Epigenetic regulation of resistance to treatments in triple negative and HER2+ breast cancer: miRNAs involved

[ES] El cáncer de mama es el cáncer más común en mujeres en todo el mundo y la principal causa de muerte por cáncer en mujeres junto al cáncer de pulmón. Este cáncer tiene muy buen pronóstico en general, con una supervivencia del 80%. Sin embargo, el pronóstico del cáncer de mama triple negativo es mucho peor, al no conocerse ninguna diana farmacológica y tratarse de forma inespecífica. La metformina, fármaco prescrito para la diabetes, ha mostrado algunos buenos resultados preliminares como potencial terapia. Por otro lado, el principal tratamiento dirigido de las pacientes HER2+ es el trastuzumab, que neutraliza al receptor HER2 amplificado; sin embargo, un elevado número de pacientes desarrollan resistencias al tratamiento. Los microRNAs son pequeños RNAs no codificantes capaces de regular la expresión génica epigenéticamente, y pueden ser secretados de la célula en vesículas llamadas exosomas. El objetivo de este trabajo es abordar estas dos problemáticas en cáncer de mama. Son necesarios estudios de los mecanismos de acción o resistencia de estos fármacos a través de la regulación epigenética por microRNAs. Queremos determinar la relación del miR-26a y sus dianas con el efecto de la metformina en cáncer de mama triple negativo y estudiar las diferencias de expresión de microRNAs que generan resistencias a trastuzumab en cáncer de mama HER2+, así como estudiar su modo de transmisión entre células. Se realizaron ensayos celulares tratando con metformina las líneas MDA-MB-231, MDA-MB-468 y MCF-7 así como sobreexpresando o inhibiendo miR-26a y se midieron sus dianas teóricas por qPCR. Para las líneas HER2+ se realizó un Affymetrix Genechip miRNA 4.0 microarray comparando líneas SKBR-3wt y BT-474wt con sus respectivas líneas con resistencia generada a trastuzumab y HCC-1954 como resistente innata. Se estudiaron los microRNAs más relevantes del array en las líneas celulares y en pacientes y se comprobó su presencia en exosomas, así como el efecto de los exosomas en la transmisión de la resistencia. La sobreexpresión de miR-26a resultó en una reducción en la viabilidad celular que se recuperó parcialmente al inhibirla. E2F3, MCL-1, EZH2, MTDH y PTEN fueron regulados negativamente por miR-26a y la proteína PTEN también se redujo tras la sobreexpresión de miR-26a. El tratamiento con metformina redujo la viabilidad de las células de cáncer de mama, aumentó la expresión de miR-26a y condujo a una reducción en la expresión de BCL-2, EZH2 y PTEN. La inhibición de miR-26a previene parte del efecto en viabilidad de la metformina y la reducción de la expresión de PTEN y EZH2. En las líneas HER2+, miR-23b-3p y miR-146a-5p fueron los principales candidatos extraídos del array. miR-23b-3p inhibió PTEN significativamente en la línea BT-474. miR-146a-5p aumentó la resistencia de las células SKBR-3 al trastuzumab y su inhibición redujo la resistencia de las SKBR-3r. El aumento de miR-146a-5p en SKBR-3wt tuvo un efecto en ciclo celular aumentando la fase S y la G2/M, inhibiendo la expresión de CDKN1A y aumentando la de CCNB1. Los exosomas de las SKBR-3 contenían miR-146a-5p, con mayores niveles en los de las resistentes (exoR). Los exoR aumentaron la resistencia a trastuzumab, la transición epitelio-mesenquimal y la migración al co-cultivarse con SKBR-3wt, y la angiogénesis en las HUVEC. Nuestros resultados sugieren que el efecto de la metformina está mediado por una mayor expresión de miR-26a y reducción de sus dianas, PTEN y EHZ2. Por tanto, el uso de metformina en el tratamiento del cáncer de mama constituye una prometedora potencial terapia. En HER2+, miR-23b parece provocar resistencia a trastuzumab vía PTEN y miR-146a a través del ciclo celular. Además, miR-146a se transmite en exosomas, que son capaces de reducir la sensibilidad al trastuzumab de las células sensibles y aumentar la TEM, migración y angiogénesis.[EN] Breast cancer is the most common cancer in women worldwide and the leading cause of cancer death in women along with lung cancer. This cancer has a very good general prognosis, with a survival of 80%. However, the prognosis for triple negative breast cancer is much worse, as it has no pharmacological target and treats it nonspecifically. Metformin, a prescribed diabetes drug, has shown some good preliminary results as potential therapy. On the other hand, the main targeted treatment for HER2 + patients is trastuzumab, which neutralizes the amplified HER2 receptor, but a large number of patients experienced resistance to treatment. MicroRNAs are small non-coding RNAs that are part of epigenetics and are capable of regulating gene expression, and which can be secreted from the cell into vesicles called exosomes. The objective of this work is to address these two problems in breast cancer, which need to study the mechanism of action or resistance of these drugs, through the epigenetics of microRNAs. We want to determine the relationship of miR-26a and its targets with the effect of metformin in triple negative breast cancer and to study the differences in the expression of microRNAs that process resistance to trastuzumab in HER2 + breast cancer, as well as to study its mode of transmission between cells. Cellular assays were performed treating the MDA-MB-231, MDA-MB-468 and MCF-7 lines with metformin as well as overexpressing or inhibiting miR-26a, and their theoretical targets were measured by qPCR. For the HER2+ cell lines, an Affymetrix Genechip miRNA 4.0 microarray was performed comparing SKBR-3wt and BT-474wt lines with their respective cell lines with generated resistance to trastuzumab and HCC-1954 as innate resistance. The most relevant microRNAs of the array in cell lines and in patients were studied and their presence in exosomes was verified, as well as the effect of exosomes in the transmission of resistance. The overexpression of miR-26a resulted in a reduction in cell viability that was partially recovered by inhibiting it. E2F3, MCL-1, EZH2, MTDH, and PTEN were down-regulated by miR-26a, and the PTEN protein was also reduced after overexpression of miR-26a. Metformin treatment reduced the viability of breast cancer cells, increased miR-26a expression, and led to a reduction in BCL-2, EZH2, and PTEN expression. Inhibition of miR-26a partly prevents the effect of metformin in viability and the reduction of the expression of PTEN and EZH2. In the HER2+ lines, miR-23b-3p and miR-146a-5p were the main candidates extracted from the array. miR-23b-3p was shown to significantly inhibit PTEN in the BT-474 cell line. miR-146a-5p increased resistance of SKBR-3wt cells to trastuzumab and its inhibition reduced resistance of SKBR-3r. The increase of miR-146a-5p in SKBR-3wt had effect on the cell cycle by increasing the S phase and the G2/M, inhibiting the expression of CDKN1A and increasing CCNB1 levels. Exosomes isolated from SKBR-3 cell lines contained miR-146a-5p, with higher levels in exosomes from the resistant cell line (exoR). The exoR were shown to increase trastuzumab resistance, EMT, and migration when co-cultivated with SKBR-3wt, and angiogenesis when in culture with HUVEC. Our results indicate that metformin effectively reduces breast cancer cell viability and suggests that the effects of the drug are mediated by an increase in miR-26a expression and a reduction of its targets, PTEN and EHZ2. Thus, the use of metformin constitutes a promising potential triple negative breast cancer therapy. In HER2+ breast cancer, miR-23b appears to elicit resistance to trastuzumab via PTEN and miR-146a throughout the cell cycle. Furthermore, miR-146a is transmitted in exosomes, which have been shown to reduce the sensitivity to trastuzumab of sensitive cells and increase EMT, migration, and angiogenesis.[CA] El càncer de mama és el càncer més comú en dones arreu del món i la principal causa de mort per càncer en dones junt amb el càncer de pulmó. Aquest càncer té molt bon pronòstic en general, amb una supervivència del 80%. No obstant això, el pronòstic del càncer de mama triple negatiu és molt pitjor, al no conèixer-se'n cap diana farmacològica i tractar-se de forma inespecífica. La metformina, fàrmac prescrit per a la diabetis, ha mostrat alguns bons resultats preliminars com a potencial teràpia. D'altra banda, el principal tractament dirigit de les pacients HER2+ és el trastuzumab, que neutralitza el receptor HER2 amplificat; tanmateix, un elevat nombre de pacients desenvolupen resistències al tractament. Els microRNAs són xicotets RNAs no codificants capaços de regular l'expressió gènica epigenèticament, i poden ser secretats de la cèl·lula en vesícules anomenades exosomes. L'objectiu d'aquest treball és abordar aquestes dues problemàtiques en càncer de mama. Són necessaris estudis dels mecanismes d'acció o resistència d'aquests fàrmacs a través de la regulació epigenètica per microRNAs. Volem determinar la relació del miR-26a i les seues dianes amb l'efecte de la metformina en càncer de mama triple negatiu i estudiar les diferències d'expressió dels microRNAs que generen resistències al trastuzumab en càncer de mama HER2+, així com estudiar la seua manera de transmissió entre cèl·lules. Es van realitzar assajos cel·lulars tractant amb metformina les línies MDA-MB-231, MDA-MB-468 i MCF-7 així com sobreexpressant o inhibint miR-26a i es van mesurar les seues dianes teòriques per qPCR. Per a les línies HER2+ es va realitzar un Affymetrix Genechip miRNA 4.0 microarray comparant línies SKBR-3wt i BT-474wt amb les seues respectives línies amb resistència generada a trastuzumab i HCC-1954 com resistent innata. Es van estudiar els microRNAs més rellevants de l'array en les línies cel·lulars i en pacients i es va comprovar la seua presència a exosomes, així com l'efecte dels exosomes en la transmissió de la resistència. La sobreexpressió de miR-26a resultà en una reducció de la viabilitat cel·lular que es recuperà parcialment en inhibir-la. E2F3, MCL-1, EZH2, MTDH i PTEN foren regulats negativament per miR-26a i la proteïna PTEN també es va reduir en sobreexpressar miR-26a. El tractament amb metformina va reduir la viabilitat de les cèl·lules de càncer de mama, va augmentar l'expressió de miR-26a i va conduir a una reducció en l'expressió de BCL-2, EZH2 i PTEN. La inhibició de miR-26a prevé part de l'efecte en la viabilitat de la metformina i la reducció de l'expressió de PTEN i EZH2. En les línies HER2+, miR-23b-3p i miR-146a-5p foren els principals candidats extrets de l'array. miR-23b-3p va inhibir PTEN significativament en la línia BT-474. miR-146a-5p va augmentar la resistència de les cèl·lules SKBR-3 al trastuzumab i la seua inhibició va reduir la resistència de les SKBR-3r. L'augment de miR-146a-5p en SKBR-3wt va tindre un efecte en cicle cel·lular augmentant la fase S i la G2/M, inhibint l'expressió de CDKN1A i augmentant la de CCNB1. Els exosomes de les SKBR-3 contenien miR-146a-5p, amb majors nivells en els de les resistents (exoR). Els exoR van augmentar la resistència a trastuzumab, la transició epiteli-mesenquimal i la migració en co-cultivar-los amb SKBR-3wt, i l'angiogènesi de les HUVEC. Els nostres resultats suggereixen que l'efecte de la metformina està intervingut per una major expressió de miR-26a i reducció de les seues dianes, PTEN i EHZ2. Per tant, l'ús de metformina al tractament de el càncer de mama constitueix una prometedora potencial teràpia. En HER2+, miR-23b sembla provocar resistència a trastuzumab mitjançant PTEN i miR-146a a través del cicle cel·lular. A més, miR-146a es transmet en exosomes, que són capaços de reduir la sensibilitat al trastuzumab de les cèl·lules sensibles i augmentar la TEM, migració i angiogènesi.Cabello Navarro, P. (2020). Epigenetic regulation of resistance to treatments in triple negative and HER2+ breast cancer: miRNAs involved [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153807TESI

An assessment of different relay network topologies to improve Earth-Mars communications

The future of deep space communications encompasses a challenging situation where the current facilities used to communicate with different spacecraft may become saturated as a result of an increasing number of missions and their complexity. From this forecast, the present study intends to provide a solution to saturation problems through strategically-located upgradable relays for Earth-Mars communications. The foremost goal of this paper is to quantitatively uncover the potential enhancements coming from relay placement in strategic orbits between Earth and Mars. Herein, two relay configurations –a.k.a. network topologies– are analyzed: the Lagrange-relays network topology and a circular, homogeneously-distributed satellite constellation, acknowledged here as pearl constellation. The first uses the Earth-Sun system Lagrange points L3, L4 and L5 as potential locations for the relays, whilst the second defines an optimized orbit between Earth and Mars with 3 or 4 relay satellites. To aid in the analysis, the authors developed an open-sourced piece of software that obtains the link availability as well as the data rate at which two nodes may communicate, taking as a reference the Deep Space Network for Earth, and the Mars Reconnaissance Orbiter for Mars. For complex topologies with more than two communicating nodes, the software outputs the end-to-end bit rate and optimal communication route at each time step. Moreover, this product is extensible to analyze and optimize any network topology and could be adapted to be used for contact management and mission planning in the future. The results show that the network-topology proposals are an advantageous option to significantly increase the link availability of Earth-Mars communications. Nevertheless, the Direct-To-Earth link always outperforms the multi-hop path due to the limited telecommunication system’s capabilities of both the spacecraft and the relays. As a result of this, the study includes an analysis on the requirements of the relay’s design in order to make the constellation a beneficial and comparable alternative to the DTE link. This way, the proposed network topologies become a suitable option whom to share with the DSN communications workload, providing enhanced bit rates and data volumes as well as higher availability of the communication.Peer ReviewedPostprint (published version

An 11 mA Capacitor-Less LDO With 3.08 nA Quiescent Current and SSF-Based Adaptive Biasing

This brief presents an ultra-low power low-dropout (LDO) regulator with an experimental total quiescent current consumption of only 3.08 nA. The circuit is designed to operate with a load current in the range 0 - 11 mA. A novel adaptive biasing scheme based on a super source follower (SSF) structure is proposed, which measures the absolute voltage difference between the two inputs of the LDO’s error amplifier and modifies the biasing current accordingly. Thus, the transient response of the regulator is improved by counteracting the effect of using such a low bias current. The proposed LDO has been fabricated in a standard CMOS 180 nm process and the experimental characterization showed an outstanding performance in terms of maximum load current over quiescent current consumption ratio.S

Aproximación a la fruta en una clase de 5 años de Infantil a través de los sentidos

Durante el desarrollo de este trabajo de fin de grado, se ha llevado a cabo una propuesta de innovación que trata de promover una alimentación sana a través de las frutas. La mala alimentación es un tema de especial relevancia en la etapa de Educación Infantil, un problema que se manifiesta tanto en el ámbito familiar como en el ámbito educativo. En la actualidad, los malos hábitos ligados a la alimentación están presentes en las aulas año tras año. Nuestra propuesta teórica será llevada a la práctica durante un día en un aula de cinco años y nos centraremos en la presentación de cuatro frutas populares: el plátano, el melocotón, el limón y la fresa. A través de este contenido, pretendemos que los niños y niñas conozcan más estos alimentos a través de los sentidos, para darles la oportunidad de conocerlas, probarlas y manipularlas de una manera diferente.During the development of this final degree project, an innovative proposal has been carried out, which tries to promote a healthy diet through fruits. Poor nutrition is a topic of special relevance for students of Early Childhood Education, a problem that manifests itself both in the family environment and in the educational field. Currently, bad habits linked to food are present in the classrooms year after year. Our theoretical proposal will be put into practice for one day in a five - years classroom, and we will focus on the explanation and development of four common fruits: banana, peach, lemon and strawberry. Through this content, we intend the kids to know more about these foods using the senses to give them the opportunity to know, test and manipulate them through the different activities that will be developed throughout this educational workshop

Refugios de montaña del pirineo. Análisis arquitectónico e impacto en el medio

En el seno más profundo de los orígenes de la arquitectura, encontramos la cabaña primitiva, la cueva, como refugio del ser humano. Desde el momento en el que tenemos la necesidad de poner un techo sobre nuestra cabeza para protegernos de los elementos, la arquitectura queda vinculada a nuestra propia naturaleza. El refugio, es la expresión arquitectónica actual que podemos encontrar en esta inercia de protegernos, de crear un pequeño espacio controlable entre cuatro paredes y como tal, puede entenderse como una de las construciones más sinceras. El objetivo de este trabajo es poner sobre la mesa el panorama actual de los refugios de montaña en el Pirineo, sus distintas tipologías y continuar con el debate formal y de empleo de materiales que en estos momentos tienen las federaciones de montaña, así como hacer hincapié en la metodología del Análisis del Ciclo de Vida (ACV) aplicado a estos edificios y de que forma esta información puede ser útil en la toma de decisiones tanto proyectuales como constructivas de los arquitectos

Ultralow power voltage reference circuit for implantable devices in standard CMOS technology

This is the peer reviewed version of the following article: Óscar Pereira-Rial, Paula López, Juan M. Carrillo, Victor M. Brea and Diego Cabello (2019) Ultralow power voltage reference circuit for implantable devices in standard CMOS technology. International journal of circuit theory and applications, 47 (7), 991-1005, which has been published in final form at https://doi.org/10.1002/cta.2643. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsAn ultralow power CMOS voltage reference for body implantable devices is presented in this paper. The circuit core consists of only regular threshold voltage PMOS transistors, thus leading to a very reduced output voltage dispersion, defined as σ/μ, and extremely low power consumption. A mathematical model of the generated reference voltage was obtained by solving circuit equations, and its numerical solution has been validated by extensive electrical simulations using a commercial circuit simulator. The proposed solution incorporates a passive RC low‐pass filter, to enhance power supply rejection (PSR) over a wide frequency range, and a speed‐up section, to accelerate the switching‐on of the circuit. The prototype was implemented in 0.18 μm standard CMOS technology and is able to operate with supply voltages ranging from 0.7 to 1.8 V providing a measured output voltage value of 584.2 mV at the target temperature of 36° C. The measured σ/μ dispersion of the reference voltage generated is 0.65% without the need of trimming. At the minimum supply of 0.7 V, the experimental power consumption is 64.5 pW, while the measured PSR is kept below –60 dB from DC up to the MHz frequency rangeThis work has been partially funded by the Spanish government projects TEC2015‐66878‐C3‐3‐R (MINECO/FEDER) and RTI2018‐097088‐B‐C32 (FEDER), by the Xunta de Galicia under project ED431C2017/69, by the Consellería de Cultura, Educación e Ordenación Universitaria (accreditation 2016‐2019, ED431G/08 and reference competitive group 2017‐2020, ED431C 2017/69), by the Junta de Extremadura R&D Plan, and the European Fund for Regional Development (EFRD) under Grant IB18079S

Distance Measurement Error in Time-of-Flight Sensors Due to Shot Noise

Unlike other noise sources, which can be reduced or eliminated by different signal processing techniques, shot noise is an ever-present noise component in any imaging system. In this paper, we present an in-depth study of the impact of shot noise on time-of-flight sensors in terms of the error introduced in the distance estimation. The paper addresses the effect of parameters, such as the size of the photosensor, the background and signal power or the integration time, and the resulting design trade-offs. The study is demonstrated with different numerical examples, which show that, in general, the phase shift determination technique with two background measurements approach is the most suitable for pixel arrays of large resolutionThis work has been partially funded by Spanish government Project TEC2012-38921-C02-02 MINECO(FEDER) and by the Xunta de Galicia with EM2013/038 and EM2014/012, AE CITIUS(CN2012/151, (FEDER)) and GPC2013/040 (FEDER)S

Wireless Sensor Network With Perpetual Motes for Terrestrial Snail Activity Monitoring

Wireless sensor networks (WSNs) are increasingly adopted in agriculture to monitor environmental variables to predict the presence of pests. Differently from these approaches, this paper introduces aWSN to detect the presence of snails in the field. The network can be used to both trigger an alarm of early pest presence and to further elaborate statistical models with the addition of environmental data as temperature or humidity to predict snail presence. In this paper we also design our own WSN simulator to account for real-life conditions as an uneven spacing of motes in the field or different currents generated by solar cells at the motes. This allows achieving more realistic network deployment in the field. Experimental tests are included in this paper, showing that our motes are perpetual in terms of energy consumptionConsellería de Cultura, Educación e Ordenación Universitaria (accreditation 2016-2019); European Regional Development Fund; Ministerio de Economía, Industria y Competitividad (TEC2015-66878-C3-3-R)S