Tesis doctorales y Tesinas
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- PLA2G4E: Bridging Synaptic Function, Brain Maturation, and Neurological Disorders with Potential Resilience in Alzheimer's Disease Treatment(2024-10-18) Badesso, S. (Sara); Garcia-Osta, A. (Ana); Cuadrado-Tejedor, M. (Mar)Alzheimers disease (AD) is the primary cause of dementia in the rapidly growing elderly population, thereby posing a significant healthcare challenge. Indeed, despite of the recent approval of new drugs, to date AD has no curative treatment. In response to this problem, the investigation on an alternative therapeutic approach, focused on protective factors against AD, is gaining ground. Consequently, the identification of the genetic and environmental basis of cognitive resilience provides the opportunity not only to unveiling the mechanisms underlying this disease but also and most important, to identify innovative therapeutic strategies. In this context, we evaluated the efficacy of two therapeutic interventions based on a lifestyle modification and a genetic protective factor. Specifically, on one side we demonstrated that the dietary DHA supplementation is capable of generating beneficial, albeit modest, effects in a preclinical AD model. On the other hand, we validated that a gene therapy involving PLA2G4E completely restores normal cognition in a 3rd generation AD mouse model. We then proceeded to delve into the mechanisms underlying the therapeutic effect of PLA2G4E. We explored the role of this enzyme in neuronal and brain development by describing its physiological expression in both in vitro and in vivo models. We found that its peak expression is required during the critical period for synaptogenesis. Moreover, we assessed the effects of inhibiting PLA2G4E demonstrating that a reduction in dendritic arborization and spine density, ultimately affecting neuronal function in vitro. On the other hand, complete knockout mice lacking PLA2G4E exhibited relevant behavioral alterations, particularly in memory function. These deficits were successfully reversed by overexpressing this enzyme, furtherly supporting the efficacy of a PLA2G4E-based gene therapy and expanding its potential to all neurological disorders coursing with synaptic degeneration.
- Functional study of chromatin factors uncovers strong lineage determining roles and divergent behaviours between normal and malignant haematopoiesis(Universidad de Navarra, 2024-01-18) Goñi-Salaverri, A. (Ainhoa); Pineda-Lucena, A. (Antonio); Lara-Astiaso, D. (David)Haematopoiesis relies on the coordinated activities of transcription and chromatin factors (TFs and CFs), which interact to form Genome Regulatory Complexes (GRCs). These dynamic complexes precisely control lineage-specific transcriptional patterns and steer cellular phenotypes. Furthermore, the significance of TFs and CFs is strongly supported by a wealth of evidence from recent studies, which consistently reveal the high recurrence and almost ubiquitous presence of mutations affecting TFs and CFs in haematological malignancies, including acute myeloid leukaemia (AML). During the last decade, researchers in the field of haematopoiesis have greatly benefited from a comprehensive understanding of normal differentiation roadmaps and the transformation events occurring in malignancies. However, our understanding of CFs contribution to haematopoiesis remains limited. We have yet to determine the main factors (CFs and TFs) within GRCs that govern haematopoietic differentiation, unravel their intricate functional interactions and whether CFs have specific roles or redundantly contribute to lineage determination. Furthermore, we still need to discern which epigenetic mechanisms are disrupted in leukaemia and determine their impact on disease initiation and/or maintenance. We hypothesised that CFs within GRCs exert distinct roles during haematopoietic lineage determination and that specific CF disruption might contribute to the initiation and/or maintenance of AML. Firstly, we systematically assessed the lineage-specifying potential of chromatin regulators (CFs and TFs) in murine haematopoietic differentiation trajectories, both ex vivo and in vivo. Ex vivo bulk results unveiled the nuanced and stage-specific roles of GRCs involved in fundamental epigenetic processes during haematopoietic differentiation, including COMPASS methyl-transferases and BAF remodellers. However, a higher level of functional consistency was observed among epigenetic repressor complexes and corepressors. Consistent with the ex vivo effects, in vivo findings at single-cell resolution revealed pronounced lineage-specific trends and functional variability for COMPASS and BAF subcomplexes and suggested that multiple epigenetic repressors, like NuRD, ISWI and N-CoR, help maintain progenitor diversity and balanced lineage distribution by mitigating excessive myelopoiesis. Our second goal was to unveil the potential interactions amongst TFs and CFs to unravel the composition of key GRCs, shifting the current regulatory paradigm from a single factor to a protein complex-centric view. We examined the dynamic patterns of chromatin accessibility upon TF/CF disruptions and selected representative TF motifs of haematopoietic cell fates to analyse global TF footprints upon every knockout. Disruption of COMPASS and BAF showed downregulation of myeloid TF motif accessibility. In contrast, NuRD and other repressors induced increased accessibility of myeloid TFs associated with inflammatory responses upon knockout. Lastly, we aimed to dissect the corruption of CFs function in a Npm1c/Flt3-ITD leukaemia model and provide leukaemic-specific vulnerabilities that suggest potential epigenetic therapeutic avenues. scRNA-seq and CITE-seq analyses unveiled leukaemia transcriptomic heterogeneity, with distinct subpopulations exhibiting varying degrees of growth potential. Afterwards, Perturb-seq analysis of the chromatin regulators loss of function across leukaemia clusters confirmed tumour vulnerabilities upon specific perturbations, facilitating the transition of AML-infected cells towards leukaemic differentiated populations with limited cell proliferation and fitness capacities. These observations revealed that leukaemias subvert the function of CFs involved in homeostatic differentiation by aberrantly blocking these processes to sustain their malignant state.
- Diseño e implementación de dos dispositivos biomédicos para pacientes con Síndrome de Apnea Obstructiva del Sueño (SAOS) basados en la detección de apneas e hipopneas y rehabilitación por medio de estimulación eléctrica(Universidad de Navarra, 2023-04-25) Moscoso, W. D. (William Daniel); Burguete-Mas, F.J. (Francisco Javier); Fernández-González, S. (Secundino)Esta investigación muestra el desarrollo de dos tecnologías enfocadas al Síndrome de apneahipopnea obstructiva del sueño. La primera tecnología se enfoca en el análisis de las señales del sueño y la segunda en una terapia alternativa basada en electroestimulación para el tratamiento de esta enfermedad. A continuación, se muestra un marco de referencia sobre los trastornos del sueño, los métodos diagnósticos para su evaluación y la estimulación eléctrica como una alternativa para el tratamiento de esta enfermedad.
- From transcriptomics to proteomics: Unraveling biological knowledge via Machine Learning(Universidad de Navarra, 2023-02-21) Serrano-Sanz, G. (Guillermo); Guruceaga, E. (Elizabeth); Hernaez, M. (Mikel)We start by highlighting basic concepts of both molecular biology and machine learning. This overview focuses on the key ideas that are required to comprehend the rest of the work, and thus, it does not attempt at providing a comprehensive review. We start with the basis of DNA and RNA, the genetic building bricks, until the formation of the proteins, the final actors of the genetic machinery. We also explore state-of-the-art technologies to measure those processes along with their limitations. After introducing the basic biological concepts, we will discuss the basics of machine learning methodologies and some of the most important models used in recent years to solve many biological problems.
- Generación de células humanas con potencial quimérico(Universidad de Navarra, 2023-02-16) Moya-Jódar, M. (Marta); Aranguren, X.L. (Xabier L.)En la actualidad existe escasez de órganos disponibles para trasplante, por lo que es necesario buscar alternativas para satisfacer la demanda en la clínica. La complementación de blastocisto se erige como una prometedora estrategia en la generación de órganos «humanizados» en animales grandes como el cerdo. Esta estrategia consiste en la microinyección de células pluripotentes en embriones modificados genéticamente inhabilitados para generar el órgano de interés, y una posterior transferencia embrionaria a una madre receptora para su desarrollo embrionario. En este contexto, las células exógenas que sí son capaces de generar el órgano colonizarán el nicho vacío del embrión y generarán el órgano de interés. Como resultado, al animal quimera tendrá el órgano exclusivamente formado por las células exógenas. Mediante complementación de blastocisto se han generado órganos funcionales interespecie en estadio adulto en quimeras rata-ratón. Sin embargo, la generación de dichos órganos humanizados en quimeras humano-cerdo es todavía propiamente una quimera. Las células capaces de generar órganos interespecie rata-ratón presentan un estadio de pluripotencia naïve, equivalente al epiblasto de un blastocisto preimplantacional. Sin embargo, las células humanas pluripotentes obtenidas convencionalmente presentan un estadio de pluripotencia primed, similar al epiblasto de un embrión de post-implantación incapaz de generar quimeras. En esta tesis se ha estudiado la conversión desde el estadio primed al estadio naïve de células madre pluripotentes inducidas humanas (hiPSCs) con diferentes condiciones de cultivo (5iLAF y NEHSM) y se han caracterizado adecuadamente los estadios naïve y primed. Además, mediante un análisis de scRNA-seq en las hiPSCs cultivadas con la condición 5iLAF, hemos descrito por primera vez diferentes poblaciones coexistiendo en el mismo cultivo que imitan los tipos celulares de un embrión humano de preimplantación. Estas poblaciones son; células similares al embrión humano de 8 células, epiblasto, trofectodermo y endodermo primitivo (Moya-Jódar et al., 2023, Stem Cell Reports). Gracias a estos resultados, las células convertidas con el medio 5iLAF podrían utilizarse como alternativa al uso de embriones humanos de preimplantación. Con la intención de potenciar la capacidad de quimerismo de las células humanas, en esta tesis también se han generado quimeras humano-ratón modulando la competencia celular. Dado que c-Myc es un gen clave en la competencia celular durante el desarrollo embrionario de los mamíferos, se ha utilizado el cruce de un modelo de ratón c-MycHET en combinación con un KI de una copia extra de C-MYC en las hiPSCs, consiguiendo aumentar el potencial de quimerismo humano-ratón tanto in vitro como in vivo. Además, modulando la competencia celular mediante la microinyección de hiPSCs C-MYC KI en embriones de cerdo, se ha conseguido generar quimeras humano-cerdo in vitro. Por último, en la complementación de blastocisto es necesario disponer de embriones deficientes para el gen de interés. Puesto que el sistema vascular es el principal causante del rechazo inmunológico en los xenotrasplantes, en esta tesis se ha optimizado la generación de embriones de cerdo deficientes para el gen ETV2, un factor de trascripción clave en el desarrollo del sistema vascular y hematopoyético. Se ha utilizado la tecnología de edición génica CRISPR/Cas9 directamente en cigotos porcinos consiguiendo obtener embriones de cerdo de preimplantación ETV2 KO de manera eficiente (Moya-Jódar et al., 2022, Animals).
- Application of single cell transcriptomics to characterize the progression of hematopoietic cells to myeloid malignancies(Universidad de Navarra, 2023-01-31) Ainciburu-Fernández, M. (Marina); Smerdou, C. (Cristian); Vanrell, L. (Lucía)Hematopoiesis is the process by which blood cells are formed. It has been a broadly studied system since the second half of the 20th century. Attention was drawn to this process after the discovery, in 1951, of the protective role that bone marrow intravenous infusion had against radiation. In the mid-1950s, it was subsequently discovered that this protection was caused by transplanted stem cells contained in the bone marrow. Multipotency of the hematopoietic stem cells (HSCs) was further proven during the 1960s, with the development of clonal in vivo repopulation assays. Following clonal in vitro assays, together with the study of cell surface markers and flow sorting, have configured the current view of the hematopoietic system, as a tree-like hierarchy with the HSCs at the top and mature cells at the bottom (Figure 1.1) [1, 2]. Hematopoiesis is active throughout life and it maintains the pool of mature blood cells, which constitute one of the human tissues with highest turnover. Broadly speaking, specialized blood cells can carry out four main functions: 1) The innate immune system, composed, among others, by macrophages, dendritic cells and granulocytes, constitutes the first defense against infection 2) The adaptive immune system, composed by B and T lymphocytes, carries out a delayed and specialized response against pathogens. 3) Red blood cells transport oxygen to every cell 4) Platelets take care of wound healing.
- Spatial and temporal patterns of plant community assembly in teo biogeographic regions: a taxonomic and functional approach.(Universidad de Navarra, 2022-07-13) Valerio-Galán, M. (Mercedes); Ibañez-Gaston, R. (Ricardo); Gazol, A. (Antonio)One of the main aims of plant community ecology throughout its history has been to understand the processes governing the assembly of plant communities. In the last decades, and in the face of the threat in biodiversity posed by global change, this interest has renewed. Despite the great number of studies carried out to understand plant community assembly, still many questions remain unclear, such as the relative influence of different processes on community assembly patterns across space and time, or the relationship between different characteristics of plant communities and environmental factors. In this regard, the aim of this thesis has been to increase our understanding of how plant communities work, by describing the patterns found in plant communities and trying to infer the processes driving them. To achieve this goal, we studied the spatial and temporal variation of plant communities in two ecosystems located in different biogeographic regions in Navarra (north of Spain): a temperate forest located in the Señorío de Bertiz Natural park, in the Atlantic region, and the Special Conservation Area of Ugarra (locality of Napal), in the Mediterranean region. In the Bertiz forest the study design consists on 102 permanent plots of 400 m2 each that were established throughout a basin of 132 ha, and which were sampled in 2006 and 2016. In the Napal grassland there were 12 permanent plots of 1m2 that were sampled annually for 14 years (2004-2017). Half of the plots in Napal were fertilized in 2003. In addition, from 2019 to 2021, we carried out extensive inventories around Napal to obtain the regional species pool. In all studies we applied a taxonomic and a functional approach, using functional traits. In the Bertiz forest we studied the spatial variation of the forest understory throughout an environmental gradient of light and soil moisture. Further, we studied changes in the forest understory in response to canopy gap dynamics over a decade. In Napal grassland, we carried out two temporal studies to analyze the effect of fertilization on semi-natural Mediterranean grasslands. Firstly, we studied plant community stability over 14 years and tried to discover its main drivers in control and fertilized plots, distinguishing year-to-year variation from long-term trends to avoid spurious conclusions when interpreting temporal patterns. Secondly, we studied over 14 years the dynamics of annual communities in response to their interactions with perennial herbs and woody species, and in response to inter-annual weather fluctuations. We found that in Bertiz forest light is the most limiting resource. Topography leads to spatial resource heterogeneity, while canopy gaps lead to spatial and temporal resource heterogeneity, which is key for maintaining the taxonomic and functional diversity of understory communities. Understory species composition also changes following gap dynamics, but it does it slowly, lasting more than a decade. In the Napal grassland community stability is driven by species richness, asynchrony and conservative traits. However, the relationship between stability and its drivers differs between year-to-year and long-term scale. Fertilization protects annuals from drought and cold and slows-down shrub encroachment in the long-term, but strengthens competition, decreases species richness and reduces stability in Mediterranean grasslands. In both study sites, abiotic filtering and competition drive community assembly, but the patterns they produce depend on the characteristic of the community analysed, the approach used, the functional trait studied and time span. Other processes such as dispersal also influence understory species composition following gap dynamics, seed bank longevity, species asynchrony and community stability. Studying spatio-temporal patterns in different regions and combining the taxonomic and functional approach can effectively advance our understanding of plant communities.
- Arañas de Navarra. Diversidad y distribución. Métodos digitales de estudio(Universidad de Navarra, 2021-12-16) Biurrun, G. (Gabriel) de; Baquero-Martin, E. (Enrique)La idea primera que llevó a iniciar este trabajo de tesis fue la de hacer un gran muestreo de arañas de Navarra; escribir algo imposible que contuviera improbablemente todas las arañas dentro. Si estuvieran quietas. Se diseñó un esquema de muestreo casi redondo, un abanico de posibles combinaciones que luciera bien y sonara coherente. Se hicieron luego miles de kilómetros por carreteras y caminos, cientos de litros de agua llovida en las trampas y en las botas. Laboratorio, lupa, microscopio, 23 000 arañas. Y un día el muestreo se acaba, y cuando está uno analizando muestras piensa si no sería estupendo poner trampas de caída en los árboles, porque no están quietas, en verdad; y cuántas debe de haber bajo el suelo, y qué distintas. Y se da uno cuenta de que, efectivamente, tener todas las arañas es improbable, y escribirlo imposible.
- Proteogenómica computacional para la detección y caracterización de proteínas en el contexto del Proyecto Proteoma Humano(Universidad de Navarra, 2021-07-12) González-Gomariz, J. (José); Guruceaga, E. (Elizabeth); Segura, V. (Víctor)En los últimos años, la consolidación de la tecnología de secuenciación masiva (NGS) en el área de la Genómica está ampliando la información que es posible obtener a partir de este tipo de experimentos (Trapnell, 2012). La posibilidad de obtener la secuencia completa de ADN de un genoma, o de ARN de un transcriptoma mediante un único experimento ha supuesto grandes avances en los estudios de genotipado y en la identificación de nuevas alteraciones cromosómicas y mutaciones puntuales. Tras los avances y resultados obtenidos en Genómica, el estudio de las proteínas codificadas por el genoma (proteoma) es el siguiente paso para comprender la fisiología humana, tanto en individuos sanos como enfermos. El estudio del proteoma, a pesar de presentar similitudes con el estudio del genoma, supone un gran desafío que la comunidad científica debe acometer. Los avances en espectrometría de masas, actualmente la tecnología más potente disponible en Proteómica (Nilsson, 2010), están generando un volumen de datos enorme que hacen imprescindible la participación de otras áreas como la Estadística, la Minería de datos y la Computación de Altas Prestaciones para poder analizarlos. Sin embargo, el análisis preciso de estos datos es un reto para el cual se requiere el desarrollo de nuevas herramientas. Otro de los desafíos pendientes dentro del campo de la Proteómica es la caracterización de proteínas detectadas experimentalmente de las que se desconoce su función, localización celular ó su implicación en determinadas enfermedades, para los que desde la comunidad científica se han propuesto distintos retos. La presente tesis describe varios flujos de trabajo, implementados para la identificación de proteínas de las que no existe evidencia experimental y para la caracterización funcional de proteínas de las que no se conoce su función, localización e implicación en enfermedades. Además, se hace una incursión en el campo del procesado de señal aplicando la Transformada Wavelet a distintos tipos de señales, tanto de experimentos de secuenciación de nueva generación (NGS) como de experimentos de espectrometría de masas, desarrollados con el objetivo de mejorar el análisis de este tipo de señales.
- Elucidating the mechanisms involved in Hepatitis Delta virus (HDV) pathogenesis(Universidad de Navarra, 2021-07-09) Maestro-Galilea, S. (Sheila); González-Aseguinolaza, G. (Gloria); Aldabe, R. (Rafael)Hepatitis Delta virus (HDV) infection is associated with the most severe form of viral hepatitis. Despite this, little is known about the molecular mechanism involved in HDV-induced liver pathology and the limited availability of small animal models hamper the development of specific therapies to ameliorate liver damage. We have recently developed an HDV mouse model based on the delivery of HDV and hepatitis B virus (HBV) replication-competent genomes using a hepatotropic adenoassociated virus (AAV). This model mimics several important characteristics of the human disease and led us to the identification of TNF-α as a critical factor involved in HDV-induced pathology. The initial part of this thesis was focused on the identification of the main cell population in charge of TNF-α production. By RNA-Fluorescent In Situ Hybridization (FISH), we identified macrophages as the main cell population responsible for TNF-α production, representing 78% of TNF-α-positive cells, while HDV-infected hepatocytes and non-identified cells represent 6% and 16% of TNF-α-producing cells, respectively. In addition, the second aim of this research project was to identify the main viral factors that are involved in HDV-induced liver damage. For that, we generated different HDV mutants by Site-Directed Mutagenesis (SDM) to avoid Hepatitis Delta Antigens (HDAgs) expression, their post-translational modifications, and to alter their subcellular localization in the infected cell. First, HDV mutants were characterized in Huh7 cells to analyse the levels of viral replication, HDAgs expression, and the ability to produce HDV infective particles. Then, AAV8 vectors were constructed carrying the HDV wild-type (WT) or HDV mutated genomes to characterize their effect in terms of liver damage and liver inflammation. On the one hand, we found that the lack of L-HDAg exacerbates liver damage, which was associated with an overexpression of S- HDAg and with an increase in viral replication. On the other hand, the lack of isoprenylation of L-HDAg at Cys-211 resulted in lower levels of this isoform, leading to a favored expression of S- HDAg over L-HDAg that was also associated with more severe liver damage. Finally, the analysis of HDV mutants with alterations in HDAgs subcellular localization led us to associate the accumulation of HDAgs in the cell nucleus with a more acute liver injury. In conclusion, our data showed that both host and viral factors are involved in HDV-induced pathology. On one side, TNF-α, produced by macrophages that become activated during HBV/HDV coinfection. On the other side, an increment in the S-HDAg/L-HDAg ratio and the accumulation of HDAgs in the infected cell is associated with a more severe liver damage.