Analytic real-time analysis and timed automata: a hybrid methodology for the performance analysis of embedded real-time systems

This paper presents a compositional and hybrid approach for the performance analysis of distributed real-time systems. The developed methodology abstracts system components by either flow-oriented and purely analytic descriptions or by state-based models in the form of timed automata. The interaction among the heterogeneous components is modeled by streams of discrete events. In total this yields a hybrid framework for the compositional analysis of embedded systems. It supplements contemporary techniques for the following reasons: (a) state space explosion as intrinsic to formal verification is limited to the level of isolated components; (b) computed performance metrics such as buffer sizes, delays and utilization rates are not overly pessimistic, because coarse-grained analytic models are used only for components that conform to the stateless model of computation. For demonstrating the usefulness of the presented ideas, a corresponding tool-chain has been implemented. It is used to investigate the performance of a two-staged computing system, where one stage exhibits state-dependent behavior that is only coarsely coverable by a purely analytic and stateless component abstraction. Finally, experiments are performed to ascertain the scalability and the accuracy of the proposed approac

Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution

Influence of different abstractions on the performance analysis of distributed hard real-time systems

System level performance analysis plays a fundamental role in the design process of hard real-time embedded systems. Several different approaches have been presented so far to address the problem of accurate performance analysis of distributed embedded systems in early design stages. The existing formal analysis methods are based on essentially different concepts of abstraction. However, the influence of these different models on the accuracy of the system analysis is widely unknown, as a direct comparison of performance analysis methods has not been considered so far. We define a set of benchmarks aimed at the evaluation of performance analysis techniques for distributed systems. We apply different analysis methods to the benchmarks and compare the results obtained in terms of accuracy and analysis times, highlighting the specific effects of the various abstractions. We also point out several pitfalls for the analysis accuracy of single approaches and investigate the reasons for pessimistic performance prediction

Performance Evaluation of Components Using a Granularity-based Interface Between Real-Time Calculus and Timed Automata

To analyze complex and heterogeneous real-time embedded systems, recent works have proposed interface techniques between real-time calculus (RTC) and timed automata (TA), in order to take advantage of the strengths of each technique for analyzing various components. But the time to analyze a state-based component modeled by TA may be prohibitively high, due to the state space explosion problem. In this paper, we propose a framework of granularity-based interfacing to speed up the analysis of a TA modeled component. First, we abstract fine models to work with event streams at coarse granularity. We perform analysis of the component at multiple coarse granularities and then based on RTC theory, we derive lower and upper bounds on arrival patterns of the fine output streams using the causality closure algorithm. Our framework can help to achieve tradeoffs between precision and analysis time.Comment: QAPL 201

Bioelectric Signaling Regulates Size in Zebrafish Fins

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

<div><p>One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 μm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.</p></div

Kaliumkanäle und Wachstumskontrolle: Identifizierung und Charakterisierung von Mutationen im Karpfenfisch Danio rerio, welche das proportionale Wachstum der Flossen beeinflussen

Growth plays an essential role during development, as it provides the raw material upon which morphogenesis and cell differentiation can act. For an organism to achieve final shape and the right proportions between its body parts, such process has to be highly coordinated. However, to date, only very little is known about how growth and organ size are regulated. In this study, I used a forward genetic approach in the zebrafish Danio rerio to identify genes that can affect organ size and alter proportional growth. I analysed several N-ethyl-N-nitrosourea (ENU) induced adult viable mutants that develop enlarged fins. Through positional cloning I show that two of these, another longfin (alf) and pfau, bear a missense mutation in kcnk5b, a gene encoding for a 2-pore domain potassium (K+) channel. The altered protein residues affect transmembrane domains of the channel and electrophysiological assays in Xenopus oocytes indicate that these mutations cause an increase in K+ conductance, which leads to hyperpolarisation of the membrane potential (Vm) in ovo. Transplantation experiments suggest that the mutations in kcnk5b act within the fin tissues. By misexpression in somatic clones, I prove that the mutant channel is sufficient to promote local growth of the fin. A second class of mutations, which cause a specific overgrowth of the dorsal fin in the mutants segel (sgl), segel-like (sllk) and flagge (fgg), was identified as harbouring defects in another K+ channel encoding gene, kcnh1a. The independent isolation of several fin overgrowth mutants that all alter the function of K+ channels suggests that ion channels play an important role in organ growth and fin size determination in the zebrafish. In this context, it will be interesting to consider whether K+ channels play a major role in size regulation also in fish species where exaggerated fin growth is occurring naturally. Preliminary data from poecilid species displaying sex-specific fin enlargement presented in this work support this notion.Wachstum spielt während der Entwicklung eine zentrale Rolle, da es die Grundlage für Morphogene&#729;se und Zelldifferentierung schafft. Um die endgültige Form eines Organismus zu erreichen und die richtigen Proportionen zwischen dessen Körperteilen zu gewährleisten, muss dieser Vorgang bis ins Detail koordiniert werden. Bis heute weiß man nur sehr wenig darüber, wie die Größe eines Organs bestimmt wird. In der vorliegenden Arbeit habe ich anhand klassischer Mutagenese im Zebrabärbling Danio rerio Gene identifiziert, welche die Dimension eines Organs beeinflussen und zu wachstumsbedingten Veränderungen der Proportionen führen. Ich analysiere hier unterschiedliche N-Ethyl-N-nitrosourea (ENU) induzierte, adult-lebensfähige Mutanten, welche vergrößerte Flossen zeigen. Durch positionelle Klonierung, konnte ich für zwei dieser Mutanten, another longfin (alf ) und pfau, die eine Vergrößerung von Flossen und Barteln vorweisen, zeigen, dass diese eine nicht-synonyme Mutation in kcnk5b tragen, einem Gen, welches für einen 2-Porendomänen Kaliumkanal codiert. Die betroffenen Aminosäuren befinden sich in den Transmembrandomänen des Kanals. Elektrophysiologische Analysen in Xenopus Oozyten zeigen, dass dies zu einer Zunahme des elektrischen Leitwerts für Kaliumionen führt. Dies führt zu einer Hyperpolarisierung des Membranpotentials (Vm) der Oocyte. Transplantationsexperimente weisen darauf hin, dass diese Mutationen in der Flosse selbst agieren. Durch Missexpression der mutanten Version des Kanals in somatischen Klonen, zeige ich dass die Mutation in kcnk5b ausreichend ist, um lokales Flossenwachstum zu verursachen. Ferner wurden die drei Mutanten segel (sgl), segel-like (sllk ) and flagge (fgg) untersucht, welche vergrößerte Rückenflossen entwickeln. Die hier vorgelegten Indizien, deuten darauf hin, dass diese Mutanten Defekte im spannungsabhängigen Kaliumkanal Kcnh1a haben. Die unabhängige Isolierung mehrerer Flossenmutanten, die alle Defekte in Kaliumkanälen vorweisen, weist darauf hin, dass Ionenkanäle eine wichtige Rolle in der Kontrolle des Flossenwachstums des Zebrabärblings spielen. In diesem Zusammenhang wird es interessant sein zu prüfen, ob Kaliumkanäle an der Größensteuerung von jenen Fischarten beteiligt sind, die sich durch ausgeprägtes Flossenwachstum auszeichnen. Vorläufige Befunde aus lebendgebärenden Zahnkarpfen mit geschlechtsspezifischem Flossenwachstum, die in dieser Arbeit vorgestellt werden, weisen in diese Richtung