3D Culture o Multiple Myeloma Cell Line Using Microgel Environments

[ES] El mieloma múltiple es una neoplasia hematológica caracterizada por una expansión descontrolada de células plasmáticas monoclonales (mPCs) en medula ósea que producen, en la mayoría de los casos, un componente monoclonal secretado en el suero y/o en orina. En la actualidad, se sigue considerando una enfermedad incurable con la constante aparición de recaídas en los pacientes. Una de las causas que condicionan esta situación, radica en la generación de resistencia frente a fármacos por parte de las mPCs. Este mecanismo de resistencia a fármacos (DR) se ha visto que no solo depende de factores intracelulares, sino que la propia interacción de las mPCs con el microambiente medular juega un papel fundamental para su supervivencia, crecimiento y desarrollo de DR. Entre los componentes del microambiente tumoral, destaca la adhesión de las mPCs a componentes de la matriz extracelular (ECM) que se ha visto relacionada con la generación de DR. Por este motivo el desarrollo de esta tesis doctoral consistió en la elaboración y validación de una plataforma de cultivo 3D basado en la síntesis de un microgel. Este sistema estará constituido por microesferas funcionalizadas con componentes de la ECM como son la fibronectina (FN), colágeno tipo I (COL), heparina (Hep), heparan sulfato (HS) y ácido hialurónico (HA), generando un entorno 3D biomimético con la capacidad de poder analizar la respuesta celular desencadenada por la interacción de las mPCs con los componentes de la ECM, así como la DR generada por la adhesión de las mPCs a estas biomoléculas. El primer estudio consistió en la realización y puesta a punto de varios protocolos para la síntesis de distintos microgeles; un primer sistema se produjo mediante la polimerización por vía radical en bloque de co-polímeros de poliacrilato de etilo (EA) y polimetacrilato de etilo (EMA) o bien por EA, EMA y ácido acrílico (AAc). Mediante una emulsión del tipo aceite en agua se consiguió producir con estos copolímeros, microesferas de un tamaño próximo al de las mPCs. Un segundo sistema se basó en microesferas de alginato. Estas microesferas se obtuvieron en un dispositivo de microfluidica produciéndose la gelificación externa de las micro-gotas con la incorporación de iones de calcio consiguiendo microesferas de un tamaño medio de 177 µm. Debido a la gran variedad de microesferas sintetizadas con diferentes grupos químicos en sus superficies, se consiguió establecer protocolos de funcionalización similares a los establecidos en la literatura, teniendo en cuenta la estabilidad de la biomolécula a lo largo del tiempo del cultivo celular. Este enfoque, permitió la funcionalización con una gran variedad de biomoléculas disponiendo así de microgeles funcionalizados con FN, COL, Hep, HS y HA. Una vez desarrollados los microgeles, en un segundo estudio se procedió a evaluar la respuesta celular en un entorno 3D basado en microgel, valorando la interacción con los componentes de la ECM. Entre los resultados observados se pudo determinar como el tamaño de las microesferas afecta al crecimiento celular incluso en ausencia de cualquier funcionalización. Con los microgeles constituidos por microesferas de un tamaño próximo al de las mPCs se obtuvo un mayor crecimiento celular que con los microgeles formados por partículas de mayor tamaño, y en ambos el crecimiento fue superior al del cultivo en suspensión. Se plantea la hipótesis de que la presencia de las microesferas favorece en gran medida que se produzca un mayor contacto célula-célula que se ve incrementado cuanto mayor es la superficie específica del microgel. Entre los componentes de la ECM estudiados, mientras que el COL no genera ninguna respuesta celular diferente al control (microgel no funcionalizado), el HA favorece la proliferación celular. La adhesión de las mPCs a la FN condiciona el bloqueo de las células en la fase G0-G1 del ciclo celular. Esta adhesión está mediada[CA] El mieloma múltiple és una neoplàsia hematològica caracteritzada per una expansió descontrolada de cèl·lules plasmàtiques monoclonals (mPCs) en medul·la òssia que produeixen, en la majoria dels casos, un component monoclonal secretat en el sèrum i/o en orina. En l'actualitat, es continua considerant una malaltia incurable, amb la constant aparició de recaigudes en els pacients. Una de les causes que condicionen aquesta situació, radica en la generació de resistència enfront de fàrmacs per part de les mPCs. Aquest mecanisme de resistència a fàrmacs (DR) s'ha vist que no sols depèn de factors intracel·lulars, sinó que la mateixa interacció de les mPCs amb el microambient medul·lar juga un paper fonamental per a la seua supervivència, creixement i desenvolupament de DR. Entre els components del microambient tumoral, destaca l'adhesió de les mPCs a components de la matriu extracel·lular (ECM) que s'ha vist relacionada amb la generació de DR. Per aquest motiu, el desenvolupament d'aquesta tesi doctoral va consistir en l'elaboració i validació d'una plataforma de cultiu 3D basada en la síntesi d'un microgel. Aquest sistema estarà constituït per microesferes funcionalitzades amb components de l'ECM com són la fibronectina (FN), col·lagen tipus I (COL), heparina (Hep), heparan sulfat (HS) i àcid hialurònic (HA), generant un entorn 3D biomimètic amb la capacitat de poder analitzar la resposta cel·lular desencadenada per la interacció de les mPCs amb els components de la ECM, així com la DR generada per l'adhesió de les mPCs a aquestes biomolècules. El primer estudi va consistir en la realització i posada a punt de diversos protocols per a la síntesi de diferents microgels; un primer sistema es va produir mitjançant la polimerització per via radical en bloc de copolímers de poliacrilat d'etil (EA) i polimetacrilat d'etil (EMA), o bé per EA, EMA i àcid acrílic (AAc). Mitjançant una emulsió del tipus oli en aigua es va aconseguir produir amb aquests copolímers, microesferes d'una grandària pròxima al de les mPCs. Un segon sistema es va basar en microesferes d'alginat. Aquestes microesferes es van obtenir en un dispositiu de microfluidica produint-se la gelificació externa de les microgotes amb la incorporació d'ions de calci aconseguint microesferes d'una grandària mitjana de 177 ¿m. A causa de la gran varietat de microesferes sintetitzades amb diferents grups químics en les seues superfícies, es va aconseguir establir protocols de funcionalització similars als establerts en la literatura, tenint en compte l'estabilitat de la biomolècula al llarg del temps del cultiu cel·lular. Aquest enfocament va permetre la funcionalització amb una gran varietat de biomolècules disposant així de microgels funcionalitzats amb FN, COL, Hep, HS y HA. Una vegada desenvolupats els microgels, en un segon estudi es va procedir a avaluar la resposta cel·lular en un entorn 3D basat en microgel, valorant la interacció amb els components de l'ECM. Entre els resultats observats es va poder determinar com la grandària de les microesferes afecta el creixement cel·lular fins i tot en absència de qualsevol funcionalització. Amb els microgels constituïts per microesferes d'una grandària pròxima al de les mPCs es va obtenir un major creixement cel·lular que amb els microgels formats per partícules de major grandària, i en tots dos el creixement va ser superior al del cultiu en suspensió. Es planteja la hipòtesi que la presència de les microesferes afavoreix en gran manera que es produïsca un major contacte cèl·lula-cèl·lula que es veu incrementat com més gran és la superfície específica del microgel. Entre els components de l'ECM estudiats, mentre que el COL no genera cap resposta cel·lular diferent del control (microgel no funcionalitzat), l'HA afavoreix la proliferació cel·lular. L'adhesió de les mPCs a la FN condiciona el bloqueig de les cèl·lules en la fase G0-G1 del cic[EN] Multiple myeloma is a haematological neoplasm characterized by an uncontrolled expansion of monoclonal plasma cells (mPCs) in bone marrow that produce, in most cases, a monoclonal component secreted in serum and/or urine. At present, it is still considered an incurable disease with the constant appearance of relapses in patients. One of the causes that condition this situation lies in the generation of drug resistance by the mPCs. This mechanism of drug resistance (DR) has been seen to depend not only on intracellular factors, but the very interaction of mPCs with the medullary microenvironment plays a fundamental role in their survival, growth and development of DR. Among the components of the tumor microenvironment, the adhesion of the mPCs to components of the extracellular matrix (ECM) stands out, which has been related to the generation of DR. For this reason, the development of this doctoral thesis consisted in the elaboration and validation of a 3D culture platform based on the synthesis of a microgel. This system will be made up of micropsheres functionalized with the components of the ECM such as fibronectin (FN), collagen type I (COL), heparin (Hep), heparan sulphate (HS) and hyaluronic acid (HA), generating a 3D biomimetic environment with the ability to analyse the cellular response triggered by the interaction of mPCs with the ECM components, as well as the DR generated by the adhesion of the mPCs to these biomolecules. The first study consisted in the realization and development of several protocols for the synthesis of different microgels. A first system was produced by the radical block polymerization of polyethylene acrylate (EA) and polymethacrylate (EMA) co-polymers or by EA, EMA and acrylic acid (AAc). By means of an oil-in-water emulsion technique, it was possible to produce, with these copolymers, microspheres of a size close to that of the mPCs. A second system was based on alginate microspheres. These microspheres were obtained in a microfluidic device producing the external gelification of the micro-drops with the incorporation of calcium ions, obtaining microspheres with an average size of 177 µm. Due to the great variety of microspheres synthesized with different chemical groups on their surfaces, it was possible to establish functionalization protocols similar to those established in the literature, taking into account the stability of the biomolecule along with the time of cell culture. This approach allowed for functionalization with a great variety of biomolecules, having in this way functionalized microgels with FN, COL, Hep, HS and HA. Once the microgels were developed, a second study was carried out to evaluate the cell response in a 3D microgel-based environment, assessing the interaction with the components of the ECM. Among the results observed, it was possible to determine how the size of the microspheres affects cell growth even in the absence of any functionalization. With the microgels constituted by microspheres close to the size of the mPCs, a greater cellular growth was obtained than with the microgels formed by larger particles, and in both the growth was higher than in suspended culture. It is hypothesized that the presence of microspheres greatly favours a greater cell-cell contact, which is increased the larger the specific surface area of the microgel. Among the components of the ECM studied, while the COL does not generate any cellular response different from the control (non-functionalized microgel), HA favours cell proliferation. The adhesion of mPCs to FN conditions the blocking of cells in the G0-G1 phase of the cell cycle. This adhesion is mediated by the integrin ¿4ß1.La presente tesis doctoral no se podría haber realizado sin la financiación del proyecto PROMETEO/2016/063, trabajo que también estuvo parcialmente financiado con fondos FEDER (CIBERONC (CB16/12/00284)). La iniciativa CIBER-BBN está financiada por el proyecto VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. Las acciones CIBER están financiadas por el Instituto de Salud Carlos III con ayuda del Fondo Europeo de Desarrollo Regional.Marin Paya, JC. (2021). 3D Culture o Multiple Myeloma Cell Line Using Microgel Environments [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/167427TESI

Feasability of Bluetooth 5.0 connectionless communications for I2V applications

[EN] Vehicle communications are one of the pillars of the development of Intelligent Transport Systems. This is a complex scenario, where several types of entities interact with each other, using a range of communication infrastructure. In this area there are several applicable technologies, with different advantages and disadvantages. Therefore, flexible and ubiquitous communication, capable of using any communication type, will allow Smart vehicles to use the most appropriate one to improve traffic movement systems. Although the use of Bluetooth has been considered in this field, it has normally been done in intra-vehicle communications, or if it has been done outside this field, for inter-vehicle communications, it has been done based on standard 4.2 or earlier versions. The Bluetooth BLE 5.0 standard has features that allow the use of this technology in previously unimaginable scenarios; in particular, the increase in range, and greater robustness in communications allowing the analysis of its use as an infrastructure to vehicle network. The preliminary results obtained demonstrate this viability and the potential of this technology in this fieldThis work has been supported by the project PRYSTINE which has received funding within the Electronic Components and Systems for European Leadership Joint Undertaking (ECSELJU) in collaboration with the European Union¿s H2020 Framework Programme and National Authorities, under grant agreement No. 783190, and from the ¿Instituto Valenciano de Competitividad Empresarial" ¿ IVACE. It has also been funded by the MCyU (Spanish Ministry of Science and Universities) under the project ATLAS (PGC2018-094151-B-I00), which is partially funded by AEI, FEDER and EUGarcía Ortíz, JC.; Silvestre-Blanes, J.; Sempere Paya, VM.; Ponce Tortajada, R. (2020). Feasability of Bluetooth 5.0 connectionless communications for I2V applications. IEEE. 1-5. https://doi.org/10.1109/ETFA46521.2020.9211984S1

Cell-level pathway scoring comparison with a biologically constrained variational autoencoder

This preprint has not undergone peer review or any post-submission improvements or corrections. The Version of Record of this contribution is published in: Pang, J., Niehren, J. (eds) Computational Methods in Systems Biology. CMSB 2023. Lecture Notes in Computer Science, vol 14137. Springer, Cham. Available online at https://doi.org/10.1007/978-3-031-42697-1_

Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization

[EN] Stem cells reside in niches, specialized microenvironments that sustain and regulate their fate. Extracellular matrix (ECM), paracrine factors or other cells are key niche regulating elements. As the conventional 2D cell culture lacks these elements, it can alter the properties of naive stem cells. In this work we designed a novel biomimetic microenvironment for cell culture, consisting of magnetic microspheres, prepared with acrylates and acrylic acid copolymers and functionalized with fibronectin or hyaluronic acid as ECM coatings. To characterize cell proliferation and adhesion, porcine mesenchymal stem cells (MSCs) were grown with the different microspheres. The results showed that the 3D environments presented similar proliferation to the 2D culture and that fibronectin allows cell adhesion, while hyaluronic acid hinders it. In the 3D environments, cells reorganize the microspheres to grow in aggregates, highlighting the advantages of microspheres as 3D environments and allowing the cells to adapt the environment to their requirements.PROMETEO/2016/063 project is acknowledged. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities trough Sandra Clara-Trujillo FPU17/05810 grant.Clara-Trujillo, S.; Marin-Paya, JC.; Cordón, L.; Sempere, A.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2019). Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization. Colloids and Surfaces B Biointerfaces. 177:68-76. https://doi.org/10.1016/j.colsurfb.2019.01.050S687617

QSI methods for determining the quality of the surface finish of concrete

[EN] The surface finish of a concrete element may become an index of its quality, relating the external and internal porosity with the mechanical and durability properties. Few methods are used to determine the surface quality of concrete elements. Mention must be made the Quality Surface Index (QSI) proposes a simplified method to quantify the surface occupied by the pores in relation with the total surface inspected, analyzing groups of pores by their diameter. The method of the CIB W29 (Commission W29 "Concrete Surface Finishings") proposes an inspection of the concrete element and its visual comparison with some standard templates. Finally, the digital processing of images allows the zones with surface defects to be delimited and quantified according to premises of quality introduced into the control software. These three methods are employed in this work and are applied in three concrete walls situated three meters from the observer (M-1, M-2 and M-3). Following the conversion of the results of the method with ImageJ and QSI, the results suppose differences that go from 0.1 tenths (2%) for M-3 up to 0.3 tenths (8%) for M-1. All values are within the obtained range with CIB W29 templates. This can validate the QSI and digital processing methods and allows a quick verification of the results. With the digital method, it is obtained that 23.5% of the total pores of M-1 have a diameter of less than 10 mm(2) and 44% of less than 100 mm(2). For M-2 and M-3 the proportions of pores with a dimension below 10 mm(2) is of 43.1% and 27.7%, respectively, and that 77.5% and 60.7% are smaller than 100 mm2. From all the above it can be highlighted that M-1 is the one with the lowest amount of pores, however the proportion of the largest is greater than for M-2 and M-3. In the case of M-3, although it has a lower proportion of larger pores than M-1, its greater amount means it is the worst in terms of surface finish of the three.Benito-Saorín, FJ.; Miñano-Belmonte, I.; Parra Costa, C.; Rodríguez-López, C.; Valcuende Payá, MO. (2018). QSI methods for determining the quality of the surface finish of concrete. Sustainability. 10(4):931-1-931-14. https://doi.org/10.3390/su10040931S931-1931-1410

Septal rupture with right ventricular wall dissection after myocardial infarction

BACKGROUND: In patients with inferior myocardial infarction, septal rupture generally involves basal inferoposterior septum, and the communicating tract between left and right ventricle is often serpiginous with a variable degree of right ventricular wall extension. Right ventricular wall dissection following septal rupture related with previous myocardial infarction has been reported in a very few cases, in many of them this condition has been diagnosed in post-mortem studies. In a recent report long-term survival has been achieved after promptly echocardiographic diagnosis and surgical repair. CASE PRESENTATION: We present a case of a 59-year-old man who had a septal rupture with right ventricular wall dissection after inferior and right ventricular myocardial infarction. Transthoracic echocardiography, as first line examination, established the diagnosis, and prompt surgical repair allowed long-term survival in our patient. CONCLUSION: Outcomes after right ventricular intramyocardial dissection following septal rupture related to myocardial infarction has been reported to be dismal. Early recognition of this complication using transthoracic echocardiography at patient bedside, and prompt surgical repair are the main factors to achieve long-term survival in these patients