TY - CONF AB - We design fast deterministic algorithms for distance computation in the CONGESTED CLIQUE model. Our key contributions include: - A (2+ε)-approximation for all-pairs shortest paths problem in O(log²n / ε) rounds on unweighted undirected graphs. With a small additional additive factor, this also applies for weighted graphs. This is the first sub-polynomial constant-factor approximation for APSP in this model. - A (1+ε)-approximation for multi-source shortest paths problem from O(√n) sources in O(log² n / ε) rounds on weighted undirected graphs. This is the first sub-polynomial algorithm obtaining this approximation for a set of sources of polynomial size. Our main techniques are new distance tools that are obtained via improved algorithms for sparse matrix multiplication, which we leverage to construct efficient hopsets and shortest paths. Furthermore, our techniques extend to additional distance problems for which we improve upon the state-of-the-art, including diameter approximation, and an exact single-source shortest paths algorithm for weighted undirected graphs in Õ(n^{1/6}) rounds. AU - Censor-Hillel, Keren AU - Dory, Michal AU - Korhonen, Janne AU - Leitersdorf, Dean ID - 6933 SN - 9781450362177 T2 - Proceedings of the 2019 ACM Symposium on Principles of Distributed Computin TI - Fast approximate shortest paths in the congested clique 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 - GEN AU - Kim, Olena AU - Borges Merjane, Carolina AU - Jonas, Peter M ID - 11222 IS - Suppl. 1 KW - hippocampus KW - mossy fibers KW - readily releasable pool KW - electron microscopy SN - 2309-8503 T2 - Intrinsic Activity TI - Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy VL - 7 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 - JOUR AB - A process of restorative patterning in plant roots correctly replaces eliminated cells to heal local injuries despite the absence of cell migration, which underpins wound healing in animals. Patterning in plants relies on oriented cell divisions and acquisition of specific cell identities. Plants regularly endure wounds caused by abiotic or biotic environmental stimuli and have developed extraordinary abilities to restore their tissues after injuries. Here, we provide insight into a mechanism of restorative patterning that repairs tissues after wounding. Laser-assisted elimination of different cells in Arabidopsis root combined with live-imaging tracking during vertical growth allowed analysis of the regeneration processes in vivo. Specifically, the cells adjacent to the inner side of the injury re-activated their stem cell transcriptional programs. They accelerated their progression through cell cycle, coordinately changed the cell division orientation, and ultimately acquired de novo the correct cell fates to replace missing cells. These observations highlight existence of unknown intercellular positional signaling and demonstrate the capability of specified cells to re-acquire stem cell programs as a crucial part of the plant-specific mechanism of wound healing. AU - Marhavá, Petra AU - Hörmayer, Lukas AU - Yoshida, Saiko AU - Marhavy, Peter AU - Benková, Eva AU - Friml, Jiří ID - 6351 IS - 4 JF - Cell SN - 00928674 TI - Re-activation of stem cell pathways for pattern restoration in plant wound healing VL - 177 ER - TY - JOUR AB - Plants as sessile organisms are constantly under attack by herbivores, rough environmental situations, or mechanical pressure. These challenges often lead to the induction of wounds or destruction of already specified and developed tissues. Additionally, wounding makes plants vulnerable to invasion by pathogens, which is why wound signalling often triggers specific defence responses. To stay competitive or, eventually, survive under these circumstances, plants need to regenerate efficiently, which in rigid, tissue migration-incompatible plant tissues requires post-embryonic patterning and organogenesis. Now, several studies used laser-assisted single cell ablation in the Arabidopsis root tip as a minimal wounding proxy. Here, we discuss their findings and put them into context of a broader spectrum of wound signalling, pathogen responses and tissue as well as organ regeneration. AU - Hörmayer, Lukas AU - Friml, Jiří ID - 6943 JF - Current Opinion in Plant Biology SN - 1369-5266 TI - Targeted cell ablation-based insights into wound healing and restorative patterning VL - 52 ER - TY - JOUR AB - Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. AU - Tabata, Shigekazu AU - Jevtic, Marijo AU - Kurashige, Nobutaka AU - Fuchida, Hirokazu AU - Kido, Munetsugu AU - Tani, Kazushi AU - Zenmyo, Naoki AU - Uchinomiya, Shohei AU - Harada, Harumi AU - Itakura, Makoto AU - Hamachi, Itaru AU - Shigemoto, Ryuichi AU - Ojida, Akio ID - 7391 IS - 12 JF - iScience SN - 2589-0042 TI - Electron microscopic detection of single membrane proteins by a specific chemical labeling VL - 22 ER - TY - JOUR AB - Proton-translocating transhydrogenase (also known as nicotinamide nucleotide transhydrogenase (NNT)) is found in the plasma membranes of bacteria and the inner mitochondrial membranes of eukaryotes. NNT catalyses the transfer of a hydride between NADH and NADP+, coupled to the translocation of one proton across the membrane. Its main physiological function is the generation of NADPH, which is a substrate in anabolic reactions and a regulator of oxidative status; however, NNT may also fine-tune the Krebs cycle1,2. NNT deficiency causes familial glucocorticoid deficiency in humans and metabolic abnormalities in mice, similar to those observed in type II diabetes3,4. The catalytic mechanism of NNT has been proposed to involve a rotation of around 180° of the entire NADP(H)-binding domain that alternately participates in hydride transfer and proton-channel gating. However, owing to the lack of high-resolution structures of intact NNT, the details of this process remain unclear5,6. Here we present the cryo-electron microscopy structure of intact mammalian NNT in different conformational states. We show how the NADP(H)-binding domain opens the proton channel to the opposite sides of the membrane, and we provide structures of these two states. We also describe the catalytically important interfaces and linkers between the membrane and the soluble domains and their roles in nucleotide exchange. These structures enable us to propose a revised mechanism for a coupling process in NNT that is consistent with a large body of previous biochemical work. Our results are relevant to the development of currently unavailable NNT inhibitors, which may have therapeutic potential in ischaemia reperfusion injury, metabolic syndrome and some cancers7,8,9. AU - Kampjut, Domen AU - Sazanov, Leonid A ID - 6848 IS - 7773 JF - Nature SN - 0028-0836 TI - Structure and mechanism of mitochondrial proton-translocating transhydrogenase VL - 573 ER - TY - JOUR AB - Grid cells with their rigid hexagonal firing fields are thought to provide an invariant metric to the hippocampal cognitive map, yet environmental geometrical features have recently been shown to distort the grid structure. Given that the hippocampal role goes beyond space, we tested the influence of nonspatial information on the grid organization. We trained rats to daily learn three new reward locations on a cheeseboard maze while recording from the medial entorhinal cortex and the hippocampal CA1 region. Many grid fields moved toward goal location, leading to long-lasting deformations of the entorhinal map. Therefore, distortions in the grid structure contribute to goal representation during both learning and recall, which demonstrates that grid cells participate in mnemonic coding and do not merely provide a simple metric of space. AU - Boccara, Charlotte N. AU - Nardin, Michele AU - Stella, Federico AU - O'Neill, Joseph AU - Csicsvari, Jozsef L ID - 6194 IS - 6434 JF - Science SN - 0036-8075 TI - The entorhinal cognitive map is attracted to goals VL - 363 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 - JOUR AB - Aberrant proteostasis of protein aggregation may lead to behavior disorders including chronic mental illnesses (CMI). Furthermore, the neuronal activity alterations that underlie CMI are not well understood. We recorded the local field potential and single-unit activity of the hippocampal CA1 region in vivo in rats transgenically overexpressing the Disrupted-in-Schizophrenia 1 (DISC1) gene (tgDISC1), modeling sporadic CMI. These tgDISC1 rats have previously been shown to exhibit DISC1 protein aggregation, disturbances in the dopaminergic system and attention-related deficits. Recordings were performed during exploration of familiar and novel open field environments and during sleep, allowing investigation of neuronal abnormalities in unconstrained behavior. Compared to controls, tgDISC1 place cells exhibited smaller place fields and decreased speed-modulation of their firing rates, demonstrating altered spatial coding and deficits in encoding location-independent sensory inputs. Oscillation analyses showed that tgDISC1 pyramidal neurons had higher theta phase locking strength during novelty, limiting their phase coding ability. However, their mean theta phases were more variable at the population level, reducing oscillatory network synchronization. Finally, tgDISC1 pyramidal neurons showed a lack of novelty-induced shift in their preferred theta and gamma firing phases, indicating deficits in coding of novel environments with oscillatory firing. By combining single cell and neuronal population analyses, we link DISC1 protein pathology with abnormal hippocampal neural coding and network synchrony, and thereby gain a more comprehensive understanding of CMI mechanisms. AU - Käfer, Karola AU - Malagon-Vina, Hugo AU - Dickerson, Desiree AU - O'Neill, Joseph AU - Trossbach, Svenja V. AU - Korth, Carsten AU - Csicsvari, Jozsef L ID - 5949 IS - 9 JF - Hippocampus TI - Disrupted-in-schizophrenia 1 overexpression disrupts hippocampal coding and oscillatory synchronization VL - 29 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 - JOUR AB - Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response. AU - Castro, João Pl AU - Yancoskie, Michelle N. AU - Marchini, Marta AU - Belohlavy, Stefanie AU - Hiramatsu, Layla AU - Kučka, Marek AU - Beluch, William H. AU - Naumann, Ronald AU - Skuplik, Isabella AU - Cobb, John AU - Barton, Nicholas H AU - Rolian, Campbell AU - Chan, Yingguang Frank ID - 6713 JF - eLife TI - An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice VL - 8 ER - TY - GEN AB - We study double quantum dots in a Ge/SiGe heterostructure and test their maturity towards singlet-triplet ($S-T_0$) qubits. We demonstrate a large range of tunability, from two single quantum dots to a double quantum dot. We measure Pauli spin blockade and study the anisotropy of the $g$-factor. We use an adjacent quantum dot for sensing charge transitions in the double quantum dot at interest. In conclusion, Ge/SiGe possesses all ingredients necessary for building a singlet-triplet qubit. AU - Hofmann, Andrea C AU - Jirovec, Daniel AU - Borovkov, Maxim AU - Prieto Gonzalez, Ivan AU - Ballabio, Andrea AU - Frigerio, Jacopo AU - Chrastina, Daniel AU - Isella, Giovanni AU - Katsaros, Georgios ID - 10065 T2 - arXiv TI - Assessing the potential of Ge/SiGe quantum dots as hosts for singlet-triplet qubits ER - TY - JOUR AB - 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 AU - Biebl, Julia AU - Roblek, Marko AU - Emtenani, Shamsi AU - György, Attila AU - Misova, Michaela AU - Ratheesh, Aparna AU - Rodrigues, Patricia AU - Shkarina, Katerina AU - Larsen, Ida Signe Bohse AU - Vakhrushev, Sergey Y AU - Clausen, Henrik AU - Siekhaus, Daria E ID - 6187 JF - eLife SN - 2050-084X TI - A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion VL - 8 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 - CONF AB - In this work, we consider the almost-sure termination problem for probabilistic programs that asks whether a given probabilistic program terminates with probability 1. Scalable approaches for program analysis often rely on modularity as their theoretical basis. In non-probabilistic programs, the classical variant rule (V-rule) of Floyd-Hoare logic provides the foundation for modular analysis. Extension of this rule to almost-sure termination of probabilistic programs is quite tricky, and a probabilistic variant was proposed in [16]. While the proposed probabilistic variant cautiously addresses the key issue of integrability, we show that the proposed modular rule is still not sound for almost-sure termination of probabilistic programs. Besides establishing unsoundness of the previous rule, our contributions are as follows: First, we present a sound modular rule for almost-sure termination of probabilistic programs. Our approach is based on a novel notion of descent supermartingales. Second, for algorithmic approaches, we consider descent supermartingales that are linear and show that they can be synthesized in polynomial time. Finally, we present experimental results on a variety of benchmarks and several natural examples that model various types of nested while loops in probabilistic programs and demonstrate that our approach is able to efficiently prove their almost-sure termination property AU - Huang, Mingzhang AU - Fu, Hongfei AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar ID - 6780 T2 - Proceedings of the 34th ACM International Conference on Object-Oriented Programming, Systems, Languages, and Applications TI - Modular verification for almost-sure termination of probabilistic programs VL - 3 ER - TY - JOUR AB - There is a huge gap between the speeds of modern caches and main memories, and therefore cache misses account for a considerable loss of efficiency in programs. The predominant technique to address this issue has been Data Packing: data elements that are frequently accessed within time proximity are packed into the same cache block, thereby minimizing accesses to the main memory. We consider the algorithmic problem of Data Packing on a two-level memory system. Given a reference sequence R of accesses to data elements, the task is to partition the elements into cache blocks such that the number of cache misses on R is minimized. The problem is notoriously difficult: it is NP-hard even when the cache has size 1, and is hard to approximate for any cache size larger than 4. Therefore, all existing techniques for Data Packing are based on heuristics and lack theoretical guarantees. In this work, we present the first positive theoretical results for Data Packing, along with new and stronger negative results. We consider the problem under the lens of the underlying access hypergraphs, which are hypergraphs of affinities between the data elements, where the order of an access hypergraph corresponds to the size of the affinity group. We study the problem parameterized by the treewidth of access hypergraphs, which is a standard notion in graph theory to measure the closeness of a graph to a tree. Our main results are as follows: We show there is a number q* depending on the cache parameters such that (a) if the access hypergraph of order q* has constant treewidth, then there is a linear-time algorithm for Data Packing; (b)the Data Packing problem remains NP-hard even if the access hypergraph of order q*-1 has constant treewidth. Thus, we establish a fine-grained dichotomy depending on a single parameter, namely, the highest order among access hypegraphs that have constant treewidth; and establish the optimal value q* of this parameter. Finally, we present an experimental evaluation of a prototype implementation of our algorithm. Our results demonstrate that, in practice, access hypergraphs of many commonly-used algorithms have small treewidth. We compare our approach with several state-of-the-art heuristic-based algorithms and show that our algorithm leads to significantly fewer cache-misses. AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar AU - Okati, Nastaran AU - Pavlogiannis, Andreas ID - 6380 IS - POPL JF - Proceedings of the ACM on Programming Languages SN - 2475-1421 TI - Efficient parameterized algorithms for data packing VL - 3 ER - TY - CONF AB - In today's programmable blockchains, smart contracts are limited to being deterministic and non-probabilistic. This lack of randomness is a consequential limitation, given that a wide variety of real-world financial contracts, such as casino games and lotteries, depend entirely on randomness. As a result, several ad-hoc random number generation approaches have been developed to be used in smart contracts. These include ideas such as using an oracle or relying on the block hash. However, these approaches are manipulatable, i.e. their output can be tampered with by parties who might not be neutral, such as the owner of the oracle or the miners.We propose a novel game-theoretic approach for generating provably unmanipulatable pseudorandom numbers on the blockchain. Our approach allows smart contracts to access a trustworthy source of randomness that does not rely on potentially compromised miners or oracles, hence enabling the creation of a new generation of smart contracts that are not limited to being non-probabilistic and can be drawn from the much more general class of probabilistic programs. AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar AU - Pourdamghani, Arash ID - 6056 T2 - IEEE International Conference on Blockchain and Cryptocurrency TI - Probabilistic smart contracts: Secure randomness on the blockchain ER - TY - CONF AB - In today's cryptocurrencies, Hashcash proof of work is the most commonly-adopted approach to mining. In Hashcash, when a miner decides to add a block to the chain, she has to solve the difficult computational puzzle of inverting a hash function. While Hashcash has been successfully adopted in both Bitcoin and Ethereum, it has attracted significant and harsh criticism due to its massive waste of electricity, its carbon footprint and environmental effects, and the inherent lack of usefulness in inverting a hash function. Various other mining protocols have been suggested, including proof of stake, in which a miner's chance of adding the next block is proportional to her current balance. However, such protocols lead to a higher entry cost for new miners who might not still have any stake in the cryptocurrency, and can in the worst case lead to an oligopoly, where the rich have complete control over mining. In this paper, we propose Hybrid Mining: a new mining protocol that combines solving real-world useful problems with Hashcash. Our protocol allows new miners to join the network by taking part in Hashcash mining without having to own an initial stake. It also allows nodes of the network to submit hard computational problems whose solutions are of interest in the real world, e.g.~protein folding problems. Then, miners can choose to compete in solving these problems, in lieu of Hashcash, for adding a new block. Hence, Hybrid Mining incentivizes miners to solve useful problems, such as hard computational problems arising in biology, in a distributed manner. It also gives researchers in other areas an easy-to-use tool to outsource their hard computations to the blockchain network, which has enormous computational power, by paying a reward to the miner who solves the problem for them. Moreover, our protocol provides strong security guarantees and is at least as resilient to double spending as Bitcoin. AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar AU - Pourdamghani, Arash ID - 6378 SN - 9781450359337 T2 - Proceedings of the 34th ACM Symposium on Applied Computing TI - Hybrid Mining: Exploiting blockchain’s computational power for distributed problem solving VL - Part F147772 ER - TY - CONF AB - We consider the problem of expected cost analysis over nondeterministic probabilistic programs, which aims at automated methods for analyzing the resource-usage of such programs. Previous approaches for this problem could only handle nonnegative bounded costs. However, in many scenarios, such as queuing networks or analysis of cryptocurrency protocols, both positive and negative costs are necessary and the costs are unbounded as well. In this work, we present a sound and efficient approach to obtain polynomial bounds on the expected accumulated cost of nondeterministic probabilistic programs. Our approach can handle (a) general positive and negative costs with bounded updates in variables; and (b) nonnegative costs with general updates to variables. We show that several natural examples which could not be handled by previous approaches are captured in our framework. Moreover, our approach leads to an efficient polynomial-time algorithm, while no previous approach for cost analysis of probabilistic programs could guarantee polynomial runtime. Finally, we show the effectiveness of our approach using experimental results on a variety of programs for which we efficiently synthesize tight resource-usage bounds. AU - Wang, Peixin AU - Fu, Hongfei AU - Goharshady, Amir Kafshdar AU - Chatterjee, Krishnendu AU - Qin, Xudong AU - Shi, Wenjun ID - 6175 KW - Program Cost Analysis KW - Program Termination KW - Probabilistic Programs KW - Martingales T2 - PLDI 2019: Proceedings of the 40th ACM SIGPLAN Conference on Programming Language Design and Implementation TI - Cost analysis of nondeterministic probabilistic programs ER - TY - CONF AB - Smart contracts are programs that are stored and executed on the Blockchain and can receive, manage and transfer money (cryptocurrency units). Two important problems regarding smart contracts are formal analysis and compiler optimization. Formal analysis is extremely important, because smart contracts hold funds worth billions of dollars and their code is immutable after deployment. Hence, an undetected bug can cause significant financial losses. Compiler optimization is also crucial, because every action of a smart contract has to be executed by every node in the Blockchain network. Therefore, optimizations in compiling smart contracts can lead to significant savings in computation, time and energy. Two classical approaches in program analysis and compiler optimization are intraprocedural and interprocedural analysis. In intraprocedural analysis, each function is analyzed separately, while interprocedural analysis considers the entire program. In both cases, the analyses are usually reduced to graph problems over the control flow graph (CFG) of the program. These graph problems are often computationally expensive. Hence, there has been ample research on exploiting structural properties of CFGs for efficient algorithms. One such well-studied property is the treewidth, which is a measure of tree-likeness of graphs. It is known that intraprocedural CFGs of structured programs have treewidth at most 6, whereas the interprocedural treewidth cannot be bounded. This result has been used as a basis for many efficient intraprocedural analyses. In this paper, we explore the idea of exploiting the treewidth of smart contracts for formal analysis and compiler optimization. First, similar to classical programs, we show that the intraprocedural treewidth of structured Solidity and Vyper smart contracts is at most 9. Second, for global analysis, we prove that the interprocedural treewidth of structured smart contracts is bounded by 10 and, in sharp contrast with classical programs, treewidth-based algorithms can be easily applied for interprocedural analysis. Finally, we supplement our theoretical results with experiments using a tool we implemented for computing treewidth of smart contracts and show that the treewidth is much lower in practice. We use 36,764 real-world Ethereum smart contracts as benchmarks and find that they have an average treewidth of at most 3.35 for the intraprocedural case and 3.65 for the interprocedural case. AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar AU - Goharshady, Ehsan Kafshdar ID - 6490 SN - 9781450359337 T2 - Proceedings of the 34th ACM Symposium on Applied Computing TI - The treewidth of smart contracts VL - Part F147772 ER - TY - JOUR AB - Interprocedural analysis is at the heart of numerous applications in programming languages, such as alias analysis, constant propagation, and so on. Recursive state machines (RSMs) are standard models for interprocedural analysis. We consider a general framework with RSMs where the transitions are labeled from a semiring and path properties are algebraic with semiring operations. RSMs with algebraic path properties can model interprocedural dataflow analysis problems, the shortest path problem, the most probable path problem, and so on. The traditional algorithms for interprocedural analysis focus on path properties where the starting point is fixed as the entry point of a specific method. In this work, we consider possible multiple queries as required in many applications such as in alias analysis. The study of multiple queries allows us to bring in an important algorithmic distinction between the resource usage of the one-time preprocessing vs for each individual query. The second aspect we consider is that the control flow graphs for most programs have constant treewidth. Our main contributions are simple and implementable algorithms that support multiple queries for algebraic path properties for RSMs that have constant treewidth. Our theoretical results show that our algorithms have small additional one-time preprocessing but can answer subsequent queries significantly faster as compared to the current algorithmic solutions for interprocedural dataflow analysis. We have also implemented our algorithms and evaluated their performance for performing on-demand interprocedural dataflow analysis on various domains, such as for live variable analysis and reaching definitions, on a standard benchmark set. Our experimental results align with our theoretical statements and show that after a lightweight preprocessing, on-demand queries are answered much faster than the standard existing algorithmic approaches. AU - Chatterjee, Krishnendu AU - Goharshady, Amir Kafshdar AU - Goyal, Prateesh AU - Ibsen-Jensen, Rasmus AU - Pavlogiannis, Andreas ID - 7158 IS - 4 JF - ACM Transactions on Programming Languages and Systems SN - 0164-0925 TI - Faster algorithms for dynamic algebraic queries in basic RSMs with constant treewidth VL - 41 ER - TY - JOUR AB - We study the problem of developing efficient approaches for proving worst-case bounds of non-deterministic recursive programs. Ranking functions are sound and complete for proving termination and worst-case bounds of nonrecursive programs. First, we apply ranking functions to recursion, resulting in measure functions. We show that measure functions provide a sound and complete approach to prove worst-case bounds of non-deterministic recursive programs. Our second contribution is the synthesis of measure functions in nonpolynomial forms. We show that non-polynomial measure functions with logarithm and exponentiation can be synthesized through abstraction of logarithmic or exponentiation terms, Farkas' Lemma, and Handelman's Theorem using linear programming. While previous methods obtain worst-case polynomial bounds, our approach can synthesize bounds of the form $\mathcal{O}(n\log n)$ as well as $\mathcal{O}(n^r)$ where $r$ is not an integer. We present experimental results to demonstrate that our approach can obtain efficiently worst-case bounds of classical recursive algorithms such as (i) Merge-Sort, the divide-and-conquer algorithm for the Closest-Pair problem, where we obtain $\mathcal{O}(n \log n)$ worst-case bound, and (ii) Karatsuba's algorithm for polynomial multiplication and Strassen's algorithm for matrix multiplication, where we obtain $\mathcal{O}(n^r)$ bound such that $r$ is not an integer and close to the best-known bounds for the respective algorithms. AU - Chatterjee, Krishnendu AU - Fu, Hongfei AU - Goharshady, Amir Kafshdar ID - 7014 IS - 4 JF - ACM Transactions on Programming Languages and Systems TI - Non-polynomial worst-case analysis of recursive programs VL - 41 ER - TY - JOUR AB - Based on a novel control scheme, where a steady modification of the streamwise velocity profile leads to complete relaminarization of initially fully turbulent pipe flow, we investigate the applicability and usefulness of custom-shaped honeycombs for such control. The custom-shaped honeycombs are used as stationary flow management devices which generate specific modifications of the streamwise velocity profile. Stereoscopic particle image velocimetry and pressure drop measurements are used to investigate and capture the development of the relaminarizing flow downstream these devices. We compare the performance of straight (constant length across the radius of the pipe) honeycombs with custom-shaped ones (variable length across the radius) and try to determine the optimal shape for maximal relaminarization at minimal pressure loss. The optimally modified streamwise velocity profile is found to be M-shaped, and the maximum attainable Reynolds number for total relaminarization is found to be of the order of 10,000. Consequently, the respective reduction in skin friction downstream of the device is almost by a factor of 5. The break-even point, where the additional pressure drop caused by the device is balanced by the savings due to relaminarization and a net gain is obtained, corresponds to a downstream stretch of distances as low as approximately 100 pipe diameters of laminar flow. AU - Kühnen, Jakob AU - Scarselli, Davide AU - Hof, Björn ID - 6486 IS - 11 JF - Journal of Fluids Engineering SN - 00982202 TI - Relaminarization of pipe flow by means of 3D-printed shaped honeycombs VL - 141 ER - TY - JOUR AB - Following the recent observation that turbulent pipe flow can be relaminarised bya relatively simple modification of the mean velocity profile, we here carry out aquantitative experimental investigation of this phenomenon. Our study confirms thata flat velocity profile leads to a collapse of turbulence and in order to achieve theblunted profile shape, we employ a moving pipe segment that is briefly and rapidlyshifted in the streamwise direction. The relaminarisation threshold and the minimumshift length and speeds are determined as a function of Reynolds number. Althoughturbulence is still active after the acceleration phase, the modulated profile possessesa severely decreased lift-up potential as measured by transient growth. As shown,this results in an exponential decay of fluctuations and the flow relaminarises. Whilethis method can be easily applied at low to moderate flow speeds, the minimumstreamwise length over which the acceleration needs to act increases linearly with theReynolds number. AU - Scarselli, Davide AU - Kühnen, Jakob AU - Hof, Björn ID - 6228 JF - Journal of Fluid Mechanics SN - 00221120 TI - Relaminarising pipe flow by wall movement VL - 867 ER - TY - JOUR AB - Polar auxin transport plays a pivotal role in plant growth and development. PIN auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis thaliana. PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport. AU - Oochi, A AU - Hajny, Jakub AU - Fukui, K AU - Nakao, Y AU - Gallei, Michelle C AU - Quareshy, M AU - Takahashi, K AU - Kinoshita, T AU - Harborough, SR AU - Kepinski, S AU - Kasahara, H AU - Napier, RM AU - Friml, Jiří AU - Hayashi, KI ID - 6260 IS - 2 JF - Plant Physiology SN - 0032-0889 TI - Pinstatic acid promotes auxin transport by inhibiting PIN internalization VL - 180 ER - TY - JOUR AB - Segregation of maternal determinants within the oocyte constitutes the first step in embryo patterning. In zebrafish oocytes, extensive ooplasmic streaming leads to the segregation of ooplasm from yolk granules along the animal-vegetal axis of the oocyte. Here, we show that this process does not rely on cortical actin reorganization, as previously thought, but instead on a cell-cycle-dependent bulk actin polymerization wave traveling from the animal to the vegetal pole of the oocyte. This wave functions in segregation by both pulling ooplasm animally and pushing yolk granules vegetally. Using biophysical experimentation and theory, we show that ooplasm pulling is mediated by bulk actin network flows exerting friction forces on the ooplasm, while yolk granule pushing is achieved by a mechanism closely resembling actin comet formation on yolk granules. Our study defines a novel role of cell-cycle-controlled bulk actin polymerization waves in oocyte polarization via ooplasmic segregation. AU - Shamipour, Shayan AU - Kardos, Roland AU - Xue, Shi-lei AU - Hof, Björn AU - Hannezo, Edouard B AU - Heisenberg, Carl-Philipp J ID - 6508 IS - 6 JF - Cell SN - 00928674 TI - Bulk actin dynamics drive phase segregation in zebrafish oocytes VL - 177 ER - TY - JOUR AU - Schwayer, Cornelia AU - Shamipour, Shayan AU - Pranjic-Ferscha, Kornelija AU - Schauer, Alexandra AU - Balda, M AU - Tada, M AU - Matter, K AU - Heisenberg, Carl-Philipp J ID - 7001 IS - 4 JF - Cell SN - 0092-8674 TI - Mechanosensation of tight junctions depends on ZO-1 phase separation and flow VL - 179 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 - JOUR AB - During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1,2,3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some—but not all—cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion7, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion. AU - Renkawitz, Jörg AU - Kopf, Aglaja AU - Stopp, Julian A AU - de Vries, Ingrid AU - Driscoll, Meghan K. AU - Merrin, Jack AU - Hauschild, Robert AU - Welf, Erik S. AU - Danuser, Gaudenz AU - Fiolka, Reto AU - Sixt, Michael K ID - 6328 JF - Nature TI - Nuclear positioning facilitates amoeboid migration along the path of least resistance VL - 568 ER - TY - JOUR AU - Kopf, Aglaja AU - Sixt, Michael K ID - 6877 IS - 1 JF - Cell SN - 0092-8674 TI - The neural crest pitches in to remove apoptotic debris VL - 179 ER - TY - JOUR AU - Contreras, Ximena AU - Hippenmeyer, Simon ID - 6830 IS - 5 JF - Neuron SN - 08966273 TI - Memo1 tiles the radial glial cell grid VL - 103 ER - TY - JOUR AB - Cortical microtubule arrays in elongating epidermal cells in both the root and stem of plants have the propensity of dynamic reorientations that are correlated with the activation or inhibition of growth. Factors regulating plant growth, among them the hormone auxin, have been recognized as regulators of microtubule array orientations. Some previous work in the field has aimed at elucidating the causal relationship between cell growth, the signaling of auxin or other growth-regulating factors, and microtubule array reorientations, with various conclusions. Here, we revisit this problem of causality with a comprehensive set of experiments in Arabidopsis thaliana, using the now available pharmacological and genetic tools. We use isolated, auxin-depleted hypocotyls, an experimental system allowing for full control of both growth and auxin signaling. We demonstrate that reorientation of microtubules is not directly triggered by an auxin signal during growth activation. Instead, reorientation is triggered by the activation of the growth process itself and is auxin-independent in its nature. We discuss these findings in the context of previous relevant work, including that on the mechanical regulation of microtubule array orientation. AU - Adamowski, Maciek AU - Li, Lanxin AU - Friml, Jiří ID - 6627 IS - 13 JF - International Journal of Molecular Sciences TI - Reorientation of cortical microtubule arrays in the hypocotyl of arabidopsis thaliana is induced by the cell growth process and independent of auxin signaling VL - 20 ER - TY - JOUR AB - We propose a novel generic shape optimization method for CAD models based on the eXtended Finite Element Method (XFEM). Our method works directly on the intersection between the model and a regular simulation grid, without the need to mesh or remesh, thus removing a bottleneck of classical shape optimization strategies. This is made possible by a novel hierarchical integration scheme that accurately integrates finite element quantities with sub-element precision. For optimization, we efficiently compute analytical shape derivatives of the entire framework, from model intersection to integration rule generation and XFEM simulation. Moreover, we describe a differentiable projection of shape parameters onto a constraint manifold spanned by user-specified shape preservation, consistency, and manufacturability constraints. We demonstrate the utility of our approach by optimizing mass distribution, strength-to-weight ratio, and inverse elastic shape design objectives directly on parameterized 3D CAD models. AU - Hafner, Christian AU - Schumacher, Christian AU - Knoop, Espen AU - Auzinger, Thomas AU - Bickel, Bernd AU - Bächer, Moritz ID - 7117 IS - 6 JF - ACM Transactions on Graphics SN - 0730-0301 TI - X-CAD: Optimizing CAD Models with Extended Finite Elements VL - 38 ER - TY - JOUR AB - Suspended particles can alter the properties of fluids and in particular also affect the transition fromlaminar to turbulent flow. An earlier study [Mataset al.,Phys. Rev. Lett.90, 014501 (2003)] reported howthe subcritical (i.e., hysteretic) transition to turbulent puffs is affected by the addition of particles. Here weshow that in addition to this known transition, with increasing concentration a supercritical (i.e.,continuous) transition to a globally fluctuating state is found. At the same time the Newtonian-typetransition to puffs is delayed to larger Reynolds numbers. At even higher concentration only the globallyfluctuating state is found. The dynamics of particle laden flows are hence determined by two competinginstabilities that give rise to three flow regimes: Newtonian-type turbulence at low, a particle inducedglobally fluctuating state at high, and a coexistence state at intermediate concentrations. AU - Agrawal, Nishchal AU - Choueiri, George H AU - Hof, Björn ID - 6189 IS - 11 JF - Physical Review Letters SN - 00319007 TI - Transition to turbulence in particle laden flows VL - 122 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 - JOUR AB - In this paper, we evaluate clock signals generated in ring oscillators and self-timed rings and the way their jitter can be transformed into random numbers. We show that counting the periods of the jittery clock signal produces random numbers of significantly better quality than the methods in which the jittery signal is simply sampled (the case in almost all current methods). Moreover, we use the counter values to characterize and continuously monitor the source of randomness. However, instead of using the widely used statistical variance, we propose to use Allan variance to do so. There are two main advantages: Allan variance is insensitive to low frequency noises such as flicker noise that are known to be autocorrelated and significantly less circuitry is required for its computation than that used to compute commonly used variance. We also show that it is essential to use a differential principle of randomness extraction from the jitter based on the use of two identical oscillators to avoid autocorrelations originating from external and internal global jitter sources and that this fact is valid for both kinds of rings. Last but not least, we propose a method of statistical testing based on high order Markov model to show the reduced dependencies when the proposed randomness extraction is applied. AU - Allini, Elie Noumon AU - Skórski, Maciej AU - Petura, Oto AU - Bernard, Florent AU - Laban, Marek AU - Fischer, Viktor ID - 10286 IS - 3 JF - IACR Transactions on Cryptographic Hardware and Embedded Systems TI - Evaluation and monitoring of free running oscillators serving as source of randomness VL - 2018 ER - TY - CONF AB - Solving parity games, which are equivalent to modal μ-calculus model checking, is a central algorithmic problem in formal methods, with applications in reactive synthesis, program repair, verification of branching-time properties, etc. Besides the standard compu- tation model with the explicit representation of games, another important theoretical model of computation is that of set-based symbolic algorithms. Set-based symbolic algorithms use basic set operations and one-step predecessor operations on the implicit description of games, rather than the explicit representation. The significance of symbolic algorithms is that they provide scalable algorithms for large finite-state systems, as well as for infinite-state systems with finite quotient. Consider parity games on graphs with n vertices and parity conditions with d priorities. While there is a rich literature of explicit algorithms for parity games, the main results for set-based symbolic algorithms are as follows: (a) the basic algorithm that requires O(nd) symbolic operations and O(d) symbolic space; and (b) an improved algorithm that requires O(nd/3+1) symbolic operations and O(n) symbolic space. In this work, our contributions are as follows: (1) We present a black-box set-based symbolic algorithm based on the explicit progress measure algorithm. Two important consequences of our algorithm are as follows: (a) a set-based symbolic algorithm for parity games that requires quasi-polynomially many symbolic operations and O(n) symbolic space; and (b) any future improvement in progress measure based explicit algorithms immediately imply an efficiency improvement in our set-based symbolic algorithm for parity games. (2) We present a set-based symbolic algorithm that requires quasi-polynomially many symbolic operations and O(d · log n) symbolic space. Moreover, for the important special case of d ≤ log n, our algorithm requires only polynomially many symbolic operations and poly-logarithmic symbolic space. AU - Chatterjee, Krishnendu AU - Dvořák, Wolfgang AU - Henzinger, Monika H AU - Svozil, Alexander ID - 10883 SN - 2398-7340 T2 - 22nd International Conference on Logic for Programming, Artificial Intelligence and Reasoning TI - Quasipolynomial set-based symbolic algorithms for parity games VL - 57 ER - TY - CONF AB - We report on a novel strategy to derive mean-field limits of quantum mechanical systems in which a large number of particles weakly couple to a second-quantized radiation field. The technique combines the method of counting and the coherent state approach to study the growth of the correlations among the particles and in the radiation field. As an instructional example, we derive the Schrödinger–Klein–Gordon system of equations from the Nelson model with ultraviolet cutoff and possibly massless scalar field. In particular, we prove the convergence of the reduced density matrices (of the nonrelativistic particles and the field bosons) associated with the exact time evolution to the projectors onto the solutions of the Schrödinger–Klein–Gordon equations in trace norm. Furthermore, we derive explicit bounds on the rate of convergence of the one-particle reduced density matrix of the nonrelativistic particles in Sobolev norm. AU - Leopold, Nikolai K AU - Pickl, Peter ID - 11 TI - Mean-field limits of particles in interaction with quantised radiation fields VL - 270 ER - TY - JOUR AB - Two generalizations of Itô formula to infinite-dimensional spaces are given. The first one, in Hilbert spaces, extends the classical one by taking advantage of cancellations when they occur in examples and it is applied to the case of a group generator. The second one, based on the previous one and a limit procedure, is an Itô formula in a special class of Banach spaces having a product structure with the noise in a Hilbert component; again the key point is the extension due to a cancellation. This extension to Banach spaces and in particular the specific cancellation are motivated by path-dependent Itô calculus. AU - Flandoli, Franco AU - Russo, Francesco AU - Zanco, Giovanni A ID - 1215 IS - 2 JF - Journal of Theoretical Probability TI - Infinite-dimensional calculus under weak spatial regularity of the processes VL - 31 ER - TY - CONF AB - We resolve in the affirmative conjectures of A. Skopenkov and Repovš (1998), and M. Skopenkov (2003) generalizing the classical Hanani-Tutte theorem to the setting of approximating maps of graphs on 2-dimensional surfaces by embeddings. Our proof of this result is constructive and almost immediately implies an efficient algorithm for testing whether a given piecewise linear map of a graph in a surface is approximable by an embedding. More precisely, an instance of this problem consists of (i) a graph G whose vertices are partitioned into clusters and whose inter-cluster edges are partitioned into bundles, and (ii) a region R of a 2-dimensional compact surface M given as the union of a set of pairwise disjoint discs corresponding to the clusters and a set of pairwise disjoint "pipes" corresponding to the bundles, connecting certain pairs of these discs. We are to decide whether G can be embedded inside M so that the vertices in every cluster are drawn in the corresponding disc, the edges in every bundle pass only through its corresponding pipe, and every edge crosses the boundary of each disc at most once. AU - Fulek, Radoslav AU - Kynčl, Jan ID - 185 SN - 978-3-95977-066-8 TI - Hanani-Tutte for approximating maps of graphs VL - 99 ER - TY - CONF AB - Smallest enclosing spheres of finite point sets are central to methods in topological data analysis. Focusing on Bregman divergences to measure dissimilarity, we prove bounds on the location of the center of a smallest enclosing sphere. These bounds depend on the range of radii for which Bregman balls are convex. AU - Edelsbrunner, Herbert AU - Virk, Ziga AU - Wagner, Hubert ID - 188 TI - Smallest enclosing spheres and Chernoff points in Bregman geometry VL - 99 ER - TY - JOUR AB - A cornerstone of statistical inference, the maximum entropy framework is being increasingly applied to construct descriptive and predictive models of biological systems, especially complex biological networks, from large experimental data sets. Both its broad applicability and the success it obtained in different contexts hinge upon its conceptual simplicity and mathematical soundness. Here we try to concisely review the basic elements of the maximum entropy principle, starting from the notion of ‘entropy’, and describe its usefulness for the analysis of biological systems. As examples, we focus specifically on the problem of reconstructing gene interaction networks from expression data and on recent work attempting to expand our system-level understanding of bacterial metabolism. Finally, we highlight some extensions and potential limitations of the maximum entropy approach, and point to more recent developments that are likely to play a key role in the upcoming challenges of extracting structures and information from increasingly rich, high-throughput biological data. AU - De Martino, Andrea AU - De Martino, Daniele ID - 306 IS - 4 JF - Heliyon TI - An introduction to the maximum entropy approach and its application to inference problems in biology VL - 4 ER - TY - BOOK AB - This book first explores the origins of this idea, grounded in theoretical work on temporal logic and automata. The editors and authors are among the world's leading researchers in this domain, and they contributed 32 chapters representing a thorough view of the development and application of the technique. Topics covered include binary decision diagrams, symbolic model checking, satisfiability modulo theories, partial-order reduction, abstraction, interpolation, concurrency, security protocols, games, probabilistic model checking, and process algebra, and chapters on the transfer of theory to industrial practice, property specification languages for hardware, and verification of real-time systems and hybrid systems. The book will be valuable for researchers and graduate students engaged with the development of formal methods and verification tools. AU - Clarke, Edmund M. AU - Henzinger, Thomas A AU - Veith, Helmut AU - Bloem, Roderick ID - 3300 SN - 978-3-319-10574-1 TI - Handbook of Model Checking ER - TY - CHAP AB - Developmental processes are inherently dynamic and understanding them requires quantitative measurements of gene and protein expression levels in space and time. While live imaging is a powerful approach for obtaining such data, it is still a challenge to apply it over long periods of time to large tissues, such as the embryonic spinal cord in mouse and chick. Nevertheless, dynamics of gene expression and signaling activity patterns in this organ can be studied by collecting tissue sections at different developmental stages. In combination with immunohistochemistry, this allows for measuring the levels of multiple developmental regulators in a quantitative manner with high spatiotemporal resolution. The mean protein expression levels over time, as well as embryo-to-embryo variability can be analyzed. A key aspect of the approach is the ability to compare protein levels across different samples. This requires a number of considerations in sample preparation, imaging and data analysis. Here we present a protocol for obtaining time course data of dorsoventral expression patterns from mouse and chick neural tube in the first 3 days of neural tube development. The described workflow starts from embryo dissection and ends with a processed dataset. Software scripts for data analysis are included. The protocol is adaptable and instructions that allow the user to modify different steps are provided. Thus, the procedure can be altered for analysis of time-lapse images and applied to systems other than the neural tube. AU - Zagórski, Marcin P AU - Kicheva, Anna ID - 37 SN - 1064-3745 T2 - Morphogen Gradients TI - Measuring dorsoventral pattern and morphogen signaling profiles in the growing neural tube VL - 1863 ER -