Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 angstrom resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

Double Hybrid Functionals and the Π-System Bond Length Alternation Challenge: Rivaling Accuracy of Post-HF Methods

Predicting accurate bond length alternations (BLAs) in long conjugated oligomers has been a significant challenge for electronic-structure methods for many decades, made particularly important by the close relationships between BLA and the rich optoelectronic properties of π-delocalized systems. Here, we test the accuracy of recently developed, and increasingly popular, double hybrid (DH) functionals, positioned at the top of Jacobs Ladder of DFT methods of increasing sophistication, computational cost, and accuracy, due to incorporation of MP2 correlation energy. Our test systems comprise oligomeric series of polyacetylene, polymethineimine, and polysilaacetylene up to six units long. MP2 calculations reveal a pronounced shift in BLAs between the 6-31G(d) basis set used in many studies of BLA to date and the larger cc-pVTZ basis set, but only modest shifts between cc-pVTZ and aug-cc-pVQZ results. We hence perform new reference CCSD(T)/cc-pVTZ calculations for all three series of oligomers against which we assess the performance of several families of DH functionals based on BLYP, PBE, and TPSS, along with lower-rung relatives including global- and range-separated hybrids. Our results show that DH functionals systematically improve the accuracy of BLAs relative to single hybrid functionals. xDH-PBE0 (N4 scaling using SOS-MP2) emerges as a DH functional rivaling the BLA accuracy of SCS-MP2 (N5 scaling), which was found to offer the best compromise between computational cost and accuracy the last time the BLA accuracy of DFT- and wave function-based methods was systematically investigated. Interestingly, xDH-PBE0 (XYG3), which differs to other DHs in that its MP2 term uses PBE0 (B3LYP) orbitals that are not self-consistent with the DH functional, is an outlier of trends of decreasing average BLA errors with increasing fractions of MP2 correlation and HF exchange.

Test of a Smock System on CPR Primary Emergency Measures and Medical Errors During Simulated Emergencies

Rates of survival of victims of sudden cardiac arrest (SCA) using cardio pulmonary resuscitation (CPR) have shown little improvement over the past three decades. Since registered nurses (RNs) comprise the largest group of healthcare providers in U.S. hospitals, it is essential that they are competent in performing the four primary measures (compression, ventilation, medication administration, and defibrillation) of CPR in order to improve survival rates of SCA patients. The purpose of this experimental study was to test a color-coded SMOCK system on:1) time to implement emergency patient care measures 2) technical skills performance 3) number of medical errors, and 4) team performance during simulated CPR exercises. The study sample was 260 RNs (M 40 years, SD=11.6) with work experience as an RN (M 7.25 years, SD=9.42).Nurses were allocated to a control or intervention arm consisting of 20 groups of 5-8 RNs per arm for a total of 130 RNs in each arm. Nurses in each study arm were given clinical scenarios requiring emergency CPR. Nurses in the intervention group wore different color labeled aprons (smocks) indicating their role assignment (medications, ventilation, compression, defibrillation, etc) on the code team during CPR. Findings indicated that the intervention using color-labeled smocks for pre-assigned roles had a significant effect on the time nurses started compressions (t=3.03, p=0.005), ventilations (t=2.86, p=0.004) and defibrillations (t=2.00, p=.05) when compared to the controls using the standard of care. In performing technical skills, nurses in the intervention groups performed compressions and ventilations significantly better than those in the control groups. The control groups made significantly (t=-2.61, p=0.013) more total errors (7.55 SD 1.54) than the intervention group (5.60, SD 1.90). There were no significant differences in team performance measures between the groups. Study findings indicate use of colored labeled smocks during CPR emergencies resulted in: shorter times to start emergency CPR; reduced errors; more technical skills completed successfully; and no differences in team performance.

Hybrid Power System Intelligent Operation and Protection Involving Distributed Architectures and Pulsed Loads

Efficient and reliable techniques for power delivery and utilization are needed to account for the increased penetration of renewable energy sources in electric power systems. Such methods are also required for current and future demands of plug-in electric vehicles and high-power electronic loads. Distributed control and optimal power network architectures will lead to viable solutions to the energy management issue with high level of reliability and security. This dissertation is aimed at developing and verifying new techniques for distributed control by deploying DC microgrids, involving distributed renewable generation and energy storage, through the operating AC power system. To achieve the findings of this dissertation, an energy system architecture was developed involving AC and DC networks, both with distributed generations and demands. The various components of the DC microgrid were designed and built including DC-DC converters, voltage source inverters (VSI) and AC-DC rectifiers featuring novel designs developed by the candidate. New control techniques were developed and implemented to maximize the operating range of the power conditioning units used for integrating renewable energy into the DC bus. The control and operation of the DC microgrids in the hybrid AC/DC system involve intelligent energy management. Real-time energy management algorithms were developed and experimentally verified. These algorithms are based on intelligent decision-making elements along with an optimization process. This was aimed at enhancing the overall performance of the power system and mitigating the effect of heavy non-linear loads with variable intensity and duration. The developed algorithms were also used for managing the charging/discharging process of plug-in electric vehicle emulators. The protection of the proposed hybrid AC/DC power system was studied. Fault analysis and protection scheme and coordination, in addition to ideas on how to retrofit currently available protection concepts and devices for AC systems in a DC network, were presented. A study was also conducted on the effect of changing the distribution architecture and distributing the storage assets on the various zones of the network on the system’s dynamic security and stability. A practical shipboard power system was studied as an example of a hybrid AC/DC power system involving pulsed loads. Generally, the proposed hybrid AC/DC power system, besides most of the ideas, controls and algorithms presented in this dissertation, were experimentally verified at the Smart Grid Testbed, Energy Systems Research Laboratory. All the developments in this dissertation were experimentally verified at the Smart Grid Testbed.

The Impact of a Classroom Performance System on Learning Gains in a Biology Course for Science Majors

This study was conducted to determine if the use of the technology known as Classroom Performance System (CPS), specifically referred to as “Clickers”, improves the learning gains of students enrolled in a biology course for science majors. CPS is one of a group of developing technologies adapted for providing feedback in the classroom using a learner-centered approach. It supports and facilitates discussion among students and between them and teachers, and provides for participation by passive students. Advocates, influenced by constructivist theories, claim increased academic achievement. In science teaching, the results have been mixed, but there is some evidence of improvements in conceptual understanding. The study employed a pretest-posttest, non-equivalent groups experimental design. The sample consisted of 226 participants in six sections of a college biology course at a large community college in South Florida with two instructors trained in the use of clickers. Each instructor randomly selected their sections into CPS (treatment) and non-CPS (control) groups. All participants filled out a survey that included demographic data at the beginning of the semester. The treatment group used clicker questions throughout, with discussions as necessary, whereas the control groups answered the same questions as quizzes, similarly engaging in discussion where necessary. The learning gains were assessed on a pre/post-test basis. The average learning gains, defined as the actual gain divided by the possible gain, were slightly better in the treatment group than in the control group, but the difference was statistically non-significant. An Analysis of Covariance (ANCOVA) statistic with pretest scores as the covariate was conducted to test for significant differences between the treatment and control groups on the posttest. A second ANCOVA was used to determine the significance of differences between the treatment and control groups on the posttest scores, after controlling for sex, GPA, academic status, experience with clickers, and instructional style. The results indicated a small increase in learning gains but these were not statistically significant. The data did not support an increase in learning based on the use of the CPS technology. This study adds to the body of research that questions whether CPS technology merits classroom adaptation.

System-on-a-Chip (SoC) based Hardware Acceleration in Register Transfer Level (RTL) Design

Today, modern System-on-a-Chip (SoC) systems have grown rapidly due to the increased processing power, while maintaining the size of the hardware circuit. The number of transistors on a chip continues to increase, but current SoC designs may not be able to exploit the potential performance, especially with energy consumption and chip area becoming two major concerns. Traditional SoC designs usually separate software and hardware. Thus, the process of improving the system performance is a complicated task for both software and hardware designers. The aim of this research is to develop hardware acceleration workflow for software applications. Thus, system performance can be improved with constraints of energy consumption and on-chip resource costs. The characteristics of software applications can be identified by using profiling tools. Hardware acceleration can have significant performance improvement for highly mathematical calculations or repeated functions. The performance of SoC systems can then be improved, if the hardware acceleration method is used to accelerate the element that incurs performance overheads. The concepts mentioned in this study can be easily applied to a variety of sophisticated software applications. The contributions of SoC-based hardware acceleration in the hardware-software co-design platform include the following: (1) Software profiling methods are applied to H.264 Coder-Decoder (CODEC) core. The hotspot function of aimed application is identified by using critical attributes such as cycles per loop, loop rounds, etc. (2) Hardware acceleration method based on Field-Programmable Gate Array (FPGA) is used to resolve system bottlenecks and improve system performance. The identified hotspot function is then converted to a hardware accelerator and mapped onto the hardware platform. Two types of hardware acceleration methods – central bus design and co-processor design, are implemented for comparison in the proposed architecture. (3) System specifications, such as performance, energy consumption, and resource costs, are measured and analyzed. The trade-off of these three factors is compared and balanced. Different hardware accelerators are implemented and evaluated based on system requirements. 4) The system verification platform is designed based on Integrated Circuit (IC) workflow. Hardware optimization techniques are used for higher performance and less resource costs. Experimental results show that the proposed hardware acceleration workflow for software applications is an efficient technique. The system can reach 2.8X performance improvements and save 31.84% energy consumption by applying the Bus-IP design. The Co-processor design can have 7.9X performance and save 75.85% energy consumption.

Physics-Based Modeling of Power System Components for the Evaluation of Low-Frequency Radiated Electromagnetic Fields

The low-frequency electromagnetic compatibility (EMC) is an increasingly important aspect in the design of practical systems to ensure the functional safety and reliability of complex products. The opportunities for using numerical techniques to predict and analyze system’s EMC are therefore of considerable interest in many industries. As the first phase of study, a proper model, including all the details of the component, was required. Therefore, the advances in EMC modeling were studied with classifying analytical and numerical models. The selected model was finite element (FE) modeling, coupled with the distributed network method, to generate the model of the converter’s components and obtain the frequency behavioral model of the converter. The method has the ability to reveal the behavior of parasitic elements and higher resonances, which have critical impacts in studying EMI problems. For the EMC and signature studies of the machine drives, the equivalent source modeling was studied. Considering the details of the multi-machine environment, including actual models, some innovation in equivalent source modeling was performed to decrease the simulation time dramatically. Several models were designed in this study and the voltage current cube model and wire model have the best result. The GA-based PSO method is used as the optimization process. Superposition and suppression of the fields in coupling the components were also studied and verified. The simulation time of the equivalent model is 80-100 times lower than the detailed model. All tests were verified experimentally. As the application of EMC and signature study, the fault diagnosis and condition monitoring of an induction motor drive was developed using radiated fields. In addition to experimental tests, the 3DFE analysis was coupled with circuit-based software to implement the incipient fault cases. The identification was implemented using ANN for seventy various faulty cases. The simulation results were verified experimentally. Finally, the identification of the types of power components were implemented. The results show that it is possible to identify the type of components, as well as the faulty components, by comparing the amplitudes of their stray field harmonics. The identification using the stray fields is nondestructive and can be used for the setups that cannot go offline and be dismantled

Electrochemical Immunosensing of Cortisol in an Automated Microfluidic System Towards Point-of-Care Applications

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Fiber, a component for health gastrointestinal endocrine system

Dietary fiber was classified according to its solubility in an attempt to relate physiological effects to chemical types of fiber. Soluble fibers (B-glucans, gums, wheat dextrin, psyllium, pectin, inulin) were considered to have benefits on serum lipids, while insoluble fibers (cellulose, lignin, pectins, hemicelluloses) were linked with laxation benefits. More important characteristics of fiber in terms of physiological benefits are viscosity and fermentability. Viscous fibers (pectins, B-glucans, gums, psyllium) are those that have gel-forming properties in the intestinal tract, and fermentable fibers (wheat dextrin, pectins, B-glucans, gum, inulin) are those that can be metabolized by colonic bacteria. Objective: To summarize the beneficial effects of dietary fiber, as nutraceuticals, in order to maintain a healthy gastrointestinal system. Methods: Our study is a systematic review. Electronic databases, including PubMed, Medline, with supplement of relevant websites, were searched. We included randomized and non-randomized clinical trials, epidemiological studies (cohort and case-control). We excluded case series, case reports, in vitro and animal studies. Results: The WHO, the U.S. Food and Drug Administration (FDA), the Heart Foundation and the Romanian Dietary Guidelines recommends that adults should aim to consume approximately 25–30 g fiber daily. Dietary fiber is found in the indigestible parts of cereals, fruits and vegetables. There are countries where people don’t eat enough food fibers, these people need to take some kind of fiber supplement. Evidence has been found that dietary fiber from whole foods or supplements may (1) reduce the risk of cardiovascular disease by improving serum lipids and reducing serum total and low-density lipoprotein (LDL) cholesterol concentrations, (2) decreases the glycaemic index of foods, which leads to an improvement in glycemic response, positive impact on diabetes, (3) protect against development of obesity by increasing satiety hormone leptin concentrations, (4) reduced risk of developing colorectal cancer by normalizes bowel movements, improve the integrity of the epithelial layer of the intestines, increase the resistance against pathogenic colonization, have favorable effects on the gut microbiome, wich is the second genomes of the microorganisms, (5) have a positive impact on the endocrine system by gastrointestinal polypeptide hormonal regulation of digestion, (6) have prebiotic effect by short-chain fatty acids (SCFA) production; butyrate acid is the preferred energy source for colonic epithelial cells, promotes normal cell differentiation and proliferation, and also help regulate sodium and water absorption, and can enhance absorption of calcium and other minerals. Although all prebiotics are fiber, not all fiber is prebiotic. This generally refers to the ability of a fiber to increase the growth of bifidobacteria and lactobacilli, which are beneficial to human health, and (7) play a role in improving immune function via production of SCFAs by increases T helper cells, macrophages, neutrophils, and increased cytotoxic activity of natural killer cells. Conclusion: Fiber consumption is associated with high nutritional value and antioxidant status of the diet, enhancing the effects on human health. Fibers with prebiotic properties can also be recommended as part of fiber intake. Due to the variability of fiber’s effects in the body, it is important to consume fiber from a variety of sources. Increasing fiber consumption for health promotion and disease prevention is a critical public health goal.

Investigating the Roles and Requirements, Manifestations and Management of Metadata in the Creation of Reliable and Preservation of Authentic Electronic Entities Created by Dynamic, Interactive and Experiential Systems: Report on the Work and Findings of the Interpares 2 Description Cross Domain Group

Metadata that is associated with either an information system or an information object for purposes of description, administration, legal requirements, technical functionality, use and usage, and preservation, plays a critical role in ensuring the creation, management, preservation and use and re-use of trustworthymaterials, including records. Recordkeeping1 metadata, of which one key type is archival description, plays a particularly important role in documenting the reliability and authenticity of records and recordkeeping systemsas well as the various contexts (legal-administrative, provenancial, procedural, documentary, and technical) within which records are created and kept as they move across space and time. In the digital environment, metadata is also the means by which it is possible to identify how record components – those constituent aspects of a digital record that may be managed, stored and used separately by the creator or the preserver – can be reassembled to generate an authentic copy of a record or reformulated per a user’s request as a customized output package.Issues relating to the creation, capture, management and preservation of adequate metadata are, therefore, integral to any research study addressing the reliability and authenticity of digital entities, regardless of the community, sector or institution within which they are being created. The InterPARES 2 Description Cross-Domain Group (DCD) examined the conceptualization, definitions, roles, and current functionality of metadata and archival description in terms of requirements generated by InterPARES 12. Because of the needs to communicate the work of InterPARES in a meaningful way across not only other disciplines, but also different archival traditions; to interface with, evaluate and inform existing standards, practices and other research projects; and to ensure interoperability across the three focus areas of InterPARES2, the Description Cross-Domain also addressed its research goals with reference to wider thinking about and developments in recordkeeping and metadata. InterPARES2 addressed not only records, however, but a range of digital information objects (referred to as “entities” by InterPARES 2, but not to be confused with the term “entities” as used in metadata and database applications) that are the products and by-products of government, scientific and artistic activities that are carried out using dynamic, interactive or experiential digital systems. The nature of these entities was determined through a diplomatic analysis undertaken as part of extensive case studies of digital systems that were conducted by the InterPARES 2 Focus Groups. This diplomatic analysis established whether the entities identified during the case studies were records, non-records that nevertheless raised important concerns relating to reliability and authenticity, or “potential records.” To be determined to be records, the entities had to meet the criteria outlined by archival theory – they had to have a fixed documentary format and stable content. It was not sufficient that they be considered to be or treated as records by the creator. “Potential records” is a new construct that indicates that a digital system has the potential to create records upon demand, but does not actually fix and set aside records in the normal course of business. The work of the Description Cross-Domain Group, therefore, addresses the metadata needs for all three categories of entities.Finally, since “metadata” as a term is used today so ubiquitously and in so many different ways by different communities, that it is in peril of losing any specificity, part of the work of the DCD sought to name and type categories of metadata. It also addressed incentives for creators to generate appropriate metadata, as well as issues associated with the retention, maintenance and eventual disposition of the metadata that aggregates around digital entities over time.

Synthetic portfolio for event studies: Estimating the effects of volatility call auctions

We propose a method denoted as synthetic portfolio for event studies in market microstructure that is particularly interesting to use with high frequency data and thinly traded markets. The method is based on Synthetic Control Method and provides a robust data driven method to build a counterfactual for evaluating the effects of the volatility call auctions. We find that SMC could be used if the loss function is defined as the difference between the returns of the asset and the returns of a synthetic portfolio. We apply SCM to test the performance of the volatility call auction as a circuit breaker in the context of an event study. We find that for Colombian Stock Market securities, the asynchronicity of intraday data reduces the analysis to a selected group of stocks, however it is possible to build a tracking portfolio. The realized volatility increases after the auction, indicating that the mechanism is not enhancing the price discovery process.

Optimization of the power electronics system associated with ocean wave electric generators

The present thesis is focused on wave energy, which is a particular kind of ocean energy, and is based on the activity carried out during the EU project SEA TITAN. The main scope of this work is the design of a power electronic section for an innovative wave energy extraction system based on a switched-reluctance machine. In the first chapter, the general features of marine wave energy harvesting are treated. The concept of Wave Energy Converter (WEC) is introduced as well as the mathematical description of the waves, their characterization and measurement, the WEC classification, the operating principles and the standardization framework. Also, detailed considerations on the environmental impact are presented. The SEA TITAN project is briefly described. The second chapter is dedicated to the technical issues of the SEA TITAN project, such as the operating principle, the performance optimization carried out in the project, the main innovations as well as interesting demonstrations on the behavior of the generator and its control. In the third chapter, the power electronics converters of SEA TITAN are described, and the design choices, procedures and calculations are shown, with a further insight into the application given by analyzing the MATLAB Simulink model of the system and its control scheme. Experimental tests are reported in the fourth chapter, with graphs and illustrations of the power electronic apparatus interfaced with the real machine. Finally, the conclusion in the fifth chapter offers a global overview of the project and opens further development pathways.

Rapid Control Prototyping of an L2+ Highway Assist System

Advanced Driver Assistance Systems (ADAS) are proving to have huge potential in road safety, comfort, and efficiency. In recent years, car manufacturers have equipped their high-end vehicles with Level 2 ADAS, which are, according to SAE International, systems that combine both longitudinal and lateral active motion control. These automated driving features, while only available in highway scenarios, appear to be very promising towards the introduction of hands-free driving. However, as they rely only on an on-board sensor suite, their continuative operation may be affected by the current environmental conditions: this prevents certain functionalities such as the automated lane change, other than requiring the driver to keep constantly the hands on the steering wheel. The enabling factor for hands-free highway driving proposed by Mobileye is the integration of high-definition maps, thus leading to the so-called Level 2+. This thesis was carried out during an internship in Maserati's Virtual Engineering team. The activity consisted of the design of an L2+ Highway Assist System following the Rapid Control Prototyping approach, starting from the definition of the requirements up to the real-time implementation and testing on a simulator of the brand new compact SUV Maserati Grecale. The objective was to enhance the current Level 2 highway driving assistance system with hands-free driving capability; for this purpose an Autonomous Lane Change functionality has been designed, proposing a Model Predictive Control-based decision-maker, in charge of assessing both the feasibility and convenience of performing a lane-change maneuver. The result is a Highway Assist System capable of driving the vehicle in a traffic scenario safely and efficiently, never requiring driver intervention.

Hardware-In-The Loop setup and test of an air spring control system

In this work, a Hardware-in-the-loop test bench is designed. The bench is used to test the behaviour of an electronic control unit used in Maserati to control the dynamics of an air spring system. First the mathematical model of the plant has been defined, then the simulation enviroment and the test environment have been set up. The performed tests succesfully highlighted some bugs in the device under test.

Model-Based validation of Driver Drowsiness Detection System for ADAS

The work described in this Master’s Degree thesis was born after the collaboration with the company Maserati S.p.a, an Italian luxury car maker with its headquarters located in Modena, in the heart of the Italian Motor Valley, where I worked as a stagiaire in the Virtual Engineering team between September 2021 and February 2022. This work proposes the validation using real-world ECUs of a Driver Drowsiness Detection (DDD) system prototype based on different detection methods with the goal to overcome input signal losses and system failures. Detection methods of different categories have been chosen from literature and merged with the goal of utilizing the benefits of each of them, overcoming their limitations and limiting as much as possible their degree of intrusiveness to prevent any kind of driving distraction: an image processing-based technique for human physical signals detection as well as methods based on driver-vehicle interaction are used. A Driver-In-the-Loop simulator is used to gather real data on which a Machine Learning-based algorithm will be trained and validated. These data come from the tests that the company conducts in its daily activities so confidential information about the simulator and the drivers will be omitted. Although the impact of the proposed system is not remarkable and there is still work to do in all its elements, the results indicate the main advantages of the system in terms of robustness against subsystem failures and signal losses.