@inproceedings{6933, abstract = {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.}, author = {Censor-Hillel, Keren and Dory, Michal and Korhonen, Janne and Leitersdorf, Dean}, booktitle = {Proceedings of the 2019 ACM Symposium on Principles of Distributed Computin}, isbn = {9781450362177}, location = {Toronto, ON, Canada}, pages = {74--83}, publisher = {ACM}, title = {{Fast approximate shortest paths in the congested clique}}, doi = {10.1145/3293611.3331633}, year = {2019}, } @phdthesis{6392, abstract = {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. }, author = {Lukacisin, Martin}, isbn = {978-3-99078-001-5}, issn = {2663-337X}, pages = {103}, publisher = {IST Austria}, title = {{Quantitative investigation of gene expression principles through combinatorial drug perturbation and theory}}, doi = {10.15479/AT:ISTA:6392}, year = {2019}, } @phdthesis{6435, abstract = {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.}, author = {Casillas Perez, Barbara E}, issn = {2663-337X}, keywords = {Social Immunity, Sanitary care, Social Insects, Organisational Immunity, Colony development, Multi-target tracking}, pages = {183}, publisher = {Institute of Science and Technology Austria}, title = {{Collective defenses of garden ants against a fungal pathogen}}, doi = {10.15479/AT:ISTA:6435}, year = {2019}, } @phdthesis{6269, abstract = {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. }, author = {Narasimhan, Madhumitha}, issn = {2663-337X}, pages = {138}, publisher = {Institute of Science and Technology Austria}, title = {{Clathrin-Mediated endocytosis, post-endocytic trafficking and their regulatory controls in plants }}, doi = {10.15479/at:ista:th1075}, year = {2019}, } @inproceedings{11222, author = {Kim, Olena and Borges Merjane, Carolina and Jonas, Peter M}, booktitle = {Intrinsic Activity}, issn = {2309-8503}, keywords = {hippocampus, mossy fibers, readily releasable pool, electron microscopy}, location = {Innsbruck, Austria}, number = {Suppl. 1}, publisher = {Austrian Pharmacological Society}, title = {{Functional analysis of the docked vesicle pool in hippocampal mossy fiber terminals by electron microscopy}}, doi = {10.25006/ia.7.s1-a3.27}, volume = {7}, year = {2019}, }