing gene expression, with the goal of making computational predictions of single-cell gene expression responses to combinatorial perturbations based on the individual perturbations possible. This ability would substantially facilitate the design of drug combination treatments and, in the long term, lead to our increased ability to more generally design targeted manipulations to any biological system. AU - Lukacisin, Martin ID - 6392 SN - 2663-337X TI - Quantitative investigation of gene expression principles through combinatorial drug perturbation and theory ER - TY - THES AB - Social insect colonies tend to have numerous members which function together like a single organism in such harmony that the term ``super-organism'' is often used. In this analogy the reproductive caste is analogous to the primordial germ cells of a metazoan, while the sterile worker caste corresponds to somatic cells. The worker castes, like tissues, are in charge of all functions of a living being, besides reproduction. The establishment of new super-organismal units (i.e. new colonies) is accomplished by the co-dependent castes. The term oftentimes goes beyond a metaphor. We invoke it when we speak about the metabolic rate, thermoregulation, nutrient regulation and gas exchange of a social insect colony. Furthermore, we assert that the super-organism has an immune system, and benefits from ``social immunity''. Social immunity was first summoned by evolutionary biologists to resolve the apparent discrepancy between the expected high frequency of disease outbreak amongst numerous, closely related tightly-interacting hosts, living in stable and microbially-rich environments, against the exceptionally scarce epidemic accounts in natural populations. Social immunity comprises a multi-layer assembly of behaviours which have evolved to effectively keep the pathogenic enemies of a colony at bay. The field of social immunity has drawn interest, as it becomes increasingly urgent to stop the collapse of pollinator species and curb the growth of invasive pests. In the past decade, several mechanisms of social immune responses have been dissected, but many more questions remain open. I present my work in two experimental chapters. In the first, I use invasive garden ants (*Lasius neglectus*) to study how pathogen load and its distribution among nestmates affect the grooming response of the group. Any given group of ants will carry out the same total grooming work, but will direct their grooming effort towards individuals carrying a relatively higher spore load. Contrary to expectation, the highest risk of transmission does not stem from grooming highly contaminated ants, but instead, we suggest that the grooming response likely minimizes spore loss to the environment, reducing contamination from inadvertent pickup from the substrate. The second is a comparative developmental approach. I follow black garden ant queens (*Lasius niger*) and their colonies from mating flight, through hibernation for a year. Colonies which grow fast from the start, have a lower chance of survival through hibernation, and those which survive grow at a lower pace later. This is true for colonies of naive and challenged queens. Early pathogen exposure of the queens changes colony dynamics in an unexpected way: colonies from exposed queens are more likely to grow slowly and recover in numbers only after they survive hibernation. In addition to the two experimental chapters, this thesis includes a co-authored published review on organisational immunity, where we enlist the experimental evidence and theoretical framework on which this hypothesis is built, identify the caveats and underline how the field is ripe to overcome them. In a final chapter, I describe my part in two collaborative efforts, one to develop an image-based tracker, and the second to develop a classifier for ant behaviour. AU - Casillas Perez, Barbara E ID - 6435 KW - Social Immunity KW - Sanitary care KW - Social Insects KW - Organisational Immunity KW - Colony development KW - Multi-target tracking SN - 2663-337X TI - Collective defenses of garden ants against a fungal pathogen ER - TY - THES AB - Clathrin-Mediated Endocytosis (CME) is an aspect of cellular trafficking that is constantly regulated for mediating developmental and physiological responses. The main aim of my thesis is to decipher the basic mechanisms of CME and post-endocytic trafficking in the whole multicellular organ systems of Arabidopsis. The first chapter of my thesis describes the search for new components involved in CME. Tandem affinity purification was conducted using CLC and its interacting partners were identified. Amongst the identified proteins were the Auxilin-likes1 and 2 (Axl1/2), putative uncoating factors, for which we made a full functional analysis. Over-expression of Axl1/2 causes extreme modifications in the dynamics of the machinery proteins and inhibition of endocytosis altogether. However the loss of function of the axl1/2 did not present any cellular or physiological phenotype, meaning Auxilin-likes do not form the major uncoating machinery. The second chapter of my thesis describes the establishment/utilisation of techniques to capture the dynamicity and the complexity of CME and post-endocytic trafficking. We have studied the development of endocytic pits at the PM – specifically, the mode of membrane remodeling during pit development and the role of actin in it, given plant cells possess high turgor pressure. Utilizing the improved z-resolution of TIRF and VAEM techniques, we captured the time-lapse of the endocytic events at the plasma membrane; and using particle detection software, we quantitatively analysed all the endocytic trajectories in an unbiased way to obtain the endocytic rate of the system. This together with the direct analysis of cargo internalisation from the PM provided an estimate on the endocytic potential of the cell. We also developed a methodology for ultrastructural analysis of different populations of Clathrin-Coated Structures (CCSs) in both PM and endomembranes in unroofed protoplasts. Structural analysis, together with the intensity profile of CCSs at the PM show that the mode of CCP development at the PM follows ‘Constant curvature model’; meaning that clathrin polymerisation energy is a major contributing factor of membrane remodeling. In addition, other analyses clearly show that actin is not required for membrane remodeling during invagination or any other step of CCP development, despite the prevalent high turgor pressure. However, actin is essential in orchestrating the post-endocytic trafficking of CCVs facilitating the EE formation. We also observed that the uncoating process post-endocytosis is not immediate; an alternative mechanism of uncoating – Sequential multi-step process – functions in the cell. Finally we also looked at one of the important physiological stimuli modulating the process – hormone, auxin. auxin has been known to influence CME before. We have made a detailed study on the concentration-time based effect of auxin on the machinery proteins, CCP development, and the specificity of cargoes endocytosed. To this end, we saw no general effect of auxin on CME at earlier time points. However, very low concentration of IAA, such as 50nM, accelerates endocytosis of specifically PIN2 through CME. Such a tight regulatory control with high specificity to PIN2 could be essential in modulating its polarity. AU - Narasimhan, Madhumitha ID - 6269 SN - 2663-337X TI - Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants ER - TY - THES AB - Lymph nodes are es s ential organs of the immune s ys tem where adaptive immune responses originate, and consist of various leukocyte populations and a stromal backbone. Fibroblastic reticular cells (FRCs) are the main stromal cells and form a sponge-like extracellular matrix network, called conduits , which they thems elves enwrap and contract. Lymph, containing s oluble antigens , arrive in lymph nodes via afferent lymphatic vessels that connect to the s ubcaps ular s inus and conduit network. According to the current paradigm, the conduit network dis tributes afferent lymph through lymph nodes and thus provides acces s for immune cells to lymph-borne antigens. An elas tic caps ule s urrounds the organ and confines the immune cells and FRC network. Lymph nodes are completely packed with lymphocytes and lymphocyte numbers directly dictates the size of the organ. Although lymphocytes cons tantly enter and leave the lymph node, its s ize remains remarkedly s table under homeostatic conditions. It is only partly known how the cellularity and s ize of the lymph node is regulated and how the lymph node is able to swell in inflammation. The role of the FRC network in lymph node s welling and trans fer of fluids are inves tigated in this thes is. Furthermore, we s tudied what trafficking routes are us ed by cancer cells in lymph nodes to form distal metastases.We examined the role of a mechanical feedback in regulation of lymph node swelling. Using parallel plate compression and UV-las er cutting experiments we dis s ected the mechanical force dynamics of the whole lymph node, and individually for FRCs and the caps ule. Physical forces generated by packed lymphocytes directly affect the tens ion on the FRC network and capsule, which increases its resistance to swelling. This implies a feedback mechanism between tis s ue pres s ure and ability of lymphocytes to enter the organ. Following inflammation, the lymph node swells ∼10 fold in two weeks . Yet, what is the role for tens ion on the FRC network and caps ule, and how are lymphocytes able to enter in conditions that resist swelling remain open ques tions . We s how that tens ion on the FRC network is important to limit the swelling rate of the organ so that the FRC network can grow in a coordinated fashion. This is illustrated by interfering with FRC contractility, which leads to faster swelling rates and a dis organized FRC network in the inflamed lymph node. Growth of the FRC network in turn is expected to releas e tens ion on thes e s tructures and lowers the res is tance to swelling, thereby allowing more lymphocytes to enter the organ and drive more swelling. Halt of swelling coincides with a thickening of the caps ule, which forms a thick res is tant band around the organ and lowers tens ion on the FRC network to form a new force equilibrium.The FRC and conduit network are further believed to be a privileged s ite of s oluble information within the lymph node, although many details remain uns olved. We s how by 3D ultra-recons truction that FRCs and antigen pres enting cells cover the s urface of conduit s ys tem for more than 99% and we dis cus s the implications for s oluble information exchangeat the conduit level.Finally, there is an ongoing debate in the cancer field whether and how cancer cells in lymph nodes s eed dis tal metas tas es . We s how that cancer cells infus ed into the lymph node can utilize trafficking routes of immune cells and rapidly migrate to blood vessels. Once in the blood circulation, these cells are able to form metastases in distal tissues. AU - Assen, Frank P ID - 6947 SN - 2663-337X TI - Lymph node mechanics: Deciphering the interplay between stroma contractility, morphology and lymphocyte trafficking ER - TY - THES AB - Brain function is mediated by complex dynamical interactions between excitatory and inhibitory cell types. The Cholecystokinin-expressing inhibitory cells (CCK-interneurons) are one of the least studied types, despite being suspected to play important roles in cognitive processes. We studied the network effects of optogenetic silencing of CCK-interneurons in the CA1 hippocampal area during exploration and sleep states. The cell firing pattern in response to light pulses allowed us to classify the recorded neurons in 5 classes, including disinhibited and non-responsive pyramidal cell and interneurons, and the inhibited interneurons corresponding to the CCK group. The light application, which inhibited the activity of CCK interneurons triggered wider changes in the firing dynamics of cells. We observed rate changes (i.e. remapping) of pyramidal cells during the exploration session in which the light was applied relative to the previous control session that was not restricted neither in time nor space to the light delivery. Also, the disinhibited pyramidal cells had higher increase in bursting than in single spike firing rate as a result of CCK silencing. In addition, the firing activity patterns during exploratory periods were more weakly reactivated in sleep for those periods in which CCK-interneuron were silenced than in the unaffected periods. Furthermore, light pulses during sleep disrupted the reactivation of recent waking patterns. Hence, silencing CCK neurons during exploration suppressed the reactivation of waking firing patterns in sleep and CCK interneuron activity was also required during sleep for the normal reactivation of waking patterns. These findings demonstrate the involvement of CCK cells in reactivation-related memory consolidation. An important part of our analysis was to test the relationship of the identified CCKinterneurons to brain oscillations. Our findings showed that these cells exhibited different oscillatory behaviour during anaesthesia and natural waking and sleep conditions. We showed that: 1) Contrary to the past studies performed under anaesthesia, the identified CCKinterneurons fired on the descending portion of the theta phase in waking exploration. 2) CCKinterneuron preferred phases around the trough of gamma oscillations. 3) Contrary to anaesthesia conditions, the average firing rate of the CCK-interneurons increased around the peak activity of the sharp-wave ripple (SWR) events in natural sleep, which is congruent with new reports about their functional connectivity. We also found that light driven CCK-interneuron silencing altered the dynamics on the CA1 network oscillatory activity: 1) Pyramidal cells negatively shifted their preferred theta phases when the light was applied, while interneurons responses were less consistent. 2) As a population, pyramidal cells negatively shifted their preferred activity during gamma oscillations, albeit we did not find gamma modulation differences related to the light application when pyramidal cells were subdivided into the disinhibited and unaffected groups. 3) During the peak of SWR events, all but the CCK-interneurons had a reduction in their relative firing rate change during the light application as compared to the change observed at SWR initiation. Finally, regarding to the place field activity of the recorded pyramidal neurons, we showed that the disinhibited pyramidal cells had reduced place field similarity, coherence and spatial information, but only during the light application. The mechanisms behind such observed behaviours might involve eCB signalling and plastic changes in CCK-interneuron synapses. In conclusion, the observed changes related to the light-mediated silencing of CCKinterneurons have unravelled characteristics of this interneuron subpopulation that might change the understanding not only of their particular network interactions, but also of the current theories about the emergence of certain cognitive processes such as place coding needed for navigation or hippocampus-dependent memory consolidation. AU - Rangel Guerrero, Dámaris K ID - 6849 SN - 2663-337X TI - The role of CCK-interneurons in regulating hippocampal network dynamics 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 - 7132 SN - 2663-337X TI - Design and characterization of methods and biological components to realize synthetic neurotransmission ER - TY - THES AB - The solving of complex tasks requires the functions of more than one brain area and their interaction. Whilst spatial navigation and memory is dependent on the hippocampus, flexible behavior relies on the medial prefrontal cortex (mPFC). To further examine the roles of the hippocampus and mPFC, we recorded their neural activity during a task that depends on both of these brain regions. With tetrodes, we recorded the extracellular activity of dorsal hippocampal CA1 (HPC) and mPFC neurons in Long-Evans rats performing a rule-switching task on the plus-maze. The plus-maze task had a spatial component since it required navigation along one of the two start arms and at the maze center a choice between one of the two goal arms. Which goal contained a reward depended on the rule currently in place. After an uncued rule change the animal had to abandon the old strategy and switch to the new rule, testing cognitive flexibility. Investigating the coordination of activity between the HPC and mPFC allows determination during which task stages their interaction is required. Additionally, comparing neural activity patterns in these two brain regions allows delineation of the specialized functions of the HPC and mPFC in this task. We analyzed neural activity in the HPC and mPFC in terms of oscillatory interactions, rule coding and replay. We found that theta coherence between the HPC and mPFC is increased at the center and goals of the maze, both when the rule was stable or has changed. Similar results were found for locking of HPC and mPFC neurons to HPC theta oscillations. However, no differences in HPC-mPFC theta coordination were observed between the spatially- and cue-guided rule. Phase locking of HPC and mPFC neurons to HPC gamma oscillations was not modulated by maze position or rule type. We found that the HPC coded for the two different rules with cofiring relationships between cell pairs. However, we could not find conclusive evidence for rule coding in the mPFC. Spatially-selective firing in the mPFC generalized between the two start and two goal arms. With Bayesian positional decoding, we found that the mPFC reactivated non-local positions during awake immobility periods. Replay of these non-local positions could represent entire behavioral trajectories resembling trajectory replay of the HPC. Furthermore, mPFC trajectory-replay at the goal positively correlated with rule-switching performance. Finally, HPC and mPFC trajectory replay occurred independently of each other. These results show that the mPFC can replay ordered patterns of activity during awake immobility, possibly underlying its role in flexible behavior. AU - Käfer, Karola ID - 6825 SN - 2663-337X TI - The hippocampus and medial prefrontal cortex during flexible behavior ER - TY - THES AB - Invasive migration plays a crucial role not only during development and homeostasis but also in pathological states, such as tumor metastasis. Drosophila macrophage migration into the extended germband is an interesting system to study invasive migration. It carries similarities to immune cell transmigration and cancer cell invasion, therefore studying this process could also bring new understanding of invasion in higher organisms. In our work, we uncover a highly conserved member of the major facilitator family that plays a role in tissue invasion through regulation of glycosylation on a subgroup of proteins and/or by aiding the precise timing of DN-Cadherin downregulation. Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis. AU - Valosková, Katarina ID - 6546 SN - 2663-337X TI - The role of a highly conserved major facilitator superfamily member in Drosophila embryonic macrophage migration ER - TY - THES AB - Distinguishing between similar experiences is achieved by the brain in a process called pattern separation. In the hippocampus, pattern separation reduces the interference of memories and increases the storage capacity by decorrelating similar inputs patterns of neuronal activity into non-overlapping output firing patterns. Winners-take-all (WTA) mechanism is a theoretical model for pattern separation in which a "winner" cell suppresses the activity of the neighboring neurons through feedback inhibition. However, if the network properties of the dentate gyrus support WTA as a biologically conceivable model remains unknown. Here, we showed that the connectivity rules of PV+interneurons and their synaptic properties are optimizedfor efficient pattern separation. We found using multiple whole-cell in vitrorecordings that PV+interneurons mainly connect to granule cells (GC) through lateral inhibition, a form of feedback inhibition in which a GC inhibits other GCs but not itself through the activation of PV+interneurons. Thus, lateral inhibition between GC–PV+interneurons was ~10 times more abundant than recurrent connections. Furthermore, the GC–PV+interneuron connectivity was more spatially confined but less abundant than PV+interneurons–GC connectivity, leading to an asymmetrical distribution of excitatory and inhibitory connectivity. Our network model of the dentate gyrus with incorporated real connectivity rules efficiently decorrelates neuronal activity patterns using WTA as the primary mechanism. This process relied on lateral inhibition, fast-signaling properties of PV+interneurons and the asymmetrical distribution of excitatory and inhibitory connectivity. Finally, we found that silencing the activity of PV+interneurons in vivoleads to acute deficits in discrimination between similar environments, suggesting that PV+interneuron networks are necessary for behavioral relevant computations. Our results demonstrate that PV+interneurons possess unique connectivity and fast signaling properties that confer to the dentate gyrus network properties that allow the emergence of pattern separation. Thus, our results contribute to the knowledge of how specific forms of network organization underlie sophisticated types of information processing. AU - Espinoza Martinez, Claudia ID - 6363 SN - 2663-337X TI - Parvalbumin+ interneurons enable efficient pattern separation in hippocampal microcircuits ER - TY - THES AB - While cells of mesenchymal or epithelial origin perform their effector functions in a purely anchorage dependent manner, cells derived from the hematopoietic lineage are not committed to operate only within a specific niche. Instead, these cells are able to function autonomously of the molecular composition in a broad range of tissue compartments. By this means, cells of the hematopoietic lineage retain the capacity to disseminate into connective tissue and recirculate between organs, building the foundation for essential processes such as tissue regeneration or immune surveillance. Cells of the immune system, specifically leukocytes, are extraordinarily good at performing this task. These cells are able to flexibly shift their mode of migration between an adhesion-mediated and an adhesion-independent manner, instantaneously accommodating for any changes in molecular composition of the external scaffold. The key component driving directed leukocyte migration is the chemokine receptor 7, which guides the cell along gradients of chemokine ligand. Therefore, the physical destination of migrating leukocytes is purely deterministic, i.e. given by global directional cues such as chemokine gradients. Nevertheless, these cells typically reside in three-dimensional scaffolds of inhomogeneous complexity, raising the question whether cells are able to locally discriminate between multiple optional migration routes. Current literature provides evidence that leukocytes, specifically dendritic cells, do indeed probe their surrounding by virtue of multiple explorative protrusions. However, it remains enigmatic how these cells decide which one is the more favorable route to follow and what are the key players involved in performing this task. Due to the heterogeneous environment of most tissues, and the vast adaptability of migrating leukocytes, at this time it is not clear to what extent leukocytes are able to optimize their migratory strategy by adapting their level of adhesiveness. And, given the fact that leukocyte migration is characterized by branched cell shapes in combination with high migration velocities, it is reasonable to assume that these cells require fine tuned shape maintenance mechanisms that tightly coordinate protrusion and adhesion dynamics in a spatiotemporal manner. Therefore, this study aimed to elucidate how rapidly migrating leukocytes opt for an ideal migratory path while maintaining a continuous cell shape and balancing adhesive forces to efficiently navigate through complex microenvironments. The results of this study unraveled a role for the microtubule cytoskeleton in promoting the decision making process during path finding and for the first time point towards a microtubule-mediated function in cell shape maintenance of highly ramified cells such as dendritic cells. Furthermore, we found that migrating low-adhesive leukocytes are able to instantaneously adapt to increased tensile load by engaging adhesion receptors. This response was only occurring tangential to the substrate while adhesive properties in the vertical direction were not increased. As leukocytes are primed for rapid migration velocities, these results demonstrate that leukocyte integrins are able to confer a high level of traction forces parallel to the cell membrane along the direction of migration without wasting energy in gluing the cell to the substrate. Thus, the data in the here presented thesis provide new insights into the pivotal role of cytoskeletal dynamics and the mechanisms of force transduction during leukocyte migration. Thereby the here presented results help to further define fundamental principles underlying leukocyte migration and open up potential therapeutic avenues of clinical relevance. AU - Kopf, Aglaja ID - 6891 KW - cell biology KW - immunology KW - leukocyte KW - migration KW - microfluidics SN - 978-3-99078-002-2 TI - The implication of cytoskeletal dynamics on leukocyte migration ER - TY - THES AB - Decades of studies have revealed the mechanisms of gene regulation in molecular detail. We make use of such well-described regulatory systems to explore how the molecular mechanisms of protein-protein and protein-DNA interactions shape the dynamics and evolution of gene regulation. i) We uncover how the biophysics of protein-DNA binding determines the potential of regulatory networks to evolve and adapt, which can be captured using a simple mathematical model. ii) The evolution of regulatory connections can lead to a significant amount of crosstalk between binding proteins. We explore the effect of crosstalk on gene expression from a target promoter, which seems to be modulated through binding competition at non-specific DNA sites. iii) We investigate how the very same biophysical characteristics as in i) can generate significant fitness costs for cells through global crosstalk, meaning non-specific DNA binding across the genomic background. iv) Binding competition between proteins at a target promoter is a prevailing regulatory feature due to the prevalence of co-regulation at bacterial promoters. However, the dynamics of these systems are not always straightforward to determine even if the molecular mechanisms of regulation are known. A detailed model of the biophysical interactions reveals that interference between the regulatory proteins can constitute a new, generic form of system memory that records the history of the input signals at the promoter. We demonstrate how the biophysics of protein-DNA binding can be harnessed to investigate the principles that shape and ultimately limit cellular gene regulation. These results provide a basis for studies of higher-level functionality, which arises from the underlying regulation. AU - Igler, Claudia ID - 6371 KW - gene regulation KW - biophysics KW - transcription factor binding KW - bacteria SN - 2663-337X TI - On the nature of gene regulatory design - The biophysics of transcription factor binding shapes gene regulation ER - TY - THES AB - Nowadays, quantum computation is receiving more and more attention as an alternative to the classical way of computing. For realizing a quantum computer, different devices are investigated as potential quantum bits. In this thesis, the focus is on Ge hut wires, which turned out to be promising candidates for implementing hole spin quantum bits. The advantages of Ge as a material system are the low hyperfine interaction for holes and the strong spin orbit coupling, as well as the compatibility with the highly developed CMOS processes in industry. In addition, Ge can also be isotopically purified which is expected to boost the spin coherence times. The strong spin orbit interaction for holes in Ge on the one hand enables the full electrical control of the quantum bit and on the other hand should allow short spin manipulation times. Starting with a bare Si wafer, this work covers the entire process reaching from growth over the fabrication and characterization of hut wire devices up to the demonstration of hole spin resonance. From experiments with single quantum dots, a large g-factor anisotropy between the in-plane and the out-of-plane direction was found. A comparison to a theoretical model unveiled the heavy-hole character of the lowest energy states. The second part of the thesis addresses double quantum dot devices, which were realized by adding two gate electrodes to a hut wire. In such devices, Pauli spin blockade was observed, which can serve as a read-out mechanism for spin quantum bits. Applying oscillating electric fields in spin blockade allowed the demonstration of continuous spin rotations and the extraction of a lower bound for the spin dephasing time. Despite the strong spin orbit coupling in Ge, the obtained value for the dephasing time is comparable to what has been recently reported for holes in Si. All in all, the presented results point out the high potential of Ge hut wires as a platform for long-lived, fast and fully electrically tunable hole spin quantum bits. AU - Watzinger, Hannes ID - 49 SN - 2663-337X TI - Ge hut wires - from growth to hole spin resonance ER - 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 -