II JORNADAS CIENTIFICAS GENYO

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1 LIBRO DE ABSTRACTS COMUNICACIONES SELECCIONADAS PARA PRESENTACIÓN ORAL SESIÓN DE INVESTIGADORES POSTDOCTORALES Abstract Nº1 Título: The Notch ligand DLL4 specifically marks Human Hematoendothelial Progenitors and Regulates their Hematopoietic Fate. Ponente: Verónica Ayllón Cases Contacto: Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hescs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a subpopulation of bipotent hemato-endothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4high HEPs are enriched in endothelial potential, while DLL4low/- HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4high HEPs are located close to DLL4low/- HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4low/- cells within hemogenic endothelium receive Notch-activating signals from DLL4high cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells. Abstract Nº2 Título: New strategies in relationship between lncrnas and the SWI/SNF complex. Ponente: Antonio Herrera Contacto: antonio.herrera@genyo.es Over the last 10 years we have come to appreciate the dynamics state of genomes. The emergence of deep sequencing techniques, along with the ENCODE [1] and FANTOM [2] projects, revealed that only a small part of the DNA (2%) coding for protein, being the remaining 98% considered initially as 'junk' DNA, whose function was attributed to protect coding DNA from damage and mutations. In the last few years, it was discovered that within this non-coding DNA, there was the presence of RNA. This RNA non-coding for proteins and is called noncoding RNA, including micrornas (mirnas), short interfering RNA (sirnas), Piwi interacting RNA (pirnas), small nucleolar RNA (sornas) and the Long Non-Coding RNA (LncRNAs). These small RNAs, regulates the expression of genes, by attaching to the promoter region, activating or silencing genes, and also affecting the stability of the messenger RNA (mrna) [3]. In this sense, it has been described in different publications mirnas as tumor suppressor and oncogenic role [4]. In our case, we focused our attention on LncRNAs, which have an important role in development processes [5], homeostasis [6], apoptosis [7], metastasis [8], angiogenesis [9] and cancer [10]. However how this LncRNAs acts and its identification remain largely unknown. As in the case of mirna, it has been described LncRNAs with tumor suppressor function and with oncogene function in several types of tumors, as we can see in the following table. In spite of this ignorance, there have been made some classification, the first group of LncRNAs are those who "kidnapped" proteins or RNA but not exert additional effects, acting as molecular traps. These LncRNAs negatively regulates the expression of their targets. A second group, function as cement" that bind different units spatially and temporally, like ANRIL for the epigenetic complex PRC1 and PRC2. The third group acts as a sort of "Guide" for the proteins to anchorage, giving rise to the complex that is directly guide to its target.

2 And finally, in the fourth group it is included those LncRNAs that can reinforce the expression of genes through the formation of loops between DNA and proteins. Papers recently published had shown the ability to detect some of these LncRNAs in peripheral blood of tumors such as prostate, gastric or lung, being useful as biomarkers for diagnosis, prognosis and response to treatment. Abstract Nº3 Título: New spectrophotometric approach to determine nanoparticles concentration and its application in cellbased assays multiplicity on nanofection. Ponente: Juan Diego Unciti Broceta Contacto: Engineered nanoparticles (ENPs) for biological applications are produced from functionalized nanoparticles (NPs) after undergoing multiple coupling and cleaning steps, giving rise to an inevitable loss of NPs in final compositions. Herein, we present a simple method to quantify the number of ENPs per microliter using standard spectrophotometers and volumes of up to one microliter. Light going through NP suspensions is scattered via reflection, refraction and diffraction phenomenon and the amount of the scattered light depend on the number of NPs found in suspensions. By measuring optical densities (OD) at 600 nm of different polystyrene NP suspensions of three different sizes (100 nm, 200 nm and 460 nm), linear correlations between OD600 and number of NPs were found for each NP size. These calibration curves can then be applied to estimate the number of ENP compositions of a particular NP size and material (Figure 1). To exemplify the method, we introduced the number of ENPs versus number of cells as a new parameter to report cellular uptake assays where capacities of cells to uptake beads or NPs ( nanofection ) need to be assessed. This parameter allows us to introduce multiplicity of nanofection 50 (MNF 50 ) index, which is defined as the number of NPs per cell needed to nanofect 50% of a given cell type, as a measure of the capacity of a cell type to uptake certain ENPs. Three mammalian cell lines were tested with 200 nm Cy5-PEG-NPs and, following flow cytometry analysis, each of them presented different MNF50, being MDA MB 231 mammalian breast cancer cell line the one with a lower MNF 50 and therefore with a higher uptaking capacity of these ENPs (Figure 2). Median of fluorescence intensity (MFI) of Cy5 positive cells analysis showed a linear behavior with different slopes for each cell line which is also a parameter to assess cell capacities for NPs uptaking (Figure 3). Furthermore, if we compare MFI increments (ΔMFI=MFI sample/mfi untreated) same results were obtained (Figure 4). A deeper study of ΔMFI showed a surprising data, from the closest ratio to their MNF 50, the increase of the ΔMFI is doubled when the NPs number are doubled, something which is not observed when ratios lowers than their MNF 50 are used. Importantly, this effect is the same for the three studied cell lines. Therefore, when MNF 50 is reached, the nanofection rate is constant and proportional to the number of nanoparticles used with cell lines presenting similar behavior. Nowadays the efficiency of many NPs-based delivery systems of bioactive cargoes are related to solid content (w/v) of NPs per cell. This method allows introducing a new parameter to analyze cellular uptake by reporting nanoparticle number versus cells number (multiplicity of nanofection). Based on these data we believe that the number of NPs per cell could be reported rather than weight of NPs per cell in any cell-based assays using NPs. The implementation of the Multiplicity of nanofection (MNF) will improve dramatically the efficiency of any nanoparticle-based devices.

3 SESIÓN DE INVESTIGADORES PREDOCTORALES CON MÁS DE 3 AÑOS DE DOCTORADO. Abstract Nº1 Título: Exome Sequencing Identifies FAM136A and DTNA as Candidate Genes in Familial Meniere s Disease. Ponente: María Teresa Requena Navarro Contacto: mariateresa.requena@genyo.es Meniere s disease (MD) is a complex disorder defined by cochlear and vestibular symptoms. Familial MD is found in 5-15% of cases in European populations. Although genetic heterogeneity is observed, most of the families have an autosomal dominant (AD) pattern of inheritance with incomplete penetrance. We have used whole-exome sequencing (WES) five families with two or more cases to identify rare variants in the family and functional analysis to assess their pathogenicity. DNA was isolated from peripheral blood and WES of genomic and mitochondrial DNA was carried out in a SOLiD 5500xl platform. Bioinformatic analyses to filter and prioritize were performed obtaining seven candidate variants. We have identified and validated by Sanger sequencing two novel single nucleotide variants, one in the FAM136A gene causing a nonsense mutation and another in the DTNA gene causing a missense mutation. qpcr revealed two mrna transcripts of FAM136A in lymphoblasts from patients and protein products were confirmed by immunoblotting. Carriers of the FAM136A mutation showed a significant decrease in the expression levels of both transcripts in lymphoblastoid cell lines. Using immunolabeling and confocal microscopy in inner ear rat tissue, we have found that FAM136A co-localizes with the mitochondrial marker COX IV in the basal zone of hair cells in the crista ampullaris. Moreover alpha-dystrobrevin was located in supporting cells in the crista, close to the stromal region. Localization of FAM136A and alpha-dystrobrevin in the vestibular crista suggest a functional role for both proteins in the clinical phenotype observed in the MD family. Abstract Nº2 Título: Host-derived but not tumoral ADAMTS1 promotes tumor growth through induction of vascular development. Ponente: Rubén Fernández Rodríguez Contacto: ruben.fernandez@genyo.es Tumor growth, development and metastasis are directly linked to the formation of a new vascular network. Sprouting angiogenesis is an invasive process where proteolytic activities are required in order to remodel vessel-adjacent extracellular matrix (ECM). ECM degrading enzyme Adamts1 has been directly implicated in cancer progression. Furthermore, it has been described as an anti-angiogenic molecule despite other studies have proven ADAMTS1 as a pro-tumorigenic and pro-metastatic protease. We tried to address this dilemma using ADAMTS1 knockout mice and producing syngenic subcutaneous tumors with B16F1 melanoma cells. Tumor morphometric analysis showed a clear reduction of tumor volume in KO mice accompanied with an increase in vessel number but a smaller vessel size. Gene expression analysis of endothelial markers, ADAMTS1 substrates and hypoxia markers showed upregulation in KO mice tumors for most of the studied genes. Coimmunostaining of Endomucin and VE-cadherin exhibited higher fluorescence intensity for VE-cadherin in vessel-distant areas in KO mice tumors. Since VE-cadherin has been described to be over-expressed in hypoxic areas we thought of a possible phenomenon of vascular permeability impairment. Further analysis with a proper hypoxic marker revealed a higher number of hypoxic regions within KO tumors compared with those in WT littermates. Also, metastasis was dramatically reduced in KO animals. Stable transduction of B16F1 cells with a lentivirus carrying a shrna against Adamts1 caused no reduction of tumor size nor any significant change in any of the parameters analysed previously. All these findings indicate that ADAMTS1 modulates vessel development favouring tumor growth and metastasis. In addition, shrna experiments showed that impairment of tumor angiogenesis is only related to host-derived ADAMTS1 whilst tumor-derived ADAMTS1 seems to play an irrelevant role.

4 Abstract Nº3 Título: Line-1 as a possible epimutagen in pluripotent genomes. Ponente: Juan David Cano Martínez Contacto: david.cano@genyo.es Long INterspersed Element class 1 (LINE-1 or L1) elements are non-ltr autonomous retrotransposons that are currently active in the human genome. It has been estimated that there are around active LINE-1s per average genome. As selfish DNA, new insertions will be transmitted to the next generation when new retrotransposition events accumulate in germ cells or during early stages of embryogenesis. In fact, it s been demonstrated new LINE-1 insertions mostly occur during early embryonic development (Kano et al, 2009). In fact, it is been already shown that human embryonic stem cells (hescs) can accommodate engineered LINE1 retrotransposition (García-Perez, et al., 2007). Controlling retrotransposition in hescs is required since uncontrolled retrotransposition could be deleterious for the developing human being. For instance, as a mechanism to restrict retrotransposition, it was shown that new engineered LINE-1 insertions are epigenetically silenced in pluripotent cells (human embryonic carcinoma cells (hecs))(garcía-pérez et al., 2010) by histones modifications. Taking into account that a new LINE-1 insertion can epigenetically impact the integration site, we would like to gain insight into the epigenetic status of the flanking genomic areas.. In order to demonstrate how a new LINE-1 event can epigenetically affect the flanking genomic areas we have generated a battery of clonal cell lines in both human embryonic carcinoma cells (PA-1) and human embryonic stem cells (hescs) containing at least one retrotransposition event. The next step would be the epigenetic characterization of both the insertion site and the flanking genomic areas before/after integration by performing ChIP with antibodies that recognize repressive and activating marks. So far, we have fully characterized 16 out of 20 insertions in PA-1. Regarding hescs, we have isolated around 20 neomycin-resistant colonies meaning they may have at least one insertion. In fact, we are currently expanding these clonal cell lines. On the other hand, we have just begun to perform ChIP but we are still in a very preliminary stage since we are testing the antibodies and we are improving the protocol.

5 SESIÓN DE INVESTIGADORES POSTDOCTORALES CON MENOS DE 3 AÑOS DE DOCTORADO. Abstract Nº1 Título: Mesenchymal Stromal Cells express GARP/LRRC32 on their Surface: Effects on their Biology and Immunomodulatory Capacity. Ponente: Ana Carrillo Gálvez Contacto: anabelen.carrillo@genyo.es Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation and autoimmune disease due to their trophic and immunomodulatory activities. However we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3 + regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript we show that human and mouse MSCs express GARP, which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. Abstract Nº2 Título: Papel de los micrornas en la inactivación de SMARCA4 en cáncer de pulmón. Ponente: Isabel Fernández Coira Contacto: isabel.fernandez@genyo.es SMARCA4 es la subunidad catalítica del complejo remodelador de la cromatina SWI/SNF que altera las interacciones entre el ADN y las histonas y modifica la capacidad del ADN para la transcripción. Los últimos resultados de secuenciación profunda del genoma de tumores han reforzado la importancia y ubicuidad del papel como supresor tumoral del complejo SWI/SNF en cáncer. Sin embargo, aunque el complejo juega un papel clave en la expresión génica, la regulación de este en sí es poco conocida. Cabe destacar cómo la regulación de la expresión de SMARCA4 ha adquirido mayor importancia debido a las recientes propuestas que lo incorporan en estrategias terapéuticas que utilizan interacciones sintéticas letales entre SMARCA4-MAX y SMARCA4-SMARCA2. Hemos encontramos que la pérdida de expresión de SMARCA4, vista en algunos tumores primarios de pulmón y cuyo mecanismo era en gran parte desconocido, puede ser explicada, aunque sea parcialmente, por la actividad de los micrornas. Hemos demostrado que la expresión de SMARCA4 está regulada por mir-101, mir-199 y especialmente por mir-155 que tiene diana en las dos 3 UTRs de SMARCA4. Nuestros experimentos sugieren que las propiedades oncológicas de mir-155 en cáncer de pulmón pueden explicarse ampliamente por su rol inhibiendo SMARCA4. Este nuevo descubrimiento, relacionándolos funcionalmente, puede explicar el mal pronóstico mostrado por los pacientes con altos niveles de mir-155 y bajos niveles de expresión de SMARCA4. Además, estos resultados pueden llevar a la aplicación de la reciente tecnología de los micrornas a las estrategias de interacciones sintéticas letales antes mencionadas.

6 Abstract Nº3 Título: Células madre embrionarias humanas WAS KO como modelo para el síndrome de Wiskott-Aldrich: Una nueva herramienta para el estudio de estrategias de terapia génica. Ponente: Almudena Sánchez Gilabert Contacto: El syndrome de Wiskott-Aldrich (WAS) es una inmunodeficiencia ligada al cromosoma X, caracterizada por inmunodeficiencia y trombocitopenia, causada por mutaciones en el gen WAS. Los defectos inmunológicos en WAS están bien caracterizados, sin embargo los mecanismos que causan tromocitopenia y leucemia aún permanecen contradictorios. Hoy día el trasplante de médula ósea es el único tratamiento curativo, sin embargo no siempre es posible y puede tener efectos secundarios. Es por ello que es necesario desarrollar terapias alternativas. La terapia génica es, cada vez más, una alternativa terapéutica prometedora para varias inmunodeficiencias primarias, incluyendo WAS. A pesar del los éxitos iniciales, los ensayos clínicos han mostrado varios efectos secundarios (leucemia, silenciamiento del transgen) que no se habían observado en los modelos animales. Haciendo uso de las Zinc Fingers Nucelasas (ZFNs) dirigidas contra el primer intrón del locus WAS, desarrollamos dos líneas celulares de células madre embrionarias humanas (hescs) deficientes para la proteína WASp (AND1_WASKO). Hemos observado que megacariocitos(mk) y plaquetas(plt) derivadas de AND1_WASKO mimetizan varios de los defectos encontrados en MK y PLT de pacientes de WAS. También hemos generado líneas AND1_WASKO trasnducidas con vectores lentivirales(lv) expresando WASp. Muchas de las alteraciones fenotípicas observadas en AND1_WASKO disminuyeron tras la expresión de WAS tras la transducción con el LV. La respuesta de MK y PLT derivadas de AND1_WASKO, a diferentes agonistas, fue restaurada tras la expresión de WASp. Consideramos que esta línea de trabajo, que dispone del primer modelo celular, en células pluripotentes humanas, es una herramienta potente para el estudio del papel de WASp durante los estadios tempranos de la diferenciación megacariocítica, así como una buena oportunidad para estudiar diferentes estrategias terapéuticas para el síndrome de WAS.

7 SESIÓN DE INVESTIGADORES EXTERNOS. Abstract Nº1 Título: Desarrollo de magnetoliposomas como sistemas transportadores de fármacos. Ponente: Cristina Lorente Guirado Contacto: Actualmente, el desarrollo de diferentes coloides como sistemas transportadores de fármacos antitumorales o cualquier otro tipo de fármaco, ha alcanzado un gran interés en Biomedicina. El grupo de investigación, Farmacia Práctica (CTS-205), tiene una amplia e importante experiencia en el desarrollo de nanosistemas magnéticos. Dentro de esta línea de investigación, se ha realizado un estudio exhaustivo de las condiciones óptimas para la síntesis de nanocompuestos óxido de hierro/recubrimiento liposomal biodegradables y biocompatibles (magnetoliposomas), con el objetivo de diseñar un vehículo coloidal eficaz que posibilite el transporte activo de fármacos hasta las células diana. Este tipo de sistemas se benefician de la capacidad de transporte y de liberación controlada de fármacos desarrollados por el componente vesicular, y de la capacidad de respuesta a gradientes magnéticos aplicados que aportan los núcleos magnéticos embebidos en esta matriz liposomal. Así, mediante este tipo de transportadores, se puede lograr que todo el principio activo se concentre en el lugar diana (gracias al uso de un gradiente magnético para guiar a las nanopartículas en el organismo) y que la concentración de fármaco en éste sea la óptima (gracias a su vehiculización en la matriz vesicular). La eficacia del recubrimiento y las ventajas que aporta a la capacidad de estas partículas para transportar el fármaco objeto de estudio, se determinó tras un análisis comparativo de la estructura y composición química de los tres tipos de materiales [óxido de hierro, liposoma y magnetoliposomas], así como de las propiedades eléctricas superficiales. La caracterización de las propiedades magnéticas de los magnetoliposomas, permitió determinar la capacidad de estos sistemas para responder a gradientes magnéticos aplicados y el grado de influencia del recubrimiento vesicular sobre estas propiedades. Por tanto, esta caracterización preliminar permite decir que se ha desarrollado una síntesis de magnetoliposomas reproducible y eficaz. Abstract Nº2 Título: The adipose niche upregulates cytokines that drive Src dependent breast tumor progression via SOX2 and mir302b. Ponente: Manuel Picón Ruíz Contacto: mpicon@ugr.es As it has been shown, obesity confers increased cancer risk and poor outcome, however mechanisms thereof are obscure. Here we show that prolonged contact between adipocytes and breast cancer cells upregulates pro-inflammatory cytokines, such as IL6, IL8, IP10, CCL2 and CCL5, which increase breast cancer sphere and colony formation ability in vitro, ALDH activity, and tumor initiation and metastasis in vivo. This effect is mediated by Src activation which is required for SOX2, cmyc and NANOG upregulation, transcription factors that confer pluripotency in embryonic stem cells and are used to generate induced pluripotent stem cells. In addition, our results indicate that differs mirnas are involved in this process, including mir302b. mir302b upregulation is Sox2-dependent, and also participates in cell self-renewal modulating cmyc and SOX2 expression. On the other hand, Src is also required for sustained induction of the pro-inflammatory cytokines mentioned above. In conclusion, our work reveal that upon cancer invasion into local fat, these interactions would establish feed-forward loops to maintain cytokine production and increase tumor initiating cell abundance, tumor growth and metastasis; and provide a novel rationale for Src inhibitors in cancer therapy.

8 Abstract Nº3 Título: Bozepinib, a new small compound against cancer and cancer stem-like cells. Ponente: Alberto Ramírez Rivera Contacto: arrivera@ujaen.es The appearance of resistance in tumoral cells and the high risk of relapse in patients due to the presence of cancer stem-like cells (CSCs) subpopulations involve the identification of new anticancer drugs targeted to more than one molecular pathways involved in cancer. In this work we show the selectivity of Bozepinib, a small anti-tumor compound on cancer cells and its inhibitory effect over kinases involved in different pathways altered in cancer, such as carcinogenesis, proliferation and angiogenesis. In this study, the cytotoxic effects of Bozepinib were observed in different breast and colon cancer cell lines expressing different receptor patterns. The inhibition of HER-2 signaling pathway and JNK and ERKs kinases was observed, in addition with an inhibitory effect on AKT and VEGF together with anti-angiogenic and antimigratory activities. Microarrays analysis also showed the modulation of pathways involved in tumorigenesis by Bozepinib. Moreover, Bozepinib was able to exert an inhibitory activity against both mamo- and colono-spheres formation and eliminated ALDH+ CSCs subpopulations at a low micromolar range similar to Salinomycin, a described anti CSCs compound. Moreover, the inhibitory effect of Bozepinib on CSCs pathways was highlighted by the down-regulation of c-myc, β-catenin and SOX2 proteins and the up-regulation of the GLI-3 hedgehog-signaling repressor. Finally, in the in vivo assays, Bozepinib shows anti-tumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting sub-acute toxicity.

9 SESIÓN DE INVESTIGADORES DE MÁSTER Abstract Nº1 Título: Exome sequencing and bioinformatic analysis in identical twins with Ménière's Disease. Ponente: María del Carmen Martín Sierra Contacto: Meniere s disease (MD) has a strong familial aggregation with a prevalence of 8-10% of familial cases [1, 2]. Moreover, the presence of the disease in monozygotic twins increases the evidences that MD could have a genetic component. We performed whole-exome sequencing (WES) analysis in a family with monozygotic twins with MD and two healthy individuals to identify candidate variants affecting this family. After process the data from SOLiD 5500xl platform, we obtained different types of files containing ~ single nucleotide variants (SNVs) per exome. To prioritize pathogenic variants we annotated a score according to: a) the effect in protein structure and phylogenetic conservation by using a seven points scoring system (SIFT (Sort Intolerant from Tolerant), PolyPhen2 (Polymorphism Phenotyping v2), Graham s Matrix, GERP+ (Genomic Evolutionary Rate Profiling), Mutation taster, PhastCons and PhyloP); b) cross species phenotype comparison according to the inheritance pattern and mouse as model organism phenotype by the Exomizer software [3]; c) minor allelic frequency (MAF) <0.01. Since, a homozygous recessive variant may affect both twins, we also performed homozygosity mapping to discard the possibility of a recessive inheritance pattern [4]. Copy Number Variant (CNVs) were analyzed by using Conifer software, but we did not find any CNV with significant Z-score associated with MD phenotype. Five variants remained after prioritization process and filtering. Four of them, were discarded because of his low level of expression in the inner ear and by their biological function. THAP1, a gene associated with dystonia 6, encoding a THAP domain-containing protein considered to be involved in endothelial cell proliferation and proapoptotic processes, and assumed to act as a transcription factor, was considered the best candidate gene in these twins. It has normal level of expression in the inner ear and mutations in this gene have cause loss of DNA binding and transcriptional dysregulation of downstream targets. Abstract Nº2 Título: Role of HDAC inhibitors in megakaryopoiesis from hescs. Ponente: Xiomara Guerrero Carreño Contacto: Xiomara.guerrero@genyo.es Human embryonic stem cells (hescs) are an exceptional in vitro model to study human development and a potential source of cells and tissues for their use in Regenerative Medicine. One of the potential applications is the production of human platelets from hescs, although the molecular mechanisms that control megakaryocytic differentiation in vitro are still undefined. We have recently shown that SCL overexpression in hescs accelerates the emergence of hemato-endothelial progenitors and their subsequent in vitro differentiation into mature megakaryocytes (MKs) and platelets. In order to improve the efficiency of this differentiation process we looked for chemical compounds that mimic the effect showed by SCL overexpression using the bioinformatics tool Connectivity map. We found that the Histones Deacetylases Inhibitors (HDACis) TSA and SAHA, activate a transcriptional program similar to the gene expression profile present in SCL-overexpressing megakaryocytic progenitors. In addition, we also studied VPA, another HDACi, recently described as an in vitro enhancer of megakaryopoiesis from hematopoietic stem cells. Our results show that the HDACis have specific dose-dependent effects throughout megakaryopoiesis. TSA and SAHA are involved in the early differentiation stages; enhancing the appearance of megakaryocytic progenitors and mainly improving megakaryopoiesis. On the other hand, VPA improves the generation of mature MKs and the production of platelets, mainly improving trombopoiesis. Our results will allow us to optimize the differentiation protocol to megacaryocytes and plateles from hescs using HDACis a more rationalised way. In addition, a more detailed study of the effects of these HDACis could help us explain the epigenetic mechanisms involved in the process of hematopoietic differentiation.

10 Abstract Nº3 Título: Hidrogeles supramoleculares como vehículos proteicos y celulares. Hacia novedosas aplicaciones biomédicas y biotecnológicas. Ponente: Rafael Contreras Montoya Contacto: rafael7_89@hotmail.com Los hidrogeles supramoleculares basados en la auto-asociación de pequeños péptidos son materiales que recientemente han encontrado una gran variedad de aplicaciones en biotecnología y biomedicina, debido a su naturaleza biocompatible y biodegradable. Estos materiales se han empleado en ingeniería tisular, catálisis, cristalización y como biosensores. Teniendo en cuenta los antecedentes expuestos, el diseño de nuevos hidrogeles supramoleculares basados en la auto-asociación de pequeños péptidos, así como el estudio de sus propiedades y aplicaciones, supone un reto interesante con una gran potencialidad. El trabajo realizado en este proyecto se ha centrado en el diseño, síntesis y aplicación de nuevos hidrogeles como medios para la cristalización de proteínas. Al estar constituidos por moléculas de bajo peso molecular basados en amino ácidos naturales se han podido obtener hidrogeles que tienen una relación de enantiomería a nivel molecular, es decir, materiales que son imágenes especulares entre sí. Al comparar los resultados obtenidos en la cristalización de proteínas en los distintos hidrogeles enantiómeros, se ha podido observar que la quiralidad del medio afecta al proceso de cristalizacion de proteínas y que ha dado lugar, entre otras cosas al descubrimiento de un nuevo polimorfo, hasta ahora no descrito, de una proteína de interés biotecnológico. Estos hidrogeles han servido también como vehículo de cristalización de una proteína de alto valor terapéutico. Este nuevo material está siendo estudiado para determinar su viabilidad como una nueva forma de administración de dicha proteína, que permitiría una liberación de dicha proteína más duradera. Estos resultados no tienen precedentes bibliográficos, y por tanto, se abre un nuevo campo de investigación con posibles aplicaciones en el estudio de proteínas y en el diseño de nuevos sistemas de liberación controlada de biológicos en el campo terapéutico incluyendo su aplicación para terapia antitumoral. Parte de los resultados obtenidos en este trabajo fin de máster se expusieron como comunicación oral en la XXV Reunión Bienal de Química Orgánica, Alicante Título: Supramolecular Hydrogels for Protein Crystallization.

11 COMUNICACIONES SELECCIONADAS PARA PRESENTACIÓN EN FORMATO PÓSTER Abstract Nº1 Título: Deciphering the possible role of LINE retrotransposition during tissue regeneration Ponente: Ana Ariza Cosano Contacto: Urodele amphibians and teleost fish are able to regenerate different parts of their body after amputation. Regeneration is typically subdivided into three steps; (a) formation of a wound epidermis at the site of damage, (b) disorganization and dedifferentiation of tissue near the wound resulting in the creation of blastema and (c) finally, proliferation of blastema cells concomitant with patterning and differentiation, resulting in the regeneration of the amputated tissue. A hallmark of this phenomenon is the reactivation of different developmental regulatory genes that previously have shown a function during embryonic patterning. Data in our lab showed that also transposable elements are highly expressed during early development. Transposable elements are endogenous pieces of mobile DNA able to jump and duplicate in the host genome. In zebrafish, two retrotransposons, called zfl2-1 and zfl2-2, have been shown to be highly expressed during early development. Our preliminary data show that during fin regeneration zfl2-1 and zfl2-2 are also overexpressed, resembling the embryonic phenotype. However, it is not known, how they are regulated. In humans, a similar mobile element, LINE-1, is regulated by several epigenetic modifications, such as promoter methylation. Our on-going experiments will shed light on if the methylation of zfl2-1 and zfl2-2 promoter they have a function in the regeneration process. With these studies we will determine the role of actives retrotransposons during regeneration and we will open a new ways to understand this important biological process. Abstract Nº2 Título: The Role of Somatic Mosaicism Generated by LINEs in Zebrafish. Ponente: Meriem Benkaddour Boumzaouad Contacto: meriem.benkaddour@genyo.es 17% of the human genome is comprised of retrotransposons called LINE-1 (Long INterspersed Elements Class 1). Today these are the only autonomously active elements in the human genome. As in humans, zebrafish also contains transposables elements in its genome. LINE elements represent 11% of the genome. Zebrafish LINE-2 elements are similar to human LINE-1. Zfl2-1 and Zfl2-2, two LINE-2 elements, have been shown to be active in HeLa cells. In our studies, we characterized the endogenous expression of Zfl2-1 and Zfl2-2 elements in zebrafish. We demonstrated their expression in different stages of zebrafish embryonic development. Higher expression levels are found in brain and neural tissues. We also were able to generate an in vivo model of engineered retrotransposition in zebrafish. Using this model, we could observe insertions in practically all tissues like neural tissue, skin, muscles, heart and digestive tract. Recently, high levels of retrotransposition have been detected in a somatic tissue, the human brain. To this aim, a new technique called Retrotransposition Capture Sequencing (RC-Seq) was used. In three different individuals, genomic DNA from brain was compared with genomic DNA from liver and blood. Numerous de novo insertions were found in the brain not present in liver or blood. Therefore, these insertions must have been occurred during the ontology of the individual and therefore they are somatic insertions. They cannot be transmitted to the next generation. However, it is not clear whether these insertions are functional or whether they are just systemic noise. To answer this question, we propose to use our zebrafish retrotransposition model. In the first place, we will map endogenous insertions of Zfl2-1 and Zfl2-2 elements in brain versus muscles, heart and other tissues. The aim is to detect somatic insertions present in brain but not in other tissues. In another part of the project, we will inhibit somatic retrotransposition in the zebrafish brain to see if a reduced level of retrotransposition negatively affects learning, memory or behavior of zebrafish. We will use the APOBEC3A protein known to inhibit retrotransposition of LINE elements in both human and zebrafish. Molecular, behavioural or memory tests will tell us whether these somatic insertions are required for the correct functioning of the brain.

12 Abstract Nº3 Título: Role of Hypoxia in Megakaryocytic Differentiation of Human Embryonic Stem Cells. Ponente: María del Mar Bonillo Lamolda Contacto: Platelet transfusions are essential for the treatment of patients with thrombocytopenia who display low numbers of platelets due to liver failure, leukemia, secondary effects of chemotherapy or genetic defects. Currently, there is no effective therapy for this disease. Human embryonic stem cells (hescs) could represent an alternative source for blood transfusion therapies and a promising tool for studying human hematopoietic development. The aim of this study was to generate functional megakaryocytes and platelets in vitro from wild type hescs and SCL over-expressing hescs under normoxia (po2=21%) and hypoxia (po2=2%; po2=5%) conditions. We also mimicked the bone marrow s physiological environment combining low oxygen tensions to favor megakaryopoiesis and thrombopoiesis. Our results demonstrated that hypoxia reduced cellular viability selecting progenitors with a higher megakaryopoietic potential, increasing the number of both mature megakaryocytes and platelets, especially under physiological condition. In contrast, in the SCL over-expressing cell line normoxia improved megakaryopoiesis. However, thrombopoiesis was increased under hypoxic conditions. These data will allow us not only to understand the molecular mechanisms controlling human megakaryopoiesis, but also to improve the current differentiation protocols from hescs. Abstract Nº4 Título: Functional analysis of Plakophilin-1 (PKP1) gene in Non-Small Cell Lung Cancer (NSCLC). Ponente: Laura Boyero Corral Contacto: laura.boyero@genyo.es Desmosomes provide mechanical resistance and contribute to tissue homeostasis by anchoring intermediate filaments to sites of cell cell contact. This type of adhering junctions avoids cell migration, invasion and metastasis. For this reason, desmosomal components have been traditionally considered as tumoral supressors although current evidences, which assign desmosomes a role in intracellular signaling such as in Wnt and Akt pahtways-, are changing that tendency. PKP1, a member of the desmosomes, is up-regulated in primary SCC suggesting a putative contribution in tumorigenesis. Besides its well-known structural role, its dual subcellular localization in desmosome and nucleus, the interaction with singlestranded DNA and translation initiation factors, etc. suggest other functions of PKP1 in cellular regulation and cancer. In order to gain greater insight into the roles of PKP1 in NSCLC, we are performing transient inhibition assays of PKP1 in several SCC lung cancer lines. We have obtained a decrease in cell growth, an arrest of cell cycle characterized by an increase of mitotic round shapes and apoptosis*. We are also working in PKP1-EGFP over-expression and shrna-pkp1 stable knockdown by constructing inducible lentiviral vectors. They will allow us to performe functional assays and expression arrays of stable cell lines in order to identify its subcellular location and the potential pro-oncogenic role of PKP1 in NSCLC carcinogenesis which may lead to improvements in the treatment and prevention of this disease. Immunocytochemistry s results revealed a PKP1 location on desmosome, nucleus and cell structures specific of cell division such as kinetochore and midbody*, which have not been previously reported, although it still requires further investigations. Our future plans include to immunoprecipitate endogenous nuclear PKP1 in order to identify new binding-partners by MALDI-TOF and clarify PKP1 role at the nucleus. In a long-term perspective, xenografts will lead to understand the molecular origins and progression of lung cancer in regard to PKP1.

13 Abstract Nº5 Título: Variantes genéticas en genes inmunomoduladores influyen sobre el riesgo a desarrollar artritis reumatoide y la respuesta a fármacos anti-tnf: estudio de casos-controles en dos fases. Ponente: Luz María Canet Antequera Contacto: luz.canet@genyo.es La Artritis Reumatoide (AR) es un desorden autoinmune con mayor prevalencia en mujeres y de etiología aún desconocida. Una respuesta inmune exacerbada y un desequilibrio entre citoquinas pro- y antiinflamatorias provocan una inflamación crónica de las articulaciones y la destrucción progresiva del cartílago y del hueso. Un gran número de estudios evidencian el componente genético de la AR y demuestran que la presencia de polimorfismos (SNPs) en genes inmunomoduladores influyen en el riesgo a desarrollar AR, así como en la respuesta a fármacos y la progresión de la enfermedad. El objetivo de nuestra línea de trabajo es desarrollar estudios GWAS y de genes candidato para identificar marcadores genéticos subyacentes a estos procesos, mejorar la estrategia terapéutica y esclarecer las bases moleculares de la enfermedad. En el contexto de colaboraciones internacionales, centramos nuestra investigación en el análisis de variantes genéticas en genes relacionados con las vías Th 1, Th 2 y Th 17. Nuestros resultados han permitido identificar que SNPs en Dectin-2, MCP-1, DC-SIGN, IL4, IL8RB, VEGFA y TNFR2 así como ciertos haplotipos (IL4 TTTCA, IL8 TA, TNFR2 GGCT ) que están asociados con el riesgo a desarrollar AR. A través de meta-ánalisis con estudios previos, pudimos confirmar algunas de las asociaciones descritas (IL4: N=7150; P=1.00E-03). Además, algunas de estas variantes mostraban un efecto específico de género (Dectin-2, MCP-1, DC-SIGN, IL4R, IL13 y VEGFA), lo que parecía sugerir su implicación en las determinación de las diferencias sexuales existentes en la incidencia de la enfermedad. Basándonos en estos resultados estratificados por género y en el papel de los estrógenos en la modulación de la respuesta inmune en AR, estamos también investigando la asociación de ciertos SNPs en genes relacionados con la síntesis, degradación y metabolismo de las hormonas esteroideas. Finalmente, identificamos ciertos SNPs en TNFR2 e IFNG que influían en la respuesta a tratamientos biológicos, lo que nos animó a desarrollar un GWAS (Immunochip, SNPs) para la identifiación de biomarcadores asociados a la respuesta a tratamiento y la progresión de la enfermedad. Abstract Nº6 Título: Nueva estrategia para la liberación selectiva mediante el uso de nonopartículas. Ponente: Victoria Cano Cortés Contacto: victoria.cano@genyo.es El objetivo primordial de este proyecto de investigación es la aplicación, explotación y desarrollo de un sistema de liberación celular genérico basado en el uso de nanopartículas poliméricas que permitan el transporte y la liberación intracelular de un amplio rango de materiales con actividad biológica y/o terapéutica. Se han desarrollado varias estrategias para la introducción de diferentes moléculas con actividad terapéutica, entre ellas se encuentran diferentes estrategias químicas para la unión y la liberación selectiva de macromoléculas biológicamente relevantes, tales como el DNA y las proteínas. La liberación intracelular controlada de los cargos asociados a estos transportadores celulares, permiten la modulación y el seguimiento en tiempo real de una gran variedad de procesos celulares al mismo tiempo que proporcionan una herramienta para el marcado y localización celular. Buscando la especificidad en la liberación intracelular de macromoléculas y así conseguir esta liberación selectiva hemos utilizado los homing peptides, péptidos que reconocen específicamente receptores celulares que se encuentran sobreexpresados en procesos patológicos tales como el cáncer. Actualmente, esta estrategia ha sido empleada para la liberación dirigida de diferentes moléculas como fármacos u oligonucleótidos. Concretamente, estamos evaluando la eficiencia de este sistema de liberación efectivo mediante la utilización del homing peptide RGD que reconoce receptores específicos sobreexpresados durante la angiogénesis del cáncer de mama. Para ello hemos unido el RGD a nanopartículas marcadas con un fluoróforo y hemos comprobado como estas nanopartículas son internalizadas específicamente en varias líneas celulares de cáncer de mama comparadas con nanopartículas unidas a una secuencia peptídica inespecífica.

14 Abstract Nº7 Título: Detección de mirna-21 en células tumorales circulantes mediante hibridación in situ. Ponente: Diego de Miguel Pérez Contacto: Antecedentes: El cáncer de mama supone la segunda causa de muerte relacionada con cáncer en el mundo y su metástasis es la responsable de la mayoría estas muertes. Las células tumorales circulantes (CTCs), diseminadas a partir del tumor van a sufrir un proceso de transición epitelio-mesénquima (EMT), por el que pierden su fenotipo epitelial en favor de uno mesenquimal. Esto les permite la intravasación y circulación hasta un órgano distante donde establecer metástasis. Actualmente, los métodos de detección de CTCs se basan en marcadores epiteliales, por lo que surge la necesidad de encontrar un biomarcador que se mantenga durante el proceso EMT. Objetivos: -Desarrollar una metodología capaz de determinar la expresión de mirnas in situ en líneas celulares tumorales, con fenotipo epitelial y fenotipo mesenquimal. -Identificar un mirna que permita la detección de células tumorales circulantes en sangre y se mantenga durante el proceso de EMT. Métodos: Se indujo el proceso EMT en células tumorales mamarias cultivadas mediante factor de crecimiento transformante β (TGF-β) factor de crecimiento epitelial (EGF) y se procedió a la hibridación in situ de mirna-21 en células con fenotipo epitelial y fenotipo mesenquimal, además de en células tumorales mamarias añadidas en sangre periférica de pacientes sanos. Resultados: Ambos tipos celulares cultivados, epitelial y mesenquimal, expresaron el mirna-21 y el protocolo de aislamiento de CTCs junto con el de hibridación in situ de mirnas, permitieron distinguir células epiteliales tumorales de células epiteliales no tumorales en sangre. Conclusión: El mirna-21 es un biomarcador eficaz para la detección de células tumorales circulantes mamarias, tanto con fenotipo epitelial como mesenquimal, además de para la identificación específica de células tumorales en sangre. Abstract Nº8 Título: The SLE susceptibility variant rs (Ala71Thr) in BLK causes constitutive phosphorylation, reduced protein abundance, and loss of interaction with BANK1. Ponente: Alejandro Díaz Barreiro Contacto: alejandro.diaz@genyo.es The B lymphocyte kinase (BLK) gene is associated genetically with several human autoimmune diseases including systemic lupus erythematosus. We recently described that the genetic risk is given by two haplotypes: one covering several strongly linked SNPs within the promoter of the gene and correlated with low transcript levels and a second haplotype that includes an uncommon non-synonymous variant (Ala71Thr). Here we show that this uncommon variant is a major determinant of BLK protein levels. In vitro analyses show that the 71Thr isoform is constitutively phosphorylated and confers a lower protein half-life. We show that in heterozygotes, the presence of the variant significantly contributed to the total amount of protein. Because the Ala71Thr substitution is located in a protein interaction domain we sought for differences between the two isoforms in trafficking and binding to protein partners. We found that binding of the 71Thr isoform to the adaptor protein BANK1 is severely reduced. Our data suggests that the abundance of BLK is determined by both, polymorphisms in the promoter affecting mrna level and the Ala71Thr variation affecting the quality of the protein. Finally, the uncommon variant also impairs BLK interaction with BANK1, an adaptor protein that is known to play a role in B-cell signaling.

15 Abstract Nº9 Título: Influencia de los polimorfismos CYP2C19 y ABCB1 en el tratamiento antiagregante con Clopidogrel en pacientes con patología vascular periférica de miembros inferiors sometidos a angioplastia transluminal percutánea. Ponente: Xando Díaz Villamarín Contacto: xandodv@gmail.com INTRODUCCIÓN: Clopidogrel es un profármaco que se metaboliza a través del isoenzima CYP2C19, a metabolito activo responsable de la inhibición de la agregación plaquetaria. La glicoproteína P, codificada por el gen ABCB1, es clave en la absorción del fármaco. Los efectos antiagregantes del clopidogrel difieren conforme al genotipo ABCB1 y al CYP2C19, estableciéndose así metabolizadores normales, intermedios y lentos. Los metabolizadores intermedios y lentos y los malos transportadores son los responsables de la mala respuesta antiagregante del fármaco (CYP2C19*2 (*1/*2 y *2/*2) ni en ABCB1 TT). OBJETIVOS: Determinar la aparición de eventos cardiovasculares en pacientes sometidos a ATP (+stent) durante un año de seguimiento y estudiar la asociación con la presencia de los polimorfismos genéticos CYP2C19 y ABCB1. Determinar la asociación entre los polimorfismos genéticos y el grado de Fontaine (clasificación de isquemia). MATERIALES Y MÉTODOS: Se seleccionaron 45 pacientes con patología vascular periférica de miembros inferiores sometidos a ATP (+- stent) y en tratamiento con clopidogrel a los que se genotipo los polimorfismos CYP2C19*2 (rs ) y ABCB1 (rs ) empleando sondas Taqman y la técnica de discriminación alélica. RESULTADOS: De los 45 pacientes incluidos, 15 (33.3%) presentaron algún evento durante el seguimiento. Los pacientes con al menos algún alelo con pérdida de función presentaron una mayor tasa de eventos frente a los pacientes con alelos con buen metabolismo (OR=6.0, IC 95% , p=0.010). Los pacientes con algún alelo con pérdida de función se asociaron a una peor evolución según el grado de Fontaine (OR= 21.35, IC 95% , p<0.0001). CONCLUSIONES: -Los pacientes con mal metabolismo del clopidogrel presentan mayor riesgo de eventos cardiovasculares y una peor evolución según el grado de Fontaine. Abstract Nº 10 Título: New target genes implicated in retrotransposon activity. Ponente: Laura García Corzo Contacto: laura.garcia@genyo.es Long Interspersed Element-1 (LINE-1 or L1) are non-ltr retrotransposable elements able to replicate by a copy-and-paste mechanism that comprise a fifth of our genome. L1 elements belong to the autonomous retrotransposon class because they encode the proteins required for their replication in the host genome. Although more than 99% of L1 copies are inactive, an average human genome contains ~ active retrotransposition-competent L1s (RC-L1s). When L1s integrate in genes, they are harmful to their hosts. Therefore, human cells have developed several strategies to regulate L1 retrotransposition. These include transcriptional silencing via DNA methylation and posttranscriptional silencing via RNA interference (RNAi). Some specific proteins like APOBEC3 and SAMHD1 show inhibitory activity against a variety of endogenous retroviruses including retrotransposons such as LINE-1, as well as exogenous retroviruses such as human immunodeficiency virus (HIV). Further studies based on a whole genome sirna screen have identified 114 genes that are involved in restriction of HIV replication during the early stages of the viral life cycle. It is unknown so far, whether some of these new identified gene products are implicated in the regulation of LINE-1 retrotransposition, similarly to APOBEC3 and SAMHD1 proteins. To perform this study we have selected two of these proteins from the screen, one with the capacity to interact with RNA:DNA or dsdna products of HIV reverse transcription, called RNA-associated Earlystage Antiviral Factor (REAF), and the other without any known function called C3orf63. Based on their potential mechanism of action, we hypothesize that these genes may be potent inhibitors of LINE-1

16 retrotransposition. To evaluate this hypothesis, we are performing LINE-1 retrotransposition assays in HeLa cells. Each gene, REAF or C3orf63, is co-transfected with one of the following constructs: human wild-type LINE-1 (JJL1.3), human mutant LINE-1 (D702A, D205A), mouse wild-type LINE-1 (L1SM), mouse mutant LINE-1 (L1SMN21A), and toxicity control (pcdna6.1). This study will provide new insights in the regulation of LINE-1 elements and will contribute to the elaboration of new therapeutical strategies. Abstract Nº11 Título: LINE-1 Retrotransposition and Protein Stability are Regulated by the Mammalian Genome-Defence Gene TEX19.1 Ponente: Marta García Cañadas Contacto: LINE-1 retrotransposons are a major component of mammalian genomes, and contribute to germline genomic instability and genetic disease in mammals. Developing germ cells possess a group of germline genome defence genes that help protect against the mutagenic activity of these transposable genetic elements, although the mechanisms by which different genome defence genes achieve this is often poorly understood. Here we demonstrate that the methylation-sensitive germline genome defence gene TEX19.1 acts to promote instability of LINE-1 ORF1p in mouse testes. We show that TEX19.1 physically interacts with both LINE-1 ORF1p and the E3 ubiquitin ligase UBR2, thereby promoting ubiquitylation and ubiquitindependent proteolysis of this retrotransposon-encoded protein. Furthermore, the human ortholog of TEX19.1, TEX19, acts in a similar manner to destabilise human LINE-1 ORF1p. Ectopic expression of TEX19.1 in somatic cell lines decreases LINE-1 ORF1p stability and inhibits LINE-1 retrotransposition, whereas loss of TEX19.1 function in embryonic stem cells results in increased LINE-1 ORF1p stability and increased LINE-1 retrotransposition. Thus, TEX19.1 appears to act post-transcriptionally as a host factor to restrict LINE-1 retrotransposition in germ cells and their pluripotent precursors. Abstract Nº12 Título: Improving gene edition tools for Wiskott-Aldrich Syndrome gene therapy. Ponente: Alejandra Gutiérrez Guerrero Contacto: alejandra.gutierrez@genyo.es Specific nucleases, including ZFNs, TALENs and CRISPR (Clustered Regulatory Interspaced Palindromic Repeats) are powerful tools for genome editing. Gene therapist can now aim for genetic rescue instead of gene addition to treat diseases. However, their efficiency and safety should be analyzed in detail before translation into clinic. Primary inmunodeficiencies (PID) are a main target for gene editing strategies since a small number of corrected cells could cure the patient. Our final aim is to developed gene edition tools for efficient genetic rescue of human hematopoietic stem cells (hhscs) from Wiskott-Aldrich Syndrome (WAS) patients. With this aim we have designed: 1- different WAS-specific CRISPR-based nucleases expressed by Lentiviral vectors (LV) plasmids. These plasmids will allow the comparison of different delivery systems using the same backbone. 2- A reporter cellular model harboring GFP coding sequences disrupted by WASP sequences (K562-GF-WASP-P). This cellular model will allow an easy readout for the efficiency and safety of different gene editing tools for the WAS locus. 3- A donor DNA containing wt GFP inside the homology arms and an expression cassette for dsred outside the homology arms. This donor will allow us to measure the unspecific insertion of the donor outside the targeted locus (red cells). Using these tools we showed efficient genetic rescue of the K562-GF-I1-P model generating up to 4% of egfp positive cells upon nucleofection of the different components. A similar efficiency was obtained for gene addition inside the endogenous locus using a donor harboring an egfp expression cassette and wt K562 cells. In summary we have developed new CRISPR based tools for efficient gene edition of the WASP locus and a new cellular model to study efficiency and safety of WASP gene edition.

17 Abstract Nº13 Título: Nuevos biomarcadores en adenocarcinoma ductal pancreático: estudio de expresión diferencial en sangre periférica Ponente: Cristina Jiménez Luna Contacto: El adenocarcinoma ductal pancreático (PDAC) es el tipo de cáncer de páncreas más común, detectándose en el 90% de los casos. Esta neoplasia se correlaciona con tasas de supervivencia realmente bajas, de hecho, sólo el 5% de los pacientes sobreviven a los 5 años. El pobre pronóstico al que se enfrentan estos pacientes se asocia, principalmente, a la detección de la enfermedad en estadios avanzados, de modo que la detección temprana del PDAC podría ser una buena estrategia para alargar la supervivencia de estos pacientes. El objetivo de este estudio fue la identificación de nuevos biomarcadores, en sangre periférica, capaces de diferenciar pacientes con PDAC de individuos sanos, con objeto de hacer posible una rápida detección de la neoplasia. Para ello, contamos con muestras de sangre periférica procedentes de una población de estudio constituida por 36 sujetos (18 pacientes con PDAC y 18 sujetos sanos), cuyo perfil de expresión génica fue analizado mediante microarray. Finalmente, los genes más relevantes se testaron, mediante PCR cuantitativa a tiempo real (RT-qPCR), en un nuevo conjunto de muestras (n=30) bajo estudio ciego. Identificamos 87 genes con expresión diferencial entre ambas poblaciones de estudio, 9 de los cuales presentaron valores de fold change iguales o superiores a 2. Seleccionamos 4 genes, cuya diferencia de expresión fue más notable, para su posterior análisis mediante RT-qPCR. Con objeto de evaluar su capacidad diagnóstica, estos 4 genes se validaron en el estudio ciego (n=30). Los resultados obtenidos con el uso combinado de los genes CLEC4D e IRAK3, muestran una precisión en el diagnóstico del 93%, una sensibilidad del 86% y una especificidad del 100%. De acuerdo con estos datos, proponemos un conjunto de 4 genes (ANKRD22, CLEC4D, VNN1, IRAK3) que podría constituir una herramienta útil en el diagnóstico de PDAC a partir de muestras de sangre periférica. Abstract Nº14 Título: Generating PKP1 knock-down, knock-out and over-expressed Non-Small-Cell Lung Cancer cell lines. Ponente: Joel Martín Padrón Contacto: joel.martin@genyo.es Despite advances in early detection and standard treatment, Non-Small-Cell Lung Cancer (NSCLC) is often diagnosed at an advanced stage and carries a poor prognosis. Greater knowledge of the molecular origins and progression of lung cancer may lead to improvements in the treatment and prevention of the disease. There is considerable evidence for the importance of desmosomes and their constituents in cancer. A role has been proposed for these adhesion genes in susceptibility to cancer and metastasis, and there is increasing evidence that the modulation of desmosomes is an important step in the initiation and invasiveness of human malignancies. In addition to the well-known structural role of desmosome genes, evidences such as their dual subcellular localization, the interaction with single-stranded DNA and translation initiation factors, etc. suggest there is a poorly understood role of these proteins in signaling pathways. PKP1 is up-regulated in primary Squamous-cell lung cancer (SCC) tissues suggesting a putative contribution in tumorigenesis. In order to gain greater insight into the multifuntional role of some desmosomal proteins in NSCLC tumours, we are generating stable cell lines with inducible overexpression (introduction of a new copy of the gene) and inhibition (by shrna) systems of PKP1 by two approximations: 1- Clones generation of selected NSCLC cell lines that comprise the T-Rex system, inducible by doxycycline, and the TetO2 regulator. 2- Transduction of selected NSCLC cell lines with the integrative plasmid plvpt-ttr-krab. Once obtained these stable cell lines, an array of expression will reveal the effect of overexpression and inhibition of pkp1 in regulating the expression of other genes whose alteration presumably could be involved in tumor development. Furthermore, we are conducting a Knockout of selected NSCLC cell lines for PKP1 by the genome-editing system "CRISPR Cas9" in order to confirm the specificity of the

18 antibodies in our immunohistochemical tests and validate the different subcellular localizations of pkp1 that our group has been observed in different stages of cell cycle that could have implications in cellular division and cancer. Also, this knock out, just like the knock down, will provide data about changes in expression of other genes and their possible implications in cancer. Abstract Nº15 Título: Condrogenic differentiation of infrapatellar fat pad-derived stem cells by chondrocytes extract from patients with osteoarthritis. Ponente: Elena López Ruíz Contacto: Infrapatellar fat pad of patients with osteoarthritis (OA) contains multipotent and highly clonogenic adipose-derived stem cells that can be isolated by low invasive methods. Moreover, nuclear and cytoplasmic cellular extracts have been showed to be effective in induction of cell differentiation and reprogramming. Objective: The aim of this study was to induce chondrogenic differentiation of autologous mesenchymal stem cells (MSCs) obtained from infrapatellar fat pad (IFPSCs) of patients with OA using cellular extracts-based transdifferentiation method. Methods: IFPSCs and chondrocytes were isolated and characterized by flow cytometry. IFPSCs were permeabilized with Streptolysin O and then exposed to a cell extract obtained from chondrocytes. Then, IFPSCs were cultured for 2 weeks and chondrogenesis was evaluated by morphologic and ultrastructural observations, immunologic detection, gene expression analysis and growth on 3-D poly (DL-lactic-coglycolic acid) (PLGA) scaffolds. Results: After isolation, both chondrocytes and IFPSCs displayed similar expression of MSCs surface makers. Collagen II was highly expressed in chondrocytes and showed a basal expression in IFPSCs. Cells exposed to chondrocyte extracts acquired a characteristic morphological and ultrastructural chondrocyte phenotype that was confirmed by the increased proteoglycan formation and enhanced collagen II immunostaining. Moreover, chondrocyte extracts induced an increase in mrna expression of chondrogenic genes such as Sox9, L- Sox5, Sox6 and Col2a1. Interestingly, chondrocytes, IFPSCs and transdifferentiated IFPSCs were able to grow, expand and produce extracellular matrix (ECM) on 3D PLGA scaffolds. Conclusions: We demonstrate for the first time that extracts obtained from chondrocytes of osteoarthritic knees promote chondrogenic differentiation of autologous IFPSCs. Moreover, combination of transdifferentiated IFPSCs with biodegradable PLGA 3D scaffolds can serve as an efficient system for the maintenance and maturation of cartilage tissue. These findings suggest its usefulness to repair articular surface in OA. Abstract Nº16 Título: Polimorfismos en genes inmunomoduladores y riesgo a desarrollar aspergilosis invasiva: Resultados del consorcio aspbiomics. Ponente: Carmen Belén Lupiañez Muñoz Contacto: carmen.lupianez@genyo.es La Aspergilosis Invasiva (AI) es una infección causada por hongos del género Aspergillus que tiene una elevada tasa de mortalidad (50-70%) en pacientes onco-hematológicos. Su difícil diagnóstico obliga al uso generalizado de profilaxis anti-fúngica lo que la convierte en la complicación clínica más cara para los sistemas sanitarios europeos. El objetivo de nuestro trabajo radica en la identificación de marcadores genéticos asociados al riesgo a desarrollar AI que nos permita predecir qué pacientes son más susceptibles de contraer la infección y adaptar la estrategia profiláctica al riesgo de cada paciente. En el contexto del consorcio aspbiomics, realizamos estudios GWAS (Immunochip, SNPs) y de genes candidatos que nos han permitido identificar regiones genómicas asociadas tanto con un mayor riesgo a desarrollar AI (IL4R, IL8, IL10, IL2-IL21, VEGFA, MAPKAPK2, C1orf106, C14orf145, LEMD2 y CD80) como con su resistencia (IL12B e IFNG). Estudios funcionales nos están permitiendo además demostrar el efecto de ciertas variantes genéticas en estos genes sobre la modulación de la respuesta inmune frente a Aspergillus. Ensayos de expresión génica en células mononucleares de sangre periférica revelaron que los individuos portadores del alelo IL4R rs g en el promotor del gen presentaban mayores niveles del RNA m (P=0.0065) y mayores niveles de este receptor en linfocitos B (P=0.07) pero no en linfocitos T (P=0.25) o macrófagos (P=0.51), lo que indica que esta variante puede tener un papel

19 importante en la modulación de la respuesta Th 2 mediada por células B. Estudios de estimulación in vitro además nos permitieron observar que los individuos portadores del alelo IL12 rs c presentaban mayores niveles de IL12B (24h-48h) tras la estimulación con antígenos fúngicos (P= y ) y una mayor estimulación de IFNG (P=0.066), lo que sugiere que dicha variante puede tener un efecto clave en la activación de la vía IL12B/IFNG frente a hongos. Finalmente, aunque los portadores del alelo IFNG rs t no presentaban mayores niveles de IFNG tras la estimulación con PHA y LPS, dichos pacientes presentaban mayores niveles de TNFa (96h; P=0.068), lo que coincidía con los datos genéticos que indicaban que esta variante induce resistencia a la infección. En su conjunto, nuestros datos sugieren que polimorfismos genéticos en genes inmunoreguladores están implicados en la modulación de la respuesta inmune frente a hongos y que su genotipado puede resultar de utilidad para el diseño de estrategias profilácticas individualizadas. Abstract Nº17 Título: Modulación del microambiente tumoral en las Cancer stem cells de Glioblastoma y Neuroblastoma. Ponente: Jaime Antonio Oliver Esteve Contacto: er_moho@hotmail.com / jaoe@correo.ugr.es Las Cancer stem cell (CSC) es una de las causas de la progresión tumoral en Glioblastoma (GBM) y Neuroblastoma (NB). El estudio del microambiente tumoral ha sido de especial interés ya que determina la transición desde tejido mesenquimal a epitelial y viceversa. En este campo, los elementos más analizados son los componentes de la matriz extracelular (CME). Los CME desempeñan múltiples funciones en la remodelación celular y la homeostasis siendo la Decorina (DCN) y el Lumican (LUM) los más destacados. Estos se han relacionado a la diferenciación celular de las células madre, la migración e invasión de las células cancerosas, así como a la quimiorresistencia. Estudios preliminares nos han mostrado que DCN y LUM podrían tener implicación en la formación de las neurosferas-3d en GBM y NB, modelo ampliamente aceptado de CSC. En nuestro estudio sometimos las líneas SK-N-SH y SF-268 de GBM y NB a medio de enriquecimiento de CSC y analizamos la expresión de ARNm, mediante qpcr, y proteica, mediante Western-Blot, tanto de DCN como LUM. Además analizamos la invasividad mediante agar-blando y la resistencia a la temozolamida (TMZ) mediante MTT. Nuestros resultados mostraron: 1. Incremento significativo en las CSC tanto a nivel de ARNm como proteico de DCN y LUM, 2. Crecimiento celular exclusivo de las CSC en agar-blando y 3. Resistencia significativa a la TMZ únicamente a las dosis más altas. De este modo, probamos la relevancia de los CME en la formación de las CSC en GBM y NB y la posible implicación en la quimiorresistencia frente a dosis altas de TMZ. Podemos concluir que la quiescencia relacionada a los CME podría ser uno de los puntos clave de la quimiorresistencia e invasividad de las CSC y que conocer mejor este fenómeno podría permitir el diseño de nuevas terapias que mejoren el pronóstico. Abstract Nº18 Título: Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast. Ponente: Francisco Gabriel Ortega Sánchez Contacto: gabriel.ortega@genyo.es In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast, which is due to the higher endocytic activity of tumor cells comparing to normal cells. In this way, we are demonstrating that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

20 Abstract Nº19 Título: Development of a celular reporter for WAS gene editing. Ponente: Sabina Sánchez Hernández Contacto: sabina.sanchez@genyo.es El síndrome de Wiskott-Aldrich (WAS) es una inmunodeficiencia primaria causada por mutaciones en el gen WAS. Actualmente, la única terapia eficiente para WAS es el trasplante de progenitores hematopoyéticos (HSCs), no siempre disponible. Como alternativa prometedora surge la terapia génica, donde la estrategia ideal sería dirigir la integración del gen funcional al sitio deseado o poder corregir la mutación que causa la enfermedad mediante edición génica. Nosotros contamos con Zinc-finger nucleasas (ZFN) y CRISPR específicos para edición de WAS, lo que nos ha permitido comparar la eficiencia de ambos sistemas. Hemos comprobado que CRISPR es tan eficiente como las mejores ZFN, tanto en cuanto a eficiencia de corte, como en cuanto a su capacidad de inducir recombinación homóloga (RH). Estos datos nos abrían la puerta a la utilización de CRISPR para terapia génica de WAS, siendo necesario estudiar en profundidad eficiencia y seguridad de estos sistemas. Para ello, desarrollamos un modelo celular que permitiría llevar a cabo tales estudios. El modelo tiene integrado en su genoma varias copias defectuosas de GFP interrumpido por el primer intrón de WAS. Este sistema reportero permite evaluar la frecuencia de NHEJ, así como de rescate de expresión de GFP, utilizando ZFN y CRISPR dirigidos frente al primer intrón de WAS. Esto nos permitirá investigar el efecto de la longitud de los donadores en la eficiencia de RH, la utilidad de vectores IDLV concentrados como método de edición génica, el uso de inhibidores del mecanismo NHEJ para favorecer RH, etc. En conclusión, hemos demostrado que el sistema CRISPR es tan eficiente como las ZFN para edición de WAS y hemos desarrollado una línea reportera que permite analizar eficiencia y seguridad de nucleasas especificas del intrón 1 de WAS, modelo que será de utilidad para estudiar la aplicabilidad del sistema CRISPR para terapia génica de WAS. Abstract Nº20 Título: Trans-ethnic genome-wide analysis reveals MHC variability in rheumatoid arthritis genetic susceptibility. Ponente: María Teruel Artacho Contacto: maria.teruel@genyo.es We have conducted a meta-analysis of Amerindian-enriched Latin American and Spanish rheumatoid arthritis cohorts with the aim of finding novel shared genetic risk loci and loci unique to each cohort. A total of 5,533 subjects were genotyped with the immunochip custom array and included in the final data set. We confirm the association in this population at genome-wide significant level of the HLA and PTPN22. We performed imputation of the MHC region with a dense reference panel. This analysis showed that four amino acids in positions 11, 13, 70 and 86 of the HLA-DRβ1 explain all the association in our combined analysis. Interestingly, we observed differential associations in each cohort separately. In the Spanish cohort, only amino acids 13 and 86 of HLA-DRβ1 were necessary to explain all the association in the MHC region, while for the Latin American population, amino acids at positions 13 and 70 were the important ones. Outside of the HLA region, the most interesting signals observed were on 2p25.3, 3q25.33 and 7p12.2 where the MYTL1, IL12A and IKZF1 genes are located, respectively. Other loci recently described as risk for RA susceptibility were also associated in our data such as SH2B3-PTPN11 and SYNGR1. Our results show that the comparative use of two populations provides clues on the diversity and the history of the genetic susceptibility behind a complex disease such as RA. Abstract Fuera de Categoría. Título: Fundacion MEDINA your partner for the Discovery of Novel Molecules. Ponente: Nuria De Pedro Contacto: Nuria.de.pedro@medinaandalucia.es