Application of Higher-Order Statistics and Subspace-Based Techniques to the Analysis and Diagnosis of Electrocardiogram Signals

The first and main contribution of this research work is the higher-order statistics (HOS)-based non-linear analysis and subsequent diagnosis of abnormal electrocardiogram (ECG) signals, particularly myocardial ischaemia. In the time domain; the second-, third-, and the fourth-order cumulants have been used in the analysis. In the frequency domain; up to the tenth-order polyspectra have been exploited. This HOS-based analysis of normal and ischaemic electrocardiogram signals has led to the identification of certain key discriminant features for the two physiological states of the heart. These features are then fed to different backpropagation-based multiple layer perceptrons for classification. The second contribution is a proposed new methodology to discriminate patients with angina pectoris or with old myocardial infarction (MI) during the first 60 seconds of stress test (or in some cases using rest ECG). It is based on the pseudo-spectral Multiple Signal Classification (MUSIC) and has the potential of being highly sensitive diagnostic signal processing tool. The third contribution is the development of a novel higher-order statistics, high-resolution estimator for quadratically coupled frequencies based on subspace spectral estimation. Extensive studies of cumulants, bispectra and bicoherence-squared of normal and ischaemic ECG signals collected from MIT and ST-T European databases has enabled us to see key discriminant features in both the third- and fourth-order cumulant domains. In the frequency domain, the polyspectral study has been extended to the lOth-order poly spectra. By calculating one-dimensional polyspectrum slices using an algorithm developed by Zhou and Giannakis (1995) a considerable reduction in the CPU time has been achieved. Furthermore, Zhou’s algorithm has been further extended to estimate the polycoherency slices which are used to characterise non-linearities in normal and ischaemic ECG signals. An important finding in this thesis is the decrease of the order of non-linearity representing the electrocardiogram signals of ischaemic patients. This thesis also includes the results of a pilot study involving eighteen healthy subjects (MIT database) and confirmed that the ECG signal is non-Gaussian, cyclostationary and quasi periodic. Combined spectral and bispectral analysis of the signal revealed that there are unique harmonic characteristics for the P-wave, QRS complex and T-wave and other frequencies due to harmonic interactions. In this work three linear and one non-linear adaptive filtering/predictions techniques have been applied to noisy ECG signals and their respective performances appraised. It is shown that the Kalman filter gives the best mean-square error MSE error but its comparatively long execution time and problems arising from ill-conditioning of the state-error covariance matrix render it of limited use in ECG applications. It is also shown that the LMS-based quadratic and cubic Volterra filters are the most superior for the ECG signal prediction. For ECG classifications; three multi-layer perceptrons employing back-propagation and modified back-propagation algorithms, and using two sets from the higher-order most discriminant features as their inputs, have yielded fairly high classification rates

Biomedical Signal and Image Processing

Written for senior-level and first year graduate students in biomedical signal and image processing, this book describes fundamental signal and image processing techniques that are used to process biomedical information. The book also discusses application of these techniques in the processing of some of the main biomedical signals and images, such as EEG, ECG, MRI, and CT. New features of this edition include the technical updating of each chapter along with the addition of many more examples, the majority of which are MATLAB based

Tissue-level Mechanisms Driving Cardiac Progenitor and Extracellular Matrix Movements during Early Vertebrate Heart Development

Vertebrate cardiogenesis involves heart progenitor cell movements from their initial lateral positions to the embryonic midline, where they assemble into a primitive heart. This early heart tube consists of an outer myocardium, a medial extracellular matrix (ECM), and an endocardial lining. Cardiac morphogenesis in avians and mammals is inseparable from development of the foregut, which provides molecular cues to regulate endocardial and myocardial differentiation from mesodermal progenitors. Concomitantly with the initiation of midline-directed cardiac progenitor movements, foregut endoderm undergoes dramatic folding and elongation. Following their initial assembly, the heart and foregut are transiently connected through a mesentery. Previous research focused on the molecular factors involved in guiding cardiac progenitors to the midline, yet cellular and tissue mechanisms coordinating these movements remain poorly understood. This work investigates movements of all three early heart constituents - the endocardial and myocardial progenitors, and surrounding ECM - in live quail embryos using a combination of time-lapse microscopy, chemical and mechanical perturbations, computational analysis and modeling. By visualizing the tissue environment for cell displacements, we distinguish the active (tissue-independent) movements from those cells undergo in a manner coordinated with the surrounding tissues. First, we analyzed the movements of endocardial progenitors and fluorescently-labeled ECM (fibronectin, fibrillin-2) fibrils. We found the bulk of midline-directed movement of pre-endocardial cells is coordinated with their surrounding ECM. Further, that ECM from extracardiac sources is transferred to and incorporated into the growing heart. By assessing the contributions of active cell motility to the observed midline endocardial displacements we found its role to be secondary to that of convective tissue movement within the anterior embryo. Second, we assessed myocardial progenitor movements relative to fibronectin ECM and endoderm. We discovered that observed antero-medial myocardial displacements are driven by a combination of: 1) medial tissue motion, and 2) anterior movement, accomplished via a coordinated deformation of myocardial progenitors, organized into a continuous epithelial sheet. Finally, we investigated the effects of VEGF overexposure on progenitor movements during early cardiogenesis. We found a dramatic VEGF-induced increase in cardiac inflow region size, which affected the coordinated movements/deformations displayed by myocardial progenitors, and resulted in heart tube elongation defects

Development, characterization and evaluation of advanced therapies for the treatment of cardiac pathologies

Cardiovascular diseases (CVDs) are the leading cause of disease burden and mortality in the world, as well as a major cause of disability and health care costs. With the average lifespan of the human population continuously increasing, it is expected that the problem of CVDs will only continue to grow in the following years. Current pharmacological treatments for age-associated cardiac pathologies such as heart failure and atrial fibrillation present severe clinical efficacy and safety problems and are not regarded as definitive cures. This makes it necessary to develop new treatment strategies that target the involved molecular pathways and trigger endogenous reparative responses. Contrary to current molecular treatments, advanced therapy medicinal products (ATMPs) such as stem cells, extracellular vesicles (EVs) and biomaterials such as hydrogels could have the potential to treat cardiac aging-associated pathologies from a more fundamental level. However, many problems and unknowns still need to be solved before they can reach the clinical scenario. Some of the most highlighted limitations we focus on in this work are: (i) the lack of deep understanding of their mechanism of action (MoA), (ii) their large variability and lack of standardization (including inadequate potency tests) and (iii) low in vivo retention at the site of interest. Therefore, the main objective of this thesis is to develop, characterize and evaluate advanced therapies for the treatment of cardiac pathologies solving some of their current limitations to enhance their therapeutic potential. To achieve this aim, we first focus on improving standardization and development of potency assays. We describe the main characteristics and challenges for a cell therapy based potency test in the cardiovascular field and we review and propose different types of assays that could be taken into consideration based on the product’s expected MoA and the target cardiovascular disease. Secondly, as cardiosphere-derived cells (CDCs) and their secreted EVs (CDC-EVs) have previously reported to have anti-senescent effects and this is considered important in aging-related cardiac diseases, we explore potential predictors of rejuvenating potency with a special focus on the chronological age of the CDC-donors and CDC-senescence, among others. Multiple in vitro tests allow us to conclude that more than cell particular biological markers or characteristics, the cell bioactivity relative to the expected MoA should be a better predictor for the ATMP potency. Thus, we evaluate if the in vitro anti-senescent and pro-angiogenic effect of the CDC-EVs, scored with a matrix assay, can be used to predict the in vivo potency of the CDC-EVs in an animal model of cardiac aging. Our results show that EVs classified in vitro as potent with the matrix assay have more cardiac reparative potential in vivo than EVs classified as non-potent. After further validation, the matrix assay proposed here could be a suitable in vitro potency test for discerning suitable allogenic biological products in the cardiac aging clinical scenario. Next, with the purpose of improving EV retention at the site of interest, we develop an optimized product combining hydrogels from cardiac extracellular matrix (cECM), polyethylene glycol and EVs to overcome some of their individual limitations: long gelation time of the cECM and poor retention of the EVs. We conclude that the combined product rapidly gels at physiological temperature and presents improved mechanical properties while maintaining the injectability, the biodegradability, and the bioactivity of its individual components. In addition, it serves to better retain the EVs on-site in vivo. Finally, we explore the electrophysiological modifications induced by CDC-EVs on arrhythmogenic tissue to better understand the mechanisms behind their antiarrhythmic effect. We found that CDC-EVs reduce spontaneous activation complexity and increase conduction velocity of cardiomyocytes leading to a less arrhythmogenic profile. If validated in other cellular models, CDC-EVs may be used specifically as antiarrhythmic agents in a wide range of cardiac pathologies. Although future work should aim to further validate these results both at preclinical and clinical level, these findings together partially overcome some of the main challenges for the therapeutic use of cellular therapies and open a new horizon for the treatment of cardiac-aging related pathologies, some still considered as unmet medical needs.Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidenta: Eva Delpón Mosquera.- Secretaria: Marta García Díez.- Vocal: Javier Bermejo Thoma

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

Background: An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment ap-proaches for patients with thyroid disease. Summary: Important clinical practices in use today for the treatment of patients with hypothy-roidism, hyperthyroidism, or thyroid cancer, are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a se-ries of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings. Conclusions: It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes

Computational methods for the analysis of functional 4D-CT chest images.

Medical imaging is an important emerging technology that has been intensively used in the last few decades for disease diagnosis and monitoring as well as for the assessment of treatment effectiveness. Medical images provide a very large amount of valuable information that is too huge to be exploited by radiologists and physicians. Therefore, the design of computer-aided diagnostic (CAD) system, which can be used as an assistive tool for the medical community, is of a great importance. This dissertation deals with the development of a complete CAD system for lung cancer patients, which remains the leading cause of cancer-related death in the USA. In 2014, there were approximately 224,210 new cases of lung cancer and 159,260 related deaths. The process begins with the detection of lung cancer which is detected through the diagnosis of lung nodules (a manifestation of lung cancer). These nodules are approximately spherical regions of primarily high density tissue that are visible in computed tomography (CT) images of the lung. The treatment of these lung cancer nodules is complex, nearly 70% of lung cancer patients require radiation therapy as part of their treatment. Radiation-induced lung injury is a limiting toxicity that may decrease cure rates and increase morbidity and mortality treatment. By finding ways to accurately detect, at early stage, and hence prevent lung injury, it will have significant positive consequences for lung cancer patients. The ultimate goal of this dissertation is to develop a clinically usable CAD system that can improve the sensitivity and specificity of early detection of radiation-induced lung injury based on the hypotheses that radiated lung tissues may get affected and suffer decrease of their functionality as a side effect of radiation therapy treatment. These hypotheses have been validated by demonstrating that automatic segmentation of the lung regions and registration of consecutive respiratory phases to estimate their elasticity, ventilation, and texture features to provide discriminatory descriptors that can be used for early detection of radiation-induced lung injury. The proposed methodologies will lead to novel indexes for distinguishing normal/healthy and injured lung tissues in clinical decision-making. To achieve this goal, a CAD system for accurate detection of radiation-induced lung injury that requires three basic components has been developed. These components are the lung fields segmentation, lung registration, and features extraction and tissue classification. This dissertation starts with an exploration of the available medical imaging modalities to present the importance of medical imaging in today’s clinical applications. Secondly, the methodologies, challenges, and limitations of recent CAD systems for lung cancer detection are covered. This is followed by introducing an accurate segmentation methodology of the lung parenchyma with the focus of pathological lungs to extract the volume of interest (VOI) to be analyzed for potential existence of lung injuries stemmed from the radiation therapy. After the segmentation of the VOI, a lung registration framework is introduced to perform a crucial and important step that ensures the co-alignment of the intra-patient scans. This step eliminates the effects of orientation differences, motion, breathing, heart beats, and differences in scanning parameters to be able to accurately extract the functionality features for the lung fields. The developed registration framework also helps in the evaluation and gated control of the radiotherapy through the motion estimation analysis before and after the therapy dose. Finally, the radiation-induced lung injury is introduced, which combines the previous two medical image processing and analysis steps with the features estimation and classification step. This framework estimates and combines both texture and functional features. The texture features are modeled using the novel 7th-order Markov Gibbs random field (MGRF) model that has the ability to accurately models the texture of healthy and injured lung tissues through simultaneously accounting for both vertical and horizontal relative dependencies between voxel-wise signals. While the functionality features calculations are based on the calculated deformation fields, obtained from the 4D-CT lung registration, that maps lung voxels between successive CT scans in the respiratory cycle. These functionality features describe the ventilation, the air flow rate, of the lung tissues using the Jacobian of the deformation field and the tissues’ elasticity using the strain components calculated from the gradient of the deformation field. Finally, these features are combined in the classification model to detect the injured parts of the lung at an early stage and enables an earlier intervention

The Role of MicroRNA Regulation of Cardiac Ion Channel in Arrhythmia

La fibrillation auriculaire (FA) est le trouble du rythme le plus fréquemment observé en pratique clinique. Elle constitue un risque important de morbi-mortalité. Le traitement de la FA reste un défi majeur en lien avec les nombreux effets secondaires associés aux approches thérapeutiques actuelles. Dans ce contexte, une meilleure compréhension des mécanismes sous-jacents à la FA est essentielle pour le développement de nouvelles thérapies offrant un meilleur rapport bénéfice/risque pour les patients. La FA est caractérisée par i) un remodelage électrique délétère associé le plus souvent ii) à un remodelage structurel du myocarde favorisant la récurrence et le maintien de l’arythmie. La diminution de la période réfractaire effective au sein du tissu auriculaire est un élément clef du remodelage électrique. Le remodelage structurel, quant à lui, se manifeste principalement par une fibrose tissulaire qui altère la propagation de l’influx électrique dans les oreillettes. Les mécanismes moléculaires impliqués dans la mise en place de ces deux substrats restent mal connus. Récemment, le rôle des microARNs (miARNs) a été pointé du doigt dans de nombreuses pathologies notamment cardiaques. Dans ce contexte les objectifs principaux de ce travail ont été i) d'acquérir une compréhension approfondie du rôle des miARNs dans la régulation de l’expression des canaux ioniques et ii) de mieux comprendre le rôle de ces molécules dans l’installation d’un substrat favorable a la FA. Nous avons, dans un premier temps, effectué une analyse bio-informatique combinée à des approches expérimentales spécifiques afin d’identifier clairement les miARNs démontrant un fort potentiel de régulation des gènes codant pour l’expression des canaux ioniques cardiaques humains. Nous avons identifié un nombre limité de miARNs cardiaques qui possédaient ces propriétés. Sur la base de ces résultats, nous avons démontré que l’altération de l'expression des canaux ioniques, observée dans diverse maladies cardiaques (par exemple, les cardiomyopathies, l’ischémie myocardique, et la fibrillation auriculaire), peut être soumise à ces miARNs suggérant leur implication dans l’arythmogénèse. La régulation du courant potassique IK1 est un facteur déterminant du remodelage électrique auriculaire associée à la FA. Les mécanismes moléculaires sous-jacents sont peu connus. Nous avons émis l’hypothèse que l'altération de l’expression des miARNs soit corrélée à l’augmentation de l’expression d’IK1 dans la FA. Nous avons constaté que l’expression de miR-26 est réduite dans la FA et qu’elle régule IK1 en modulant l’expression de sa sous-unité Kir2.1. Nous avons démontré que miR-26 est sous la répression transcriptionnelle du facteur nucléaire des lymphocytes T activés (NFAT) et que l’activité accrue de NFATc3/c4, aboutit à une expression réduite de miR-26. En conséquence IK1 augmente lors de la FA. Nous avons enfin démontré que l’interférence in vivo de miR-26 influence la susceptibilité à la FA en régulant IK1, confirmant le rôle prépondérant de miR-26 dans le remodelage auriculaire électrique. La fibrose auriculaire est un constituant majeur du remodelage structurel associé à la FA, impliquant l'activation des fibroblastes et l’influx cellulaire du Ca2 +. Nous avons cherché à déterminer i) si le canal perméable au Ca2+, TRPC3, jouait un rôle dans la fibrose auriculaire en favorisant l'activation des fibroblastes et ii) étudié le rôle potentiel des miARNs dans ce contexte. Nous avons démontré que les canaux TRPC3 favorisent l’influx du Ca2 +, activant la signalisation Ca2 +-dépendante ERK et en conséquence activent la prolifération des fibroblastes. Nous avons également démontré que l’expression du TRPC3 est augmentée dans la FA et que le blocage in vivo de TRPC3 empêche le développement de substrats reliés à la FA. Nous avons par ailleurs validé que miR-26 régule les canaux TRPC3 en diminuant leur expression dans les fibroblastes. Enfin, nous avons montré que l'expression réduite du miR-26 est également due à l’activité augmentée de NFATc3/c4 dans les fibroblastes, expliquant ainsi l’augmentation de TRPC3 lors de la FA, confirmant la contribution de miR-26 dans le processus de remodelage structurel lié à la FA. En conclusion, nos résultats mettent en évidence l'importance des miARNs dans la régulation des canaux ioniques cardiaques. Notamment, miR-26 joue un rôle important dans le remodelage électrique et structurel associé à la FA et ce, en régulant IK1 et l’expression du canal TRPC3. Notre étude démasque ainsi un mécanisme moléculaire de contrôle de la FA innovateur associant des miARNs. miR-26 en particulier représente apres ces travaux une nouvelle cible thérapeutique prometteuse pour traiter la FA.Atrial fibrillation (AF) is the most frequently-encountered arrhythmia in clinical practice and constitutes a major cause of cardiac morbidity and mortality. The management of AF remains a major challenge as current therapeutic approaches are limited by potential adverse effects and high rate of AF recurrence/persistence. A better understanding of the mechanisms underlying AF is of great importance to improve AF therapy. AF is characterized by impaired electrical and structural remodeling, both of which favors the recurrence and maintenance of the arrhythmia. A key feature in electrical remodeling is the reduced atrial effective refractory period, due to ion channel alteration. Structural remodeling, on the other hand, mainly results from atrial fibrosis. However, the precise molecular mechanisms underlying these remodeling processes are still incompletely understood. The importance of microRNAs (miRNAs) in various pathophysiological conditions of the heart has been well established, but little is known with regard to cardiac arrhythmias. Emerging evidence suggests that dysregulation of miRNAs may underlie heart rhythm disturbances. The aim of the present work was to acquire a comprehensive understanding of miRNA-mediated regulation of ion channels in cardiac arrhythmias. Notably, we will focus on the mechanistic insights of miRNAs related to the control of AF. Currently available experimental approaches do not permit thorough characterization of miRNA targeting. For this purpose, we performed bioinformatic analyses in conjunction with experimental approaches to identify miRNAs from the database that potentially regulate human cardiac ion channel genes. We found that only a subset of miRNAs target cardiac ion channel genes. Based on these results, we further demonstrated that the dysregulation of ion channel gene expression observed in various cardiac disorders (e.g. cardiomyopathy, myocardial ischemia, and atrial fibrillation) can be explained by the dysregulation of miRNAs. These findings further support the potential implication of miRNAs in arrhythmogenesis under these cardiac conditions. The upregulation of the cardiac inward rectifying potassium current, IK1, is a key determinant of adverse atrial electrical remodeling associated with AF. The molecular mechanisms underlying this ionic remodeling are poorly understood. We hypothesized that altered miRNA expression is responsible for IK1 upregulation in AF. We found that miR-26 is significantly downregulated in AF and regulates IK1 by controlling the expression of its underlying subunit Kir2.1. Moreover, we demonstrated that miR-26 is under the transcriptional repression of the nuclear factor of activated T cells (NFAT) and enhanced activities of members of the NFAT family, NFATc3/c4, results in miR-26 downregulation, which accounts for IK1 enhancement in AF. Furthermore, we observed that in vivo interference of miR-26 affects AF susceptibility via the regulation of IK1, suggesting an important role of miR-26 in atrial electrical remodeling. Atrial fibrosis is a major constituent in AF-associated adverse atrial structural remodeling, involving the activation of fibroblasts and cellular Ca2+ entry. Here, we sought to determine whether the Ca2+ permeable channel, TRPC3, plays a role in AF-induced fibrosis by promoting fibroblast activation. Furthermore, we investigated the potential role of miRNAs in this context. We found that TRPC3 channels promote Ca2+-entry, which results in activation of Ca2+-dependent ERK-signaling and consequently fibroblast activation. We also demonstrated that TRPC3 is upregulated in AF and in vivo TRPC3 blockade suppresses the development of AF-promoting substrate. Furthermore, we observed that miR-26 regulates TRPC3 channels via controlling the expression of the underlying channel subunit and is downregulated in AF-fibroblasts. Finally, we showed that the reduced expression of miR-26 is also due to the enhanced NFATc3/c4 activities in AF-fibroblasts and accounts for AF-induced upregulation of TRPC3, suggesting the potential contribution of miR-26 in AF-related adverse structural remodeling process. In conclusion, our findings emphasize the importance of miRNAs in the regulation of cardiac ion channels. Notably, miR-26 plays a crucial role in AF-associated electrical and structural remodeling via the regulation of IK1 and TRPC3 channel genes. Thus, our study unravels a novel molecular control mechanism of AF at the miRNA level, suggesting miR-26 as a new and promising therapeutic target for AF