Development of milk exosome-based probes for biomedical imaging

Exosomes are extracellular vesicles naturally secreted by living cells, intended for the exchange of information between them. Their physicochemical characteristics, featured by nanometric size and lipid bilayer structure, make them similar to the synthetic liposomes designed to be applied in the field of biomedicine. In addition, exosomes have associated an intrinsic biological role, which confers them natural tropism to certain tissues and pathological processes. Taken together, these characteristics suggest the potential use of exosomes as natural platforms to design new nanoprobes for molecular imaging, overcoming the limitations presented by nanoparticles traditionally used in this research field, generally associated with their toxicity and in vivo stability. As source of exosomes, milk stands out due to its high content of these nanovesicles, with proven functionality as carriers of molecules and drugs. The main goal of this thesis was to develop milk exosome-based probes for the detection of several pathologies by molecular imaging. To reach this aim, the project was divided in four specific objectives, as follows: i) establishing an optimal protocol for the isolation of exosomes from, in this case, commercial goat milk, according to the milk characteristics and the resources available in the research laboratory, ii) exploring alternative strategies for the radioactive and fluorescent labeling of exosomes, overcoming the limitations of current approaches, iii) determining the in vivo pharmacokinetics and biodistribution of the exosome-based probes, to elucidate their natural behavior in healthy mice and iv) evaluating the capacity of the exosome-based probes to target inflammatory processes, as potential nanoplatforms for diagnosis. Thus, first contribution of this thesis is to describe for first time an isolation protocol based on the combination of physical and biological approaches to obtain pure and homogeneous milk exosome samples. By means of physicochemical, proteomic and biological characterization, the efficacy of the biophysical protocol was demonstrated, as well as the exosomal nature of the isolated vesicles and their non-toxicity in healthy mice. The second contribution of this thesis is based on the development of new chemical strategies for the labeling of exosomes with both radioisotopes and fluorophores. In the case of the radiochemical labeling, exosomes were labeled with Technetium-99, by the passive incorporation of this isotope into the nanovesicle structure. The main advantages of this straightforward approach is that it does not require a chelator for the coordination of the radiometal and employs mild conditions that avoid the chemical modification or degradation of the exosomes. The second chemical approach presented in this thesis is the fluorescent labeling of exosomes. Current methodologies for the fluorescent labeling of exosomes involve advanced knowledge of genetic engineering or result in unstable fluorescent probes that can lead to false positives. This thesis proposes a labeling strategy based on the covalent binding of commercial fluorophores to the functional groups available on the exosome surface. This strong chemical bond ensures the stability of the nanoprobe, preventing the release of the fluorophore. In both cases, physicochemical characterization of the resulting molecular probes showed that the original properties of the exosomes are not altered after the corresponding labeling. The third contribution of this work is the in vivo evaluation by nuclear and optical techniques of the natural behavior of labeled goat milk exosomes after exogenous administration in healthy mice. By means of SPECT/CT imaging and ex vivo evaluation, it was determined that the way of administering the exosome-based probe significantly alters their pharmacokinetic and biodistribution parameters. On the other hand, both optical and nuclear imaging revealed the high accumulation of the nanoprobes in liver after intravenous injection, due to its high uptake by hepatocytes and Kupffer cells. This finding could endorse their future used in the field of hepatic diseases. Last contribution of this thesis deals with the evaluation of the exosome-based probes for diagnostic of inflammatory pathologies. Based on the literature, goat milk exosomes are involved in the immune response and inflammation. For this reason, a fluorescent exosome-based probe developed in this thesis was evaluated in vitro against cells related to the inflammatory process and in vivo in a peritonitis mouse model, by different optical imaging techniques. Fluorescent exosomes demonstrated their ability to detect the inflammatory process underlying the disease, presenting particular uptake by the proinflammatory population of macrophages. In conclusion, this thesis provides novel methodologies to the labeling and characterization of milk exosomes for use as natural nanoprobes for molecular imaging. These approaches improve the current labeling protocols, enabling the development of stable molecular probes that do not alter the original properties of the exosomes. Pharmacokinetic and biodistribution evaluation of the developed nanoprobes highlight the importance of choosing the appropriate route of administration of exosomes according to their purpose, as well as their potential applicability in liver pathologies. Finally, studies performed in the in vivo model of peritonitis support their use as imaging probes for the detection of inflammatory response, enabling the evaluation of the process at cellular level as well as the in vivo localization of the inflammatory focus.The work developed in this PhD thesis was framed within several national and international research projects, supported by the following funding: - Instituto de Salud Carlos III, through the project “PI16/02037”, co-funded by European Regional Development Fund (ERDF), “A way to make Europe” - Comunidad de Madrid, project “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/BMD-3867 (RENIM-CM)”, co-funded by European Structural and Investment Fund. During part of the pre-doctoral period (2022 – 2023), the author of this thesis has been also funded by Instituto de Investigación Sanitaria Gregorio Marañón, Intramural Programme for the Promotion of R&D&I 2021, Sub-programme "Pre-doctoral training contract".Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidente: Carlos Pérez Medina.- Secretario: Benito Jorge Rubio Retama.- Vocal: Francisca Mulero Aniort

Los cambios en las relaciones con la sociedad de las AUC a las BACRIM. El caso de Córdoba

Luego de la desmovilización de las Autodefensas Unidas de Colombia que finalizó en 2006-, agrupación paramilitar que operaba en casi la totalidad del territorio colombiano para hacer frente a las guerrillas y sustituir los vacíos estatales, el proceso de reinserción de los ex combatientes a la sociedad civil no fue exitoso. Los mandos medios se rearmaron y tomaron partes de las estructuras criminales ya existentes en las zonas para reconfigurarse y reincidir en el delito. Estas bandas emergentes están conformadas por reductos paramilitares, por distintas estructuras criminales y tienen lazos pre establecidos con el narcotráfico. El escenario actual en los lugares donde operan estos grupos se caracteriza por enfrentamientos entre dos o más bandas que intentan controlar territorios que son importantes para el negocio del narcotráfico, que tienen significativos recursos para su sustento económico como los mineros-, o que cuentan con una ubicación estratégica para la configuración territorial. Es imprescindible tener en cuenta la relación entre dichos actores armados con la sociedad, en tanto esta es la puerta para el acceso al territorio en el que se mueven, en ella se encuentran los medios para que permanezcan y crezcan y por ella pueden verse favorecidos o relegados. Este análisis permitirá conocer las características de ambos fenómenos, saber cómo se comportan, las relaciones que se configuran entre estos actores armados y la sociedad, determinar los usos de la violencia en contra de la población y de qué forma la sociedad saca provecho o no de estas relaciones, en la medida en que así se puede dar cuenta de la caracterización de las AUC y de las BACRIM.After the demobilization of the Autodefensas Unidas de Colombia - that finished in 2006-, paramilitary group that operated in almost the totality of the Colombian territory to face to the guerrillas groups and to replace the state emptinesses, the process of rehabilitation of the ex-soldiers to the civil society was not successful. The middle controls went rearmed and took parts of the already existing criminal structures in the zones for realign and to relapse in the crime. These emergent bands are shaped by paramilitary redoubts, by different criminal structures and have pre established ties with the drug trafficking. The current scene in the places where these groups are characterizes for clashes between two or more bands that try to control territories that are important for the the drug trafficking business, which significant resources for his economic sustenance - as the mineral-, or that possess an strategic location for the territorial configuration. It is indispensable to take into account the relation between the above mentioned actors armed with the society, while this one is the door for the access to the territory in which they move, where they find the ways in order that they remain and grow and for it they can turn favored or relegated. This analysis will allow to know the characteristics of both phenomena, to know how they behave, the relations that are formed between these armed actors and the society, to determine the uses of the violence in opposition to the population and in which ways the society benefits or not from these relations, in the measure in which it is possible to realize the characterization of the AUC and the BACRIM.Politólogo (a)Pregrad

Síndrome polen-alimento (síndrome de alergia por vía oral)

El síndrome de alergia a alimentos y pólenes, también conocido como síndrome polen-alimento o síndrome de alergia oral, se caracteriza por una reacción local en la boca y faringe después de ingerir ciertos alimentos vegetales crudos, en individuos sensibilizados al polen de hierbas, malezas y árboles. El abedul-manzana es el prototipo de este síndrome, siendo la manzana, pera y ciruela los alimentos más comúnmente asociados. Los síntomas suelen limitarse a la cavidad oral, pero pueden incluir reacciones sistémicas, incluida la anafilaxia. La sensibilización a alérgenos de polen, como las proteínas de transferencia de lípidos, profilina y proteínas PR-10, desencadena este síndrome. Su prevalencia varía según la región geográfica y el tipo de polen predominante, afectando entre el 30% y el 60% de las alergias alimentarias. El diagnóstico implica historia clínica, pruebas cutáneas y, en casos ambiguos, pruebas de provocación alimentaria oral. El tratamiento consiste principalmente en evitar los alimentos desencadenantes

Relation between diagnosis of atheromatous plaque from orthopantomographs and cardiovascular risk factors. A study of cases and control subjects

Background: In recent years the use of orthopantomography has been proposed as a low-cost, reliable and noninvasive diagnostic medium for detecting atheromatous plaque. The purpose of this study was to correlate the presence of carotid calcifications (atheroma) in orthopantomographs with specific risk factors for cerebrovascular accidents (previous cerebrovascular accidents, arterial hypertension, and diabetes). Patient and Methods: The methods used in this observational study of cases and control subjects followed STROBE (Strengthening the Reporting of Observational studies in Epidemiology) recommendations. The study analyzed a total of 1,602 panoramic radiographs taken for dental diagnostic purposes between January 2010 and February 2014. The main variables analyzed were the incidence of atheromatous plaque and other cardiovascular risk factors. Epidat 3.1 statistical software was used to determine minimum sample sizes and the results were analyzed using PASW (Predictive Analytics Software) Statistics 10.0.0. Results: For all the variables analyzed, the correlation between radiographic detection of atheromatous plaque and the presence of cardiovascular disease risk factors was found to be statistically significant (RR>1.5). Conclusions: The presence of cardiovascular risk factors is related to the incidence of radiopaque lesions at the carotid artery bifurcation, indicating the presence of atheromatous plaque

Inferior Alveolar Nerve Paresthesia After Overfilling of Endodontic Sealer Into the Mandibular Canal

The present study describes a case of endodontic sealer(AH Plus) penetration within and along the mandibular canal from the periapical zone of a lower second molar after endodontic treatment. The clinical manifestations comprised anesthesia of the left side of the lower lip, paresthesia and anesthesia of the gums in the third quadrant, and paresthesia and anesthesia of the leftmental nerve, appearing immediately after endodontic treatment. The paresthesia and anesthesia of the lip and gums were seen to decrease, but the mental nerve paresthesia and anesthesia persisted after 3.5 years. This case illustrates the need to expend great care with all endodontic techniques when performing nonsurgical root canal therapy, especially when the root apices are in close proximity to vital anatomic struc tures such as the inferior alveolar cana

Rescue surgery (surgical repositioning) of impacted lower second molars

La impactación de los segundos molares inferiores es una complicación de la erupción dentaria muy infrecuente, dado que su incidencia se cifra de un 0,03 a un 0,21 %. Se ha detectado en mayor frecuencia de forma unilateral que bilateral y es más usual en mandíbula que en maxilar. Presenta una ligera predicción por el sexo masculino, y la inclinación mesial es la más habitual. Se han publicado una amplia variedad de aproximaciones terapéuticas, fundamentalmente apelando a técnicas quirúrgicas únicas o ayudadas de técnicas ortodóncicas, con el objeto de llevar al diente a su correcta posición, y que se engloban bajo el concepto de cirugía de rescate. En los casos resueltos con una reubicación del diente impactado,la extracción profiláctica del germen se ha propugnado como obligatoria. Presentamos el caso de una paciente de 12 años y 6 meses derivado al Master de Cirugía Bucal de la Universidad de Sevilla por presentar falta de erupción del segundo molar inferior izquierdo. El paciente fue derivado por su ortodoncista, quien detecta la impactación del diente, antes de iniciar el tratamiento ortodóncico.Dicho compañero nos indica que, si es posible, no extraigamos el germen del tercer molar, pues prevé que será viable su erupción en el futuro (dispondrá de espacio en la arcada suficiente). Medidos los espacios de que disponemos, decidimos intentar la reubicación del diente impactado sin extraer el germen del cordal, que se llevó a cabo de forma exitosa.The impaction of lower second molars, given that its incidence is 0.03 to 0.21%, is a rare complication in tooth eruption. It has been detected more often in unilateral form than bilateral and is more common in the mandible than in the maxillary. It has a slight predilection for males, and mesial inclination ismore usual. A wide variety of therapeutic approaches have been published,basically referring to surgical techniques, independent or complemented by means of orthodontic technical aids, with the aim of placing the tooth in the correct position, and which are encompassed under the concept of surgical rescue.In cases resolved with repositioning of an impacted tooth, prophylactic root extraction has been proposed as obligatory.We present a case of a 12 and a half year old patient referred to the University of Seville due to non-eruption of the left lower second molar.The patient was referred by her orthodontist, who detected the impaction before starting orthodontic treatment. The orthodontist requested that, if it was possible, we did not extract the root of the third molar, because its eruption would be feasible in the future (there would be sufficient space in the arch). The spaces available were measured and we decided to attempt the repositioning of the impacted tooth without extracting the root of the wisdom tooth, which was carried out successfully

Alergia alimentaria no mediada por IgE

La alergia alimentaria es una respuesta inmunitaria a las proteínas de los alimentos. Suele afectar al 8% de los niños y al 2% de los adultos en países occidentales. La alergia alimentaria no mediada por IgE afecta, principalmente, el aparato gastrointestinal. Las alergias alimentarias gastrointestinales se clasifican, por su patogenia subyacente, en: mediadas por IgE, no mediadas por IgE, o mixtas. Los síntomas de pacientes con proctocolitis alérgica inducida por proteínas alimentarias se originan por la inflamación local del colon distal, que causa hematoquecia en neonatos. Puede afectar todo el conducto gastrointestinal y provocar síntomas de emesis intratable, con subsiguientes trastornos metabólicos y choque hipovolémico. El síndrome de enterocolitis inducida por proteínas alimentarias es una alergia no mediada por IgE que suele aparecer en la infancia, con vómito prolongado repetitivo, que inicia entre 1 a 4 horas después de la ingestión de alimentos. La manifestación en adultos suele desencadenarse por el consumo de mariscos. Las enfermedades atópicas afectan del 40-60% de los pacientes con síndrome de enterocolitis inducida por proteínas alimentarias, incluso al 40-50% de quienes padecen enteropatía y proctocolitis inducidas por proteínas alimentarias. Los probióticos (Lactobacillus GG) pueden aliviar los síntomas de proctocolitis alérgica inducida por proteínas alimentarias, al alterar la composición de la microbiota intestinal. El trasplante de microbiota fecal (TMF) puede cambiar la microecología intestinal de manera eficiente comparada con los alimentos o probióticos

aZIBO Shape Descriptor for Monitoring Tool Wear in Milling

El objetivo de este trabajo es estimar eficientemente el desgaste del mecanizado de metales y mejorar las operaciones de sustitución de la herramienta. El procesamiento de imágenes y la clasificación se utilizan para automatizar la toma de decisiones sobre el tiempo adecuado para el reemplazo dela herramienta. Específicamente, el descriptor de forma aZIBO (momentos absolutos de Zernike con orientación de contorno invariable) se ha utilizado para caracterizar el desgaste de la plaquita y garantizar su uso óptimo. Se ha creado un conjunto de datos compuesto por 577 regiones con diferentes niveles de desgaste. Se han llevado a cabo dos procesos de clasificación diferentes: el primero con tres clases diferentes (desgaste bajo, medio y alto -L, M y H, respectivamente) y el segundo con sólo dos clases: Low (L) y High (H). La clasificación se llevó a cabo utilizando por un lado kNN con cinco distancias diferentes y cinco valores de k y, por otra parte, una máquina de vectores de soporte (SVM). El rendimiento de aZIBO se ha comparado con descriptores de forma clásicos como los momentos de Hu y Flusser. Los supera, obteniendo tasas de éxito de hasta el 91,33% para la clasificación L-H y 90,12% para la clasificación L-M-H

Weak Interactions in Cocrystals of Isoniazid with Glycolic and Mandelic Acids

This research was funded by the Network of Excellence “Metallic Ions in Biological Systems” CTQ2017-90802-REDT [Ministerio de Economía y Competitividad (Spain) and European Regional Development Fund (EU)], and the Xunta de Galicia (Spain) [Rede de Excelencia MetalBIO ED431D 2017/01].Acknowledgments: We thank the “Centre de Tecnologies de la Informació” (CTI) at the Univeritat de les Illes Baleares for computational facilities.This work deals with the preparation of pyridine-3-carbohydrazide (isoniazid, inh) cocrystals with two -hydroxycarboxylic acids. The interaction of glycolic acid (H2ga) or d,l-mandelic acid (H2ma) resulted in the formation of cocrystals or salts of composition (inh) (H2ga) (1) and [Hinh]+[Hma]– (H2ma) (2) when reacted with isoniazid. An N0-(propan-2-ylidene)isonicotinic hydrazide hemihydrate, (pinh) 1/2(H2O) (3), was also prepared by condensation of isoniazid with acetone in the presence of glycolic acid. These prepared compounds were well characterized by elemental analysis, and spectroscopic methods, and their three-dimensional molecular structure was determined by single crystal X-ray crystallography. Hydrogen bonds involving the carboxylic acid occur consistently with the pyridine ring N atom of the isoniazid and its derivatives. The remaining hydrogen-bonding sites on the isoniazid backbone vary based on the steric influences of the derivative group. These are contrasted in each of the molecular systems. Finally, Hirshfeld surface analysis and Density-functional theory (DFT) calculations (including NCIplot and QTAIM analyses) have been performed to further characterize and rationalize the non-covalent interactions.Network of Excellence “Metallic Ions in Biological Systems” CTQ2017-90802-REDT [Ministerio de Economía y Competitividad (Spain) and European Regional Development Fund (EU)]Xunta de Galicia (Spain) [Rede de Excelencia MetalBIO ED431D 2017/01

A new roman fish-salting workshop in the Saltes Island (Tinto-Odiel Estuary, SW Spain): La Cascajera and its archaeological and geological context

The southwestern Atlantic coast of the Iberian Peninsula presents an important Roman heritage that includes numerous fish-salting workshops, with an industrial activity that went on for almost a millennium (1st century BC-7th century AD). Nevertheless, a future broad research is still necessary to determine the geologic substratum on which they are based, their palaeoenvironmental evolution, their main economic objectives and the byproducts derived from their activities. This paper is focused on the geology, dating and the archaeological record of La Cascajera, a new site located in the Tinto-Odiel estuary (SW Spain). This new cetaria occupied the northwestern end of La Cascajera ridge (Saltés Island), constituted by sandy, bioclastic deposits of previous washover fans (1st-2th centuries AD). During the main period of activity (middle of 4th century AD-5th century AD), the existence of a certain typology of amphorae as well as the documentation of a shell deposit formed mostly by Glycymeris suggest that this factory was oriented to the production of mixed fish sauces and the handling of edible bivalves. The main features of this deposit (texture, paleontology, taphonomy) could be used to differentiate middens from natural shelly ridges.La costa suratlántica de la Península Ibérica posee un importante legado romano que incluye numerosos talleres halieúticos, con una actividad que se desarrolló durante casi un milenio (siglo I a.C.-siglo VII d.C.). Sin embargo, se precisan nuevas investigaciones que determinen el contexto geológico en el que se desarrollaron, su evolución paleoambiental, sus principales objetivos económicos y los subproductos derivados de sus actividades. Este trabajo se centra en la geología, datación y registro arqueológico de La Cascajera, un nuevo yacimiento localizado en el estuario de los ríos Tinto y Odiel (S.O. de España). Esta nueva cetaria ocupaba el extremo noroccidental de la cresta de La Cascajera (Isla de Saltés), constituida por depósitos bioclásticos arenosos de abanicos de derrame previos (siglos I-II d.C.). Durante su principal periodo de actividad (mitad del siglo IV d.C.-siglo V d.C.), la existencia de una cierta tipología de ánforas así como la existencia de un conchero formado principalmente por Glycymeris sugieren que esta factoría estaba dedicada a la producción de salsas mixtas de pescado y la extracción de bivalvos comestibles. Las principales características de este conchero (textura, paleontología, tafonomía) podrían ser usadas para diferenciar concheros de acumulaciones naturales de conchas.Ministerio de Innovación, Ciencia y Empresa de España. Plan Nacional de I+D+i DEATLANTIR II-HAR2017-89154-PJunta de Andalucía-HUM-132, RNM-238 y RNM-29