EVOLUTION OF THE CIRCADIAN CLOCK IN EXTREME ENVIRONMENT: LESSONS FROM CAVEFISH.

Evolution has been strongly influenced by the daily cycles of temperature and light imposed by the rotation of the Earth. Fascinating demonstrations of this are seen in extreme environments such as caves where some animals have remained completely isolated from the day-night cycle for millions of years. Most of these species show convergent evolution, sharing a range of striking physical properties such as eye loss. One fundamental issue is whether “hypogean” species retain a functional circadian clock. This highly conserved, physiological timing mechanism allows organisms to anticipate daily environmental changes and is synchronized primarily by light. The Somalian cavefish, Phreatichthys andruzzii does possess a circadian clock that is entrained by a daily regular feeding time but strikingly, not by light. Under constant conditions the P. andruzzii clock oscillates with an extremely long period and also lacks normal temperature compensation. We document multiple mutations affecting a light-induced clock gene, Period2 as well as the genes encoding the extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin. Remarkably, we show that ectopic expression of zebrafish homologs of these opsins rescues light induced clock gene expression in P. andruzzii cells. Thus, by studying this natural mutant we provide direct evidence for a peripheral light-sensing function of extra-retinal opsins in vertebrates. Furthermore, the properties of this cavefish illustrate that evolution in constant darkness leads not only to anatomical changes but also to loss of gene function linked with the detection and anticipation of the day-night cycle

Assessment of ventricular repolarization instability and cardiac risk stratification in different pathological and abnormal conditions

Cardiovascular diseases (CVDs) represents the leading cause of mortality worldwide [1,2]. These pathological conditions are mainly characterized by a structurally abnormal heart, that is, a vulnerable substrate, prone to the abnormal generation and/or propagation of the electrical impulse, determining the onset of ventricular arrhythmias, which can result in sudden cardiac death (SCD) [3]. In this context, the assessment of ventricular repolarization from the electrocardiogram (ECG) signal has been shown to provide with valuable information for risk stratification and several electrocardiographic indices have been proposed in the literature [4]. The main objective of this thesis is to propose methodological advances for the assessment of ventricular repolarization instability in pathological and abnormal conditions. These contributions are aimed at improving the prediction of ventricular arrhythmias and, consequently, better identifying SCD risk. In particular, we have addressed this objective by developing robust methodologies for the assessment of T-wave alternans (TWA) and ventricular repolarization instability, in invasive and non-invasive cardiac signals, that have been evaluated in both experimental and clinical conditions. In the first part of the thesis, TWA was simultaneously characterized (prevalence, magnitude, time-course, and alternans waveform) in body-surface ECG and intracardiac electrograms (EGMs) signals during coronary artery occlusion. Signals from both body surface ECG and intracardiac EGMs recorded from 4 different anatomical heart locations (coronary sinus, epicardial space and left and right ventricles) were analyzed following a multilead strategy. Leads were linearly combined using the periodic component analysis (πCA) [5], which maximizes the 2-beat periodicity (TWA periodicity) content present on the available leads. Then the Laplacian Likelihood Ratio method (LLRM) [6] was applied for TWA detection and estimation. A sensitivity study for TWA detection from the 5 different locations of leads was performed, revealing that it is the combination of the ECG leads that better performs. In addition, this multilead approach allowed us to find the optimal combination of intracardiac leads usable for in-vivo monitorization of TWA directly from an implantable device, with a sensitivity comparable to the ECG analysis. These results encourage further research to determine the feasibility of predicting imminent VT/VF episodes by TWA analysis implemented in implantable cardioverter defibrillator’s (ICD) technology.Then, we have studied the potential changes induced by a prolonged exposure to simulated microgravity on ventricular repolarization in structurally normal hearts. It is well known that this environmental condition affects the control of autonomic and cardiovascular systems [7], with a potential increase on cardiac electrical instability. The effects of short- (5 days), mid- (21 days) and long- (60 days) exposure to simulated microgravity on TWA using the head-down bed-rest (HDBR) model [8] were assessed. TWA was evaluated before (PRE), during and after (POST) the immobilization period, by the long-term averaging technique in ambulatory ECG Holter recordings [9]. Additionally, we proposed an adapted short-term averaging approach for shorter, non-stationary ECG signals obtained during two stress manoeuvres (head-up tilt-table and bicycle exercise tests). Both approaches are based on the multilead analysis used in the previous study. The absence of significant changes between PRE and POST-HDBR on TWA indices suggests that a long-term exposure to simulated microgravity is not enough to induce alterations in healthy myocardial substrate up to the point of reflecting electrical instability in terms of TWA on the ECG. Finally, methodological advances were proposed for the assessment of ventricular repolarization instability from the ECG signal in the presence of sporadic (ventricular premature contractions, VPCs) and sustained (atrial fibrillation) rhythm disturbances.On the one hand, a methodological improvement for the estimation of TWA amplitude in ambulatory ECG recordings was proposed, which deals with the possible phase reversal on the alternans sequence induced by the presence of VPCs [10]. The performance of the algorithm was first evaluated using synthetic signals. Then, the effect of the proposed method in the prognostic value of TWA amplitude was assessed in real ambulatory ECG recordings from patients with chronic heart failure (CHF). Finally, circadian TWA changes were evaluated as well as the prognostic value of TWA at different times of the day. A clinical study demonstrated the enhancement in the predictive value of the index of average alternans (IAA) [9] for SCD stratification. In addition, results suggested that alternans activity is modulated by the circadian pattern, preserving its prognostic information when computed just during the morning, which is also the day interval with the highest reported SCD incidence. Thus, suggesting that time of the day should be considered for SCD risk prediction. On the other hand, the high irregularity of the ventricular response in atrial fibrillation (AF) limits the use of the most common ECG-derived markers of repolarization heterogeneity, including TWA, under this clinical condition [11]. A new method for assessing ventricular repolarization changes based on a selective averaging technique was developed and new non-invasive indices of repolarization variation were proposed. The positive impact in the prognostic value of the computed indices was demonstrated in a clinical study, by analyzing ECG Holter recordings from CHF patients with AF. To the best of our knowledge, this is the first study that attempts a non-invasive SCD stratification of patients under AF rhythm by assessing ventricular repolarization instability from the ECG signal. To conclude, the research presented in this thesis sheds some light in the identification of pro-arrhythmic factors, which plays an important role in adopting efficient therapeutic strategies. In particular, the optimal configuration for real-time monitoring of repolarization alternans from intracardiac EGMs, together with the prognostic value of the proposed non-invasive indices of alternans activity and ventricular instability variations in case of AF rhythms demonstrated in two clinical studies, would increase the effectiveness of (ICD) therapy. Finally, the analysis of ECG signals recorded during HDBR experiments in structurally healthy hearts, also provides interesting information on cardiovascular alterations produced in immobilized or bedridden patients.<br /

An Optimal Time for Treatment-Predicting Circadian Time by Machine Learning and Mathematical Modelling

Tailoring medical interventions to a particular patient and pathology has been termed personalized medicine. The outcome of cancer treatments is improved when the intervention is timed in accordance with the patient's internal time. Yet, one challenge of personalized medicine is how to consider the biological time of the patient. Prerequisite for this so-called chronotherapy is an accurate characterization of the internal circadian time of the patient. As an alternative to time-consuming measurements in a sleep-laboratory, recent studies in chronobiology predict circadian time by applying machine learning approaches and mathematical modelling to easier accessible observables such as gene expression. Embedding these results into the mathematical dynamics between clock and cancer in mammals, we review the precision of predictions and the potential usage with respect to cancer treatment and discuss whether the patient's internal time and circadian observables, may provide an additional indication for individualized treatment timing. Besides the health improvement, timing treatment may imply financial advantages, by ameliorating side effects of treatments, thus reducing costs. Summarizing the advances of recent years, this review brings together the current clinical standard for measuring biological time, the general assessment of circadian rhythmicity, the usage of rhythmic variables to predict biological time and models of circadian rhythmicity

Novel Approaches to ECG-Based Modeling and Characterization of Atrial Fibrillation

This thesis deals with signal processing algorithms for analysis of the electrocardiogram (ECG) during atrial fibrillation (AF). Such analysis can be used for diagnosing patients, and for monitoring and predicting their response to various treatment. The thesis comprises an introduction and five papers describing methods for ECG-based modeling and characterization of AF. Paper I--IV deal with methods for characterization of the atrial activity, whereas Paper V deals with modeling of the ventricular response, both problems with the assumption that AF is present. In Paper I, a number of measures characterizing the atrial activity in the ECG, obtained using time-frequency analysis as well as nonlinear methods, are evaluated for their ability to predict spontaneous termination of AF. The AF frequency, i.e, the repetition rate of the atrial fibrillatory waves of the ECG, proved to be a significant factor for discrimination between terminating and non-terminating AF. Noise is a common problem in ECG signals, particularly in long-term ambulatory recordings. Hence, robust algorithms for analysis and characterization are required. In Paper II, a robust method for tracking the AF frequency in noisy signals is presented. The method is based on a hidden Markov model (HMM), which takes the harmonic pattern of the atrial activity into account. Using the HMM-based method, the average RMS error of the frequency estimates at high noise levels was significantly lower compared to existing methods. In Paper III, the HMM-based method is employed for analysis of 24-h ambulatory ECG signals in order to explore circadian variation in AF frequency. Circadian variations reflect autonomic modulation; attenuation or absence of such variations may help to diagnose patients. Methods based on curve fitting, autocorrelation, and joint variation, respectively, are employed to quantify circadian variations, showing that it is present in most patients with long-standing persistent AF, although the short-term variation is considerable. In Paper IV, 24-h ambulatory ECG recordings with paroxysmal and persistent AF are analyzed using an entropy-based method for characterization of the atrial activity. Short segments are classified based on these measures, showing that it is feasible to distinguish between patient with paroxysmal and persistent AF from 10-s ECGs; the average classification rate was above 95%. The ventricular response during AF is mainly determined by the AV nodal blocking of atrial impulses. In Paper V, a new model-based approach for analysis of the ventricular response during AF is proposed. The model integrates physiological properties of the AV node and the atrial fibrillatory rate; the model parameters can be estimated from ECG signals. Results show that ventricular response is sufficiently represented by the estimated model in a majority of the recordings; in 85.7% of the analyzed 30-min segments the model fit was considered accurate, and that changes of AV nodal properties caused by autonomic modulation could be tracked through the estimated model parameters. In summary, the work within this thesis contributes with new methods for non-invasive analysis of AF, which can be used to tailor and evaluate different strategies for AF treatment

Abnormal ECG search in long-term electrocardiographic recordings from an animal model of heart failure

Heart failure is one of the leading causes of death in the United States. Five million Americans suffer from heart failure. Advances in portable electrocardiogram (ECG) monitoring systems and large data storage space allow the ECG to be recorded continuously for long periods. Long-term monitoring could potentially lead to better diagnosis and treatment if the progression of heart failure could be followed. The challenge is to analyze the sheer mass of data. Manual analysis using the classical methods is impossible. In this dissertation, a framework for analysis of long-term ECG recording and methods for searching an abnormal ECG are presented.;The data used in this research were collected from an animal model of heart failure. Chronic heart failure was gradually induced in rats by aldosterone infusion and a high Na and low Mg diet. The ECG was continuously recorded during the experimental period of 11-12 weeks through radiotelemetry. The ECG leads were placed subcutaneously in lead-II configuration. In the end, there were 80 GB of data from five animals. Besides the massive amount of data, noise and artifacts also caused problems in the analysis.;The framework includes data preparation, ECG beat detection, EMG noise detection, baseline fluctuation removal, ECG template generation, feature extraction, and abnormal ECG search. The raw data was converted from its original format and stored in a database for data retrieval. The beat detection technique was improved from the original algorithm so that it was less sensitive to signal baseline jump and more sensitive to beat size variation. A method for estimating a parameter required for baseline fluctuation removal is proposed. It provides a good result on test signals. A new algorithm for EMG noise detection was developed using morphological filters and moving variance. The resulting sensitivity and specificity are 94% and 100%, respectively. A procedure for ECG template generation was proposed to capture gradual change in ECG morphology and manage the matching process if numerous ECG templates are created. RR intervals and heart rate variability parameters are extracted and plotted to display progressive changes as heart failure develops. In the abnormal ECG search, premature ventricular complexes, elevated ST segment, and split-R-wave ECG are considered. New features are extracted from ECG morphology. The Fisher linear discriminant analysis is used to classify the normal and abnormal ECG. The results provide classification rate, sensitivity, and specificity of 97.35%, 96.02%, and 98.91%, respectively

Circadian Genes in, EWD-8, Triple-Negative Breast Cancer Cells May Demonstrate Rhythmicity in Vitro

Studies have shown a link between greater rates and risks of breast cancer in women that work night shifts, such that this type of work has been labeled a probable carcinogen. It has been suggested that the disruption of circadian genes- PER and CRY - could be partly responsible for this increased breast cancer risk. The suprachiasmatic nucleus (SCN) in the brain is the central circadian pacemaker; however, how and if Triple-negative breast cancer (TNBC) cells, an aggressive type of breast cancer that is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), as well as HER-2 growth factor receptors, express particular circadian clock genes when disconnected from the “master clock” is not extensively researched nor understood. This study’s goal was to answer the following questions: do triple-negative breast cancer cells (TNBCs) express circadian genes? Do TNBCs exhibit rhythmic circadian gene expression in vitro? Are TNBCs and the circadian genes responsive to external red-light exposure at a wavelength shown to alter mitochondrial function? Cells were cultured in vitro, exposed or not exposed to red light, and then harvested at 6-hour time intervals for 24 hours. The harvested cells endured RNA isolation, followed by cDNA synthesis and PCR to amplify circadian genes PER1, PER2, PER3, CRY1, CRY2. The expression level was compared to constitutively expressed reference genes to determine whether circadian rhythmicity is present in TNBC cells growing in vitro

[Circadian Genes in, EWD-8, Triple-Negative Breast Cancer Cells May Demonstrate Rhythmicity in Vitro

Studies have shown a link between greater rates and risks of breast cancer in women that work night shifts, such that this type of work has been labeled a probable carcinogen. It has been suggested that the disruption of circadian genes- PER and CRY - could be partly responsible for this increased breast cancer risk. The suprachiasmatic nucleus (SCN) in the brain is the central circadian pacemaker; however, how and if Triple-negative breast cancer (TNBC) cells, an aggressive type of breast cancer that is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), as well as HER-2 growth factor receptors, express particular circadian clock genes when disconnected from the “master clock” is not extensively researched nor understood. This study’s goal was to answer the following questions: do triple-negative breast cancer cells (TNBCs) express circadian genes? Do TNBCs exhibit rhythmic circadian gene expression in vitro? Are TNBCs and the circadian genes responsive to external red-light exposure at a wavelength shown to alter mitochondrial function? Cells were cultured in vitro, exposed or not exposed to red light, and then harvested at 6-hour time intervals for 24 hours. The harvested cells endured RNA isolation, followed by cDNA synthesis and PCR to amplify circadian genes PER1, PER2, PER3, CRY1, CRY2. The expression level was compared to constitutively expressed reference genes to determine whether circadian rhythmicity is present in TNBC cells growing in vitro