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El Anexo 3 y resultados están sujetos a confidencialidad por el autor. 329 p., The study of biomarkers and their detection in human fluids represents one of the best ways to improve the diagnosis and prevention of diseases. The use of human fluids for biomarker analysis can facilitate a friendly and non-invasive diagnosis of various pathologies that affect daily life. Early and accurate detection of these diseases is crucial, otherwise they can lead to serious conditions. In addition to its role in diagnosis, the study of biomarkers also contributes to improve treatments, as it helps to monitor response, identify new drug targets and explore therapeutic alternatives. Based on this premise, bibliographic knowledge, and previous studies of -omics technologies carried out by our research group, the present PhD thesis uses the immobilization of molecules on solid supports as a tool for the detection of biomarkers in human fluids. In this PhD thesis, we developed antibody microarrays (AbMAs) directed against human MMP-9 as a useful tool to detect this biomarker of ocular surface inflammation in tear samples. Moreover, multiplexed AbMAs were used to also detect the tear biomarkers CST4 and S100A6, providing a more complete panel of biomarkers for ocular diseases and improving patient diagnosis. In addition, we applied molecule immobilization technologies to develop a rapid test that detects collagenase activity in human synovial fluid samples by digesting an immobilized fluorescently labeled gelatin. This assay could aid in the diagnosis of diseases in which elevated extracellular matrix degradation is observed and could be used as a rapid test in primary health care. Additionally, we applied the microarray platform to study protein-protein interaction, specifically analyzing the interaction between PD-1 and PD-L1. This study demonstrates the functionality of microarrays not only in biomarker detection, but also in the analysis of protein dynamics. In conclusion, the results of the present PhD thesis deepen the existing knowledge of humoral biomarke

128 p., El Trastorno del Espectro Autista (TEA) es una condición del neurodesarrollo que presenta diversos síntomas, entre los que destacan los déficits en comunicación y comportamiento social y la presencia de movimientos repetitivos/restrictivos. A día de hoy no existe tratamiento y múltiples estudios en animales y humanos se han centrado en diferentes sistemas neuronales para explicar su patofisiología. En concreto, en esta tesis se han estudiado el sistema cortical de excitación e inhibición y el circuito dopaminérgico. Para ello se ha utilizado un modelo murino de autismo, que tiene el gen Cntnap2 eliminado y refleja los mismos síntomas que se han detectado en humanos. En el primer estudio, relacionado con la parte del sistema excitación/inhibición, no se encontró ninguna alteración relevante que pudiera clarificar el papel del gen Cntnap2 en las alteraciones observadas en este modelo. Por otro lado, se estudió el sistema dopaminérgico a nivel estructural y funcional y los resultados revelaron alteraciones funcionales en el modelo de autismo, que se ven reflejadas en una menor activación de las neuronas dopaminérgicas del Área Tegmental Ventral (ATV) y una liberación anómala de dopamina en el Núcleo accumbens (NAc) durante la interacción social. Además, se pudo revertir estos déficits sociales con activación quimiogenética de las neuronas dopaminérgicas del ATV, lo que confirmaba que éstas son las responsables de dichos déficits sociales.

Los capítulos 3, 4,5,6 y 7 están sujetos a confidencialidad por la autora. 104 p., En el siglo XXI, el cáncer se ha convertido en la segunda causa de muerte en todo el mundo. Esta enfermedad es el resultado de un conjunto de mutaciones, provocando que las células crezcan, se dividan y se propaguen sin control, dando lugar a que se desarrolle metástasis.El cáncer colorrectal, (CCR) el cual se puede originar tanto en el colon como en el recto, se encuentra entre los cánceres con mayor mortalidad a nivel mundial. Varias investigaciones han demostrado que en el CCR existen bucles autocrinos, que juegan un papel importante en la iniciación y progresión del cáncer. Los factores de crecimiento están involucrados en esta estimulación autocrina, entre ellos cabe destacar los miembros de la familia del factor de crecimiento endotelial vascular (VEGF), aunque al principio se creía que su función solo se limitaba a la permeabilidad vascular y la angiogénesis de manera paracrina.El presente trabajo se ha centrado en el estudio de VEGF-C, ya que se ha demostrado su importante implicación en el microambiente tumoral del CCR. Además, nuestro laboratorio estudia las proteínas convertasas, las cuales son necesarias para para la maduración y activación del VEGFC. Las convertasas concretamente cortan entre el C-terminal silk homology domain (SHD) y el VEGF homology domain(VHD), desprendiendo el fragmento C-terminal.Hasta la fecha, se creía que el fragmento C-terminal liberado durante la escisión pro-VEGF-C no tenía ninguna función, pero hemos dedicado nuestro esfuerzo a dilucidar su papel en el CCR; siendo este el objetivo de la presente tesis. Para ello hemos realizado una serie de experimentos tanto in vitro como in vivo que han demostrado que el fragmento C terminal del VEGFC muestra características protumorales que le presentan como una diana para el CRC.

The need for new and effective treatments for neonates suffering from hypoxia–ischemia is urgent, as the only implemented therapy in clinics is therapeutic hypothermia, only effective in 50% of cases. Cannabinoids may modulate neuronal development and brain plasticity, but further investigation is needed to better describe their implication as a neurorestorative therapy after neonatal HI. The cannabinoid URB447, a CB1 antagonist/CB2 agonist, has previously been shown to reduce brain injury after HI, but it is not clear whether sex may affect its neuroprotective and/or neurorestorative effect. Here, URB447 strongly reduced brain infarct, improved neuropathological score, and augmented proliferative capacity and neurogenic response in the damaged hemisphere. When analyzing these effects by sex, URB447 ameliorated brain damage in both males and females, and enhanced cell proliferation and the number of neuroblasts only in females, thus suggesting a neuroprotective effect in males and a double neuroprotective/neurorestorative effect in females.

Background Glaucoma, a progressive neurodegenerative disease, is a leading cause of irreversible vision loss worldwide. This study aims to elucidate the critical role of Müller glia (MG) in the context of retinal ganglion cell (RGC) death, particularly focusing on the influence of peripheral MG sensitivity to high pressure (HP). Methods Co-cultures of porcine RGCs with MG were isolated from both the central and peripheral regions of pig retinas and subjected to both normal and HP conditions. Mass spectrometry analysis of the MG-conditioned medium was conducted to identify the proteins released by MG under all conditions. Results Peripheral MG were found to secrete a higher quantity of neuroprotective factors, effectively promoting RGC survival under normal physiological conditions. However, under HP conditions, co-cultures with peripheral MG exhibited impaired RGC survival. Moreover, under HP conditions, peripheral MG significantly upregulated the secretion of proteins associated with apoptosis, oxidative stress, and inflammation. Conclusions This study provides robust evidence suggesting the involvement of MG in RGC death in glaucoma, thus paving the way for future therapeutic investigations.

Renal cell carcinoma (RCC) ranks among the most prevalent malignancies in Western countries, marked by its notable heterogeneity, which contributes to an unpredictable clinical trajectory. The insufficiency of dependable biomarkers adds complexity to assessing this tumor progression. Imbalances of several components of the intrarenal renin–angiotensin system (iRAS) significantly impact patient prognoses and responses to first-line immunotherapies. In this study, we analyzed the immunohistochemical expression of the Mas-related G-protein-coupled receptor D (MrgD), which recognizes the novel RAS peptide alamandine (ALA), in a series of 87 clear cell renal cell (CCRCCs), 19 papillary (PRCC), 7 chromophobe (ChRCC) renal cell carcinomas, and 11 renal oncocytomas (RO). MrgD was expressed in all the renal tumor subtypes, with a higher mean staining intensity in the PRCCs, ChRCCs, and ROs. A high expression of MrgD at the tumor center and at the infiltrative front of CCRCC tissues was significantly associated with a high histological grade, large tumor diameter, local invasion, and locoregional node and distant metastasis. Patients with worse 5-year cancer-specific survival and a poorer response to antiangiogenic tyrosine-kinase inhibitors (TKIs) showed higher MrgD expression at the center of their primary tumors. These findings suggest a possible role of MrgD in renal carcinogenetic processes. Further studies are necessary to unveil its potential as a novel biomarker for CCRCC prognosis and response to frontline therapies.

Next-generation high-performance polymers require consideration as sustainable solutions. Here, to satisfy these criteria, we propose to combine high-performance styrenic block copolymers, a class of thermoplastic elastomer, with cellulose derivatives as a reinforcing agent with the aim of maintaining and/or improving structural and surface properties. A great advantage of the proposed blends is, besides their biocompatibility, a decrease in environmental impact due to blending with a natural polymer. Particularly, we focus on identifying the effect of different blending compounds and blend ratios on the morphological, structural, thermal, mechanical, electrical and cytotoxic characteristics of materials. This research provides, together with novel material formulations, practical guidelines for the design and fabrication of next-generation sustainable high-performance polymers.

n his worldwide best-seller Homo Deus [1], historian and philosopher Yuval Noah Harari discusses the future challenges facing humankind. In particular, he predicts that humanity will pursue three goals in the coming decades: immortality, bliss, and divinity. Biomedical engineering is pivotal to at least two of these alleged human aspirations—immortality and divinity—and it may arguably pertain to the pursuit of bliss, as well. The possibility of replacing or upgrading cellular constituents of the human body has led to an unprecedented scenario; for the first time in history, new cells and body tissues can be generated ex vivo to replace damaged or decayed body parts via cell transplant, similarly to the replacement of pieces of a LEGO®® figure. The use of stem cells for tissue/organ reconstruction and for the treatment of lesions and diseases has seen overwhelming success over the past few decades, and multiple well-validated therapies are now available for dealing with hitherto incurable conditions.

293 p., El melanoma cutáneo es un trastorno neoplásico de la piel que se origina por la transformación maligna de los melanocitos de la epidermis. Se trata de un tipo de cáncer muy agresivo con gran capacidad de metastatizar para el que no existen tratamientos eficaces en estadios avanzados, por lo que la categorización de los enfermos diagnosticados en fases tempranas en función de su pronóstico es clave para un mejor abordaje la enfermedad. Las vesículas extracelulares (EV) constituyen un mecanismo de comunicación celular de gran relevancia en la progresión del melanoma y representan un campo de estudio clave para comprender mejor los mecanismos de progresión del cáncer e identificar biomarcadores con valor pronóstico. En esta tesis doctoral a) se caracterizaron mediante LC-MS/MS los perfiles proteómicos de las EV liberadas por líneas celulares de melanocitos y melanoma metastásico en cultivo, b) se evaluó la implicación de una de las proteínas identificadas, CEMIP, en la modulación del microambiente tumoral, demostrando que la exposición de fibroblastos dérmicos a EV tumorales con alta y baja presencia de CEMIP modula su capacidad migratoria de manera CEMIP-dependiente sin llevar asociado un cambio en los niveles de ¿SMA, c) se optimizó el método de aislamiento de EV a partir de muestras de suero y d) se identificó, mediante un estudio proteómico dirigido basado en ensayo de extensión por proximidad (PEA), que niveles más altos de CXCL11, CXCL13, VEGF-A y MMP-7 contenidos en EV séricas se asocian a mal pronóstico en pacientes con melanoma cutáneo en estadio II, lo que sugiere su potencial como biomarcadores en estadios tempranos de la enfermedad.

Neonatal hypoxia-ischemia (HI) often causes hypoxic-ischemic encephalopathy (HIE), a neurological condition that can lead to overall disability in newborns. The only treatment available for affected neonates is therapeutic hypothermia; however, cooling is not always effective to prevent the deleterious effects of HI, so compounds such as cannabinoids are currently under research as new therapies. Modulating the endocannabinoid system (ECS) may reduce brain damage and/or stimulate cell proliferation at the neurogenic niches. Further, the long-term effects of cannabinoid treatment are not so clear. Here, we studied the middle- and long-term effects of 2-AG, the most abundant endocannabinoid in the perinatal period after HI in neonatal rats. At middle-term (postnatal day 14), 2-AG reduced brain injury and increased SGZ’s cell proliferation and the number of neuroblasts. At post-natal day 90, the treatment with the endocannabinoid showed global and local protection, suggesting long-lasting neuroprotective effects of 2-AG after neonatal HI in rats.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

203 p., El cáncer y las enfermedades cardiovasculares son en la actualidad las principales causas de mortalidad a nivel mundial. El rápido desarrollo socioeconómico experimentado durante el último siglo ha traído consigo la producción e introducción en el ambiente de millones de sustancias químicas sin suficiente control toxicológico. El incremento experimentado en los últimos años en la incidencia y mortalidad causadas por el cáncer y las enfermedades cardiovasculares no puede ser explicado únicamente por el aumento en el tamaño poblacional, el envejecimiento y factores como la dieta y el estilo de vida. Cada vez existe mayor evidencia de que la exposición a determinados factores ambientales incrementa el riesgo de cáncer y enfermedades cardiovasculares. Entre estos contaminantes ambientales se encuentranlos bifenilos policlorados (PCBs). Los PCBs son compuestos químicos fabricados para su comercialización desde el año 1920. Su estabilidad y propiedades propiciaron su uso comercial (en plásticos, pinturas, pesticidas...) y que se distribuyeran a nivel mundial en el medio ambiente. Su amplia distribución, su resistencia a la degradación y su lipofilicidad son las responsables de que hayan entrado en la cadena alimentaria y sigan detectándose en la actualidad en el aire, alimentos e incluso leche materna humana, en niveles que exceden los valores de referencia establecidos por la EFSA. Los PCBs son conocidos actualmente por ser contaminantes orgánicos persistentes y carcinógenos. Sin embargo, aún existe a día de hoy controversia sobre la relación entre la exposición a PCBs y su correlación con el cáncer de mama y el melanoma, y su potencial cardiotóxico durante el desarrollo embrionario apenas ha sido estudiado. Por ello, se decidió estudiar el efecto de una mezcla representativa de la contaminación ambiental por PCBs (Aroclor 1254) sobre el potencial invasivo de líneas tumorales humanas y sobre las propiedades cinemáticas y dinámicas de cardiomiocitos derivados de células E

The Dental Pulp of permanent human teeth is home to stem cells with remarkable multilineage differentiation ability: human Dental Pulp Stem Cells (DPSCs). These cells display a very notorious expression of pluripotency core factors, and the ability to give rise to mature cell lineages belonging to the three embryonic layers. For these reasons, several researchers in the field have long considered human DPSCs as pluripotent-like cells. Notably, some signaling pathways such as Notch and Wnt contribute to maintaining the stemness of these cells through a complex network involving metabolic and epigenetic regulatory mechanisms. The use of recombinant proteins and selective pharmacological modulators of Notch and Wnt pathways, together with serum-free media and appropriate scaffolds that allow the maintenance of the non-differentiated state of hDPSC cultures could be an interesting approach to optimize the potency of these stem cells, without a need for genetic modification. In this review, we describe and integrate findings that shed light on the mechanisms responsible for stemness maintenance of hDPSCs, and how these are regulated by Notch/Wnt activation, drawing some interesting parallelisms with pluripotent stem cells. We summarize previous work on the stem cell field that includes interactions between epigenetics, metabolic regulations, and pluripotency core factor expression in hDPSCs and other stem cell types.

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co‑transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.

Ocular diseases have a strong impact on individuals, the effects of which extend from milder visual impairment to blindness. Due to this and to their prevalence, these conditions constitute important health, social and economic challenges. Thus, improvements in their early detection and diagnosis will help dampen the impact of these conditions, both on patients and on healthcare systems alike. In this sense, identifying tear biomarkers could establish better non-invasive approaches to diagnose these diseases and to monitor responses to therapy. With this in mind, we developed a solid phase capture assay, based on antibody microarrays, to quantify S100A6, MMP-9 and CST4 in human tear samples, and we used these arrays to study tear samples from healthy controls and patients with Sjögren’s Syndrome, at times concomitant with rheumatoid arthritis. Our results point out that the detection of S100A6 in tear samples seems to be positively correlated to rheumatoid arthritis, consistent with the systemic nature of this autoinflammatory pathology. Thus, we provide evidence that antibody microarrays may potentially help diagnose certain pathologies, possibly paving the way for significant improvements in the future care of these patients.

Prostate cancer is one of the most common cancers among men. Although many patients respond favorably to first-line treatments, castration—and chemotherapy—resistance arises after a few years, leading to metastasis. Thus, new approaches are being investigated using natural supplements to reinforce current therapies. Ocoxin is a plant-based mixture with antitumor properties that have been proved in several cancers. Here, we evaluated the cytotoxic capacity of this compound itself and combined with Docetaxel, Enzalutamide and Olaparib as an adjuvant agent. We observed that Ocoxin reduced tumor cell viability; slowed down cell cycles; altered the expression of genes involved in DNA replication, cell cycles and the p53 signaling pathway; and reduced migratory capacity after stimulation with cancer-associated fibroblasts (CAFs) and osteoblasts in vitro and reduced tumor volume in vivo. The combination of the nutritional supplement with chemotherapy showed a higher cytotoxic effect than chemotherapy alone and reverted chemoresistance conferred by CAFs and osteoblasts. Moreover, the adjuvant therapy also improved the outcome in vivo compared to the treatment with solo chemotherapy, where mice developed smaller tumors and less angiogenesis. Therefore, Ocoxin arises as a good candidate for further studies in combination with current treatments for prostate-cancer patients.

The capture of tumour-derived extracellular vesicles (TEVs) by cells in the tumour microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumour cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumour cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lung macrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression.

Background Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. Aims In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. Results The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3′–5′ domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Conclusion Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Primary brain tumors are most commonly detected in the late stages of the disease and there are only a few treatment opportunities. Overall survival rates are significantly low and the response to chemo/radiotherapy is not sufficient, despite surgical intervention. The tumor microenvironment (TME) regulates several brain tumor hallmarks, such as cell migration, invasiveness, proliferation, therapy resistance, stemness maintenance, immune evasion, among others. Therefore, it is necessary to understand the pro-tumoral mechanisms that are regulated by the TME in order to detect and identify new biomarkers and novel therapeutic targets.

Stem cell-based therapies have shown promising results for the regeneration of the nervous system. However, the survival and integration of the stem cells in the neural circuitry is suboptimal and might compromise the therapeutic outcomes of this approach. The development of functional scaffolds capable of actively interacting with stem cells may overcome the current limitations of stem cell-based therapies. In this study, three-dimensional hydrogels based on graphene derivatives and cerium oxide (CeO2) nanoparticles are presented as prospective supports allowing neural stem cell adhesion, migration and differentiation. The morphological, mechanical and electrical properties of the resulting hydrogels can be finely tuned by controlling several parameters of the self-assembly of graphene oxide sheets, namely the amount of incorporated reducing agent (ascorbic acid) and CeO2 nanoparticles. The intrinsic properties of the hydrogels, as well as the presence of CeO2 nanoparticles, clearly influence the cell fate. Thus, stiffer adhesion substrates promote differentiation to glial cell lineages, while softer substrates enhance mature neuronal differentiation. Remarkably, CeO2 nanoparticle-containing hydrogels support the differentiation of neural stem cells to neuronal, astroglial and oligodendroglial lineage cells, promoting the in vitro generation of nerve tissue grafts that might be employed in neuroregenerative cell therapies.

Background: The main purpose of this article is to introduce a universal mathematics-aided vaccine design method against malignant melanoma based on neoantigens. The universal method can be adapted to the mutanome of each patient so that a specific candidate vaccine can be tailored for the corresponding patient. Methods: We extracted the 1134 most frequent mutations in melanoma, and we associated each of them to a vector with 10 components estimated with different bioinformatics tools, for which we found an aggregated value according to a set of weights, and then we ordered them in decreasing order of the scores. Results: We prepared a universal table of the most frequent mutations in melanoma ordered in decreasing order of viability to be used as candidate vaccines, so that the selection of a set of appropriate peptides for each particular patient can be easily and quickly implemented according to their specific mutanome and transcription profile. Conclusions: We have shown that the techniques that are commonly used for the design of personalized anti-tumor vaccines against malignant melanoma can be adapted for the design of universal rankings of neoantigens that originate personalized vaccines when the mutanome and transcription profile of specific patients is considered, with the consequent savings in time and money, shortening the design and production time.

Background: Melanoma incidence has continued to rise in the latest decades, and the forecast is not optimistic. Non-invasive diagnostic imaging techniques such as optical coherence tomography (OCT) are largely studied; however, there is still no agreement on its use for the diagnosis of melanoma. For dermatologists, the differentiation of non-invasive (junctional nevus, compound nevus, intradermal nevus, and melanoma in-situ) versus invasive (superficial spreading melanoma and nodular melanoma) lesions is the key issue in their daily routine. Methods: This work performs a comparative analysis of OCT images using haematoxylin–eosin (HE) and anatomopathological features identified by a pathologist. Then, optical and textural properties are extracted from OCT images with the aim to identify subtle features that could potentially maximize the usefulness of the imaging technique in the identification of the lesion’s potential invasiveness. Results: Preliminary features reveal differences discriminating melanoma in-situ from superficial spreading melanoma and also between melanoma and nevus subtypes that pose a promising baseline for further research. Conclusions: Answering the final goal of diagnosing non-invasive versus invasive lesions with OCT does not seem feasible in the short term, but the obtained results demonstrate a step forward to achieve this.

67 p., ¿Es posible que las ondas eléctricas del cerebro (registradas mediante EEG) y su interacción con las hemodinámicas (registradas mediante PPG) puedan determinar de alguna manera situaciones de dolor progresivo? La sinergia entre la tecnología electrónica y la instrumentación de última generación, junto con la incorporación del análisis estadístico y la ciencia de datos, ofrece enormes posibilidades en la investigación biomédica, y más concretamente, en la neurociencia.La posibilidad de monitorizar on-line sus variaciones en la percepción del dolor térmico utilizando un dispositivo y algoritmos puede ser de interés para estudiar diferentes patologías que afectan al sistema nervioso periférico, como neuropatías de fibras pequeñas, fibromialgia o neuropatía diabética dolorosa.En este estudio, se ha diseñado y desarrollado una plataforma, que por un lado genera un estímulo térmico controlado gracias al aumento o disminución de temperatura de una placa Peltier, llegando a ser "doloroso" y por otro, captura simultáneamente las señales EEG y PPG para posteriormente estudiar los posibles patrones o biomarcadores del dolor a través del análisis estadístico y la ciencia de datos

218 p., El fotoenvejecimiento es un proceso complejo caracterizado por un conjunto de alteraciones cutáneas inducidas por la exposición continuada a la radiación UV-B y el consecuente estrés oxidativo celular.Actualmente, la terapia celular con células madre mesenquimales (MSCs) surge como una alternativa prometedora en la prevención del fotoenvejecimiento, gracias a su capacidad de regeneración celular, sus propiedades pro-angiogénicas y su protección frente al estrés oxidativo. Sin embargo, a pesar de su potencialidad terapéutica, la utilización de MSCs no está exenta de dificultades, fundamentalmente relacionadas con el mantenimiento de su viabilidad, propiedades y función antes y después de la implantación in vivo. Para hacer frente a estas limitaciones se han desarrollado distintas estrategias de pre-acondicionamiento in vitro de las MSCs basadas, entre otras, en incrementar la resistencia frente al estrés oxidativo mediante su exposición durante un corto periodo de tiempo a bajas concentraciones de H2O2. Dicho pre-acondicionamiento permite además estimular la actividad paracrina de dichas células, la cual puede reducir los factores pro-inflamatorios y limitar el desarrollo de fibrosis.En este trabajo hemos demostrado, en primer lugar, que las células madre mesenquimales humanas derivadas de tejido adiposo (hASCs, unas de las más frecuentemente utilizadas en medicina regenerativa) poseen mayor resistencia frente al efecto nocivo de la radiación UV-B respecto a la de los HFFs. En segundo lugar, que el pre-acondicionamiento de las hASCs con H2O2 aumenta la capacidad de respuesta de estas células al estrés oxidativo causado por la radiación UV-B, evidenciado por la mayor actividad del sistema antioxidante celular inducido por Nrf2, el aumento de su viabilidad y de su capacidad de migración. En tercer lugar, hemos demostrado, que el tratamiento con el medio acondicionado (CM) de las hASC preacondicionadas (PC/CM) ejerce un efecto citoprotector de los HFFs frente al daño le

209 p., In the field of regenerative medicine, it has been pointed out the relevance of human adipose derived stem cells (hASCs) for cell therapy. They can be easily obtained, have low immunogenicity, and secrete soluble factors that could regulate neuroinflammation and oxidative stress. However, their transplantation at the site of injury results in a low percentage of survival and engraftment, mainly due to the harsh microenvironment they encounter. To address this issue, it is imperative to pre-adapt cells, so that they resist harmful environmental factors such as oxidative stress. In this study, we preconditioned hASCs (PC-hASCs) with low doses of H2O2 and evaluated their resistance to an oxidative stress insult. PC-hASCs displayed lower levels of ROS, apoptosis and cytotoxicity than hASCs, indicating their increased capacity to resist to oxidative stress. In addition, we analyzed the molecular and bioenergetic mechanisms underlying the survival and adaptation of PC-hASCs under oxidative stress. On these conditions, PC-hASCs i, reduce intracellular ROS levels by overexpressing the transcription factor NRF2 and their related antioxidant enzymes HO-1, SOD-1, GPx-1, and CAT; ii, reduce the secretion of pro-inflammatory molecules COX-2 and IL-1ß by attenuating the expression of NF-kB, and iii, increase the total ATP production rate by adapting their metabolism in order to meet the bioenergetic demand required to survive. Finally, we evaluated the therapeutic role of PC-hASCs to overcome the deleterious effect of oxidative stress in an oligodendroglial cell population, and proved that PC-hASCs restored cell viability and diminished the intracellular ROS levels of these damaged oligodendrocytes by promoting their antioxidant response.Altogether, these findings support that PC-hASCs, given their beneficial advantages, might be considered an important breakthrough in cell¿based therapies.

Los capítulos de Resultados, Discusion y Conclusiones están sujetos a confidencialidad. 179 p., El objetivo de esta tesis doctoral fue el de encontrar nuevos biomarcadores para el melanoma, ya que lasupervivencia de los pacientes se reduce dramáticamente cuando no se detecta en la fase temprana de laenfermedad. Para esto, se ha estudiado y comparado el contenido lipídico de numerosas líneas celularesde melanocitos de piel sanos, melanocitos extraidos de nevus, melanoma primarios y melanomametastásico, aplicando diferentes metodologías de análisis lipidómico. Los resultados demuestran que lascélulas sanas y tumorales presenta una huella lipídica diferencial. Además, aplicando diferentes análisisestadísticos, hemos detectado numerosas especies de lípidos que presentan una alteración en suintensidad al comparar los diferentes grupos de estudio. Todas estas especies lipídicas se han definidocomo potenciales biomarcadores, y la metodología de detectarlos va a ser próximamente patentada. Porotro lado, hemos comprobado que algunas de las enzimas que catabolizan algunos de los lípidos conexpresión diferencial en melanoma tienen su expresión y actividad alteradas. En concreto, hemoscomprobado que la fosfolipasa D2 ayuda en el proceso tumoral y metastásico del melanoma.

Three-dimensional bioprinting combined with natural hydrogels is a promising technology for the treatment of several pathologies and different tissue regeneration. One of the most studied tissues is cartilage, a complex and avascular tissue that displays a limited self-repair capacity after injuries. Herein, the development of alginate-based hydrogels and scaffolds containing different microstructure is presented and the printability of alginate by 3D bioprinting is studied. Rheological characterization was performed for the determination of viscosity and viscoelastic properties of hydrogels and mechanical characterization was carried out for the determination of compressive modulus of alginate hydrogels. All these characteristics were correlated with alginate behaviour during 3D bioprinting process. For the printability evaluation filament diameter, perimeter of the pores, area of the pores and shrinkage of alginate scaffolds were measured. The results demonstrate that alginate microstructure has a great influence on its printability and on hydrogels’ physicochemical properties. Molecular weight of alginate determines its viscosity while M/G ratio determines cross-linking conditions and mechanical properties that vary with cross-linking density. These results suggest the importance of an exhaustive control of the viscoelastic and mechanical properties of alginate hydrogels to obtain structures with high resolution and precision.

Cellular and tissue damage triggered after hypoxia-ischemia (HI) can be generalized and affect the neurogenic niches present in the central nervous system. As neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy, the goal of the present work was to investigate the neurogenic response to HI in the neurogenic niche of the subventricular zone (SVZ) in the neonatal piglet. A total of 13 large white male piglets aged <24 h were randomized into two groups: i) HI group (n = 7), animals submitted to transient cerebral HI and resuscitation; and ii) Control group (n = 6), non-HI animals. At 48 h, piglets were euthanized, and the SVZ and its surrounding regions, such as caudate and periventricular white matter, were analyzed for histology using hematoxylin-eosin staining and immunohistochemistry by evaluating the presence of cleaved caspase 3 and TUNEL positive cells, together with the cell proliferation/neurogenesis markers Ki67 (cell proliferation), GFAP (neural stem cells processes), Sox2 (neural stem/progenitor cells), and doublecortin (DCX, a marker of immature migrating neuroblasts). Hypoxic-ischemic piglets showed a decrease in cellularity in the SVZ independent of cell death, together with decreased length of neural stem cells processes, neuroblast chains area, DCX immunoreactivity, and lower number of Ki67 + and Ki67 + Sox2 + cells. These data suggest a reduction in both cell proliferation and neurogenesis in the SVZ of the neonatal piglet, which could in turn compromise the replacement of the lost neurons and the achievement of global repair.

[EN] Discoidin domain receptor 2 (DDR2) is arising as a promising therapeutic target in breast carcinoma (BC). The ability of DDR2 to bind to collagen promotes protumoral responses in cancer cells that influence the tumor microenvironment (TME). Nonetheless, the interrelation between DDR2 expression and TME modulation during BC progression remains poorly known. For this reason, we aim to evaluate the correlation between intratumoral expression of DDR2 and the infiltration of the main TME cell populations, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). First, collagen and DDR2 expression levels were analyzed in human invasive BC samples. Then, DDR2 status correlation with tumor aggressiveness and patient survival were retrieved from different databases. Subsequently, the main pathways, cell types, and tissues correlated with DDR2 expression in BC were obtained through bioinformatics approach. Finally, we studied the association of DDR2 expression with the recruitment of CAFs and TAMs. Our findings showed that, together with the expected overexpression of TME markers, DDR2 was upregulated in tumor samples. Besides, we uncovered that altered TME markers were linked to DDR2 expression in invasive BC patients. Consequently, DDR2 modulates the stromal reaction through CAFs and TAMs infiltration and could be used as a potential worse prognostic factor in the treatment response of invasive BC.

Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface.

[EN] The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.

[EN] The renin angiotensin system (RAS) appears to influence male fertility at multiple levels. In this work, we analyzed the relationship between the RAS and DNA integrity. Fifty male volunteers were divided into two groups (25 each): control (DNA fragmentation ≤20%) and pathological (DNA fragmentation >20%) cases. Activities of five peptidases controlling RAS were measured fluorometrically: prolyl endopeptidase (which converts angiotensin [A] I and A II to A 1-7), neutral endopeptidase (NEP/CD10: A I to A 1-7), aminopeptidase N (APN/CD13: A III to A IV), aminopeptidase A (A II to A III) and aminopeptidase B (A III to A IV). Angiotensin-converting enzyme (A I to A II), APN/CD13 and NEP/CD10 were also assessed by semiquantitative cytometry and quantitative flow cytometry assays, as were the receptors of all RAS components: A II receptor type 1 (AT1R), A II receptor type 2 (AT2R), A IV receptor (AT4R or insulin-regulated aminopeptidase [IRAP]), (pro)renin receptor (PRR) and A 1-7 receptor or Mas receptor (MasR) None of the enzymes that regulate levels of RAS components, except for APN/CD13 (decrease in fragmented cells), showed significant differences between both groups. Micrographs of RAS receptors revealed no significant differences in immunolabeling patterns between normozoospermic and fragmented cells. Labeling of AT1R (94.3% normozoospermic vs 84.1% fragmented), AT4R (96.2% vs 95.3%) and MasR (97.4% vs 87.2%) was similar between the groups. AT2R (87.4% normozoospermic vs 63.1% fragmented) and PRR (96.4% vs 48.2%) were higher in non-fragmented spermatozoa. These findings suggest that fragmented DNA spermatozoa have a lower capacity to respond to bioactive RAS peptides.

[EN] Analogue gravity systems offer many insights into gravitational phenomena, both at the classical and at the semiclassical level. The existence of an underlying Minkowskian structure (or Galilean in the non-relativistic limit) in the laboratory has been argued to directly forbid the simulation of geometries with Closed Timelike Curves (CTCs) within analogue systems. We will show that this is not strictly the case. In principle, it is possible to simulate spacetimes with CTCs whenever this does not entail the presence of a chronological horizon separating regions with CTCs from regions that do not have CTCs. We find an Analogue-gravity Chronology protection mechanism very similar in spirit to Hawking's Chronology Protection hypothesis. We identify the universal behaviour of analogue systems near the formation of such horizons and discuss the further implications that this analysis has from an emergent gravity perspective. Furthermore, we build explicit geometries containing CTCs, for instance spacetimes constructed from two warp-drive configurations, that might be useful for future analysis, both from a theoretical and an experimental point of view.

[EN] Muller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Muller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Muller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Muller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Muller glia to neurons. Here, the Muller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Muller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Muller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Muller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Muller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular ma

Nowadays, biopolymers are playing a fundamental role in our society because of the environmental issues and concerns associated with synthetic polymers. The aim of this Special Issue entitled ‘Women in Polymer Science and Technology: Biopolymers’ is highlighting the work designed and developed by women on biopolymer science and technology. In this context, this short review aims to provide an introduction to this Special Issue by highlighting some recent contributions of women around the world on the particular topic of biopolymer science and technology during the last 20 years. In the first place, it highlights a selection of important works performed on a number of well-studied natural polymers, namely, agar, chitin, chitosan, cellulose, and collagen. Secondly, it gives an insight into the discovery of new polysaccharides and enzymes that have a role in their synthesis and in their degradation. These contributions will be paving the way for the next generation of female and male scientists on this topic.

Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease. In this study, the tear proteome profile of patients with idiopathic PD (iPD, n = 24), carriers of the E46K-SNCA mutation (n = 3) and healthy control (CT, n = 27) subjects was analyzed to identify candidate biomarkers for the diagnosis of PD. An observational, prospective and case-control pilot study was carried out, analyzing the participants tear samples by nano-liquid chromatography–mass spectrometry (nLC–MS/MS) and assessing their neurological impairment. The proteomic data obtained are available at ProteomeXchange with identifier 10.6019/PXD028811. These analyses led to the identification of 560 tear proteins, some of which were deregulated in PD patients and that have been implicated in immune responses, inflammation, apoptosis, collagen degradation, protein synthesis, defense, lipid transport and altered lysosomal function. Of these proteins, six were related to neurodegenerative processes and showed a good capacity to classify patients and controls. These findings revealed that certain proteins were upregulated in the tears of PD patients, mainly proteins involved in lysosomal function. Thus, in this study, tear proteins were identified that are implicated in neurodegeneration and that may be related to an aggressive disease phenotype in PD patients.

Objective. Study the feasibility of using artificial intelligence as a sensitive and specific method for COVID-19 screening in patients with respiratory conditions, using chest CT scan images and a telemedicine platform. Methods. From March 2020 to June 2021, the authors conducted an observational descriptive multicenter feasibility study based on artificial intelligence (AI) for COVID-19 screening using chest images of patients with respiratory conditions who presented at public hospitals. The AI platform was used to diagnose chest CT scan images; this was then compared with molecular diagnosis (RT-PCR) to determine whether they matched and to analyze the feasibility of AI for screening patients with suspected COVID-19. A telemedicine platform was used to send images and diagnostic results. Results. Screening of 3 514 patients with a suspected COVID-19 diagnosis was performed in 14 hospitals around the country. Most patients were aged 27 to 59 years, followed by those over 60. The average age was 48.6 years; 52.8% were male. The most frequent findings were severe pneumonia, bilateral pneumonia with pleural effusion, bilateral pulmonary emphysema, and diffuse ground glass opacity, among others. There was an average of 93% matching and 7% mismatching between images analyzed by AI and RT-PCR. Sensitivity and specificity of the AI system, obtained by comparing AI and RT-PCR screening results, were 93% and 80% respectively. Conclusions. The use of sensitive and specific AI for stratified rapid detection of COVID-19 in patients with respiratory conditions by using chest CT scan images and a telemedicine platform in public hospitals in Paraguay is feasible.

[EN] The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Muller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep s

[EN] The Tear Film Lipid Layer (TFLL) acts primarily as an interface between the aqueous layer and air. Tear film lipid is composed of a thin layer of polar lipids that interact with the secretory layer of the underlying mucosa and a thicker layer of non-polar lipids at the air interface. The tear film has a complex structure and composition that protects the cornea, promotes wound healing, and maintains high-quality vision. Plasma Rich in Growth Factor (PRGF) eye drops emerged as an exciting new treatment for corneal epitheliopathies, including aqueous deficient dry eye. The purpose of this study was to compare the lipidomic profile of eye drops obtained from PRGF with tear lipidome to determine whether PRGF drops could be an adequate complement to tears in patients with impaired TFLL. To address this study, tears and blood was collected and processed from healthy donors to obtain PRGF eye drops. Samples were aliquoted and stored at -80°C until use. The lipid profiles of these samples were analysed by Ultrahigh Performance Liquid Chromatography (UHPLC) using a Vanquish UHPLC system to obtain untargeted lipidome profiles on a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. In PRGF eye drops, 408 lipids were identified in ESI+mode and 183 in ESI- mode, and they were grouped into 15 different lipid classes from four distinct categories. By contrast, 112 lipid species were identified from tear samples in ESI+mode and 36 in ESI- mode, belonging to 12 lipid classes from six different categories. The relative abundance of most lipid species was much greater in the PRGF eye drops than in the tear, although there were some lipids present in tears that were not found in the PRGF, such as wax esters and (O-acyl)-omega-hydroxy fatty acids. In summary, these results suggest that the lipids present in PRGF eye drops could serve as a tear supplement in individuals in whom tear lipid composition is altered, although there are differences in the lipid profile of these

Testicular Germ Cell Tumours (TGCT) are widely considered a “curable cancer” due to their exceptionally high survival rate, even if it is reduced by many years after the diagnosis due to metastases and relapses. The most common therapeutic approach to TGCTs has not changed in the last 50 years despite its multiple long-term side effects, and because it is the most common malignancy in young Caucasian men, much research is needed to better the quality of life of the many survivors. Proprotein Convertases (PC) are nine serine proteases responsible for the maturation of inactive proproteins with many diverse functions. Alterations in their expression have been associated with various diseases, including cancer and inflammation. Many of their substrates are adhesion molecules, metalloproteases and proinflammatory molecules, all of which are involved in tumour development. Inhibition of certain convertases has also been shown to slow tumour formation, demonstrating their involvement in this process. Considering the very established link between PCs and inflammation-related malignancies and the recent studies carried out into the immune microenvironment of TGCTs, the study of the involvement of PCs in testicular cancer may open up avenues for being both a biomarker for diagnosis and a therapeutic target.

[EN] Postoperative endoscopic cyclophotocoagulation (CPC) for the treatment of glaucoma in patients with Boston keratoprosthesis type II (BKPro II) was first described in 2017 by Poon et al. (Endoscopic cyclophotocoagulation for the treatment of glaucoma in Boston keratoprosthesis type ii patient. J Glaucoma. 2017 Apr;26(4):e146-9). As we do not have this device, we present a case of transscleral CPC (TSCPC), in a BKPro II patient who had graft versus host disease and developed uncontrolled glaucoma. We dissected plane by plane to expose the bare sclera and performed the procedure as traditionally described. We concluded that this is a safe, controlled, and effective option in this patient population where the glaucoma treatment options are very limited. To the best of our knowledge, this is the first case report to describe the surgical technique of TSCPC in a BKPro II patient.

308 p., El objeto de esta tesis doctoral ha sido la caracterización proteómica y lipidómica de cinco poblaciones celulares de la córnea humana para la identificación de nuevos biomarcadores de cada tipo celular. Además, se han empleado las poblaciones celulares caracterizadas para el desarrollo de un sistema de monitorización de la respuesta inmune humoral frente a injerto generada por trasplantes de córnea. Para esto, se han extraído cinco tipos celulares de corneas humanas y sus extractos se han analizados mediante ensayos de proteómica. En paralelo, se han estudiado extractos celulares, cortes de córnea y células corneales inmovilizadas sobre portas de vidrio utilizando distintos métodos lipidómicos. Así mismo, se han empleado sueros de pacientes que hayan recibido al menos un trasplante corneal para detectar anticuerpos reactivos frente al injerto, utilizando las células corneales previamente caracterizadas como dianas antigénicas. Los resultados obtenidos demuestran que cada tipo celular tiene un perfil proteómico y lipidómico único que permite diferenciarlos entre sí. Además, se ha propuesto un biomarcador proteico para la identificación de células epiteliales progenitoras del limbo esclerocorneal, relacionadas con la regeneración corneal, y que podría ser de gran interés para mejorar la terapia celular en casos de la deficiencia en células madre limbares. En cuanto al estudio lipidómico, se ha visto que los tipos de ácidos grasos son diferentes en zonas concretas de la córnea, dando lugar a una distribución específica que se podría relacionar con la funcionalidad de cada región. Por otro lado, se ha conseguido realizar una prueba de concepto del sistema de monitorización de la respuesta inmune humoral, en la que se han identificado anticuerpos reactivos en sueros de pacientes que habían recibido al menos un trasplante corneal.

The successful reprogramming of human somatic cells into induced pluripotent stem cells (hiPSCs) represented a turning point in the stem cell research field, owing to their ability to differentiate into any cell type with fewer ethical issues than human embryonic stem cells (hESCs). In mice, PSCs are thought to exist in a naive state, the cell culture equivalent of the immature pre-implantation embryo, whereas in humans, PSCs are in a primed state, which is a more committed pluripotent state than a naive state. Recent studies have focused on capturing a similar cell stage in human cells. Given their earlier developmental stage and therefore lack of cell-of-origin epigenetic memory, these cells would be better candidates for further re-differentiation, use in disease modeling, regenerative medicine and drug discovery. In this study, we used primed hiPSCs and hESCs to evaluate the successful establishment and maintenance of a naive cell stage using three different naive-conversion media, both in the feeder and feeder-free cells conditions. In addition, we compared the directed differentiation capacity of primed and naive cells into the three germ layers and characterized these different cell stages with commonly used pluripotent and lineage-specific markers. Our results show that, in general, naive culture NHSM medium (in both feeder and feeder-free systems) confers greater hiPSCs and hESCs viability and the highest naive pluripotency markers expression. This medium also allows better cell differentiation cells toward endoderm and mesoderm.

Los capítulos 3, 4,5,6 y 7 están sujetos a confidencialidad por la autora. 104 p., En el siglo XXI, el cáncer se ha convertido en la segunda causa de muerte en todo el mundo. Esta enfermedad es el resultado de un conjunto de mutaciones, provocando que las células crezcan, se dividan y se propaguen sin control, dando lugar a que se desarrolle metástasis.El cáncer colorrectal, (CCR) el cual se puede originar tanto en el colon como en el recto, se encuentra entre los cánceres con mayor mortalidad a nivel mundial. Varias investigaciones han demostrado que en el CCR existen bucles autocrinos, que juegan un papel importante en la iniciación y progresión del cáncer. Los factores de crecimiento están involucrados en esta estimulación autocrina, entre ellos cabe destacar los miembros de la familia del factor de crecimiento endotelial vascular (VEGF), aunque al principio se creía que su función solo se limitaba a la permeabilidad vascular y la angiogénesis de manera paracrina.El presente trabajo se ha centrado en el estudio de VEGF-C, ya que se ha demostrado su importante implicación en el microambiente tumoral del CCR. Además, nuestro laboratorio estudia las proteínas convertasas, las cuales son necesarias para para la maduración y activación del VEGFC. Las convertasas concretamente cortan entre el C-terminal silk homology domain (SHD) y el VEGF homology domain(VHD), desprendiendo el fragmento C-terminal.Hasta la fecha, se creía que el fragmento C-terminal liberado durante la escisión pro-VEGF-C no tenía ninguna función, pero hemos dedicado nuestro esfuerzo a dilucidar su papel en el CCR; siendo este el objetivo de la presente tesis. Para ello hemos realizado una serie de experimentos tanto in vitro como in vivo que han demostrado que el fragmento C terminal del VEGFC muestra características protumorales que le presentan como una diana para el CRC.

Objectives: To compare characteristics of caregivers of adults with acute traumatic brain injury (TBI) in the U.S. and Latin America (Mexico and Colombia). Design: Secondary data analysis of two cohorts. Cohort 1: English-speaking caregivers of adults with TBI in the U.S. (n = 80). Cohort 2: Spanish-speaking caregivers of adults with TBI in Mexico or Colombia (n = 109). Results: Similarities between the U.S. and Latin American caregiver groups, respectively, were: predominantly women (81.3%, 81.7%, respectively); spouses/domestic partners (45%, 31.2%); and motor vehicle accident (41.5%, 48.6%) followed by fall etiologies (40%, 21.1%). Differences between U.S. and Latin American caregivers were: age (49.5 years, 41.5 years, p < 0.001); employment status ((X-5(2) = 59.63, p < 0.001), full-time employment (63.7%, 25.7%), homemaker (2.5%, 31.2%), and retired (17.5%, 1.8%)); violence-related etiology (2.5%, 15.6%); and severity of depressive symptoms (M = 7.9, SD = 5.8; M = 5.8, SD = 5.7; p = 0.014). Conclusions: TBI caregivers in the U.S. were older and employed full-time or retired more often than those in Latin America. Violence-related etiology was nearly five times more common in Latin America, raising concerns for potential implications of post-traumatic stress and family adjustment after injury. Although both groups likely could use mental health support, this was particularly true of the U.S. cohort, maybe due to differential demographics, mechanisms of injury, or family and community support.

Dogs are known to be the first species domesticated by humans, although the geographic and temporal origin of this process is still under debate in different fields of knowledge. In the present study, we examined a humerus from a canid recovered in the Lower Magdalenian level of the site of Erralla (Zestoa, Gipuzkoa, Basque Country, Spain), combining morphology, radiocarbon dating and genetics. Our results confirm the identification of this specimen as Canis lupus familiaris, discarding miss-identification with a dhole (Cuon alpinus) through genetic analyses of cytochrome b gene and mtDNA haplogroup. The direct AMS 14C dating (17,410–17,096 cal. BP) indicated that the Erralla specimen represents one of the earliest domesticated dogs in Europe, in the Lower Cantabrian Magdalenian period. We discuss our results in the light of the debate of the origin of dogs, conducting a critical review of the datings of sites of Eurasia that have provided remains of Paleolithic and Mesolithic dogs, including the so-called “dog-like wolves”.

3D cell culture systems based on biological scaffold materials obtainable from both animal and human tissues constitute very interesting tools for cell therapy and personalised medicine applications. The white adipose tissue (AT) extracellular matrix (ECM) is a very promising biomaterial for tissue engineering due to its easy accessibility, malleability and proven biological activity. In the present study, human dental pulp stem cells (hDPSCs) were combined in vitro with ECM scaffolds from porcine and human decellularised adipose tissues (pDAT, hDAT) processed as 3D solid foams, to investigate their effects on the osteogenic differentiation capacity and bone matrix production of hDPSCs, compared to single-protein-based 3D solid foams of collagen type I and conventional 2D tissue-culture-treated polystyrene plates. pDAT solid foams supported the osteogenic differentiation of hDPSCs to similar levels to collagen type I, as assessed by alkaline phosphatase and alizarin red stainings, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and osteocalcin/bone gamma-carboxyglutamate protein (BGLAP) immunostaining. Interestingly, hDAT solid foams showed a markedly lower capacity to sustain hDPSC osteogenic differentiation and matrix calcification and a higher capacity to support adipogenesis, as assessed by RT-qPCR and oil red O staining. White ATs from both human and porcine origins are relatively abundant and available sources of raw material to obtain high quality ECM-derived biomedical products. These biomaterials could have promising applications in tissue engineering and personalised clinical therapy for the healing and regeneration of lesions involving not only a loss of calcified bone but also its associated soft non-calcified tissues.

The endocannabinoid (EC) system is a complex cell-signaling system that participates in a vast number of biological processes since the prenatal period, including the development of the nervous system, brain plasticity, and circuit repair. This neuromodulatory system is also involved in the response to endogenous and environmental insults, being of special relevance in the prevention and/or treatment of vascular disorders, such as stroke and neuroprotection after neonatal brain injury. Perinatal hypoxia–ischemia leading to neonatal encephalopathy is a devastating condition with no therapeutic approach apart from moderate hypothermia, which is effective only in some cases. This overview, therefore, gives a current description of the main components of the EC system (including cannabinoid receptors, ligands, and related enzymes), to later analyze the EC system as a target for neonatal neuroprotection with a special focus on its neurogenic potential after hypoxic–ischemic brain injury.

192 p., Melanoma is an extremely lethal skin cancer that arises as a result of the malignant transformation of melanocytes. The incidence of this pathology worldwide has increased in the last 30 years in greater proportion than the rest of the cancer, between 4-6% yearly, and shows substantial disparities between populations. To ensure appropriate treatment and a successful outcome, a timely and accurate diagnosis and prognosis of malignant melanoma is essential. Because of this, molecular alterations in the pathogenesis of melanoma are the subject of more active research. In terms of histology, melanoma show a wide variety of characteristics including epithelial, hematological, mesenchymal, and neural features, which can often make the diagnosis of the disease challenging. As new biomarkers candidates, in thisthesis we examined the expression of RKIP (Raf Kinase Inhibitor Protein) and Pirin. RKIP has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancer, and Pirin originally was considered to act as a transcriptional co-factor, but it has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. However, these studies were performed with a small cohort of patients, so a larger study is required for further evaluation of this marker's diagnostic or prognostic value. In this context, this doctoral thesis¿ goals were to evaluate the potential value of RKIP and Pirin as melanoma markers and their implication on melanocytic cell biology.Regarding RKIP, immunohistochemistry analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlate