Assessment of Methods for the Real-Time Simulation of Electronic and Thermal Circuits

[Abstract] Time–domain simulation of electronic and thermal circuits is required by a large array of applications, such as the design and optimization of electric vehicle powertrain components. While efficient execution is always a desirable feature of simulation codes, in certain cases like System-in-the-Loop setups, real-time performance is demanded. Whether real-time code execution can be achieved or not in a particular case depends on a series of factors, which include the mathematical formulation of the equations that govern the system dynamics, the techniques used in code implementation, and the capabilities of the hardware architecture on which the simulation is run. In this work, we present an evaluation framework of numerical methods for the simulation of electronic and thermal circuits from the point of view of their ability to deliver real-time performance. The methods were compared using a set of nontrivial benchmark problems and relevant error metrics. The computational efficiency of the simulation codes was measured under different software and hardware environments, to determine the feasibility of using them in industrial applications with reduced computational power.Ministerio de Economía y Competitividad; RYC-2016-20222Xunta de Galicia; ED431B2016/03

Functional and evolutionary analysis of sugar transporters in Kluyveromyces marxianus

Kluyveromyces marxianus is a yeast species traditionally found in fermented dairy products such as yogurt and kefir. Because of its association with these products, this yeast has a QPS/GRAS status that facilitates applications in the food industry. K. marxianus is also known for possessing remarkable physiological traits: the yeast is thermotolerant, has a rapid growth rate and is able to utilise a wide range of substrates. Since these traits are desirable from an industrial standpoint, K. marxianus has been exploited for several biotechnological applications including bioethanol, flavour molecule and organic acid production. Perhaps the best-known application involving this yeast is the production of ethanol from whey, a by-product of the dairy industry. The capacity of the yeast to utilise lactose, essential in this environment, was previously shown to be a variable trait with some strains being able to utilise the sugar better than others. To understand the genetic basis that accounts for these differences, a detailed analysis of lactose utilisation was performed. The K. marxianus genome was found to encode four copies of the lactose permease (LAC12) gene, which are present in both good and poor lactose utilising strains. To determine if these genes were responsible for the growth differences observed, the LAC12 genes from a good and a poor lactose utiliser were cloned into a replicative plasmid and expressed in S. cerevisiae. Expression of one of these gene copies (KmLAC12) conferred growth on lactose, indicating that this is the main lactose transporter in K. marxianus. However, growth on lactose was only observed when expressing the KmLAC12 gene from a good lactose utiliser, suggesting that good and poor utilisers carry a functional (KmLAC12-B) and non-functional (KmLAC12-A) version of the lactose permease gene, respectively. Overexpression of KmLAC12-B and not KmLAC12-A conferred growth on a poor lactose utiliser, supporting this idea. A comparison of KmLac12p sequences from 12 K. marxianus strains confirmed that poor and good lactose utilisers carry distinct versions of the lactose transporter. Through this analysis it was established that the KmLac12p-B and KmLac12p-A differ in 16 amino acid positions. Altogether, these results showed unequivocally that differences in the KmLAC12 sequence are the main factor accounting for variability in lactose utilisation in K. marxianus. Lactose utilisation is not the only variable trait in K. marxianus. In fact, several studies have shown that the yeast displays a high level of diversity in a range of phenotypes including thermotolerance, stress tolerance, glucose repression and sugar utilisation. While several K. marxianus genome sequences are currently available, a comparative analysis aiming to determine the level of genetic diversity between these sequences had not previously been performed. To investigate this aspect, the genomes of nine K. marxianus strains from different origins were sequenced and compared. Also, five previously published genomes were included in this analysis. Single Nucleotide Polymorphism (SNP) analyses revealed a relatively high level of genetic diversity in the species, with up to 3 % DNA sequence divergence between alleles. These data also showed that K. marxianus strains can be found in different ploidy states (i.e. haploid, diploid or triploid), a finding that was confirmed by flow cytometry experiments. Diploid and triploid strains showed large regions of loss of heterozygosity in several chromosomes. Also, sequence coverage analyses revealed differences in copy number of large regions of the genome, indicating that some of these isolates are partially aneuploid. Remarkably, ploidy was found to correlate with strain origin: all the strains isolated from dairy environments were diploid or triploid while non-dairy strains were haploid. A phylogenetic tree based on SNP data was constructed to further investigate the relationship between these isolates. The haplotype tree showed that dairy and nondairy strains form two distinct clades. In addition, all of the dairy strains utilised lactose efficiently and were found to carry the KmLAC12-B gene, encoding the functional lactose transporter. The SNP data also allowed us to determine that the diploid strains in this study were hybrids between a dairy and non-dairy haplotype. On the other hand, triploid strains contained three copies of the dairy haplotype. The data obtained in this study reveals that K. marxianus displays a high level of genetic diversity and shows that there is a relationship between genetic diversity and the environment from where the strains were isolated. The role of KmLAC12-B in lactose utilisation was clear, but the function of the rest of the LAC12 genes was not known. Since the LAC12 gene was previously shown to encode a galactose transporter in K. lactis, we tested whether any of the LAC12 genes from K. marxianus encoded a similar function. Heterologous expression of these genes revealed that three out of the four copies encoded galactose transporters – no function was identified for LAC12-3. However, single or multiple disruptions of these genes in K. marxianus did not abolish growth on galactose, indicating the presence of additional galactose transporters. A second galactose transport system, encoded by the HGT1 gene, was previously described in K. lactis. To examine if K. marxianus genome encoded any orthologues of this gene, a bioinformatics analysis was carried out. Several tandem copies of the HGT1 gene were identified in K. marxianus genome. Also, multiple tandem copies of the glucose transporters KHT1 and KHT2 were discovered through this analysis. The expansion of these sugar transporters was not found in other Kluyveromyces genomes examined, indicating that these gene expansions are specific to K. marxianus. Interestingly, copy number of the KHT and HGT1 genes was variable among K. marxianusstrain; the strains were found to encode either 6 or 5 copies of KHT and either 5 or 3 copies of HGT1. To assess the functionality of these genes, heterologous expression experiments were carried out in S. cerevisiae EBY.VW4000, a strain deleted for all hexose transporters. Expression of the K. marxianus transporters identified additional galactose transporters; 4 encoded by HGT1 orthologues and 1 encoded by a KHT orthologue. K. marxianus strains carrying mutations in these genes were constructed and tested on galactose medium. Only a mutant carrying mutations in all eight of the identified galactose transporters showed a significant growth defect on 2 % galactose medium whereas a mutant in the HGT1 locus and the three functional LAC genes failed to grow on 0.1 % galactose. These results demonstrate that galactose transport is highly redundant in K. marxianus, that different proteins appear to be involved in low or high-affinity transport and highlight important differences between the K. lactis and K. marxianus galactose transport systems. In addition to studying the expansion of sugar transporter families in K. marxianus, molecular tools to genome engineer this yeast were also developed during this PhD. While some tools to genetically manipulate this yeast are currently available, there is a need to develop new techniques that allow simple and efficient gene targeting for construction of mutants and for allelic replacement. To tackle this issue, the CRISPRCas9 system was implemented in K. marxianus. To establish the methodology, a single-plasmid system, comprising Cas9 and an sgRNA, was constructed and successfully used to target the ADE2 gene at high efficiency (> 96%) in this yeast. The system was also shown to function in K. lactis, where the ADE2 gene was targeted at similar efficiency. Mutants were obtained in both yeasts both by non-homologous end- joining (NHEJ) and homology-dependent repair (HDR) methods, though the capacity to carry out allelic replacement by homologous recombination using repair fragments was of variable efficiency in different strains. To improve gene targeting in K. marxianus, the KU80 gene involved in the NHEJ DNA repair system was disrupted using the CRISPR-Cas9 system. Homologous recombination frequency was greatly enhanced in a ku80 mutant, suggesting that the NHEJ had been inactivated. Finally, to further improve homologous recombination repair, the RAD52 gene, involved in homologous recombination in S. cerevisiae, was expressed in K. marxianus. A strain expressing this gene showed a seven-fold increase in homologous recombination-mediated repair, albeit from a low base. These tools were used throughout the thesis to facilitate the functional analysis of sugar transporters. The results presented in this thesis contribute to better understanding the function and evolution of sugar transporters in K. marxianus. Also, the tools developed in this work will help to further develop this yeast for cell factory applications

An Automated Methodology to Select Functional Co-Simulation Configurations

[Abstract] The development of machinery often requires system-level analysis, in which non-mechanical subsystems, such as hydraulics, need to be considered. Co-simulation allows analysts to divide a problem into subsystems and use tailored software solutions to deal individually with their respective dynamics. On the other hand, these subsystems must be coupled at particular instants in time, called communication points, through the exchange of coupling variables. Between communication points, each subsystem solver carries out the integration of its states without interacting with its environment. This may cause the integration to become unstable, especially when non-iterative co-simulation is used. The co-simulation configuration, i.e., the parameters and simulation options selected by the analyst, such as the way to handle the coupling variables or the choice of subsystem solvers, is often a critical factor regarding co-simulation stability. In practice it is difficult to anticipate which selection is the most appropriate for a particular problem, especially if some inputs come from external sources, such as human operators, and cannot be determined beforehand. We put forward a methodology to automatically determine a stable and computationally efficient configuration for Jacobi-scheme co-simulation. The method uses energy residuals to gain insight into co-simulation stability. The relation between energy residual and communication step-size is exploited to monitor co-simulation accuracy during a series of tests in which the external inputs are replaced with predetermined input functions. The method was tested with hydraulically actuated mechanical examples. Results indicate that the proposed method can be used to find stable and accurate configurations for co-simulation applications.Ministerio de Economía; RYC-2016-2022

On the Effect of Multirate Co-simulation Techniques in the Efficiency and Accuracy of Multibody System Dynamics

This is a post-peer-review, pre-copyedit version of an article published in Multibody System Dynamics. This version of the article has been accepted for publication, after peer review and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/ s11044-010-9234-7.[Abstract] Dynamic simulation of complex mechatronic systems can be carried out in an efficient and modular way making use of weakly coupled co-simulation setups. When using this approach, multirate methods are often needed to improve the efficiency, since the physical components of the system usually have different frequencies and time scales. However, most multirate methods have been designed for strongly coupled setups, and their application in weakly coupled cosimulation is not straightforward due to the limitations enforced by commercial simulation tools used in mechatronics design. This work describes a weakly coupled multirate method intended to be a generic multirate interface between block diagram software and multibody dynamics simulators, arranged in a co-simulation setup. Its main advantage is that it does not enforce equidistant or synchronized communication time-grids, and therefore it can be easily applied to set up weakly-coupled co-simulations using off-the-shelf commercial block diagram simulators while giving the user a great flexibility for selecting the integration scheme for each subsystem. The method is first tested on a simple, purely mechanical system with known analytical solution and variable frequency ratio (FR) of the coupled subsystems. Several synchronization schemes (fastest-first and slowest-first) and interpolation/extrapolation methods (polynomials of different orders and smoothing) have been implemented and tested. Next, the effect of the interface on accuracy and efficiency is assessed making use of a real-life co-simulation setting that links an MBS model of a kart to a thermal engine modelled in Simulink. Results show that the proposed weakly coupled multirate method can achieve considerable reductions in the execution times of the simulations without degrading the numerical solution of the proble

Energy-based monitoring and correction to enhance the accuracy and stability of explicit co-simulation

[Abstract] The simulation of complex engineering applications often requires the consideration of component-level dynamics whose nature and time-scale differ across the elements of which the system is composed. Co-simulation offers an effective approach to deal with the modelling and numerical integration of such assemblies by assigning adequate description and solution methods to each component. Explicit co-simulation, in particular, is frequently used when efficient code execution is a requirement, for instance in real-time setups. Using explicit schemes, however, can lead to the introduction of energy artifacts at the discrete-time interface between subsystems. The resulting energy errors deteriorate the accuracy of the co-simulation results and may in some cases develop into the instability of the numerical integration process. This paper discusses the factors that influence the severity of the energy errors generated at the interface in explicit co-simulation applications, and presents a monitoring and correction methodology to detect and remove them. The method uses only the information carried by the variables exchanged between the subsystems and the co-simulation manager. The performance of this energy-correction technique was evaluated in multi-rate co-simulation of mechanical and multiphysics benchmark examples.Xunta de Galicia; ED431B2016/031Xunta de Galicia; ED431F2021/04Ministerio de Economía, Industria y Competitividad; RYC-2016-20222Ministerio de Economía, Industria y Competitividad; TRA2017-86488-

Transcriptional response to lactic acid stress in the hybrid yeast Zygosaccharomyces parabailii

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH, and a fermentative metabolism with a fast growth rate. Here we used RNA-seq to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than in control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/2, previously described as lactic acid-responsive in Saccharomyces cerevisiae. Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii as compared to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.Importance Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression in conditions of interest, and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains

Propensity score matched comparison of transcatheter aortic valve implantation versus conventional surgery in intermediate and low risk aortic stenosis patients: A hint of real-world

Background: Recently, the use of transcatheter aortic valve implantation (TAVI) in inter­mediate-low risk patients has been evaluated in the PARTNER II randomized trial. However, in the last years, this therapy has been employed in this scenario with underreported results, as compared to surgical aortic valve replacement (SAVR). Methods: We enrolled 362 consecutive patients with severe symptomatic aortic stenosis and intermediate-low surgical risk (logEuroSCORE < 20%), treated in our center with TAVI (103 patients) or single SAVR (259 patients) between 2009 and 2014. Patients were matched according to age, gender, logEuroSCORE, and use of bioprosthesis. Results: Mean age of the patients was 73 ± 10.4 years, and 40.3% were women. LogEuroSCORE and Society Thoracic Surgeons score were 7.0 ± 4.4% and 4.2 ± 2.5%, respectively, with mean left ventricular ejection fraction of 52 ± 9%. There were no differences regarding other comorbidities. The length-of-hospitalization was 11 ± 5 days after TAVI vs. 17 ± 9 days after SAVR (p = 0.003). After matched comparison, no differences in terms of in-hospital mortality (5.7% after TAVI vs. 2.9% after SAVR, p = 0.687) and 1-year mortality (11.4% vs. 7.1%, p = 0.381) were found. The combined endpoint of stroke and mortality at 1-year was also similar between both groups (15.7% in TAVI patients vs. 14.4% after SAVR, p = 0.136). Multivariate analysis determined that aortic regurgitation (AR) was an independent predictor of mortality (OR = 3.623, 95% CI: 1.267–10.358, p = 0.016). Although the rate of AR was higher after TAVI, none of the patients treated with the newest generation devices (10.7%) presented more than a mild degree of AR. Conclusions: TAVI is feasible and shows comparable results to surgery in terms of early, 1-year mortality, as well as cerebrovascular events in patients with severe aortic stenosis and intermediate-low operative risk. Better transvalvular gradients, yet higher rates of AR were found, however, newer devices presented comparable rate of AR.

Detection of Aeromonas Salmonicida Subsp. Salmonicida Infection in Zebrafish by Labelling Bacteria With Gfp and a Fluorescent Probe Based on the Siderophore Amonabactin

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG[Abstract] Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.This work was supported by grants RTI2018-093634-B-C21/C22 and PID2021-122732OB-C22/C21 from MCIN/AEI/10.13039/501100011033/FEDER “A way to make Europe” (AEI, Spanish State Agency for Research and FEDER Programme from the European Union). Work in University of Santiago de Compostela and University of A Coruña was also supported by grants ED431C 2022/23 and ED431C 2022/39, respectively, from Xunta de Galicia. Funding for open access charge: Universidade da Coruña/CISUGXunta de Galicia; ED431C 2022/23Xunta de Galicia; ED431C 2022/3

Actigraphic sleep and dietary macronutrient intake in children aged 6–9 years old: A pilot study

The objective of this study was to examine the relationship between different sleep parameters and energy and macronutrient intake in school-aged children. A total of 203 children 6 to 9 years of age participated in this cross-sectional study. Anthropometric measurements were taken first. Diet was assessed with 3-day food logs and sleep was measured with a questionnaire on sleep quality and a wrist actigraph worn for at least 7 days. A decrease of 165.45 kcal was observed per each additional hour of sleep during the week (β (95% CI) = −165.45 (−274.01, −56.88); p = 0.003). This relationship was also observed for fat (β (95% CI) = −11.14 (−18.44, −3.84); p = 0.003) and protein (β (95% CI) = −13.27 (−22.52, −4.02); p = 0.005). An increase in weekend sleep efficiencies for those under the recommended threshold of 85% also had a similar association with energy (β (95% CI) = −847.43 (−1566.77, 128.09); p = 0.021) and carbohydrate (β (95% CI) = −83.96 (−161.76, −6.15); p = 0.035)) intake. An increase in habitual sleep variability was related with a slight increase in protein intake (β (95% CI) = 0.32 (0.031, 0.62); p = 0.031). Children who slept less had a higher energy intake, especially from fat and protein and those who presented inefficient sleep had a higher carbohydrate intake. Strategies to enhance sleep quality and quantity combined with dietary recommendations could help to improve energy and macronutrient intake levels in children