WCET and Priority Assignment Analysis of Real-Time Systems using Search and Machine Learning

Real-time systems have become indispensable for human life as they are used in numerous industries, such as vehicles, medical devices, and satellite systems. These systems are very sensitive to violations of their time constraints (deadlines), which can have catastrophic consequences. To verify whether the systems meet their time constraints, engineers perform schedulability analysis from early stages and throughout development. However, there are challenges in obtaining precise results from schedulability analysis due to estimating the worst-case execution times (WCETs) and assigning optimal priorities to tasks. Estimating WCET is an important activity at early design stages of real-time systems. Based on such WCET estimates, engineers make design and implementation decisions to ensure that task executions always complete before their specified deadlines. However, in practice, engineers often cannot provide a precise point of WCET estimates and they prefer to provide plausible WCET ranges. Task priority assignment is an important decision, as it determines the order of task executions and it has a substantial impact on schedulability results. It thus requires finding optimal priority assignments so that tasks not only complete their execution but also maximize the safety margins from their deadlines. Optimal priority values increase the tolerance of real-time systems to unexpected overheads in task executions so that they can still meet their deadlines. However, it is a hard problem to find optimal priority assignments because their evaluation relies on uncertain WCET values and complex engineering constraints must be accounted for. This dissertation proposes three approaches to estimate WCET and assign optimal priorities at design stages. Combining a genetic algorithm and logistic regression, we first suggest an automatic approach to infer safe WCET ranges with a probabilistic guarantee based on the worst-case scheduling scenarios. We then introduce an extended approach to account for weakly hard real-time systems with an industrial schedule simulator. We evaluate our approaches by applying them to industrial systems from different domains and several synthetic systems. The results suggest that they are possible to estimate probabilistic safe WCET ranges efficiently and accurately so the deadline constraints are likely to be satisfied with a high degree of confidence. Moreover, we propose an automated technique that aims to identify the best possible priority assignments in real-time systems. The approach deals with multiple objectives regarding safety margins and engineering constraints using a coevolutionary algorithm. Evaluation with synthetic and industrial systems shows that the approach significantly outperforms both a baseline approach and solutions defined by practitioners. All the solutions in this dissertation scale to complex industrial systems for offline analysis within an acceptable time, i.e., at most 27 hours

Distinct Mechanisms for Ctr1-mediated Copper and Cisplatin Transport

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with multimer assembly of Ctr1. Copper near the Km value of Ctr1 enhanced FRET in a manner that correlated with cellular copper transport. In vitro cross-linking of Ctr1 confirmed that copper-induced FRET reflects conformational changes within pre-existing Ctr1 complexes. FRET assays in membrane-disrupted cells and protein extracts showed that intact cell structure is necessary for Ctr1 activity. Despite Ctr1-dependent cellular accumulation, cisplatin did not change Ctr1 FRET nor did it attenuate copper-induced FRET. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs that are dispensable for copper transport play a critical role for cisplatin uptake. Taken together, our data reveal functional roles for structural remodeling of the Ctr1 multimeric complex in copper transport and suggest distinct mechanisms employed by Ctr1 for copper and cisplatin transport

Organ-specific regulation of ATP7A abundance is coordinated with systemic copper homeostasis

Copper (Cu) is an essential cofactor for various enzymatic activities including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Consequently, Cu dysregulation is associated with fatal neonatal disease, liver and cardiac dysfunction, and anemia. While the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation. Here we show, using Cu-deficient mouse models, that steadystate levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Strikingly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestinespecific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Our results support a systemic model for how a single transporter can be inversely regulated in a tissue-specific manner to maintain organismal Cu homeostasis

Fuzzing for CPS Mutation Testing

Mutation testing can help reduce the risks of releasing faulty software. For such reason, it is a desired practice for the development of embedded software running in safety-critical cyber-physical systems (CPS). Unfortunately, state-of-the-art test data generation techniques for mutation testing of C and C++ software, two typical languages for CPS software, rely on symbolic execution, whose limitations often prevent its application (e.g., it cannot test black-box components). We propose a mutation testing approach that leverages fuzz testing, which has proved effective with C and C++ software. Fuzz testing automatically generates diverse test inputs that exercise program branches in a varied number of ways and, therefore, exercise statements in different program states, thus maximizing the likelihood of killing mutants, our objective. We performed an empirical assessment of our approach with software components used in satellite systems currently in orbit. Our empirical evaluation shows that mutation testing based on fuzz testing kills a significantly higher proportion of live mutants than symbolic execution (i.e., up to an additional 47 percentage points). Further, when symbolic execution cannot be applied, fuzz testing provides significant benefits (i.e., up to 41% mutants killed). Our study is the first one comparing fuzz testing and symbolic execution for mutation testing; our results provide guidance towards the development of fuzz testing tools dedicated to mutation testing.Comment: This article is the camera-ready version for ASE 202

NCOA4 Regulates Iron Recycling and Responds to Hepcidin Activity and Lipopolysaccharide in Macrophages

Macrophages, via erythrophagocytosis, recycle iron from effete erythrocytes to newly developing red blood cells. Conversion of potentially cytotoxic levels of iron from its heme into nonheme form during iron recycling is safely accomplished via coordinated regulations of cellular iron transport and homeostasis. Herein, we demonstrate the roles and regulation of NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy in macrophages after erythrophagocytosis using the mouse macrophage cell line J774 cells. Ferritin in J774 cells increased with the rise of nonheme iron by erythrocyte ingestion and declined when total cellular iron contents subsequently decreased. NCOA4, a selective autophagic cargo receptor for ferritin, was responsible for the control of cellular ferritin and total iron contents at the later stage of erythrophagocytosis. A hepcidin analog, which limits the flux of iron through iron-recycling by inhibiting iron export at the plasma membrane, repressed NCOA4 expression and led to accumulation of ferritin in the mouse macrophages. Transcriptome analyses revealed a functional association of immune response with NCOA4-dependent gene expressions, and we confirmed repression of Ncoa4 by lipopolysaccharide (LPS) in J774 cells and the spleen of mice. Collectively, our studies indicate that NCOA4 facilitates cellular ferritin turnover and the release of iron by macrophages after erythrophagocytosis and functions as a regulatory target for molecular signals of systemic iron overload and inflammation. These identify macrophage NCOA4 as a potential therapeutic target for disorders of systemic iron dysregulation, including anemia of inflammation and hemochromatosis

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans

Copper plays key catalytic and regulatory roles in biochemical processes essential for normal growth, development, and health. Defects in copper metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of copper. The intestinal enterocyte is a key regulatory point for copper absorption into the body; however, the mechanisms by which intestinal cells transport copper to maintain organismal copper homeostasis are poorly understood. Here, we identify a mechanism by which organismal copper homeostasis is maintained by intestinal copper exporter trafficking that is coordinated with extraintestinal copper levels in Caenorhabditis elegans. Specifically, we show that CUA-1, the C. elegans homolog of ATP7A/B, localizes to lysosome-like organelles (gut granules) in the intestine under copper overload conditions for copper detoxification, whereas copper deficiency results in a redistribution of CUA-1 to basolateral membranes for copper efflux to peripheral tissues. Worms defective in gut granule biogenesis exhibit defects in copper sequestration and increased susceptibility to toxic copper levels. Interestingly, however, a splice isoform CUA-1.2 that lacks a portion of the N-terminal domain is targeted constitutively to the basolateral membrane irrespective of dietary copper concentration. Our studies establish that CUA-1 is a key intestinal copper exporter and that its trafficking is regulated to maintain systemic copper homeostasis. C. elegans could therefore be exploited as a whole-animal model system to study regulation of intra- and intercellular copper trafficking pathways

Oma1 Links Mitochondrial Protein Quality Control and TOR Signaling To Modulate Physiological Plasticity and Cellular Stress Responses

ACKNOWLEDGMENTS We thank Dennis Winge (University of Utah) and the members of the Khalimonchuk laboratory for critical comments. We also thank Christoph Schuller (University of Natural Resources, Austria) and Paul Herman (Ohio State University) for reagents. We acknowledge the expert technical assistance of Nataliya Zahayko. We also thank Donna MacCallum for help with the Candida virulence assays. This research was supported by grants from the NIH (P30GM103335 and 5R01GM108975 [O.K.], GM071775-06 and GM105781-01 [A.B.], DK079209 [J.L.]), the U.K. Biotechnology and Biological Research Council (BB/K017365/1 [A.J.P.B.]), the U.K. Medical Research Council (MR/ M026663/1 [A.J.P.B.]), and the European Research Council (C-2009- AdG-249793 [A.J.P.B.]). We declare that we have no competing financial interests. FUNDING INFORMATION This work, including the efforts of Alistair J. P. Brown, was funded by Biotechnology and Biological Research Counsil (BB/K017365/1). This work, including the efforts of Oleh Khalimonchuk, was funded by HHS | National Institutes of Health (NIH) (5R01GM108975). This work, including the efforts of Oleh Khalimonchuk, was funded by HHS | National Institutes of Health (NIH) (P30GM103335).This work, including the efforts of Antoni Barrientos, was funded by HHS | National Institutes of Health (NIH) (GM071775-06). This work, including the efforts of Antoni Barrientos, was funded by HHS | National Institutes of Health (NIH) (GM105781-01). This work, including the efforts of Jaekwon Lee, was funded by HHS | National Institutes of Health (NIH) (DK079209). This work, including the efforts of Alistair J. P. Brown, was funded by Medical Research Council (MRC) (MR/M026663/1). This work, including the efforts of Alistair J. P. Brown, was funded by EC | European Research Council (ERC) (C-2009-AdG-249793).Peer reviewedPublisher PD

Schedulability Analysis of Real-Time Systems with Uncertain Worst-Case Execution Times

Schedulability analysis is about determining whether a given set of real-time software tasks are schedulable, i.e., whether task executions always complete before their specified deadlines. It is an important activity at both early design and late development stages of real-time systems. Schedulability analysis requires as input the estimated worst-case execution times (WCET) for software tasks. However, in practice, engineers often cannot provide precise point WCET estimates and prefer to provide plausible WCET ranges. Given a set of real-time tasks with such ranges, we provide an automated technique to determine for what WCET values the system is likely to meet its deadlines, and hence operate safely. Our approach combines a search algorithm for generating worst-case scheduling scenarios with polynomial logistic regression for inferring safe WCET ranges. We evaluated our approach by applying it to a satellite on-board system. Our approach efficiently and accurately estimates safe WCET ranges within which deadlines are likely to be satisfied with high confidence

Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250–350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a degron of Pca1p sense cadmium.Anunbiased approach selecting non-functional degrons indicated a critical role of hydrophobic amino acids in the degron for its function.Asecondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the degron, which might be useful for the identification and characterization of other degrons