II JORNADAS CIENTIFICAS GENYO

Transcription

II JORNADAS CIENTIFICAS GENYO - 2014
LIBRO DE ABSTRACTS
COMUNICACIONES SELECCIONADAS PARA PRESENTACIÓN ORAL
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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: [email protected]
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
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.
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
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”
(MNF50) 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 MNF50 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 MNF50, the increase of the ΔMFI is doubled
when the NPs number are doubled, something which is not observed when ratios lowers than their MNF50
are used. Importantly, this effect is the same for the three studied cell lines. Therefore, when MNF50 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.
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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
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
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.
Abstract Nº3
Título: Line-1 as a possible epimutagen in pluripotent genomes.
Ponente: Juan David Cano Martínez
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 80-100 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.
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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
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
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.
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
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.
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SESIÓN DE INVESTIGADORES EXTERNOS.
Abstract Nº1
Título: Desarrollo de magnetoliposomas como sistemas transportadores de fármacos.
Ponente: Cristina Lorente Guirado
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
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.
Abstract Nº3
Título: Bozepinib, a new small compound against cancer and cancer stem-like cells.
Ponente: Alberto Ramírez Rivera
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.
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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
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 ~50.000
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
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.
Abstract Nº3
Título: Hidrogeles supramoleculares como vehículos proteicos y celulares. Hacia novedosas aplicaciones
biomédicas y biotecnológicas.
Ponente: Rafael Contreras Montoya
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 2014. Título: “Supramolecular Hydrogels for
Protein Crystallization”.
COMUNICACIONES SELECCIONADAS PARA PRESENTACIÓN EN FORMATO PÓSTER
Abstract Nº1
Título: Deciphering the possible role of LINE retrotransposition during tissue regeneration
Ponente: Ana Ariza Cosano
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
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.
Abstract Nº3
Título: Role of Hypoxia in Megakaryocytic Differentiation of Human Embryonic Stem Cells.
Ponente: María del Mar Bonillo Lamolda
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
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.
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
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 Th1, Th2 y
Th17. Nuestros resultados han permitido identificar que SNPs en Dectin-2, MCP-1, DC-SIGN, IL4, IL8RB,
VEGFA y TNFR2 así como ciertos haplotipos (IL4TTTCA, IL8TA, TNFR2GGCT) 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®, 200.000 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
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.
Abstract Nº7
Título: Detección de miRNA-21 en células tumorales circulantes mediante hibridación in situ.
Ponente: Diego de Miguel Pérez
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 rs55758736 (Ala71Thr) in BLK causes constitutive phosphorylation,
reduced protein abundance, and loss of interaction with BANK1.
Ponente: Alejandro Díaz Barreiro
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.
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
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 (rs4244285) y ABCB1 (rs1045642) 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% 1.53-23.53,
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% 3.85-118.35, 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
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
~80-100 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
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
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
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.
Abstract Nº13
Título: Nuevos biomarcadores en adenocarcinoma ductal pancreático: estudio de expresión diferencial en
sangre periférica
Ponente: Cristina Jiménez Luna
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
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
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
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, LSox5, 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
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®, 200.000 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 IL4Rrs2107356G en el promotor del gen presentaban
mayores niveles del RNAm (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
importante en la modulación de la respuesta Th2 mediada por células B. Estudios de estimulación in vitro
además nos permitieron observar que los individuos portadores del alelo IL12rs3212227C presentaban
mayores niveles de IL12B (24h-48h) tras la estimulación con antígenos fúngicos (P=0.0017 y 0.0060) 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
IFNGrs2069705T 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: [email protected] / [email protected]
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
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.
Abstract Nº19
Título: Development of a celular reporter for WAS gene editing.
Ponente: Sabina Sánchez Hernández
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
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
Patrocinadores:
Comité Científico:
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




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Per Anderson
Concepción Marañón
Francisco Martín
Pedro Medina
Verónica Ramos
Pedro J. Real
Juan Carlos Rodríguez-Manzaneque
Juan Sainz
Rosario María Sánchez

II JORNADAS CIENTIFICAS GENYO

Transcription

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