@phdthesis{539, abstract = {The whole life cycle of plants as well as their responses to environmental stimuli is governed by a complex network of hormonal regulations. A number of studies have demonstrated an essential role of both auxin and cytokinin in the regulation of many aspects of plant growth and development including embryogenesis, postembryonic organogenic processes such as root, and shoot branching, root and shoot apical meristem activity and phyllotaxis. Over the last decades essential knowledge on the key molecular factors and pathways that spatio-temporally define auxin and cytokinin activities in the plant body has accumulated. However, how both hormonal pathways are interconnected by a complex network of interactions and feedback circuits that determines the final outcome of the individual hormone actions is still largely unknown. Root system architecture establishment and in particular formation of lateral organs is prime example of developmental process at whose regulation both auxin and cytokinin pathways converge. To dissect convergence points and pathways that tightly balance auxin - cytokinin antagonistic activities that determine the root branching pattern transcriptome profiling was applied. Genome wide expression analyses of the xylem pole pericycle, a tissue giving rise to lateral roots, led to identification of genes that are highly responsive to combinatorial auxin and cytokinin treatments and play an essential function in the auxin-cytokinin regulated root branching. SYNERGISTIC AUXIN CYTOKININ 1 (SYAC1) gene, which encodes for a protein of unknown function, was detected among the top candidate genes of which expression was synergistically up-regulated by simultaneous hormonal treatment. Plants with modulated SYAC1 activity exhibit severe defects in the root system establishment and attenuate developmental responses to both auxin and cytokinin. To explore the biological function of the SYAC1, we employed different strategies including expression pattern analysis, subcellular localization and phenotypic analyses of the syac1 loss-of-function and gain-of-function transgenic lines along with the identification of the SYAC1 interaction partners. Detailed functional characterization revealed that SYAC1 acts as a developmentally specific regulator of the secretory pathway to control deposition of cell wall components and thereby rapidly fine tune elongation growth.}, author = {Hurny, Andrej}, issn = {2663-337X}, pages = {147}, publisher = {Institute of Science and Technology Austria}, title = {{Identification and characterization of novel auxin-cytokinin cross-talk components}}, doi = {10.15479/AT:ISTA:th_930}, year = {2018}, } @phdthesis{48, abstract = {The hippocampus is a key brain region for spatial memory and navigation and is needed at all stages of memory, including encoding, consolidation, and recall. Hippocampal place cells selectively discharge at specific locations of the environment to form a cognitive map of the space. During the rest period and sleep following spatial navigation and/or learning, the waking activity of the place cells is reactivated within high synchrony events. This reactivation is thought to be important for memory consolidation and stabilization of the spatial representations. The aim of my thesis was to directly test whether the reactivation content encoded in firing patterns of place cells is important for consolidation of spatial memories. In particular, I aimed to test whether, in cases when multiple spatial memory traces are acquired during learning, the specific disruption of the reactivation of a subset of these memories leads to the selective disruption of the corresponding memory traces or through memory interference the other learned memories are disrupted as well. In this thesis, using a modified cheeseboard paradigm and a closed-loop recording setup with feedback optogenetic stimulation, I examined how the disruption of the reactivation of specific spiking patterns affects consolidation of the corresponding memory traces. To obtain multiple distinctive memories, animals had to perform a spatial task in two distinct cheeseboard environments and the reactivation of spiking patterns associated with one of the environments (target) was disrupted after learning during four hours rest period using a real-time decoding method. This real-time decoding method was capable of selectively affecting the firing rates and cofiring correlations of the target environment-encoding cells. The selective disruption led to behavioural impairment in the memory tests after the rest periods in the target environment but not in the other undisrupted control environment. In addition, the map of the target environment was less stable in the impaired memory tests compared to the learning session before than the map of the control environment. However, when the animal relearned the task, the same map recurred in the target environment that was present during learning before the disruption. Altogether my work demonstrated that the reactivation content is important: assembly-related disruption of reactivation can lead to a selective memory impairment and deficiency in map stability. These findings indeed suggest that reactivated assembly patterns reflect processes associated with the consolidation of memory traces. }, author = {Gridchyn, Igor}, issn = {2663-337X}, pages = {104}, publisher = {Institute of Science and Technology Austria}, title = {{Reactivation content is important for consolidation of spatial memory}}, doi = {10.15479/AT:ISTA:th_1042}, year = {2018}, } @phdthesis{9, abstract = {Immune cells migrating to the sites of infection navigate through diverse tissue architectures and switch their migratory mechanisms upon demand. However, little is known about systemic regulators that could allow the acquisition of these mechanisms. We performed a genetic screen in Drosophila melanogaster to identify regulators of germband invasion by embryonic macrophages into the confined space between the ectoderm and mesoderm. We have found that bZIP circadian transcription factors (TFs) Kayak (dFos) and Vrille (dNFIL3) have opposite effects on macrophage germband infiltration: Kayak facilitated and Vrille inhibited it. These TFs are enriched in the macrophages during migration and genetically interact to control it. Kayak sets a less coordinated mode of migration of the macrophage group and increases the probability and length of Levy walks. Intriguingly, the motility of kayak mutant macrophages was also strongly affected during initial germband invasion but not along another less confined route. Inhibiting Rho1 signaling within the tail ectoderm partially rescued the Kayak mutant phenotype, strongly suggesting that migrating macrophages have to overcome a barrier imposed by the stiffness of the ectoderm. Also, Kayak appeared to be important for the maintenance of the round cell shape and the rear edge translocation of the macrophages invading the germband. Complementary to this, the cortical actin cytoskeleton of Kayak- deficient macrophages was strongly affected. RNA sequencing revealed the filamin Cheerio and tetraspanin TM4SF to be downstream of Kayak. Chromatin immunoprecipitation and immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Immunostaining revealed that the formin Diaphanous is another downstream target of Kayak. Indeed, Cheerio, TM4SF and Diaphanous are required within macrophages for germband invasion, and expression of constitutively active Diaphanous in macrophages was able to rescue the kayak mutant phenotype. Moreover, Cher and Diaphanous are also reduced in the macrophages overexpressing Vrille. We hypothesize that Kayak, through its targets, increases actin polymerization and cortical tension in macrophages and thus allows extra force generation necessary for macrophage dissemination and migration through confined stiff tissues, while Vrille counterbalances it.}, author = {Belyaeva, Vera}, issn = {2663-337X}, pages = {96}, publisher = {Institute of Science and Technology Austria}, title = {{Transcriptional regulation of macrophage migration in the Drosophila melanogaster embryo }}, doi = {10.15479/AT:ISTA:th1064}, year = {2018}, } @phdthesis{6266, abstract = {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. }, author = {Mckenzie, Catherine}, issn = {2663-337X}, pages = {95}, publisher = {Institute of Science and Technology Austria}, title = {{Design and characterization of methods and biological components to realize synthetic neurotransmission }}, doi = {10.15479/at:ista:th_1055}, year = {2018}, } @phdthesis{50, abstract = {The Wnt/planar cell polarity (Wnt/PCP) pathway determines planar polarity of epithelial cells in both vertebrates and invertebrates. The role that Wnt/PCP signaling plays in mesenchymal contexts, however, is only poorly understood. While previous studies have demonstrated the capacity of Wnt/PCP signaling to polarize and guide directed migration of mesenchymal cells, it remains unclear whether endogenous Wnt/PCP signaling performs these functions instructively, as it does in epithelial cells. Here we developed a light-switchable version of the Wnt/PCP receptor Frizzled 7 (Fz7) to unambiguously distinguish between an instructive and a permissive role of Wnt/PCP signaling for the directional collective migration of mesendoderm progenitor cells during zebrafish gastrulation. We show that prechordal plate (ppl) cell migration is defective in maternal-zygotic fz7a and fz7b (MZ fz7a,b) double mutant embryos, and that Fz7 functions cell-autonomously in this process by promoting ppl cell protrusion formation and directed migration. We further show that local activation of Fz7 can direct ppl cell migration both in vitro and in vivo. Surprisingly, however, uniform Fz7 activation is sufficient to fully rescue the ppl cell migration defect in MZ fz7a,b mutant embryos, indicating that Wnt/PCP signaling functions permissively rather than instructively in directed mesendoderm cell migration during zebrafish gastrulation.}, author = {Capek, Daniel}, issn = {2663-337X}, pages = {95}, publisher = {Institute of Science and Technology Austria}, title = {{Optogenetic Frizzled 7 reveals a permissive function of Wnt/PCP signaling in directed mesenchymal cell migration}}, doi = {10.15479/AT:ISTA:TH_1031}, year = {2018}, } @phdthesis{26, abstract = {Expression of genes is a fundamental molecular phenotype that is subject to evolution by different types of mutations. Both the rate and the effect of mutations may depend on the DNA sequence context of a particular gene or a particular promoter sequence. In this thesis I investigate the nature of this dependence using simple genetic systems in Escherichia coli. With these systems I explore the evolution of constitutive gene expression from random starting sequences at different loci on the chromosome and at different locations in sequence space. First, I dissect chromosomal neighborhood effects that underlie locus-dependent differences in the potential of a gene under selection to become more highly expressed. Next, I find that the effects of point mutations in promoter sequences are dependent on sequence context, and that an existing energy matrix model performs poorly in predicting relative expression of unrelated sequences. Finally, I show that a substantial fraction of random sequences contain functional promoters and I present an extended thermodynamic model that predicts promoter strength in full sequence space. Taken together, these results provide new insights and guides on how to integrate information on sequence context to improve our qualitative and quantitative understanding of bacterial gene expression, with implications for rapid evolution of drug resistance, de novo evolution of genes, and horizontal gene transfer.}, author = {Steinrück, Magdalena}, issn = {2663-337X}, pages = {109}, publisher = {Institute of Science and Technology Austria}, title = {{The influence of sequence context on the evolution of bacterial gene expression}}, doi = {10.15479/AT:ISTA:th1059}, year = {2018}, } @article{5816, abstract = {Solid-state qubit manipulation and read-out fidelities are reaching fault-tolerance, but quantum error correction requires millions of physical qubits and therefore a scalable quantum computer architecture. To solve signal-line bandwidth and fan-out problems, microwave sources required for qubit manipulation might be embedded close to the qubit chip, typically operating at temperatures below 4 K. Here, we perform the first low temperature measurements of a 130 nm BiCMOS based SiGe voltage controlled oscillator at cryogenic temperature. We determined the frequency and output power dependence on temperature and magnetic field up to 5 T and measured the temperature influence on its noise performance. The device maintains its full functionality from 300 K to 4 K. The carrier frequency at 4 K increases by 3% with respect to the carrier frequency at 300 K, and the output power at 4 K increases by 10 dB relative to the output power at 300 K. The frequency tuning range of approximately 20% remains unchanged between 300 K and 4 K. In an in-plane magnetic field of 5 T, the carrier frequency shifts by only 0.02% compared to the frequency at zero magnetic field.}, author = {Hollmann, Arne and Jirovec, Daniel and Kucharski, Maciej and Kissinger, Dietmar and Fischer, Gunter and Schreiber, Lars R.}, issn = {00346748}, journal = {Review of Scientific Instruments}, number = {11}, publisher = {AIP Publishing}, title = {{30 GHz-voltage controlled oscillator operating at 4 K}}, doi = {10.1063/1.5038258}, volume = {89}, year = {2018}, } @phdthesis{6263, abstract = {Antibiotic resistance can emerge spontaneously through genomic mutation and render treatment ineffective. To counteract this process, in addition to the discovery and description of resistance mechanisms,a deeper understanding of resistanceevolvabilityand its determinantsis needed. To address this challenge, this thesisuncoversnew genetic determinants of resistance evolvability using a customized robotic setup, exploressystematic ways in which resistance evolution is perturbed due to dose-responsecharacteristics of drugs and mutation rate differences,and mathematically investigates the evolutionary fate of one specific type of evolvability modifier -a stress-induced mutagenesis allele.We find severalgenes which strongly inhibit or potentiate resistance evolution. In order to identify them, we first developedan automated high-throughput feedback-controlled protocol whichkeeps the population size and selection pressure approximately constant for hundreds of cultures by dynamically re-diluting the cultures and adjusting the antibiotic concentration. We implementedthis protocol on a customized liquid handling robot and propagated 100 different gene deletion strains of Escherichia coliin triplicate for over 100 generations in tetracycline and in chloramphenicol, and comparedtheir adaptation rates.We find a diminishing returns pattern, where initially sensitive strains adapted more compared to less sensitive ones. Our data uncover that deletions of certain genes which do not affect mutation rate,including efflux pump components, a chaperone and severalstructural and regulatory genes can strongly and reproducibly alterresistance evolution. Sequencing analysis of evolved populations indicates that epistasis with resistance mutations is the most likelyexplanation. This work could inspire treatment strategies in which targeted inhibitors of evolvability mechanisms will be given alongside antibiotics to slow down resistance evolution and extend theefficacy of antibiotics.We implemented astochasticpopulation genetics model, toverifyways in which general properties, namely, dose-response characteristics of drugs and mutation rates, influence evolutionary dynamics. In particular, under the exposure to antibiotics with shallow dose-response curves,bacteria have narrower distributions of fitness effects of new mutations. We show that in silicothis also leads to slower resistance evolution. We see and confirm with experiments that increased mutation rates, apart from speeding up evolution, also leadto high reproducibility of phenotypic adaptation in a context of continually strong selection pressure.Knowledge of these patterns can aid in predicting the dynamics of antibiotic resistance evolutionand adapting treatment schemes accordingly.Focusing on a previously described type of evolvability modifier –a stress-induced mutagenesis allele –we find conditions under which it can persist in a population under periodic selectionakin to clinical treatment. We set up a deterministic infinite populationcontinuous time model tracking the frequencies of a mutator and resistance allele and evaluate various treatment schemes in how well they maintain a stress-induced mutator allele. In particular,a high diversity of stresses is crucial for the persistence of the mutator allele. This leads to a general trade-off where exactly those diversifying treatment schemes which are likely to decrease levels of resistance could lead to stronger selection of highly evolvable genotypes.In the long run, this work will lead to a deeper understanding of the genetic and cellular mechanisms involved in antibiotic resistance evolution and could inspire new strategies for slowing down its rate. }, author = {Lukacisinova, Marta}, issn = {2663-337X}, pages = {91}, publisher = {Institute of Science and Technology Austria}, title = {{Genetic determinants of antibiotic resistance evolution}}, doi = {10.15479/AT:ISTA:th1072}, year = {2018}, } @article{544, abstract = {Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid in fully understanding these processes, but are lacking. Here we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, cytoplasm or actin cytoskeleton from embryonic Stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae and adults. They permit efficient GAL4-independent FACS analysis/sorting of plasmatocytes throughout life. To facilitate genetic analysis of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that in combination with extant GAL4 drivers allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and a GAL80 line that blocks GAL4 drivers from affecting plasmatocytes, both of which function from the early embryo to the adult.}, author = {György, Attila and Roblek, Marko and Ratheesh, Aparna and Valosková, Katarina and Belyaeva, Vera and Wachner, Stephanie and Matsubayashi, Yutaka and Sanchez Sanchez, Besaiz and Stramer, Brian and Siekhaus, Daria E}, journal = {G3: Genes, Genomes, Genetics}, number = {3}, pages = {845 -- 857}, publisher = {Genetics Society of America}, title = {{Tools allowing independent visualization and genetic manipulation of Drosophila melanogaster macrophages and surrounding tissues}}, doi = {10.1534/g3.117.300452}, volume = {8}, year = {2018}, } @article{612, abstract = {Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network.}, author = {Luján, Rafael and Aguado, Carolina and Ciruela, Francisco and Cózar, Javier and Kleindienst, David and De La Ossa, Luis and Bettler, Bernhard and Wickman, Kevin and Watanabe, Masahiko and Shigemoto, Ryuichi and Fukazawa, Yugo}, journal = {Brain Structure and Function}, number = {3}, pages = {1565 -- 1587}, publisher = {Springer}, title = {{Differential association of GABAB receptors with their effector ion channels in Purkinje cells}}, doi = {10.1007/s00429-017-1568-y}, volume = {223}, year = {2018}, }