Nuevos fluoróforos con aplicación en el desarrollo de biosensores fluorescentes de proteínas

Esta tesis se ha realizado en el Instituto de Química Médica del CSIC, en el marco de los proyectos “Búsqueda de moduladores de interacciones proteína-proteína. Nuevas vías hacia fármacos novedosos” (SAF2012-32209) y “Herramientas para el estudio de interacciones proteína-proteína: Aplicación al estudio y validación de la proteína DREAM como diana terapéutica” (BFU2015-67284-R) y se planteó con el objetivo general de desarrollo de sensores de fluorescencia como herramientas para el estudio de interacciones proteína-proteína. Para la consecución de este objetivo se ha diseñado, sintetizado y caracterizado una nueva familia de fluoróforos derivados de quinolimida altamente fluorogénicos y solvatocrómicos, con propiedades fotofísicas superiores a las de fluoróforos análogos conocidos. La manipulación química de los sustituyentes sobre el esqueleto de quinolimida ha permitido modular las propiedades fotofísicas para la obtención de fluoróforos con emisión de fluorescencia por transferencia de carga (ITC), que han resultado muy solvatocrómicos, o por transferencia fotoinducida de electrones (PET), que presentan solvatocromismo inverso. Los fluoróforos solvatocrómicos, sensibles a la polaridad del medio, han mostrado alto potencial como sensores de fluorescencia de interacciones proteína-proteína, tales como la interacción de la quinasa CDK5 con su proteína reguladora p25 o la agregación del péptido -amiloide. Mientras que, los fluoróforos PET han mostrado potencial como sensores de agua y se han obtenido sensores selectivos del citoplasma o del núcleo en macrófagos, donde interaccionan como intercalantes con el ADN. Por otra parte, se han obtenido derivados de dihidrociclopenta[de]quinolin-2-ona portadoras de grupos fosfonato o carboxilato que se coordinan con el catión Eu3+ y, tras su excitación UV, transfieren energía al catión, actuando como antenas de la luminiscencia del lantánido, que, dependiendo de los sustituyentes, han mostrado alto potencial como sensores luminiscentes de agua o biotioles. La simplificación de la estructura de estas dihidrociclopenta[de]quinolin-2-onas para dar lugar a quinolin-2-onas, análogas de carbostirilo, ha conducido a antenas selectivas de la emisión de Tb3+, que también han mostrado aplicación como sensores de agua. En resumen, se ha diseñado, sintetizado y estudiado una nueva familia de fluoróforos y de antenas de lantánidos que han demostrado un alto potencial como herramientas para el desarrollo de novedosos biosensores de fluorescencia

Highly solvatochromic and tunable fluorophores based on a 4,5-quinolimide scaffold: Novel CDK5 probes

Novel 4,5-quinolimide-based fluorophores are more solvatochromic and red-shifted than known naphthalimide analogues. Conjugation of one of these fluorophores to a peptide derived from CDK5 kinase demonstrated its sensitivity for monitoring the interaction with its regulatory partner p25. Introduction of the quinolimide-labelled peptide into living glioblastoma cells probed the interaction with endogenous p25.The work was supported by the Spanish Ministerio de Economía y Competividad grant SAF2012-32209 and the CSIC grant 2012280E096. J. A. G.-V. held a JAEdoc research contract from the CSIC and now is supported by a Marie-Curie fellowship EC-FP7 Framework (PIEF-GA-2013-623151). The work was also supported by the CNRS and a grant from Canceropole GSO 2015-E03 to MCM and has benefited from the facilities and expertise of the Montpellier RIO imaging facility (www.mri.cnrs.fr) at the Centre de Recherches en Biochimie Macromoléculaire, MontpellierPeer Reviewe

Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells

This work was supported by grants CTQ2017-85658-R, BFU2015-67284-R, and PID2019-104366RB-C22 funded by MCIN/AEI/10.13039/501100011033/FEDER "Una manera de hacer Europa"; grant PID2020-114256RB-I00 funded by MCIN/AEI/10.13039/501100011033; grant A-FQM-386-UGR20 funded by FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades, and the CSIC grant 201580E073. Funding for open access charge: Universidad de Granada/CBUA.The small molecule 8-methoxy-2-oxo-1,2,4,5- tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves as a reactive non-fluorescent Michael acceptor, which after reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after self-assembling with this cation in water. This behavior makes 2b a highly selective GSH biosensor, which has demonstrated high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH can be monitored by the fluorescence of the product of addition to 2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was able to capture baseline differences in GSH intracellular levels among murine and human CD4+ T, CD8+ T, and B cells. We also successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naiv̈ e T cells into regulatory T cells (TREG).MCIN/AEI/FEDER "Una manera de hacer Europa" CTQ2017-85658-R BFU2015-67284-R PID2019-104366RB-C22MCIN/AEI PID2020-114256RB-I00FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades A-FQM-386-UGR20CSIC grant 201580E073Universidad de Granada/CBU

Smart lanthanide antennas for sensing water

Two new families of lanthanide antennas are described. 8-Methoxy- 4,5-dihydrocyclopenta[de]quinolin-2(1H)-one phosphonates or carboxylates behave as selective antennas exhibiting Eu3+ luminescence in organic solvents, while quinolin-2(1H)-one analogues selectively sensitize the Tb3+ emission. These emissions are quenched by H2O addition. Based on this behaviour, the new lanthanide antennas can be used as highly sensitive water sensors.Spanish Ministerio de Economia y Competividad SAF2012-32209 FU2015-67284-RMinisterio de Ciencia e Innovacion/Agencia Estatal de Investigacion/European Regional Development Fund CTQ2017-85658-R CTQ2015-63997-C2Consejo Superior de Investigaciones Cientificas (CSIC) 201580E07

Predictive models for the characterization of internal defects in additive materials from active thermography sequences supported by machine learning methods

The present article addresses a generation of predictive models that assesses the thickness and length of internal defects in additive manufacturing materials. These modes use data from the application of active transient thermography numerical simulation. In this manner, the raised procedure is an ad-hoc hybrid method that integrates finite element simulation and machine learning models using different predictive feature sets and characteristics (i.e., regression, Gaussian regression, support vector machines, multilayer perceptron, and random forest). The performance results for each model were statistically analyzed, evaluated, and compared in terms of predictive performance, processing time, and outlier sensibility to facilitate the choice of a predictive method to obtain the thickness and length of an internal defect from thermographic monitoring. The best model to predictdefect thickness with six thermal features was interaction linear regression. To make predictive models for defect length and thickness, the best model was Gaussian process regression. However, models such as support vector machines also had significative advantages in terms of processing time and adequate performance for certain feature sets. In this way, the results showed that the predictive capability of some types of algorithms could allow for the detection and measurement of internal defects in materials produced by additive manufacturing using active thermography as a non-destructive test.This research was funded by Ministry of Science and Innovation, Government of Spain, through the research project titled Fusion of non-destructive technologies and numerical simulation methods for the inspection and monitoring of joints in new materials and additive manufacturing processes (FaTIMA) with code RTI2018-099850-B-I00. The authors are grateful to the Fundación Universidad de Salamanca for the indirect support provided by the ITACA proof-of-concept project (PC_TCUE_18-20_047), being this helpful for some of the purposes of this article

Step heating thermography supported by machine learning and simulation for internal defect size measurement in additive manufacturing

A methodology based on step-heating thermography for predicting the length dimension of small defects in additive manufacturing from temperature data measured on thermal images is proposed. Regression learners were applied with different configurations to predict the length of the defects. These algorithms were trained using large datasets generated with Finite Element Method simulations. The different predictive methods obtained were optimized using Bayesian inference. Using predictive methods generated and based on intrinsic performance results, knowing the material characteristics, the defect length can be predicted from single temperature data in defect and non-defect zone. Thus, the developed algorithms were implemented in a laboratory set-up carried out on ad-hoc manufactured parts of Nylon and polylactic acid which include induced defects with different sizes and thicknesses. Using the trained algorithm, the deviation of the predicted results for the defect size varied between 13% and 37% for PLA and between 13% and 36% for Nylon.This research has been funded by Ministry of Science and Innovation (Government of Spain) through the research project titled Fusion of nondestructive technologies and numerical simulation methods for the inspection and monitoring of joints in new materials and additive manufacturing processes (FaTIMA) with code RTI2018-099850-B-I00

Selecting FRET pairs for visualizing amyloid aggregation

This work was supported by grant CTQ2017–85658-R funded by MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa” and grants PID2019–104366RB-C22 and PID2020–114256RBI00 funded by MCIN/AEI/10.13039/ 501100011033. A.R.-A. thanks the Spanish Ministerio de Educación y Formación Profesional for an FPU Ph.D. studentship.In a recent work, we reported a methodology for imaging the different stages of amyloid aggregation in quantitative multiparametric dual-color fluorescence lifetime imaging (FLIM) and superresolution microscopy by using a pair of dyes capable of binding aggregates and undergoing subsequent intra-aggregate energy transfer (FRET) (RuizArias et al. Sensors Actuat. B, 2022, 350:130882). In this microarticle we present the optimization process for choosing the best pair of dyes through a screening of different naphthalimides and quinolimides and other known amyloid-binding dyes.Spanish Ministerio de Educación y FormaciónGrant CTQ2017–85658-R funded by MCIN/AEI/10.13039/501100011033/FEDER “Una manera de hacer Europa”Grants PID2019–104366RB-C22 and PID2020–114256RBI00 funded by MCIN/AEI/10.13039/ 50110001103

Repositorio de material docente para el desarrollo de trabajos de fin de grado en el área de mecánica de medios continuos y teoría de estructuras

Memoria ID-135. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2018-2019

Mitochondrion targeted trypanosome alternative oxidase inhibitors as chemotherapeutic agents against T. brucei

Trabajo presentado en el XII SEQT Mini Symposium. IIIrd Spanish/Portuguese/Brazilian Meeting, celebrado en Madrid del 17 al 18 de noviembre de 2016.During their life-cycle, trypanosomes adapt their energy metabolism to the availability of nutrients in their environment. Hence, procyclic forms of T. brucei have a fully functional respiratory chain and synthesize ATP by oxidative phosphorylation in the mitochondrion. In contrast, respiration of bloodstream forms (BSF) of T. brucei (i.e. the human-infective form) relies exclusively on glycolysis for energy production. The trypanosome alternative oxidase (TAO) is the sole terminal oxidase enzyme to re-oxidize NADH accumulated during glycolysis. It is a cyanide-resistant and cytochrome-independent ubiquinol oxidase which is sensitive to the specific inhibitors salicylhydroxamic acid (SHAM) and ascofuranone. This enzyme which is essential to the viability of BSF trypanosomes and has no counterpart in the mammalian host is a potential target for chemotherapy. To boost the activity of TAO inhibitors against T. brucei, we investigated a chemical strategy consisting in the conjugation of the inhibitor with lipophilic cations (LC) that can cross lipid bilayers by non-carrier mediated transport, and thus accumulate specifically into the mitochondrion, driven by the plasma and mitochondrial transmembrane potentials (negative inside). This design afforded several LC¿TAO inhibitor conjugates active in the submicromolar to low nanomolar range against wild type and resistant strains of African trypanosomes (T. b. brucei, T. congolense). Selectivity over human cells was >500. Studies of the effects on purified TAO, parasite respiration, mitochondrial membrane potential (¿m), and cell cycle suggest that TAO is a likely target of the compounds in vivo

Exchangeable Self-Assembled Lanthanide Antennas for PLIM Microscopy

This is the published manuscript version of the following manuscript: Exchangeable Self-Assembled Lanthanide Antennas for PLIM Microscopy, Angew. Chem. Int. Ed. 2023, e202314595 doi: https://doi.org/10.1002/anie.202314595 This article may be used for non-commercial purposes in accordance with Wiley Sharing Policies. Supplementary Materials accompanying this article can be found on-line at the publisher’s site.Lanthanides have unique photoluminescence (PL) emission properties, including very long PL life- times. This makes them ideal for biological imaging applications, especially using PL lifetime imaging micro- scopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lantha- nide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous-soluble, lanthanide antenna, 8-methoxy-2-oxo- 1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self-assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self-assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well- established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.Grant PID2020-114256RB-I00 funded by AEI/10.13039/501100011033Grant PID2019- 104366RB-C22 funded by AEI/10.13039/501100011033/ FEDER “Una manera de hacer Europa”Grants P21_00212, A-FQM-386-UGR20 and 2021/00627/001-FEDER_UJA_ 2020 funded by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y UniversidadesCSIC grant 202180E073Acción 1 from Universidad de JaénFunding for open access charge: Universidad de Granada/CBUASpanish Ministerio de Educación y Formación Profesional for the FPU Ph.D. scholarshi