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218 p., During the Spanish Civil War and the posterior dictatorship, a huge number of people disappeared during the war or in episodes of repression. A large number of victims remain unidentified. One of the main limitations of these analyses is the difficulty of obtaining informative STRs profiles from the remains, limiting the identification of missing persons. With this aim, this work focuses on multidisciplinary research to investigate the relationship between the factors (physical-chemical and biological) that influence the state of preservation (anthropological and genetic) in order to understand the influence of these factors and increase the number of identifications of victims in the context of the Spanish Civil War. For this purpose, we have collected and analyzed the physicochemical composition of the graves and performed the anthropological analysis. Subsequently, the autosomal and Y-chromosome STRs of the skeletal remains were analysed. Finally, we developed a predictive statistical model to determine the most influential variable in obtaining an informative profile of autosomal STRs (>12STRs).With this, it has been possible to demonstrate that, in addition to DNA concentration and inhibition, also organic matter influence in successfully obtaining an informative autosomal STRs profile from skeletal remains buried in mass graves of Spanish Civil War.

170 p., Cystic fibrosis is the most common autosomal recessive rare disease in Caucasian populations. Despite the wide variety of mutations, patients with same mutations may present different clinical symptoms. This variability has led to the study of severity modifying genes.In the present study, a set of 33 SNPs located in 19 genes previously identified as modulatory genes for the severity of CF have been studied in 106 CF patients from the north of the Iberian Peninsula and the Balearic Islands, their parents and health controls.The main objective of this doctoral thesis is to detect the presence of gene variants and to infer a possible interaction between genetics and the common symptomatology of the disease. To carry out this objective, we proceeded to perform the association analysis (ASs) of each individual SNP with the different symptomatologies of the disease. These ASs have also shown that SNPs related to a modulator effect on a given symptomatology, also show associations with other CF symptoms. Several of these associations had not been previously described, thus constituting new findings. Considering genetically linked SNPs, haplotypes ASs were also performed. This approach has scarcely been used in previous studies in this field. The combination of the transversality of the effects of modulator gene SNPs combined with the increased robustness of association results based on haplotypic analyses determined the interest in performing multigene association analyses. This novel approach based on multigene models has allowed us to identify and objectify the impact of risk/protection genotypes, as this strategy better fits the reality of the patients' genome. The study of the modifier genes seems be a useful tool to dig deep on the pathogeny and to identify new therapeutic targets.

226 p., La aparición de nuevos conocimientos y tecnologías en microfabricación han permitido el desarrollo de nuevas herramientas miniaturizadas para ensayos de biología celular. El mayor progreso que estos dispositivos ofrecen es proveer a los investigadores con recursos para ensayos analíticos, útiles para un amplio rango de aplicaciones que pueden llegar a superar los métodos establecidos en términos de sensibilidad y rentabilidad. En la presente tesis, se han aprovechado algunas de estas microtecnologías para el desarrollo de plataformas biosensóricas simples pero versátiles y fiables, para su implementación en ensayos de alta relevancia biomédica. Concretamente, se han desarrollado dos tipos de biosensores. En primer lugar, se crearon biosensores para ensayos biomédicos de afinidad celular y citotoxicidad, basados en la adhesión de células a patrones de biomoléculas, sin y con integración en dispositivos microfluídicos. En segundo lugar, biosensores plasmónicos basados en fibras ópticas y nanopartículas de oro se desarrollaron y usaron para cuantificar agentes bioactivos como los iones de cobre y la proteína Citocromo c. Estos biosensores constituyen un paso adelante en un campo en auge que está llamado a cerrar la brecha entre la obtención de resultados cuantitativos y reproducibles y la democratización de los medios para obtener dichos resultados.

160 p., Market globalization and the growing demand for seafood products are contributing to increase supply chains¿ complexity, making traceability efforts an incredibly challenging task. Under such circumstances, there is a definite need for verification methods that occur independently of labelling claims and offer an unbiased secondary check on the data collected through traceability systems. Verifying the geographic origin is critical to enforce food safety regulations, prevent commercial fraud, guarantee legal and well managed sustainable resources, as well as fulfil present and future expectations of conscientious consumers, producers and authorities.This thesis is focused on the development of three analytical tools for verifying the geographical origin of farmed Mytilus galloprovincialis mussels in order to ensure their integrity, whether they are fresh, processed or canned. Beforehand, we verified the species following the fast SYBR-Green real-time PCR methodology developed in Chapter 1, to ensure species substitution had not occurred. Then, we evaluated the use of genetic markers (Chapter 2), trace elements (Chapter 3) and bacterial communities (Chapter 4) as a proxy to determine the provenance of this bivalve mollusc.Our findings support that these methodological approaches have the potential to trace the geographic origin of farmed M. gallo provincialis mussels and can therefore play a pivotal role in ensuring transparency and safety in the globalized mussel trade. It is also important to highlight that there is no perfect universal approach, they all have a set of limitations. Thus, according to the cultivation area and to the type of product (fresh, frozen or preserved) the right tool should be applied, independently or in combination.

235 p., Las células madre del folículo piloso humano (hHF-MSCs) se encuentran en el compartimento mesenquimal del folículo, que está formado por la papila dérmica, la vaina dérmica y la región peribulbar.Estas células se caracterizan por su alto potencial regenerativo sin la generación de procesos fibróticos en la cicatrización de heridas, y han mostrado beneficios frente al empleo de MSCs de otras fuentes de procedencia cuando han sido transplantadas. Sin embargo, su uso está aún por explorar en un amplio rango de terapias celulares de reemplazamiento y secreción de factores de crecimiento para la reactivación de células madre endógenas.El folículo piloso es una rica y fácilmente accesible fuente de MSCs. El presente trabajo muestra el desarrollo de una metodología que simplifica el aislamiento y enriquecimiento de hHF-MSCs de la región occipital del cuero cabelludo de donantes de entre 20 y 40 años, y que, a su vez, favorece su capacidad de duplicación y predice la probabilidad de éxito en la obtención de cultivos proliferativos, lo que facilita su adecuación para su futuro uso en terapia celular.

224 p., El efecto de los factores ecológicos y fisiológicos influyen de manera drástica en las respuestas biológicas medidas en organismos centinela como los mejillones (Mytilus sp.) haciendo que la evaluación de la salud medioambiental usando biomarcadores pueda ser poco fiable, esto se ve más acentuado en lugares con condiciones ambientales extremas. En el presente trabajo se ha seleccionado una batería de biomarcadores de nivel celular y tisular para descifrar los valores base y el efecto de la variabilidad natural (temperatura, disponibilidad de alimento, lesiones histopatológicas, factores relacionados con la latitud) en la capacidad de respuesta de estos biomarcadores ante la contaminación en poblaciones de mejillón en el Océano Atlántico Norte, Océano Ártico y Mar Báltico. La presente tesis doctoral ofrece información clave para entender las respuestas biológicas de los mejillones de las localizaciones estudiadas ante la contaminación y el potencial efecto de los factores naturales suponiendo esto una ventaja considerable a la hora de realizar experimentos toxicológicos y programas de biomonitoring para la contaminación.

235 p., Las células madre del folículo piloso humano (hHF-MSCs) se encuentran en el compartimento mesenquimal del folículo, que está formado por la papila dérmica, la vaina dérmica y la región peribulbar.Estas células se caracterizan por su alto potencial regenerativo sin la generación de procesos fibróticos en la cicatrización de heridas, y han mostrado beneficios frente al empleo de MSCs de otras fuentes de procedencia cuando han sido transplantadas. Sin embargo, su uso está aún por explorar en un amplio rango de terapias celulares de reemplazamiento y secreción de factores de crecimiento para la reactivación de células madre endógenas.El folículo piloso es una rica y fácilmente accesible fuente de MSCs. El presente trabajo muestra el desarrollo de una metodología que simplifica el aislamiento y enriquecimiento de hHF-MSCs de la región occipital del cuero cabelludo de donantes de entre 20 y 40 años, y que, a su vez, favorece su capacidad de duplicación y predice la probabilidad de éxito en la obtención de cultivos proliferativos, lo que facilita su adecuación para su futuro uso en terapia celular.

338 p., The demersal mixed-fisheries operating in the Bay of Biscay catch more than 150 species, but only eight of them are included in the Western Waters and adjacent waters multiannual management plan. This raises the issue of whether the current multiannual management plan is sufficient to ensure the sustainability of the whole system. In this PhD Thesis, we evaluated the impact of current and alternative management strategies on the sustainability of the system in a simulated reality. In comparison to previous works, we broadened the scope of the work to non-target and data-limited stocks. First, we applied a stock prioritization approach to identify the stocks most at risk, most exploited, and/or economically most important for the fishery. The potential risk was calculated using the Productivity-Susceptibility Analysis (PSA). Then, we evaluated by ¿self-test¿ simulation the ability of a low data-demand assessment model like Stock Reduction Analysis (SRA) to provide accurate population estimates under alternative data availability, population exploitation levels and initial population assumptions. This allowed us to identify the data-limited stocks whose dynamics could be reliably mimicked based on the SRA model. Finally, we evaluated the performance of current and alternative management strategies. The simulation included 28 stocks: 13 had age-structured population dynamics (9 data-rich stocks based on analytical assessments and 4 data-limited stocks using SRA model) and for 15 stocks the catch was proportional to the effort level and independent of their abundance. The obtained results contributed to providing a holistic evaluation of the management of the mixed-fisheries operating in the Bay of Biscay.

368 p., The rapid development of nanotechnology has led to a growing concern about the entry and possibleimpacts of nanomaterials, such as graphene family nanomaterials (GFNs) in the marine environment.Similarly, plastic pollution has become a global pollution problem and effects of nanoplastics (NPs) andmicroplastics (MPs) are particularly under studied. Further, GFNs and NPs and MPs could cause anadditional threat to marine organisms because they could act as carriers of pollutants such as polycyclicaromatic hydrocarbons (PAHs) to marine organisms, increasing their bioavailability and toxicity throughthe so-called Trojan horse effect. Thus, this PhD thesis aimed to assess the potential hazards for themarine environment posed by GFNs and NPs and MPs and associated PAHs. Overall, this workdemonstrated that GFNs, NPs and MPs can act as carriers of the PAH benzo(a)pyrene (BaP) but the roleof GFNs in reducing the bioaccumulation and toxicity of associated pollutants must be highlighted.Interactions between GFNs, NPs and MPs with sorbed BaP occur, giving rise to different toxicity patternsdepending on the response studied at molecular, cellular, tissue and organism levels. Overall the Trojanhorse effect resulted more complex than previously anticipated and further studies are needed tounderstand the hazards posed by GFNs, NPs and MPs and associated pollutants for marine organisms atboth early developmental and adult stages, especially at long-term.

249 p., The main objective of the present thesis has consisted in implementing a life cycle-oriented perspective when assessing the risks associated to synthetic chemicals including biocides and engineered nanomaterials (ENMs) as examples of conventional and emerging contaminants, respectively.In the case of biocidal products, in general, once used, biocidal active substances can easily reach aquatic compartments where they are transformed in metabolites, degradation and transformation products. However, the hazard of such substances towards trophic levels representative of the freshwater compartment is less known than that of the parent compounds.For EMNs, the different processes that nanotechnology enabled products and/or applications (NEPs/NEAs) undergo throughout their life cycle need to be simulated in a controlled environment for the assessment of their potential release and associated exposure to their released forms. We have developed two nano-release assessment studies selecting (i) a NEA in solid state, generally aimed to be used at industrial scale at the indoor compartment (polypropylene based nanocomposite samplesintegrating different types of (nano)fillers) and (ii) a NEP in liquid state, aimed to be used by consumers at the indoor compartment (nano-additivated inkjet printing ink).We have demonstrated that under a holistic, life cycle-oriented perspective that includes use and end of life stages, novel risks differing from those derived from the manufacturing stage occur.Finally, we have evaluated the possibility to derive new characterization factors for human respiratory and ecotoxicity of one of the case studies for ENMs in order to account for the specific impacts of these emerging chemicals in the life cycle assessment framework according to ISO 14040.

226 p., Los metales y metaloides están presentes de forma natural en el medioambiente y muchos de ellos son esenciales para la supervivencia de los seres vivos. El ser humano ha extraído metales a lo largo de la historia para su uso, lo que ha conllevado el desarrollo de actividades mineras en zonas con litologías naturalmente ricas en estos elementos químicos. Dichas actividades han degradado significativamente el medioambiente, reduciendo la calidad de los ecosistemas. Dada la ausencia de normas de calidad ambiental para metales en las matrices de sedimento y la biota, en esta tesis se han propuesto metodologías para el desarrollo de criterios de metales en tejido basados en la aproximación a la condición de referencia (condiciones inalteradas o prístinas).La evaluación de riesgo de los metales en zonas afectadas por actividades mineras en este estudio ha incluido varias líneas de evidencia: una evaluación del riesgo de contaminación de metales en el sedimento a través de los umbrales ecológicos calculados para un área concreta, una evaluación de la toxicidad crónica de los sedimentos, una evaluación sobre el estado de la comunidad de macroinvertebrados de campo y un estudio sobre los niveles de bioacumulación de metales. Elconocimiento y caracterización de los niveles de referencia y de niveles umbrales de efecto han hecho posible una evaluación de riesgo realista y sólida de las comunidades de macroinvertebrados expuestas a los contaminantes. Además, se ha desarrollado el transcriptoma del oligoqueto acuático Tubifex tubifex y estudiado la expresión génica de biomarcadores relacionados con rutas relevantes de respuesta al estrés.Finalmente, este estudio ha demostrado que la capacidad de los ecosistemas para mantener comunidades de macroinvertebrados saludables, en términos de composición, diversidad y organización funcional, ha sido alterada por la contaminación por metales en los sedimentos y por la bioacumulación de los mismos en los organismos del bentos. Se han dete

Seaweed are organisms rich in many bioactive compounds such as proteins, minerals, vitamins, fibers, essential amino acids, pigments, and fatty acids, which give them extraordinary antihypertensive, antidiabetic, antioxidant, anti-inflammatory, antitumoral, antiviral, and antimicrobial properties. Seaweed could potentially contribute to future global security in functional foods and nutraceuticals and could be an important compound in the pharmaceutical and biotechnological industries in drug development, among other uses. This review focuses on the gastronomic point of view and discusses the compositional and nutritional characteristics, topics related to consumption, current applications and technologies, limitations and challenges in production, and market developments in this rising market.

Los capítulos 3, 4 y 5 están sujetos a confidencialidad por el autor. 99 p., La presente Tesis Doctoral se basa en el desarrollo de tres nuevos sistemas de producción de PRP. El objetivo fue mejorar su contenido molecular, enriqueciendo, además de los factores de crecimiento plaquetarios como en el PRP convencional, también los extraplaquetarios. Así, el trabajo incluye el desarrollo del protocolo de los tres nuevos métodos de obtención de PRP, la caracterización celular y molecular del PRP resultante y el análisis de su bioactividad en cultivos celulares. La Tesis es de carácter internacional, ya que se ha realizado una estancia de investigación en la Universidad de Twente, donde se testó el nuevo PRP en un modelo de articulación osteoartrítica de rodilla en chip.

A toxicogenomic approach was used for toxicity evaluation of arsenic in the aquatic environment, and differential gene expression was investigated from 24 h and 96 h water-only acute toxicity tests with the aquatic oligochaete, Tubifex tubifex (Annelida, Clitellata). Several toxicological endpoints (survival and autotomy) of the oligochaete and tissue residues were measured, and dose-response modelling of gene expression data was studied. A reference transcriptome of the aquatic oligochaete, T. tubifex, was reconstructed for the first time, and genes related to cell stress response (Hsc70, Hsp10, Hsp60, and Hsp83), energy metabolism (COX1), oxidative stress (Cat, GSR, and MnSOD), and the genes involved in the homeostasis of organisms (CaM, RpS13, and UBE2) were identified and characterised. The potential use of the genes identified for risk assessment in freshwater ecosystems as early biomarkers of arsenic toxicity is discussed.

288 p., En esta tesis, desarrollada en el grupo de investigación de Biología Celular en Toxicología Ambiental (BCTA/UPV/EHU) y enmarcada dentro de un proyecto europeo (GRACE - 'Integrated oil spill response actions and environmental effects'), se investiga la toxicidad de tres petróleos de interés en los mares fríos de las regiones Ártica y Sub-ártica bajo distintas condiciones (temperatura y concentración de contaminante), así como la influencia de distintas estrategias previstas para responder a posibles vertidos de petróleo (aplicación de dispersantes, envejecimiento bajo hielo y la quema del petróleo) sobre la toxicidad de dichos petróleos, mediante bioensayos con embriones de erizo de mar alóctonos, utilizando tanto criterios estándar de como metodologías novedosas.En la introducción se presenta una visión general del origen y uso actual del petróleo, así como de su composición, clasificación e incidencia en vertidos de petróleo en la última década. Se exponen las distintas estrategias de respuesta a dichos incidentes, entre los que se encuentran la aplicación de dispersante químico y la quema de petróleo (in situ burning), estrategias de respuesta que se analizarán en la presente tesis. Además, dado que las condiciones ambientales pueden influenciar el comportamientodel petróleo, se plantea la pregunta de cómo se verá afectada la toxicidad en condiciones extremas como, por ejemplo, en el caso de los mares helados, cuando hay presencia de hielo y el petróleo queda por debajo de este, o/y, a un rango amplio de distintas temperaturas. Para poder evaluar esta toxicidad se utiliza la fracción acomodada al agua (WAF), que asemeja la columna de agua en la que se encuentran disueltos los hidrocarburos presentes en el petróleo y que son biodisponible para los organismos marinos y, por lo tanto, los responsables de causar efectos tóxicos sobre ellos. Se hace un breve repaso al uso de invertebrados marinos utilizados en los test de toxicidad de petróleo y se centra en el erizo d

Los apéndices 6 y 7 así como las páginas 68 a 76 están sujetos a confidencialidad por el autor, El cultivo celular es una herramienta poderosa que permite estudiar los procesos celulares de una manera sencilla. Aunque los métodos convencionales cultivo han permitido obtener inmensurable conocimiento en muchísimas áreas, incluyendo la biología celular y la biomedicina, siempre se ha requerido de estudios in vivo complementarios debido a la falta de control de las interacciones y los microambientes celulares en los métodos convencionales de cultivo. La microfabricación permite la generación de nuevos sistemas y plataformas que aporten un mayor control del cultivo celular, así como una monitorización más eficiente de sus procesos. En esta tesis, se ha combinado la microfabricación, la ciencia de materiales, la bioquímica y la biología celular para el desarrollo de una herramienta de monitorización celular. Para ello, se han desarrollado nuevos sensores para la secreción celular y patrones combinados de células y microesferas que permitan el control de las interacciones celulares, la presentación eficiente de moléculas señalizadoras a las células y la detección directa de la secreción celular incorporando los biosensores desarrollados.

Non-invasive collection of target cells is crucial for research in biology and medicine. In this work, we combine a thermo-responsive material, poly(N-isopropylacrylamide), with an electroactive material, poly(3,4-ethylene-dioxythiopene):poly(styrene sulfonate), to generate a smart and conductive copolymer for the label-free and non-invasive detection of the capture and release of cells on gold electrodes by electrochemical impedance spectroscopy. The copolymer is functionalized with fibronectin to capture tumor cells, and undergoes a conformational change in response to temperature, causing the release of cells. Simultaneously, the copolymer acts as a sensor, monitoring the capture and release of cancer cells by electrochemical impedance spectroscopy. It is expected that this platform has the potential to play a role in top-notch label-free electrical monitoring of human cells obtaining in clinic.

123 p.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

The use of Plant Protection Products (PPPs) is leading to high exposure scenarios with potential risk to soil organisms, including non-target species. Assessment of the effects of PPPs on non-target organisms is one of the most important components of environmental risk assessment (ERA) since they play crucial functions in ecosystems, being main driving forces in different soil processes. As part of the framework, EFSA is proposing the use of the ecosystem services approach for setting specific protection goals. In fact, the services provided by soil organisms can be impacted by the misuse of PPPs in agroecosystems. The aim of this work was to assess PPPs potential risk upon ecosystem services along European soils, considering impacts on earthworms and collembola. Four well-known (2 insecticides-esfenvalerate and cyclaniliprole- and 2 fungicides - picoxystrobin and fenamidone-) worst case application (highest recommended application) were studied; exploring approaches for linked observed effects with impacts on ecosystem services, accounting for their mode of action (MoA), predicted exposure, time-course effects in Eisenia fetida and Folsomia sp. and landscape variability. The selected fungicides exerted more effects than insecticides on E. fetida, whereas few effects were reported for both pesticides regarding Folsomia sp. The most impacted ecosystem services after PPP application to crops appeared to be habitat provision, soil formation and retention, nutrient cycling, biodiversity, erosion regulation, soil remediation/waste treatment and pest and disease regulation. The main factors to be taken into account for a correct PPP use management in crops are discussed.

Over the last decade, xenoestrogenic effects have been reported in populations of thicklip grey mullet Chelon labrosus from contaminated estuaries in the Bay of Biscay, resulting in intersex condition. To understand the level of gene flow in individuals of different Basque estuaries microsatellite markers were used to evaluate the population structure and connectivity of C. labrosus from estuaries of the Basque coast. 46 microsatellites were tested and 10 validated for the analysis of 204 individuals collected from 5 selected Basque estuaries and 2 outgroups in the Bay of Cadiz and Thermaic Gulf. The polymorphic microsatellites revealed 74 total alleles, 2–19 alleles per locus. The mean observed heterozygosity (0.49 ± 0.02) was lower than the expected one (0.53 ± 0.01). There was no evidence of genetic differentiation (FST = 0.0098, P = 0.0000) among individuals or sites. Bayesian clustering analysis revealed a single population in all sampled locations. The results of this study indicate widespread genetic homogeneity and panmixia of C. labrosus across the current sampling areas spanning the Atlantic and Mediterranean basins. The hypothesis of panmixia could therefore be well supported so individuals inhabiting estuaries with high prevalence of intersex condition should be considered as members of the same single genetic group as those inhabiting adjacent estuaries without incidence of xenoestrogenicity.

Introduction: Estuarine ecosystems are under high anthropogenic pressure and receive a wide Q7 variety of contaminants, including metals. They can remain trapped in estuarine sediments at high concentrations for long periods but eventually they can be released to the overlying water and become toxic for the resident benthic biota. As a consequence, biomonitoring programs surveying the health status of estuaries and coastal areas count with benthic species for the assessment of health disturbances in these specific ecosystems. The present investigation aims at recognizing toxicopathic effects elicited in estuarine life stages of the benthic fish Solea senegalensis on exposure to waterborne Cd, a model compound for metal contamination. Methods: Biological responses to metal exposure were assessed based on the application of a “biomarker + histopathology” approach. Two-year old sole juveniles were exposed to various concentrations of waterborne Cd for 3 and 7 days (Control; Low Cd: 1 µg/l; Mid Cd: 10 µg/l and High Cd: 1000 µg/l). Liver samples were collected for chemical analysis at day 7. Biological samples were collected at days 3 and 7 for biochemical (brain and liver), histochemical (liver) and histopathological analysis (liver, gills and gonad). Results: Brain acetyl cholinesterase was inhibited after 7 days exposure, indicating neurotoxic effects on exposure to 1000 µg Cd/l. Hepatic catalase and glutathione-Stransferase were induced at day 3 and inhibited at day 7, which suggests a bell-shaped response. A dose-dependent lysosomal membrane destabilization in hepatocytes was recorded at day 7. In parallel, histopathological lesions in gills, liver and gonad were more frequent at day 7 in soles exposed to high Cd concentrations. Discussion: Overall, the “biomarker + histopathology” approach revealed that waterborne Cd causes toxicopathic effects in sole juveniles upon exposure to environmentally relevant concentrations (10 µg Cd/l). The effects were clearly time dep

Analysis of fish behaviour is an effective way to indirectly identify the presence of environmental pollutants that negatively affect fish life, its production and quality. Monitoring individual and collective behaviours produces large amounts of non-linear data that require tailor-suited computational methods to interpret and manage the information. Fractal dimension (FD) and entropy are two groups of such non-linear analysing methods that serve as indicators of the complexity (FD) and predictability (entropy) of the behaviours. Since behavioural complexity and predictability may be modulated by contaminants, the changes in its FD and entropy values have a clear potential to be embedded in a biological early warning system (BEWS), which may be particularly useful in Precision Fish Farming settings and to monitor wild populations. This work presents a review of the effects of a wide range of environmental contaminants, including toxic compounds, cleaning and disinfecting agents, stimulant (caffeine), anaesthetics and antibiotics, heavy metals (lead, cupper, and mercury), selenium, pesticides and persistent environmental pollutants, on the FD and entropy values of collective and individual behavioural responses of different fish species. All the revised studies demonstrate the usefulness of both FD and entropy to indicate the presence of pollutants and underline the need to consider early changes in the trend of the evolution of their values prior to them becoming significantly different from the control values, i.e., while it is still possible to identify the contaminant and preserve the health and integrity of the fish.

Platelet-Rich Plasma (PRP) is an autologous biological product which, due to its regenerative capacity, is currently used in different fields of medicine. This biological treatment has proven to be effective in numerous research studies due to its high content of growth factors released by platelets. However, the current systems used to obtain PRP do not enrich the growth factors and cytokines outside platelets. Considering this, the present work aims to develop a new technique by which all the biomolecules present in plasma are enriched. Thus, a new method based on ultrafiltration has been developed for the obtaining of the novel PRP. By this method, ultrafiltration of the plasma water is carried out using a 3KDa filtering unit. The results showed that the technique was able to concentrate extraplatelet factors, such as IGF-1 and HGF, in contrast with conventional plasmas. Thus, the cultured cells responded with increased viability to this new PRP. These results could provide a new approach to the treatment of injuries requiring regenerative medicine, potentially improving the outcomes of the conventional PRPs.

Platelet-rich plasma (PRP) is an autologous biologic product used in several fields of medicine for tissue repair due to the regenerative capacity of the biomolecules of its formulation. PRP consists of a plasma with a platelet concentration higher than basal levels but with basal levels of any biomolecules present out of the platelets. Plasma contains extraplatelet biomolecules known to enhance its regenerative properties. Therefore, a PRP containing not only a higher concentration of platelets but also a higher concentration of extraplatelet biomolecules that could have a stronger regenerative performance than a standard PRP. Considering this, the aim of this work is to develop a new method to obtain PRP enriched in both platelet and extraplatelet molecules. The method is based on the absorption of the water of the plasma using hydroxyethyl acrylamide (HEAA)-based hydrogels. A plasma fraction obtained from blood, containing the basal levels of platelets and proteins, was placed in contact with the HEAA hydrogel powder to absorb half the volume of the water. The resulting plasma was characterized, and its bioactivity was analyzed in vitro. The novel PRP (nPRP) showed a platelet concentration and platelet derived growth factor (PDGF) levels similar to the standard PRP (sPRP), but the concentration of the extraplatelet growth factors IGF-1 (p < 0.0001) and HGF (p < 0.001) were significantly increased. Additionally, the cells exposed to the nPRP showed increased cell viability than those exposed to a sPRP in human dermal fibroblasts (p < 0.001) and primary chondrocytes (p < 0.01). In conclusion, this novel absorption-based method produces a PRP with novel characteristics compared to the standard PRPs, with promising in vitro results that could potentially trigger improved tissue regeneration capacity.

This investigation deals with how temperature influences oil toxicity, alone or combined with dispersant (D). Larval lengthening, abnormalities, developmental disruption, and genotoxicity were determined in sea urchin embryos for assessing toxicity of low-energy water accommodated fractions (LEWAF) of three oils (NNA crude oil, marine gas oil –MGO-, and IFO 180 fuel oil) produced at 5–25 °C. PAH levels were similar amongst LEWAFs but PAH profiles varied with oil and production temperature. The sum of PAHs was higher in oil-dispersant LEWAFs than in oil LEWAFs, most remarkably at low production temperatures in the cases of NNA and MGO. Genotoxicity, enhanced after dispersant application, varied depending on the LEWAF production temperature in a different way for each oil. Impaired lengthening, abnormalities and developmental disruption were recorded, the severity of the effects varying with oil, dispersant application and LEWAF production temperature. Toxicity, only partially attributed to individual PAHs, was higher at lower LEWAF production temperatures.

Glucose is an analyte of great importance, both in the clinical and sports fields. As an alternative to standard blood analysis, sweat arises as an easily accessible, non-invasive biofluid, as well as a great candidate to monitor human health and sport performance of athletes. However, there is a current need for the fabrication of low cost, easy to use technologies capable of sequential analysis of sweat biomarkers at different times. In this research, we developed a chrono-sampling microfluidic platform integrated with an alginate/TiO2 biosystem for sweat glucose determination. Glucose concentrations ranging from 10 to 1000 µM in artificial sweat were detected and quantified, both in a bead format and in the device, where the whole system was calibrated and verified. The PDMS microfluidic platform allowed chrono-sampling over more than 42 min. This research is oriented towards the development of a miniaturized device for fast analysis at the point and place of need. The integration of the chrono-sampling capacity opens the possibility for a continuous monitoring of sweat biomarkers during exercise, allowing a personalized-oriented training to improve the performance of athletes.

Biomachining has been investigated as a sustainable and effective alternative to conventional prototyping techniques for molding polymeric materials for their subsequent use as microfluidic devices. A novel and simple process based on the combination of a Pressure Sensitive Adhesive mask and a varnish has been proposed for preparing metal workpieces as an alternative to photolithography, with the latter being the most widely used technique for protecting workpieces. As far as the bioprocess is concerned, it has been applied in successive mold-etching and oxidant bio-regeneration stages. Metal solubilization has proven to be repeatable in several consecutive mold-etching stages when using the regenerated oxidant solution. As a result, the lifespan of the biomachining medium has been prolonged, contributing to process sustainability. An equation with two restrictions has been proposed to predict the time required to obtain a mold with a fixed height, as metal solubilization evolves differently between the first and subsequent hours. Finally, the bio-engraved copper pieces have acted as effective molds in the fabrication of self-powered polydimethylsiloxane microfluidic devices. This new biomachining application is therefore an effective and ecofriendly process for producing microfluidic devices.

In a non-linear system, such as a biological system, the change of the output (e.g., behaviour) is not proportional to the change of the input (e.g., exposure to stressors). In addition, biological systems also change over time, i.e., they are dynamic. Non-linear dynamical analyses of biological systems have revealed hidden structures and patterns of behaviour that are not discernible by classical methods. Entropy analyses can quantify their degree of predictability and the directionality of individual interactions, while fractal dimension (FD) analyses can expose patterns of behaviour within apparently random ones. The incorporation of these techniques into the architecture of precision fish farming (PFF) and intelligent aquaculture (IA) is becoming increasingly necessary to understand and predict the evolution of the status of farmed fish. This review summarizes recent works on the application of entropy and FD techniques to selected individual and collective fish behaviours influenced by the number of fish, tagging, pain, preying/feed search, fear/anxiety (and its modulation) and positive emotional contagion (the social contagion of positive emotions). Furthermore, it presents an investigation of collective and individual interactions in shoals, an exposure of the dynamics of inter-individual relationships and hierarchies, and the identification of individuals in groups. While most of the works have been carried out using model species, we believe that they have clear applications in PFF. The review ends by describing some of the major challenges in the field, two of which are, unsurprisingly, the acquisition of high-quality, reliable raw data and the construction of large, reliable databases of non-linear behavioural data for different species and farming conditions.

Mercury (Hg) is a toxicant of concern, particularly in aquatic food webs. Mercury can move to terrestrial systems through consumption of aquatic prey or emergence of insects with aquatic larval phases. The possible movement of Hg from sediments to wetland plants and into terrestrial food webs though primary consumers has received less attention. We investigated differences and correlations in Hg in soil, wood and leaves of willows (Salix caroliniana) and folivorous beetles from a wetland with enhanced levels of Hg. Further, we compared samples from tree islands that had enriched Hg in soil through bird guano with control islands. Hg in any sample type did not correlate with Hg in any other sample type from the same island. We found higher [Hg] in soils and significantly higher [Hg] in leaves from colony islands, while [Hg] in beetles appeared to be higher in control islands. In any case, despite comparatively high [Hg] in soil and leaves, Hg in folivorous beetles was below detection levels and lower than that reported from other studies. We conclude that movement of Hg from wetland trees to terrestrial food webs through wetland vegetation is negligible in this ecosystem.

This work presents a new architecture concept for microfluidic devices, which combines the conventional 3D printing fabrication process with the stable and precise integration of polymeric functional materials in small footprints within the microchannels in well-defined locations. The approach solves the assembly errors that normally occur during the integration of functional and/or sensing materials in hybrid microfluidic devices. The method was demonstrated by embedding four pH-sensitive ionogel microstructures along the main microfluidic channel of a complex 3D printed microfluidic device. The results showed that this microfluidic architecture, comprising the internal integration of sensing microstructures of diverse chemical compositions, highly enhanced the adhesion force between the microstructures and the 3D printed microfluidic device that contains them. In addition, the performance of this novel 3D printed pH sensor device was investigated using image analysis of the pH colour variations obtained from photos taken with a conventional camera. The device presented accurate and repetitive pH responses in the 2 to 12 pH range without showing any type of device deterioration or lack of performance over time.

BACKGROUNDDietary methyl donors might influence DNA methylation during carcinogenesis of colorectal cancer (CRC). However, whether the influence of methyl donor intake is modified by polymorphisms in such epigenetic regulators is still unclear.AIMTo improve the current understanding of the molecular basis of CRC.METHODSA literature search in the Medline database, Reference Citation Analysis (https:// www.referencecitationanalysis.com/), and manual reference screening were performed to identify observational studies published from inception to May 2022.RESULTSA total of fourteen case-control studies and five cohort studies were identified. These studies included information on dietary methyl donors, dietary components that potentially modulate the bioavailability of methyl groups, genetic variants of methyl metabolizing enzymes, and/or markers of CpG island methylator phenotype and/or microsatellite instability, and their possible interactions on CRC risk.CONCLUSIONSeveral studies have suggested interactions between methylenetetrahydrofolate reductase polymorphisms, methyl donor nutrients (such as folate) and alcohol on CRC risk. Moreover, vitamin B-6, niacin, and alcohol may affect CRC risk through not only genetic but also epigenetic regulation. Identification of specific mechanisms in these interactions associated with CRC may assist in developing targeted prevention strategies for individuals at the highest risk of developing CRC.

Global warming and acidification of the global ocean are two important manifestations of the ongoing climate change. To characterize their joint impact on Vibrio adaptation and fitness, we analyzed the temperature-dependent adaptation of Vibrio harveyi at different pHs (7.0, 7.5, 8.0, 8.3 and 8.5) that mimic the pH of the world ocean in the past, present and future. Comparison of V. harveyi growth at 20, 25 and 30 °C show that higher temperature per se facilitates the logarithmic growth of V. harveyi in nutrient-rich environments in a pH-dependent manner. Further survival tests carried out in artificial seawater for 35 days revealed that cell culturability declined significantly upon incubation at 25 °C and 30 °C but not at 20 °C. Moreover, although acidification displayed a negative impact on cell culturability at 25 °C, it appeared to play a minor role at 30 °C, suggesting that elevated temperature, rather than pH, was the key player in the observed reduction of cell culturability. In addition, analyses of the stressed cell morphology and size distribution by epifluorescent microscopy indicates that V. harveyi likely exploits different adaptation strategies (e.g., acquisition of coccoid-like morphology) whose roles might differ depending on the temperature–pH combination.

Graphene oxide (GO) has gained a great scientific and economic interest due to its unique properties. As incorporation of GO in consumer products is rising, it is expected that GO will end up in oceans. Due to its high surface to volume ratio, GO can adsorb persistent organic pollutants (POPs), such as benzo(a)pyrene (BaP), and act as carrier of POPs, increasing their bioavailability to marine organisms. Thus, uptake and effects of GO in marine biota represent a major concern. This work aimed to assess the potential hazards of GO, alone or with sorbed BaP (GO+BaP), and BaP alone in marine mussels after 7 days of exposure. GO was detected through Raman spectroscopy in the lumen of the digestive tract and in feces of mussels exposed to GO and GO+BaP while BaP was bioaccumulated in mussels exposed to GO+BaP, but especially in those exposed to BaP. Overall, GO acted as a carrier of BaP to mussels but GO appeared to protect mussels towards BaP accumulation. Some effects observed in mussels exposed to GO+BaP were due to BaP carried onto GO nanoplatelets. Enhanced toxicity of GO+BaP with respect to GO and/or BaP or to controls were identified for other biological responses, demonstrating the complexity of interactions between GO and BaP.

Oil spills pose significant environmental risks, particularly in cold seas. In the Baltic Sea, the low salinity (from 0 to 2 up to 18) affects the behaviour of the spilled oil as well as the efficiency and ecological impacts of oil spill response methods such as mechanical collection and the use of dispersants. In the present study, mussels (Mytilus trossulus) were exposed under winter conditions (5 °C) to the water-accommodated fraction (WAF) of Naphthenic North Atlantic crude oil prepared by mechanical dispersion or to the chemically enhanced fraction (CEWAF) obtained using the dispersant Finasol OSR 51 at salinities of 5.6 and 15.0. Especially at the lower salinity, high bioaccumulation of polycyclic aromatic hydrocarbons was recorded in mussels in the CEWAF treatments, accompanied by increased biomarker responses. In the WAF treatments these impacts were less evident. Thus, the use of dispersants in the Baltic Sea still needs to be carefully considered.

The presence of microplastics in the food chain is a public concern worldwide, and its analysis is an analytical challenge. In our research, we apply Raman imaging to study the presence of 1mum polystyrene microplastics in cryosections of Mytilus galloprovincialis due to its wide geographic distribution, widespread occurrence in the food web, and general high presence in the environment. Ingested microplastics are accumulated in the digestive tract, but a large number can also be rapidly eliminated. Some authors state that the translocation of microplastics to the epithelial cells is possible, increasing the risk of microplastics transmission along the food chain. However, as seen in our study, a surface imaging approach (2D) is probably not enough to confirm the internalization of particles and avoid misinterpretation. In fact, while some microplastic particles were detected in the epithelium by 2D Raman imaging, further 3D Raman imaging analysis demonstrated that those particles were dragged from the lumens to the epithelium during sample preparation due to the blade drag effect of the cryotome, and subsequently located on the surface of the analyzed cryosection, discarding the translocation to the epithelial cells. This effect can also happen when the samples are fortuitously contaminated during sample preparation. Several research articles that use similar analytical techniques have shown the presence of microplastics in different types of tissue. It is not our intention to put such results in doubt, but the present work points out the necessity of appropriate three-dimensional analytical methods including data interpretation and the need to go a step further than just surface imaging analysis.

5S rRNA is highly transcribed in fish oocytes and this transcription levels can be used to identify the presence of oocytes in the intersex testes of fish exposed to xenoestrogens. Similar to 5S rRNA, tRNAs are transcribed by RNA polymerase III (Pol-III) in eukaryotes, so this study focuses in the analysis of the levels of expression of tRNAs in the gonads (ovaries and testes) of eight teleost species as a possible new oocyte molecular marker. Total RNA extracted from gonads of six commercial teleost species in the Biscay Bay, from the pollution sentinel species thicklip grey mullet (Chelon labrosus) known present intersex testes in response to xenoestrogens in Gernika estuary and from the laboratory model species Danio rerio were analysed through capillary electrophoresis. Bioanalyzer electropherograms were used to quantify the concentrations of tRNAs, 5S and 5.8S rRNA. All studied ovaries expressed significantly higher levels of tRNAs and 5S rRNA than testes. A tRNA to 5.8S rRNA index was calculated which differentiates ovaries from testes, and identifies some intersex testes in between testes and ovaries in mullets. The tRNA/5.8S ratio was highest in ovaries in previtellogenic stage, decreasing towards maturity. Thus, strong oocyte expression of tRNAs is an additional proof of high activity levels of Pol-III during early stages of oocyte development in teleost ovaries. Incidentally, we observed that miRNA concentrations were always higher in testes than ovaries. The indexing approach developed in the present study could have multiple applications in teleost reproduction research and in the development of early molecular markers of intersex condition.

Glucose is an analyte of great importance, both in the clinical and sports fields. Since blood is the gold standard biofluid used for the analytical determination of glucose, there is high interest in finding alternative non-invasive biofluids, such as sweat, for its determination. In this research, we present an alginate-based bead-like biosystem integrated with an enzymatic assay for the determination of glucose in sweat. The system was calibrated and verified in artificial sweat, and a linear calibration range was obtained for glucose of 10–1000 µM. The colorimetric determination was investigated, and the analysis was carried out both in the black and white and in the Red:Green:Blue color code. A limit of detection and quantification of 3.8 µM and 12.7 µM, respectively, were obtained for glucose determination. The biosystem was also applied with real sweat, using a prototype of a microfluidic device platform as a proof of concept. This research demonstrated the potential of alginate hydrogels as scaffolds for the fabrication of biosystems and their possible integration in microfluidic devices. These results are intended to bring awareness of sweat as a complementary tool for standard analytical diagnosis.

The burial of multiple individuals within a single funerary monument invites speculation about the relationships between the deceased: were they chosen on the basis of status, gender or relatedness, for example? Here, the authors present the results of aDNA and isotope analyses conducted on seven individuals from an Early Iron Age barrow at Dolge njive, south-eastern Slovenia. All seven individuals are close biological relatives. While the group composition suggests strict adherence to neither patrilineal nor matrilineal structures, the funerary tradition appears highly gendered, and family links through both the male and female lines seem important in structuring of the community. The results have implications for understanding of kinship and funerary practices in late prehistoric Europe.

Sediment toxicity testing has become a crucial component for assessing the risks posed by contaminated sediments and for the development of sediment quality assessment strategies. Commonly used organisms for bioassays with estuarine sediments include amphipods, Arenicola marina polychaetes and echinoids. Among the latter, the Sea Urchin Embryo test (SET) is the most widely used. However, one relevant limitation of this bioassay is the unavailability of gametes all year-round, particularly outside the natural spawning seasons. Consequently, the establishment of an appropriate and complementary model organism for a continuous assessment of sediment quality is recommended. A reliable assessment of the hazards resulting from pollutants in sediments or pore water, can be achieved with ecologically relevant species of sediment such as the polychaete Hediste diversicolor, which is widespread in estuaries and has the capacity to accumulate pollutants. The aim of this work was to develop reliable in vivo and in vitro bioassays with H. diversicolor and its coelomocytes (immune cells) to determine the toxicity thresholds of different contaminants bounded to sediments or resuspended into water. Polychaetes were exposed to sublethal concentrations of CuCl2 (in vivo) and a non-invasive method for collection of polychaetes coelomocytes was applied for the in vitro bioassay, exposing cells to a series of CuCl2 and AgNPs concentrations. Same reference toxicants were used to expose Paracentrotus lividus following the SET (ICES N 51; Beiras et al., 2012) and obtained toxicity thresholds were compared between the two species. In vivo exposure of polychaetes to high concentrations of Cu produced weight loss and histopathological alterations. After in vitro approaches, a significant decrease in coelomocytes viability was recorded for both toxicants, in a monotonic dose-response curve, at very short-exposure times (2h). The toxicity thresholds obtained with polychaetes were in line with t

Mercury (Hg) is a globally distributed pollutant. Its sub-lethal effects on reproduction of birds have been used as indicators of contamination and of potential demographic effects. However, studies typically used single endpoints that might not be representative of entire reproductive cycle. To estimate timing and net cumulative effects of Hg exposure under field conditions, we used observational data over 11 years from >1200 nests of great egrets breeding under temporally and spatially varying food availability and Hg exposures in the Florida Everglades. We collected measures of fish biomass and availability (>100 locations annually) and used four avian reproductive endpoints that represented the entire breeding cycle. We calculated net reproductive loss by adding estimated Hg effects on failures prior to egg laying, clutch size, hatching success and nestling survival in response to food availability and Hg exposure. To validate and assess results of the observational egret study, we ran the same analyses with data of captive breeding white ibises experimentally exposed to Hg with ad libitum food over 3 years. We found large (>50 %) reductions in great egret offspring with high Hg exposure (18 mug/g dw THg nestling feather, ~0.7 mug/g ww whole egg THg) and high food availability, and even larger reductions (up to 100 %) with high Hg exposure and low food. Timing and the relative contribution of different endpoints to overall reproductive failure varied with food availability. Failures prior to egg laying were relevant at all food availabilities and proportionally most important during high food availability (~70 % of total losses). Under high food, post-hatching failures increased moderately with increasing exposure (~10 % of total losses), and under low food, hatching failures became dominant (~50 % of total losses). Patterns of failure of captive white ibis fed ad libitum resembled those of great egrets under high food availability but differed in total magnitud

Follicular atresia is an energy-saving oocyte resorption process that can allow the survival of female fish when environmental conditions are unfavourable and at the expense of fecundity. This study investigated the transcription levels of apoptosis and autophagy-related genes during atresia in the European hake that can show episodes of increased follicular atresia throughout the reproductive cycle. 169 female individuals were collected from the Bay of Biscay, and the ovaries were analysed using histological and molecular methods. Different levels of atresia were histologically detected in 73.7% of the ovaries analysed and the TUNEL assay identified apoptotic nuclei in follicles from both previtellogenic and vitellogenic stages. Transcripts of beclin-1 and ptenb were up-regulated in the ovaries containing atretic follicles, whereas p53, caspase-3, cathepsin D and dapk1 were up-regulated only in ovaries presenting vitellogenic atretic follicles. Our results indicate different implications of apoptotic vs autophagic processes leading to atresia during oocyte development, vitellogenesis being the moment of maximal apoptotic and autophagic activity in atretic hakes. The analysed genes could provide early warning biomarkers to identify follicular atresia in fish and evaluate fecundity in fish stocks.

The majority of the plastic produced in the last century is accumulated in the environment, leading to an exacerbated contamination of marine environments due to transport from land to the ocean. In the ocean, mechanical abrasion, oxidation, and photodegradation degrade large plastics into microplastics (MPs) - 0.1mum to 5mm (EFSA, 2016) which are transported through water currents reaching the water surface, water column, and sediments. Further, they can be accumulated by aquatic and benthic species, entering the trophic chain and becoming a potential threat to humans. In the present research, we aimed to decipher the accumulation and distribution time-courses between different organs or target tissues of organisms inhabiting coastal areas such as mussels Mytilus galloprovincialis and polychaetes Hediste diversicolor. Both were exposed in microcosm experiments to fluorescent polystyrene MPs (1mum) which were spiked at two doses (103 and 105 particles/mL) for 1, 4, 24, and 72h. Mussels and polychaetes were digested with 10% KOH and filtered to quantify the number of MPs incorporated. Different anatomical parts of the body were selected and processed for cryosectioning and posterior microscopic localisation of MPs. Both species accumulate MPs spiked in water column, mainly after exposure to the highest dose. In mussels, particles were found in distinct parts of the digestive tract (stomach, digestive diverticula, ducts) and gills. Even if the majority of MPs were localised in the lumen of the digestive tract, in some cases, were inside the digestive epithelium. The identification of MPs and their internalization in the digestive system was studied using Raman spectroscopy. A decreasing trend with time regarding MPs number in the digestive tract (stomach) of mussels was observed while the opposite was recorded for polychaetes and sediments. The combination of microscopical observations of frozen sections and Raman, appeared to be accurate methodologies to address MPs

271 p., La mayoría de los eucariotas son organismos unicelulares (protistas), muchos de ellos pertenecientes a linajes que divergieron temprano en la historia evolutiva de este Dominio de organismos nucleados. Microscópicos, enormemente diversos y fenotípicamente convergentes, su clasificación cladística ha sido históricamente compleja, dejando atrás un extenso registro de taxones y de términos parafiléticos y polifiléticos. Teniendo que investigar atributos estructurales, celulares, biológicos y ecológicos en un mundo de rápidas interacciones y difícilmente accesible a simple vista, la protistología es particularmente dependiente de la sistemática. Ésta permite inferir rasgos de especies crípticas a partir de especies evolutivamente relacionadas.Las moléculas de ADN (y ARN), representan un "registro" preciso de estos eventos de diversificación, que preceden incluso a los más antiguos registros fósiles. En los últimos años, la maduración de los métodos de filogenia molecular, catalizados por una mayor accesibilidad a la secuenciación de próxima generación (NGS), está permitiendo resolver preguntas e hipótesis sobre la evolución y la especiación de estos organismos micro-eucariotas que no se habían podido responder mediante otros métodos. Por una parte, arboles filogenéticos construidos mediante concatenaciones de cientos, incluso miles de genes, están permitiendo rastrear la historia evolutiva de los linajes protistas hasta el último ancestro común de todos los eucariotas (LECA). Concomitantemente, análisis moleculares basados en genes e incluso fragmentos cortos (especialmente 18S rRNA), recuperados principalmente de matrices ambientales u orgánicas (eDNA o ADN ambiental), están revelando una ¿caja de Pandora¿ de diversidad micro-eucariota. Ésta diversidad ¿oculta¿ está transformando nuestra percepción de los protistas en la cadena trófica y la estructura ecológica. En el medio marino, sus papeles como autótrofos, heterótrofos (predadores, saprófitos, parásitos) o mixótrof

217 p., Over the past 10 years, Species Distribution Models have become commonplace in the studies of biogeography, conservation biology, ecology, and climate change. However, most studies applying SDMs mainly quantify species-environment relationships and usually omit functional roles of species, biotic interactions and resource-consumer dynamics. Within this context, this thesis aimed to advance in the understanding of how biotic and abiotic factors shape species distribution and abundance by using the community of top predators of the Bay of Biscay as case study. To address this overall objective, we carried out three different studies that aimed to answer the following specific research questions:- To identify the main environmental and biotic drivers of species co-occurrences within the top predators-prey community of the Bay of Biscay. - To understand how co-occurring top predators share the environmental and trophic niche.- To define the role that prey, climatic and oceanographic variables in driving the spatio-temporal patterns of a highly mobile top predator.- To determine the relative importance of biotic and environmental drivers explaining the abundance and distribution of top predators.By this means, this thesis intended to obtain a more comprehensive understanding of the drivers governing the assemblage of top predators and a better knowledge of ecosystem functioning of the BoB.

280 p., La presencia y los efectos de micro- (<5 mm) y nanomateriales (100 nm, NM) en el medio ambiente es un tema de preocupación actual. En sistemas acuáticos que presentan un coctel de químicos debido a la alta presión de la contaminación, los micro- y NMs pueden actuar como esponjas para los contaminantes debido a su alto ratio superficie/volumen y a la hidrofobicidad de su superficie. Este fenómeno puede alterar la biodisponibilidad de los contaminantes presentes en los ecosistemas acuáticos, especialmente para los compuestos hidrófobos, y seguidamente, modular su toxicidad para los organismos acuáticos. Por ello, en la presente tesis los siguientes objetivos fueron establecidos: (1) Evaluar la biodisponibilidad y la toxicidad potencial de nanoplásticos de poliestireno (NPs), y de microplásticos (MPs) solos o con hidrocarburos aromáticos policíclicos ad/absorbidos (HAPs) para la larva de Artemia y el pez cebra; (2) Determinar la capacidad de ad/absorción de HAPs por los MPs y los NMs de óxido de grafeno (GNMs); (3) Evaluar la biodisponibilidad y toxicidad de GNMs solos o con HAPs ad/absorbidos para el pez cebra.La exposición a MPs prístinos no causó ningún impacto significativo en la supervivencia de la larva de Artemia o del embrión de pez cebra, mientras que algunos tratamientos que contenían elevadas concentraciones (mg/L) de MPs con benzo(a)pireno (B(a)P) ad/absorbido y B(a)P sólo resultaron entoxicidad aguda. Además, ambos tamaños de MPs fueron exitosos vectores de B(a)P en larvas de Artemia y embriones de pez cebra. Los resultados indicaron que los MPs pequeños (0.5 ¿m) mostraron una mayor capacidad máxima de ad/absorción de B(a)P que los MPs grandes (4.5 ¿m MPs). Para una mezcla compleja y medioambientalmente relevante de HAPs, como es la formada en la fracción acomodada al agua (WAF) de un petróleo crudo, se observó una ad/absorcíon limitada relativamente a 4.5 ¿m MPs y que dependió de la hidrofobicidad y la concentración inicial de cada HAP. En adultos de pez

Water quality monitoring of drinking, waste, continental and seawaters is of great importance to ensure safety and wellbeing for humans, fauna and flora. Researchers are developing robust water monitoring microfluidic devices but, the delivery of a cost-effective, commercially available platform has not been achieved yet. Conventional water monitoring is mainly based on laboratory instruments or on sophisticated and expensive handheld probes for on-site analysis, both requiring trained personnel and being time-consuming. As an alternative, microfluidics has emerged as a powerful tool with the capacity to displace conventional analytical systems. Nevertheless, microfluidic devices largely use conventional pumps and valves for operation, and electronics for sensing, that increment the dimensions and costs of the final platforms, diminishing their commercialization perspectives. In this review, we critically analyze the characteristics of conventional microfluidic devices for water monitoring, focusing on different water matrices (drinking, waste, continental and seawaters), towards their application in potential commercial products. Moreover, we introduce the revolutionary concept of using functional materials such as hydrogels, poly(ionic liquid) hydrogels and ionogels as alternatives to conventional fluidic handling and sensing tools for water monitoring in microfluidic devices.

246 p. (eusk) 224 p. (eng.), Tesi honetan Pirinioetako muturluzearen ekologia espaziala ikertu da bi erreka desberdinetan: bata kontserbazio-egoera bikainean (Elama), eta bestea jarduera hidroelektrikorako ur-erauzketa sistemen eraginpean egonik (Leitzaran). Helburua muturluzeak bere bizi-eremuen baitan egiten duen bazkahabitaten hautespena zehaztea zen, beren habitataren egokitasuna baldintzatzen duten faktoreak identifikatu ahal izateko. Irrati-telemetriak, Pirinioetako mutur luzeak ur-bare eta putzuen aldeanur lasterrak positiboki hautatzen zituela erakutsi zuen, bereziki ur-erauzketen eraginpean zegoen errekan.Irrati-telemetriak, ordea, ezin zuen bereizi muturluzearen habitat-hautespena urlasterretako bazkaeskuragarritasun hobearengatik edo bestelako arrazoiren batengatik gertatzen ote zen. Hortaz, aldi berean Pirinioetako muturluzearen ekologia trofikoa ikertu da. DNA metabarcoding bidezko dietarendeskribapen taxonomikoa eta ezaugarri bereizgarrien traiten araberako deskribapena aintzakotzat hartuz litekeena da bazkaren ordez beste arrazoi batzuek azaltzea animalia honen urlasterrekiko zaletasuna muturluzeen flotagarritasun naturalak, esaterako. Tesiak, oro har, Pirinioetako muturluzearen habitat-erabileraren eta dietaren espezializazio handia berresten du. Hortaz gain,muturluzearentzako ur garbiak nahikoa ez direla ere azpimarratzen du, muturluzearen kontserbaziorako ur lasterrak eta erreken konektagarritasuna funtsezko faktoreak izanik

During years sewage sludges have been worldwide poured in agricultural soils to enhance vegetal production. The "Landfill 17" located in Gernika-Lumo town (43°19'28.9"N 2°40'30.9"W) received for decades sewage sludges from the local Waste Water Treatment Plant (WWTP) with agricultural purposes. To this WWTP, several pollutants as heavy metals (Cd, Cr, Ni, Pb), PAHs (benzo(a)pyrene among many others) and pesticides (i.e. dieldrin) could have arrived from local industry and be widespread all over the landfill. Soil invertebrates like earthworms and plants are of special interest due to their close contact with the polluted matrix and their potential effects by the presence of pollutants. In this context, the aim of the present work was to determine the health status of landfill soils by evaluating the effects on model soil organisms exerted by long-lasted pollutants after on site deposition of WWTP active sludges. With such a purpose, different standard toxicity tests and cellular level endpoints were performed on lettuce and earthworms. Indeed, germination (EPA 850.4100) and root elongation (EPA 850.4230) tests were carried out in Lactuca sativa, while OECD acute toxicity test (OECD-204), reproduction test (OECD-222) and Calcein-AM viability test with coelomocytes were applied in Eisenia fetida worms. For the exposure, soils collected in the landfield containing low, medium and high concentrations of pollutants were selected, and as reference LUFA 2.3 natural standard soil was chosen. While no differences were shown in the assays with L. sativa, significant differences between sludge exposed groups and control group were recorded with E. fetida, with lower coelomocyte number and viability and higher tissue metal accumulation after 28days of exposure to polluted soils. These results confirmed the impact of contaminants to soil biota even after long periods of time.