The role of WNT ligand secretion in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease of yet unknown etiology characterized by a progressive decrease of lung function and poor prognosis. The cellular hallmarks of IPF are repetitive epithelial cell injury that leads to aberrant activity of developmental pathways, such as WNT signaling pathway, and activation of myofibroblasts which secrete extracellular matrix components in excess. Extracellular vesicles (EVs) are secreted membranous particles that transport diverse signaling mediators such as proteins, nucleic acids and lipids, over long distances thus mediating intercellular communication. The Non-canonical ligand WNT-5A is upregulated in IPF, especially in lung fibroblasts from patients in comparison to donors. Recently, it was discovered that WNT ligands are secreted on EVs which can activate WNT signaling in the receptor cells. However, the role of EVs and the secretion of WNT ligands through EVs in IPF remain largely unknown. The present work hypothesizes that EV secretion is increased in IPF and the WNT-ligand WNT-5A is transported on EVs which contributes to profibrotic lung fibroblast function. In order to characterize the EV secretion profile in IPF and elucidate the contribution of EV-bound WNT-5A to the disease, EVs were isolated from bronchoalveolar lavage fluid (BALF) from experimental lung fibrosis as well as from IPF, non IPF-ILD, non-ILD and healthy volunteer samples from two independent cohorts or from primary human lung fibroblasts (phLFs). EVs were then characterized by transmission electron microscopy, nanoparticle tracking analysis and Western Blotting (WB). These experiments reported an upregulated EV secretion in BALF from both experimental and human lung fibrosis samples with an increase in the content of the WNT-ligand WNT-5A. In addition, WNT-5A secretion on EVs was induced in primary human lung fibroblasts (phLFs) upon fibrotic stimulation by TGF-β. For functional studies, phLFs were used for EV stimulation and analysed by metabolic activity assays, qPCR and WB upon WNT-5A loss-of-function and WNT-5A stimulation studies. The phLF-derived EVs were able to promote phLF proliferation, which was attenuated by WNT-5A silencing and antibody-mediated WNT-5A inhibition. Similarly, EVs from IPF-BALF were capable of inducing phLF proliferation which was dependent on the EV-bound WNT-5A ligand. Taken together, this thesis showed that increased secretion of EVs in the IPF lung mediates WNT-5A signaling, which contributes to disease pathogenesis by increasing lung fibroblast proliferation. Future studies of EV secretion and composition, as well as their role in disease pathogenesis may lead to novel approaches for the diagnosis and treatment of pulmonary fibrosis.Die idiopathische Lungenfibrose (IPF) ist eine chronische interstitielle Lungenerkrankung mit bisher unbekannter Ätiologie, die durch eine fortschreitende Abnahme der Atemkapazität und einer sehr schlechten Prognose gekennzeichnet ist. Kennzeichenend fuer die IPF ist die wiederholt auftretende epitheliale Zellschädigung, die zu aberranten Aktivität von Entwicklungssignalwegen wie dem WNT-Signalweg und der Aktivierung von Myofibroblasten führen, die infolgedessen vermehrt extrazelluläre Matrixkomponenten sekretieren. Extrazelluläre Vesikel (EVs) sind sekretierte Vesikel, die verschiedene Signalmoleküle wie Proteine, Nukleinsäuren und Lipide über weite Strecken transportieren und so die interzelluläre Kommunikation fördern. Es wurde festgestellt, dass der nicht-kanonische WNT-5A-Ligand in der IPF hochreguliert ist, insbesondere bei Lungenfibroblasten von IPF Patienten im Vergleich zu gesunden Spendern. Vor Kurzem wurde entdeckt, dass WNT-Liganden durch EVs transportiert werden und in der Lage sind, die WNT-Signalübertragung in den Rezeptorzellen zu aktivieren. Die Rolle von EVs und die Sekretion von WNT-Liganden in EVs ist in der IPF jedoch unbekannt. Die vorliegende Arbeit basiert auf der Hypothese, dass die EV-Sekretion in IPF erhöht ist und dass WNT-5A vermehrt in EVs sekretiert wird was zu einem profibrotischen Phänotyp der Lungenfibroblasten beiträgt. Um das EV-Sekretionsprofil in IPF zu charakterisieren und den Beitrag von EV-gebundenem WNT-5A zur Entwicklung und Fortschreiten der Krankheit aufzuklären, wurden EVs aus bronchoalveolarer Lavageflüssigkeit (BALF) von experimenteller Lungenfibrose sowie von IPF , nicht IPF-ILD und nicht ILD sowie Gesunden Proben aus zwei unterschiedlichen Kohorten, isoliert. Die EVs wurden dann durch Transmissionselektronenmikroskopie, Nanopartikel-Tracking-Analyse und Western Blot (WB) charakterisiert. Primäre humane Lungenfibroblasten (phLFs) wurden zur EV-Isolierung verwendet und durch Stoffwechselaktivitätstests, qPCR und WB nach WNT-5A-Funktionsverlust- und WNT-5A-Stimulationsstudien analysiert. Diese Experimente zeigen eine hochregulierte EV-Sekretion in BALF von sowohl experimenteller als auch humaner Lungenfibrose mit einem Anstieg des WNT-Liganden WNT-5A-. Zusätzlich wurde die WNT-5A-Sekretion auf EVs in primären menschlichen Lungenfibroblasten (phLFs) nach fibrotischer Stimulation durch TGF-β induziert. Darüber hinaus konnten die von phLF sekretierten EVs die Proliferation von phLF fördern, die durch WNT-5A-Inhibierung durch siRNA oder Antikörper Behandlung abgeschwächt wurde. In ähnlicher Weise waren EVs aus IPF-BALF in der Lage, eine PhLF-Proliferation zu induzieren, die vom EV-gebundenen WNT-5A-Liganden abhängig war. In dieser Doktorarbeit konnte gezeiget werden, dass eine erhöhte Sekretion von EVs im BALF von IPF-Erkrankten die Signalübertragung von WNT-5A vermittelt, die durch Erhöhung der Proliferation von Lungenfibroblasten zur Pathogenese der Erkrankung beiträgt. Zukünftige Studien zur Sekretion und Zusammensetzung von Evs, sowie deren Rolle bei der Pathogenese der IPF, könnten zu neuen Ansätzen für die Diagnose und Behandlung von Lungenfibrose führen

Estudio del comportamiento del gas radón (222Rn) en los procesos de transferencia en agua y en aire

[ES] El radón (222Rn) es un gas radiactivo, de origen natural, generado a partir de la desintegración radiactiva del radio (226Ra). Clasificado como elemento cancerígeno por la Organización Mundial de la Salud, es la segunda causa de cáncer de pulmón en la población. La elevada peligrosidad de este gas ha fomentado e impulsado numerosos estudios y análisis dentro del ámbito de la protección radiológica. En este contexto de avance y mejora tecnológica y dada la necesidad de estudiar el comportamiento físico del gas radón, se plantea el objetivo principal de esta investigación. La presente Tesis Doctoral se basa en un análisis del comportamiento difusivo del gas radón durante su acumulación en aire y en procesos de transferencia en los que coexisten agua y aire. En primer lugar, se describe un estudio de caso real, basado en mediciones de campo que suponen el punto de partida de la tesis y que han promovido el desarrollo de la metodología necesaria para poder implementar los ensayos a escala laboratorio. Se han llevado a cabo diferentes procedimientos experimentales que permiten monitorizar la evolución de la concentración de radón en el tiempo. Por un lado, se analiza únicamente en aire, cuando el gas se acumula en el interior de depósitos de diferentes volúmenes, y por otro, se estudia la transferencia del radón del agua al aire durante los procesos de acumulación y decaimiento, tanto en condiciones de agua en estado estático como en agitación. A partir de los resultados obtenidos de concentración de radón en aire, se determina la concentración máxima alcanzable y la tasa de emisión generada por cada fuente muestreada. Por último, el tratamiento de datos de los resultados experimentales de transferencia de radón en los ensayos agua-aire, han requerido el desarrollo de modelos dinámicos que reproducen el comportamiento del gas en las diferentes condiciones ensayadas. Esto ha permitido determinar parámetros característicos del proceso difusivo del gas radón como el coeficiente de velocidad de transferencia del gas de un medio a otro y la tasa de emanación del medio acuoso. Se han validado estos resultados mediante la comparación de las concentraciones de radón en agua y en aire estimadas a partir del modelo analítico para las diferentes condiciones ensayadas, con los valores experimentales correspondientes. El ajuste preciso de estos modelos dinámicos permite predecir las concentraciones de radón en otras condiciones de ensayo.[CA] El radó (222Rn) és un gas radioactiu, d'origen natural, generat a partir de la desintegració radioactiva del radi (226Ra). Classificat com a element cancerigen per la Organització Mundial de la Salut, és la segona causa de càncer de pulmó en la població. L'elevada perillositat d'aquest gas ha promogut i impulsat nombrosos estudis i anàlisi en l'àmbit de la protecció radiològica. En aquest context d'avanç i millora tecnològica i degut a la necessitat d'estudiar el comportament físic del gas radó, es planteja l'objectiu principal d'aquesta investigació. Aquesta Tesis Doctoral es basa en una anàlisi del comportament difusiu del gas radó durant la seua acumulació en aire i en processos de transferència en els que estan en contacte agua i aire. En primer lloc, es descriu un estudi de cas real, basat en mesures de camp que suposa el punt de partida i que han permés el desenvolupament de la metodologia necessària per poder implementar els estudis a escala laboratori. S'han dut a terme diversos procediments experimentals que permeten monitoritzar l'evolució de la concentració de radó en funció del temps. Per una banda, s'analitzen els nivells únicament en aire, quan el gas s'acumula en l'interior de depòsits de diferents volums i per altra, per estudiar la transferència de radó de l'aigua a l'aire, tant en processos d'acumulació com de decaïment, quan l'aigua es troba en estat estàtic i en agitació. A partir dels resultats experimentals obtinguts de concentració de radó en aire, es determina la concentració màxima, i la taxa d'emissió generada per cada font mostrejada. Per últim, el tractament de les dades de de transferència de radó en els assaigs agua-aire, requereixen del desenvolupament de models dinàmics que reprodueixen el comportament del gas en les diferents condicions. Açò ha permés determinar paràmetres característics del procés difusiu del gas d'un medi a altre així com la taxa d'emanació de l'aigua. S'han validat els resultats mitjançant la comparació de les concentracions de radó en aigua i en aire, estimades a partir del model analític, amb els valors experimentals del laboratori. L'ajust precís d'aquests models dinàmics permet predir les concentracions de radó en altres condicions de mostreig.[EN] Radon (222Rn) is a naturally occurring radioactive gas generated from radium (226Ra) decay. Classified as a carcinogen by the World Health Organization, it is the second cause of lung cancer. The hazardous nature of this gas has encouraged numerous studies and investigations within the field of radiological protection. In hits context of technological progress and improvement, according to the importance of studying radon physical behavior, the main goal of this research is proposed. This PhD thesis provides an analysis of the diffusive behavior of radon gas during its accumulation in air and in transfer processes in which water and air coexist. First, a real study case is described, based on field measurements which are the starting point of the thesis and has driven the development of the methodology to be able to implement the tests at laboratory scale. Different experimental procedures have been carried out to evaluate the radon concentration. On the one hand, it is analyzed only in air, when the gas accumulates inside tanks of different volumes and, on the other hand, the radon transfer from water to air, during accumulation and decay processes are studied, both in conditions of water in static state and in turbulence. From the results obtained for radon concentration in air, the maximum achievable concentration and the emission rate generated by each sampled source is determined. Finally, the data processing of the experimental results of radon transfer in the water-air tests has required the development of dynamic models that reproduce the gas behavior in the different conditions tested. It has been determined characteristic parameters of the diffusive process of radon gas, such as the gas transfer rate coefficient from one medium to another and the emanation rate from water to air. These results have been validated by comparing the radon concentrations in water and air, estimated from the analytical model for the different conditions tested, with the corresponding experimental values. Accurate fitting of these dynamic models allows prediction of radon concentrations under other test conditions.Noverques Medina, A. (2022). Estudio del comportamiento del gas radón (222Rn) en los procesos de transferencia en agua y en aire [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185792TESI

El greix que aprima

Títol del póster : Brown adipose tissue

Experimental radon exhalation measurements: Comparison of different techniques

[EN] Radon is a gas produced from the radioactive natural disintegration of radium and uranium, which is present in soils and different building materials. Due to its gaseous nature, it emanates to the surface and penetrates both by convection and diffusion into the houses. According to recent studies, radon is considered the second leading cause of lung cancer, and it has prompted legislative changes to reduce exposure to this gas. In Spain, the current legislation -R.D. 783/2001 Regulation on Sanitary Radiation Protection and IS-33 Instruction of the Nuclear Safety Council, establishes uniform rules for workers¿ health protection in radioactive installations. In 2013, the European legislation was updated with Directive 2013/59/EURATOM which sets limit values and action plans for radon exposure for homes, public buildings and workplaces for any entrance roads (soil, building materials or water). This Directive must be incorporated before 18th February 2018 into Spanish legislation, so it is necessary to perform, prior to that date, appropriate protocol techniques and methodologies for the measurement radon concentrations in air. This work aims at a preventive approach to the situation. That is, to measure the concentration levels of radon exhaled prior to the design and construction of the dwellings to avoid their accumulation inside The main purpose of this work is the comparison of different radon exhalation measurement techniques in order to define the most appropriate methodology in different cases for the implementation of the new radon control Directive.Noverques-Medina, A.; Verdú Martín, GJ.; Juste Vidal, BJ.; Sancho, M. (2019). Experimental radon exhalation measurements: Comparison of different techniques. Radiation Physics and Chemistry. 155:319-322. https://doi.org/10.1016/j.radphyschem.2018.08.002S31932215

Ultra rapid in vivo screening for anti-Alzheimer anti-amyloid drugs

More than 46 million people worldwide suffer from Alzheimer's disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid β-peptide (Aβ), considered one of the main culprits in Alzheimer's disease. Limitations in monitoring the aggregation of Aβ in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aβ aggregation in vivo in realtime, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aβ in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aβ aggregation and the effect of potential antiaggregating agents

Study of potential capacity as adsorbent of Moringa oleifera substrates for treatment of radon contaminated air in indoor spaces: Preliminary test

[EN] Radon is a radioactive gas known to be a human carcinogenic element that causes lung cancer. The Directive 2013/59/EURATOM establishes action plans for its monitorization and control in water and air specially at workplaces. There are several techniques to reduce the concentration of radon in air mainly based on improving ventilation rates. However, intelligent and energy-efficient buildings are well insulated and have centralized ventilation systems where air is recirculated continuously. This strategy has a negative influence on radon accumulation at indoor spaces. So, ventilation systems should be composed by filters with suitable materials to adsorb radon from indoor air. This work studies the radon adsorption ability of the most used adsorbent (activated carbon) and some not-processed substrates coming from Moringa oleifera, a natural plant with high potential as adsorbent for heavy metals and coagulant in and water treatment. The radon adsorption efficiency of the different solids is analyzed, showing promising results for radionuclide removal from air.García-Fayos, B.; Juste Vidal, BJ.; Sancho, M.; Arnal Arnal, JM.; Noverques-Medina, A.; Verdú Martín, GJ. (2020). Study of potential capacity as adsorbent of Moringa oleifera substrates for treatment of radon contaminated air in indoor spaces: Preliminary test. Radiation Physics and Chemistry. 167:1-3. https://doi.org/10.1016/j.radphyschem.2019.04.012S13167Garcia-Fayos, B., Arnal, J. M., Piris, J., & Sancho, M. (2016). Valorization of Moringa oleifera seed husk as biosorbent: isotherm and kinetics studies to remove cadmium and copper from aqueous solutions. Desalination and Water Treatment, 57(48-49), 23382-23396. doi:10.1080/19443994.2016.1180473García-Tobar, J. (2014). Influencia de las condiciones meteorológicas sobre el nivel de radón a corto plazo en un laboratorio subterráneo. Nova Scientia, 6(12), 78. doi:10.21640/ns.v6i12.25George, A. C. (1984). Passive, Integrated Measurement of Indoor Radon Using Activated Carbon. Health Physics, 46(4), 867-872. doi:10.1097/00004032-198404000-00012Guyot, G., Sherman, M. H., & Walker, I. S. (2018). Smart ventilation energy and indoor air quality performance in residential buildings: A review. Energy and Buildings, 165, 416-430. doi:10.1016/j.enbuild.2017.12.051Karunakara, N., Sudeep Kumara, K., Yashodhara, I., Sahoo, B. K., Gaware, J. J., Sapra, B. K., & Mayya, Y. S. (2015). Evaluation of radon adsorption characteristics of a coconut shell-based activated charcoal system for radon and thoron removal applications. Journal of Environmental Radioactivity, 142, 87-95. doi:10.1016/j.jenvrad.2014.12.01

Puesta a punto de un sistema de medida de radón a partir de un dispositivo de simulación de exhalación

[EN] Radon is a radioactive gas from the uranium decay which is being considered as a carcinogenic element by the World Health Organization (WHO) since 2010. In 2013, it was published the Directive 59/2013/EURATOM which establishes new radon limit values, more restrictive, and action plans against radon in air. Since then, and due to the great concern about radon hazardous, several research centers, as well as laboratories and collaborating entities, are joining their efforts in order to develop different methodologies and protocols that allow the control and mitigation of radon gas, with the aim of reducing its concentration in air, and thus preserving population health. Within this context, this works performs the setting up of a RadonScout detector (SARAD GmbH), portable equipment that lets to know in situ and continuously the concentration of radon in air in any location. Measurements have been taken with experimental equipment designed to keep constant environmental conditions. To check the repeatability of the measurements, the results have been compared with RAD7 (Durridge) detector which has been previously used in several intercomparison and calibration procedures.[ES] El radón es un gas radiactivo procedente de la desintegración del uranio, considerado como un elemento cancerígeno por la Organización Mundial de la Salud (OMS) desde 2010. En 2013, se publicó la Directiva 59/2013/EURATOM que establecía unos nuevos valores límite, más restrictivos, y planes de acción frente al radón en aire. Desde entonces, y dada la gran preocupación por la peligrosidad de este gas, diversos centros de investigación, así como laboratorios y entidades colaboradoras, aúnan sus esfuerzos para disponer de metodologías y protocolos que permitan el control y la mitigación del gas radón, con el fin de disminuir su concentración en el aire, y así preservar la salud de la población. Dentro de este contexto, en este trabajo se realiza la puesta a punto del detector RadonScout (SARAD GmbH), un equipo portátil que permite conocer in situ y en continuo la concentración de radón en aire en cualquier emplazamiento. Las medidas se han llevado a cabo a partir de un equipo experimental diseñado para mantener las diferentes variables ambientales estables. Para comprobar la repetibilidad de las medidas, los resultados han sido comparados con los del detector RAD7 (Durridge), con el que se ha participado en diferentes intercomparaciones y calibraciones.Este trabajo ha sido financiado por el Proyecto Prometeo/2018/035 de la Generalitat ValencianaNoverques-Medina, A.; Juste Vidal, BJ.; Sancho, M.; Verdú Martín, GJ.; Arnal Arnal, JM.; García-Fayos, B. (2019). Setting up of a radon measurement system from an exhalation simulation device. AEIPRO. 1741-1753. http://hdl.handle.net/10251/181287S1741175

Dissecting the Role of Mesenchymal Stem Cells in Idiopathic Pulmonary Fibrosis:Cause or Solution

Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of idiopathic interstitial pneumonias, characterized by chronic and progressive fibrosis subverting the lung’s architecture, pulmonary functional decline, progressive respiratory failure, and high mortality (median survival 3 years after diagnosis). Among the mechanisms associated with disease onset and progression, it has been hypothesized that IPF lungs might be affected either by a regenerative deficit of the alveolar epithelium or by a dysregulation of repair mechanisms in response to alveolar and vascular damage. This latter might be related to the progressive dysfunction and exhaustion of the resident stem cells together with a process of cellular and tissue senescence. The role of endogenous mesenchymal stromal/stem cells (MSCs) resident in the lung in the homeostasis of these mechanisms is still a matter of debate. Although endogenous MSCs may play a critical role in lung repair, they are also involved in cellular senescence and tissue ageing processes with loss of lung regenerative potential. In addition, MSCs have immunomodulatory properties and can secrete anti-fibrotic factors. Thus, MSCs obtained from other sources administered systemically or directly into the lung have been investigated for lung epithelial repair and have been explored as a potential therapy for the treatment of lung diseases including IPF. Given these multiple potential roles of MSCs, this review aims both at elucidating the role of resident lung MSCs in IPF pathogenesis and the role of administered MSCs from other sources for potential IPF therapies