<p dir="ltr">In this dissertation, we utilized the zebrafish model and the human neutrophil model to investigate the novel mechanisms that regulate neutrophil motility and chemotaxis.</p>

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[ES] Las antraciclinas son fármacos antineoplásicos ampliamente utilizados en el trata-miento de varios tipos de cáncer, incluyendo tanto tumores sólidos como hematoló-gicos. A pesar de su eficacia, su uso se ve limitado por su efecto cardiotóxico. El aumento de los supervivientes de cáncer, especialmente pediátrico, ha provocado que cada vez haya más personas con cardiotoxicidad inducida por antraciclinas. Por este motivo, es necesaria la búsqueda de nuevos modelos de enfermedad relevan-tes para comprender la fisiopatología del daño cardíaco inducido por antraciclinas, así como el desarrollo de nuevas terapias cardioprotectoras que permitan el uso de las antraciclinas evitando su efecto cardiotóxico. En este trabajo se ha estudiado, por una parte, la susceptibilidad al daño por do-xorrubicina (una de las principales antraciclinas empleadas en clínica) de cardiomio-citos obtenidos a partir de células madre pluripotentes inducidas derivadas de pa-cientes pediátricos oncológicos que experimentaron cardiotoxicidad causada por an-traciclinas. Los cardiomiocitos de los pacientes fueron tratados con doxorrubicina y se evaluaron diferentes parámetros, incluyendo la viabilidad, apoptosis, estrés oxida-tivo, daño genómico, daño mitocondrial, desorganización sarcomérica, etc. compa-rándolos con cardiomiocitos control. Nuestros resultados mostraron que estos car-diomiocitos recapitulan la susceptibilidad a la doxorrubicina observada en los pacien-tes, constituyendo un buen modelo de enfermedad para estudiar los mecanismos de cardiotoxicidad de la doxorrubicina o el cribado de fármacos. Por otra parte, se ha evaluado el papel cardioprotector de un miARN, el miR-4732-3p, frente al daño inducido por antraciclinas. Este miARN está desregulado en pa-cientes con cáncer de mama que sufrieron cardiotoxicidad inducida por antraciclinas. Para comprobar su efecto cardioprotector, este miARN fue sobreexpresado en célu-las cardíacas de rata, las cuales fueron tratadas con doxorrubicina, observándose que incrementaba la supervivencia de las células y reducía el estrés oxidativo. Tam-bién se estudió la cardioprotección in vivo en un modelo de cardiotoxicidad inducida por doxorrubicina en rata, observando que mejora la función cardíaca, reduce la fi-brosis intersticial y el estrés oxidativo. Además, se hizo un estudio de los posibles genes diana de este miARN. En su conjunto, nuestros resultados muestran que el miR-4732-3p tiene un efecto cardioprotector frente al daño por doxorrubicina, y po-dría ser una herramienta terapéutica para el tratamiento del daño cardíaco causado por las antraciclinas. / [CAT] Les antraciclines són fàrmacs antineoplàstics àmpliament utilitzats en el tractament de diversos tipus de càncer, incloent tant tumors sòlids com hematològics. Tot i la seva eficàcia, el seu ús es veu limitat pel seu efecte cardiotòxic. L'augment dels su-pervivents de càncer, especialment pediàtric, ha provocat que cada cop hi hagi més persones amb cardiotoxicitat induïda per antraciclines. Per aquest motiu, cal cercar nous models de malaltia rellevants per comprendre la fisiopatologia del dany cardíac induït per antraciclines, així com el desenvolupament de noves teràpies cardiopro-tectores que permetin l'ús de les antraciclines evitant-ne l'efecte cardiotòxic. En aquest treball s'ha estudiat, d'una banda, la susceptibilitat al dany per doxorrubi-cina (una de les principals antraciclines emprades en clínica) de cardiomiòcits obtin-guts a partir de cèl¿lules mare pluripotents induïdes derivades de pacients pediàtrics oncològics que van experimentar cardiotoxicitat causada per antraciclines. Els cardi-omiòcits dels pacients van ser tractats amb doxorrubicina i es van avaluar diferents paràmetres, incloent-hi la viabilitat, apoptosi, estrès oxidatiu, dany genòmic, dany mitocondrial, desorganització sarcomèrica, etc. comparant-los amb cardiomiòcits control. Els nostres resultats van mostrar que aquests cardiomiòcits recapitulen la susceptibilitat a la doxorrubicina observada en els pacients, constituint un bon model de malaltia per estudiar els mecanismes de cardiotoxicitat de la doxorrubicina o el cribratge de fàrmacs. D'altra banda, s'ha avaluat el paper cardioprotector d'un miARN, el miR-4732-3p, davant del dany induït per antraciclines. Aquest miARN està desregulat en pacients amb càncer de mama que van patir cardiotoxicitat induïda per antraciclines. Per comprovar el seu efecte cardioprotector, aquest miARN va ser sobreexpressat en cèl·lules cardíaques de rata, les quals van ser tractades amb doxorrubicina, obser-vant-se que incrementava la supervivència de les cèl·lules i reduïa l'estrès oxidatiu. També es va estudiar la cardioprotecció in vivo en un model de cardiotoxicitat induï-da per doxorrubicina en rata, observant que millora la funció cardíaca, redueix la fi-brosi intersticial i l'estrès oxidatiu. A més, es va fer un estudi dels possibles gens di-ana d'aquest miARN. En conjunt, els nostres resultats mostren que el miR-4732-3p té un efecte cardioprotector davant el dany per doxorrubicina, i podria ser una eina terapèutica per al tractament del dany cardíac causat per les antraciclines. / [EN] Anthracyclines are drugs widely used in the treatment of several types of cancer, including both solid tumors and hematologic malignancies. Despite its proven effica-cy, its use is hampered by its cardiotoxic effect. The increase in cancer survivors, especially pediatric, has led to more and more people with anthracycline-induced cardiotoxicity. Therefore, it is necessary to search for new relevant disease models to better understand the physiopathology of cardiac damage-induced by anthracy-clines, as well as the development of new cardioprotective therapies that allow the clinic use of anthracyclines avoiding their cardiotoxic effect. In this work we have studied, on the one hand, the susceptibility against doxorubicin damage (major anthracycline used in clinic) of cardiomyocytes obtained from in-duced pluripotent stem cells derived from oncology pediatric patients that underwent cardiotoxicity-induced by anthracyclines. Cardiomyocytes from these patients were treated with doxorubicin, and we evaluated several parameters, including cell viabil-ity, apoptosis, oxidative stress, genomic damage, mitochondrial damage, sarcomere disorganization, etc. comparing the results with control cardiomyocytes. Our results showed that these cardiomyocytes recapitulate the susceptibility against doxorubicin observed in the patients, making them a good disease model to study cardiotoxicity mechanisms of doxorubicin or drug screening. On the other hand, we evaluated the cardioprotective role of one miRNA, miR-4732-3p, against doxorubicin-induced damage. This miRNA is dysregulated in breast can-cer patients that suffered cardiotoxicity-induced by anthracyclines. To test its cardio-protective effect, this miRNA was overexpressed in rat cardiac cells that were treated with doxorubicin, showing an increment of cell survival and a reduction of oxidative stress levels. We also studied in vivo cardioprotection in a doxorubicin-induced cardi-otoxicity model in rat, showing an improvement in cardiac function, reduced intersti-tial fibrosis and reduced oxidative stress levels. Moreover, we studied possible target genes of this miARN. Overall, our results showed that miR-4732-3p has a cardiopro-tective role against doxorrubicin-induced damage and could be used as a therapeutic tool for treatment of cardiac damage caused by anthracycline. / Reinal Ferré, I. (2023). Modelado de la cardiotoxicidad inducida por antraciclinas en hiPSC-CM de pacientes oncológicos pediátricos y estudio del papel cardioprotector del miR-4732-3p [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/193075

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La polyarthrite rhumatoïde (PR) est la maladie auto-immune la plus fréquente d'une prévalence de 1%. Les cellules résidentes de la cavité synoviale, les fibroblast-like synoviocytes (FLS), sont des acteurs majeurs de la PR. Leur activation par des récepteurs de l'immunité innée participe à l'acquisition d'un phénotype agressif menant à la destruction ostéo-articulaire. Dans cette étude, nous avons évalué le rôle régulateur de miARN sur les voies de signalisation des Toll-like receptors (TLR). L'activation de TLR2 et de TLR4 dans les FLS induit la diminution de l'expression de plusieurs miARN, dont miR-19a et b (miR-19), alors que TLR2 est surexprimé. Nous avons pu ainsi montrer que miR-19 régule Tlr2 et que la transfection de mir-19 dans les FLS activés induit une diminution de l'expression de TLR2 et de la synthèse d'IL-6 et de MMP-3. Mir-19 appartient au cluster miR-17~92, dont l'expression est abaissée dans les FLS. Il code pour 6 miARN dont miR-20a. miR-20a est également sous-régulé après activation de TLR2 et TLR4 dans les FLS et les THP-1. Nous avons montré que miR-20a régule directement l'expression d'Ask1, impliquée et surexprimée après activation de TLR4. La transfection de miR-20a in vitro nous a permis de montrer que miR-20a contrôle l'expression d'ASK1 et induit une inhibition de la synthèse de cytokines majeures de la PR dans les FLS et les THP-1. Des résultats équivalents ont été obtenus ex vivo chez la souris. Ces travaux ont permis d'identifier dans les FLS rhumatoïdes des miARN anti-inflammatoires dont la baisse d'expression permet une augmentation de l'expression de TLR2 et d'ASK1. Ces miARN pourraient donc constituer de nouvelles cibles thérapeutiques.

Background-Previously we found that smooth muscle cell (SMC)-specific knockout of miR-17 similar to 92 attenuates hypoxia-induced pulmonary hypertension. However, the mechanism underlying miR-17 similar to 92-mediated pulmonary artery SMC (PASMC) proliferation remains unclear. We sought to investigate whether miR-17 similar to 92 regulates hypoxia-inducible factor (HIF) activity and PASMC proliferation via prolyl hydroxylases (PHDs). Methods and Results-We show that hypoxic sm-17 similar to 92(-/-) mice have decreased hematocrit, red blood cell counts, and hemoglobin contents. The sm-17 similar to 92 (-/-) mouse lungs express decreased mRNA levels of HIF targets and increased levels of PHD2. miR-17 similar to 92 inhibitors suppress hypoxia-induced levels of HIF1 alpha, VEGF, Glut1, HK2, and PDK1 but not HIF2 alpha in vitro in PASMC. Overexpression of miR-17 in PASMC represses PHD2 expression, whereas miR-17/20a inhibitors induce PHD2 expression. The 3'-UTR of PHD2 contains a functional miR-17/20a seed sequence. Silencing of PHD2 induces HIF1a and PCNA protein levels, whereas overexpression of PHD2 decreases HIF1 alpha and cell proliferation. SMC-specific knockout of PHD2 enhances hypoxia-induced vascular remodeling and exacerbates established pulmonary hypertension in mice. PHD2 activator R59949 reverses vessel remodeling in existing hypertensive mice. PHDs are dysregulated in PASMC isolated from pulmonary arterial hypertension patients. Conclusions-Our results suggest that PHD2 is a direct target of miR-17/20a and that miR-17 similar to 92 contributes to PASMC proliferation and polycythemia by suppression of PHD2 and induction of HIF1 alpha.

Application of cytotoxic chemotherapy still remains the essential treatment strategy in advanced colorectal cancer. The intrinsic and acquired drug resistance represents one of the reasons that may even lead to failure of cancer therapy. The DNA damage response pathways have been shown to play an important role in the development of chemoresistance. There is sufficient evidence showing the high-frequency deregulated expression of many DNA repair genes across multiple cancer types. An example of such gene in colorectal cancer is MRE11, which encodes protein known as a sensor of DNA double-strand breaks. In year 2016, there was a substantial study published by our group at The Department of Molecular Biology of Cancer (IEM CAS, Prague), the study analysed the association of polymorphisms in predicted microRNA target sites of double-strand breaks (DSBs) repair genes, including MRE11, and clinical outcome and efficacy of chemotherapy in colorectal cancer. Our hypothesis, based on the mentioned study, is that specifically and exactly defined microRNAs with ability to regulate certain DNA repair proteins may not only affect the survival of colorectal cancer cells, but also the sensitivity to chemotherapy. In practical part of the submitted thesis we have identified miR-140 as a potential regulator of...

This thesis concerns with the recognition of music instruments from isolated notes. Music instrument recognition is a relatively nascent problem fast gaining importance not only because of the academic value the problem provides, but also for the potential it has in being able to realize applications like music content analysis, music transcription etc. Line spectral frequencies are proposed as features for music instrument recognition and shown to perform better than Mel filtered cepstral coefficients and linear prediction cepstral coefficients. Assuming a linear model of sound production, features based on the prediction residual, which represents the excitation signal, is proposed. Four improvements are proposed for classification using Gaussian mixture model (GMM) based classifiers. One of them involves characterizing the regions of overlap between classes in the feature space to improve classification. Applications to music instrument recognition and speaker recognition are shown. An experiment is proposed for discovering the hierarchy in music instrument in a data-driven manner. The hierarchy thus discovered closely corresponds to the hierarchy defined by musicians and experts and therefore shows that the feature space has successfully captured the required features for music instrument characterization.

Turbulent flows and coherent structures emerging within turbulent flow fields have been extensively studied for the past few decades and a wide variety of experimental and numerical techniques have been developed for measurement and analysis of turbulent flows. The complex nature of turbulence requires methods that can accurately estimate its highly chaotic spatial and temporal behavior. Some of the classical cases of turbulent flows with simpler geometries have been well characterized by means of the existing experimental techniques and numerical models. Nevertheless, since most turbulent fields are of complex geometries; there is an increasing interest in the study of turbulent flows through models with more complicated geometries. In this dissertation, characteristics of turbulent flows through two different facilities with complex geometries are studied applying two different experimental methods. The first study involves the investigation of turbulent impinging jets through a staggered array of rods with or without crossflow. Such flows are crucial in various engineering disciplines. This experiment aimed at modeling the coolant flow behavior and mixing phenomena within the lower plenum of a Very High Temperature Reactor (VHTR). Dynamic Particle Image Velocimetry (PIV) and Matched Index of Refraction (MIR) techniques were applied to acquire the turbulent velocity fields within the model. Some key flow features that may significantly enhance the flow mixing within the test section or actively affect some of the structural components were identified in the velocity fields. The evolution of coherent structures within the flow field is further investigated using a Snapshot Proper Orthogonal Decomposition (POD) technique. Furthermore, a comparative POD method is proposed and successfully implemented for identification of the smaller but highly influential coherent structures which may not be captured in the full-field POD analysis. The second experimental study portrays the coolant flow through the core of an annular pebble bed VHTR. The complex geometry of the core and the highly turbulent nature of the coolant flow passing through the gaps of fuel pebbles make this case quite challenging. In this experiment, a high frequency Hot Wire Anemometry (HWA) system is applied for velocity measurements and investigation of the bypass flow phenomena within the near wall gaps of the core. The velocity profiles within the gaps verify the presence of an area of increased velocity close to the outer reflector wall; however, the characteristics of the coolant flow profile is highly dependent on the gap geometry and to a less extent on the Reynolds number of the flow. The time histories of the velocity are further analyzed using a Power Spectra Density (PSD) technique to acquire information about the energy content and energy transfer between eddies of different sizes at each point within the gaps.

Chez les vertébrés, le battement coordonné des cils motiles présents par centaines à la surface apicale des cellules multiciliées (MCC) est requis pour propulser directionnellement les fluides biologiques à l'intérieur de certains organes (voies respiratoires, ventricules cérébraux, trompes utérines ou certaines structures embryonnaires). De nombreuses pathologies humaines sont associées à des défauts ciliaires ou à une perte des MCC (dyskinésies ciliaires, mucoviscidose, asthme,...). Dans ce contexte, mon travail de thèse a consisté à élucider les mécanismes complexes contrôlant la différenciation des MCC et donc la formation des cils motiles (multiciliogenèse). Par des approches de génomiques fonctionnelles à partir de deux modèles d'épithéliums multiciliés évolutivement éloignés (épithélium respiratoire humain et épiderme d'embryon de Xénope) nous avons identifié la famille des microARN (petits ARN non-codants régulateurs de l'expression génique) miR-449 comme majoritairement exprimée dans les MCC. Nous avons montré que miR-449 contrôle la multiciliogenèse i) en bloquant le cycle cellulaire, ii) en réprimant directement la voie de signalisation Notch et iii) en inhibant l'expression de la petite GTPase R-Ras. Enfin, nos travaux montrent que l'ensemble de ces mécanismes est conservé chez les vertébrés. En conclusion, miR-449 est un nouveau régulateur clé de la multiciliogenèse conservé au cours de l'évolution. Nos résultats pourraient ouvrir la voie à de nouvelles stratégies thérapeutiques utilisant des petits ARN régulateurs dans le traitement de certaines pathologies associées à des défauts ciliaires.