Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 153-171). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T07:55:23Z (GMT). No. of bitstreams: 1 Quaresma_AlexandreJoseChristino_D.pdf: 9068041 bytes, checksum: 9661a43a5c28440721d55ca90f6c94ac (MD5) Previous issue date: 2008 / Resumo: Os membros da família de proteínas chamada hnRNPs (heterogenous nuclear ribonuclein proteins) apresentam importantes papeis no controle da expressão gênica e no metabolismo dos mRNAs. Os membros hnRNPD (AUF1) e hnRNPQ (NSAP1) foram alvos deste estudo. AUF1 apresenta dois domínios de ligação à RNA do tipo RRM (RNA recognition motif) e participa ativamente no processo de desestabilização de uma classe de mRNAs que apresentam um motivo rico em AU na região 3' não traduzida. Demonstramos, através do sistema de duplo híbrido em levedura, que a isoforma p37 de AUF1 interagiu com as proteínas hnRNPQ, IMP-2, NSEP1 (YB-1) e UBC9. Além disso, a proteína hnRNPQ também foi pescada num outro ensaio de duplo híbrido em levedura, que utilizou como isca a proteína humana arginina metiltransferase (PRMT1). hnRNPQ apresenta, na sua região Cterminal, um ¿motivo rico em argininas e glicinas¿ (RGG box). Demonstramos que ela é alvo de metilação pela PRMT1 in vitro e in vivo. Funcionalmente, sua metilação é importante para sua localização nuclear. NSAP1 têm uma constituição modular com um domínio ácido (AcD) no seu Nterminal, seguido por três domínios de ligação à RNA do tipo RRM e o já mencionado RGG box no seu C-terminal. Funcionalmente hnRNPQ está envolvido em vários aspectos do etabolismo de RNA, incluindo a edição do mRNA da proteína humana ApoB. Para isso, ela interage não somente com o mRNA de ApoB, mas com a enzima efetora da edição Apobec1 e com a proteína que ativadora do Apobec1 (ACF1). Mostramos que o domínio ácido, de NSAP1 é capaz de interagir com Apobec1 e que sua fosforilação in vitro pela PKC inibe esta interação. Ainda identificamos que hnRNPQ interage com proteínas da família heat shock (incluindo HSP70 e BiP), e vimos que hnRNPQ é um alvo de fosforilação principalmente pela PKCd, in vitro. A localização sub-celular de hnRNPQ é modificada pela ativação in vivo das PKCs. Em conseqüência desta ativação ou da aplicação de estresse oxidativo, térmico ou indução de estresse do reticulo endoplasmático (tratamento com tapsigargina) hnRNPQ se desloca do núcleo para o citoplasma aonde se encontra em vesículas/corpúsculos definidas. Em resumo, nossos dados sugerem que as diversas funções da hnRNPQ relacionadas ao metabolismo de mRNAs, sofrem diferentes regulações, mediadas por modificações pós-traducionais (fosforilação e metilação), que interferem tanto na sua localização celular quanto na sua afinidade por determinados proteínas parceiras / Abstract: The members of the hnRNPs family (heterogenous nuclear ribonuclein proteins) play important roles in gene expression control and mRNAs metabolism. The proteins hnRNPD (AUF1) and hnRNPQ (NSAP1) were the main targets of this study. AUF1 has two RNA recognition motifs (RRM) and participates in the process of destabilization of a class of mRNAs that contain AU-rich sequences in their 3' untranslated regions (3'-UTR). We found, using the ¿yeast two-hybrid system¿ (Y2HS), that the isoform p37 of AUF1 (AUF1p37) interacts with the proteins: hnRNPQ, IMP-2, NSEP1 (YB-1) and UBC9. Moreover, the protein hnRNPQ was also identified as a prey protein in another Y2HS screen, which used as bait the human protein Arginine methyltransferase (PRMT1). HnRNPQ presents, in its C-terminal region, an "Arginine/Glicine-rich sequence" (RGG box). We are able to show that this RGG box is a target for methylation by PRMT1 in vitro and is methylated in vivo. Functionally, this methylation is important for its nuclear localization. hnRNPQ has a modular organization with an acid domain (AcD) in its N-terminal, followed by three RNA-binding domains (RRM) and the previously mentioned RGG box in its C-terminal. Functionally, hnRNPQ is involved in diverse aspects of RNA metabolism, including editing of the mRNA encoding the human protein ApoB. It has been shown previously to interact with the mRNA of ApoB, and also with the editing enzyme Apobec1 and the Apobec1 activation protein (ACF1). Here we show that the acid domain of hnRNPQ mediates the interaction with Apobec1 and that its in vitro phosphorylation (by PKC) inhibits this interaction. Furthermore, we found that hnRNPQ interacts with members the heat shock family of proteins (including HSP70 and BiP), and demonstrated that hnRNPQ can be in vitro phosphorylated by PKCd. Finally, we discovered that the sub-cellular localization of hnRNPQ undergoes modification after activation of PKC pathways. This also occurs after application of endoplasmic reticulum stress (using tarpsigargin), oxidative or heat stress. Under all of these conditions hnRNPQ translocated from the nucleus to the cytoplasm, where it is found at defined vesicles or granules. In summary, our data suggest that the diverse functions of hnRNPQ in the context of mRNA metabolism, may suffer specific regulations, by post-translational modifications, including phosphorylation and methylation, which modify both the proteins sub-cellular localizations as well as its affinity to interacting protein partners / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21030号 / 医科博第91号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 清水 章, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 122-140.

Ce travail de thèse porte sur développement de méthodes en spectrométrie de masse structurale pour l’analyse de protéines recombinantes et de leurs complexes associés. L’objectif central s’est porté sur des développements méthodologiques en échange hydrogène/deutérium couplé à la spectrométrie de masse (HDX-MS). Les techniques biophysiques de caractérisation structurale à haute résolution comme la cristallographie ou la RMN se heurtent régulièrement à des problèmes de productions de cristaux, de taille de complexes analysables ou encore de quantité de matériel nécessaire importante. Le développement de méthodes spécifiques HDX-MS a permis de réaliser une caractérisation structurale de systèmes protéiques variés, et réfractaires aux approches haute résolution. La combinaison de cette approche à différents outils de MS structurale est aussi illustrée, et montre tout son intérêt pour l’obtention d’informations à résolution augmentée. / This thesis work focuses on development of structural mass spectrometry methods for the analysis of recombinant proteins and their associated complex. The central objective has focused on the development of hydrogen/deuterium exchange coupled to mass spectrometry approaches (HDX-MS). The high resolution biophysical techniques for structural characterization such as crystallography or NMR regularly face problems of crystal productions, size analyzable complex or quantity of material required. The development of specific HDX-MS methods allowed the characterization of various, and refractory protein systems to high resolution approaches. The combination of this approach with complementary structural MS tools is also illustrated, and shows its interest to obtain increased resolution information.

Au sein des cellules, les ARNms sont liés par de nombreuses protéines, générant ainsi des particules appelées mRNPs (Ribonucléoparticules de messagers). Leur formation est cotranscriptionnelle, et leur composition va réguler l’ensemble des étapes du métabolisme des ARNms : stabilité, maturation, export, localisation et traduction. Au vu de l’importance de ces mécanismes dans la physiologie cellulaire, le contenu protéique des mRNPs est finement régulé dans le temps et l’espace et fait l’objet de nombreux remodelages. Ces changements de composition dépendent notamment des hélicases, ainsi que des modifications post-traductionnelles ; cependant, ces mécanismes demeurent à caractériser de façon plus approfondie. Une modification post-traductionnelle susceptible de moduler ces remaniements depuis la levure S. cerevisiae jusqu’aux métazoaires est la sumoylation. En effet, la SUMO-protéase Ulp1/SENP2, une enzyme clé de la machinerie de sumoylation, est localisée au panier des pores nucléaires, à proximité d’une plateforme d’ancrage des mRNPs destinées à l’export. Par ailleurs, il a été rapporté chez la levure que des mutants affectant la localisation et la stabilité d’Ulp1 présentent des défauts d’export et de localisation des mRNPs. Au vu de ces données, le laboratoire s’est intéressé aux rôles potentiels de la sumoylation dans le métabolisme de ces particules d’ARNm. Dans ce but, un crible protéomique a été réalisé chez la levure S. cerevisiae afin de comparer la composition des mRNPs entre des cellules sauvages ou mutantes pour Ulp1. Ce crible a mis en évidence un rôle d’Ulp1 dans le recrutement de deux composants des mRNPs, le complexe THO et l’hnRNP Hek2. Le complexe THO est un facteur multiprotéique qui participe à la prévention de l’instabilité génique et contribue à la transcription des ARNms, à l’assemblage des mRNPs et à leur export. L’hnRNP Hek2 est une protéine aux rôles multiples, dont l’association à un ARNm est susceptible de moduler sa stabilité, sa traduction et/ou sa localisation. Des analyses biochimiques nous ont permis de mettre en évidence l’existence de formes sumoylées de la sous-unité Hpr1 du complexe THO ainsi que de l’hnRNP Hek2. Toutes deux sont Ulp1-dépendantes, et interviennent sur la partie C-terminale de ces protéines. Nous avons également mis en évidence que chacune de ces sumoylations contrôle le recrutement de son substrat au sein des mRNPs. L’analyse fonctionnelle d’un mutant affectant la sumoylation d’Hpr1 a identifié cette modification comme nécessaire au recrutement du complexe THO sur une population d’ARNms impliqués dans la résistance au stress acide, autrement dégradés par l’exosome. Ainsi, l’absence de sumoylation d’Hpr1 diminue fortement la viabilité cellulaire en conditions de stress, un phénotype supprimé par l’inactivation de l’exosome. L’étude des effets de la sumoylation d’Hek2 suggère une modulation par SUMO de certaines de ses fonctions, notamment dans la localisation cellulaire des ARNms. L’ensemble de ces données fournit donc les deux premiers exemples de régulation du métabolisme des mRNPs par des événements de sumoylation intervenant au niveau du pore nucléaire. / Within the cells, mRNAs are associated to proteins, thereby generating particles called mRNPs (messenger ribonucleoproteins). mRNPs form in a cotranscriptional manner and their composition defines the fate of mRNAs by modulating the different steps of their metabolism, including their stability, their processing, their export, their localisation and their translation. In view of the importance of such mechanisms for cell physiology, several mechanisms ensure a tight spatio-temporal control of mRNPs composition through multiple mRNP remodelling events. These changes in the protein content of mRNPs depend on helicases and post-translational modifications, but remain to be further investigated. Sumoylation is one of the modifications that could contribute to mRNPs remodelling from yeast (S. cerevisiae) to metazoans. Indeed, it has been reported that the SUMO-protease Ulp1/SENP2, a key enzyme of the sumoylation machinery, is localized at the basket of nuclear pore complexes, in close vicinity with mRNPs committed for export. This particular localization, together with the reported defects in mRNPs export and localisation of yeast mutants affecting Ulp1, prompted the lab to ask whether sumoylation could contribute to mRNP biogenesis. In order to investigate this hypothesis, our lab compared mRNPs composition between wild-type and ulp1 mutant S. cerevisiae yeast strains using a proteomic approach. This screen identified two mRNP components that depend on Ulp1 for their recruitment onto these particles: the THO complex and the hnRNP Hek2. The THO complex is a multi-subunit factor that prevents genome instability and contributes to transcription, mRNP assembly and export. Hek2 has multiple functions in mRNA stability, translation and/or localization. Using biochemical approaches, we have been able to visualize sumoylated versions of the Hpr1 subunit of the THO complex and of the hnRNP Hek2. In both cases, this modification depends on Ulp1 activity and occurs on the C-terminal part of the protein. We further showed that these sumoylation events control THO and Hek2 recruitment onto mRNPs. Functional analysis of a mutant impairing Hpr1 sumoylation revealed that this modification is required for proper recruitment of the THO complex onto a subset of mRNAs involved in acidic stress resistance, which are otherwise degraded by the exosome. Decreased Hpr1 sumoylation results in a strong reduction of viability in acid stress conditions, a phenotype that is rescued by inactivation of the exosome. The investigation of the role of Hek2 sumoylation in mRNPs metabolism suggests that this modification regulates some of Hek2 functions, especially in mRNA localisation. All together, these results provide the two first examples of mRNPs components whose functions are regulated by sumoylation events occurring at the level of nuclear pores.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell disorders that often progress to chemotherapy-resistant secondary acute myeloid leukemia (sAML). We used whole-genome sequencing to perform an unbiased comprehensive screen to discover the somatic mutations in a sample from an individual with sAML and genotyped the loci containing these mutations in the matched MDS sample. Here we show that a missense mutation affecting the serine at codon 34 (Ser34) in U2AF1 was recurrently present in 13 out of 150 (8.7%) subjects with de novo MDS, and we found suggestive evidence of an increased risk of progression to sAML associated with this mutation. U2AF1 is a U2 auxiliary factor protein that recognizes the AG splice acceptor dinucleotide at the 3' end of introns, and the alterations in U2AF1 are located in highly conserved zinc fingers of this protein. Mutant U2AF1 promotes enhanced splicing and exon skipping in reporter assays in vitro. This previously unidentified, recurrent mutation in U2AF1 implicates altered pre-mRNA splicing as a potential mechanism for MDS pathogenesis.

No description available.

[ES] La leucemia mieloide aguda (LMA) se trata de un grupo heterogéneo de desórdenes hematológicos producidos por alteraciones genéticas en las células precursoras mieloides. Las mutaciones en la enzima Isocitrato deshidrogenasa 2 (IDH2) son unas de estas alteraciones. Las mutaciones más frecuentes en esta proteína afectan a las posiciones R140 y R172, provocando una ganancia de función con la producción del oncometabolito D-2-hidroxiglutarato (2-HG). A pesar de que ambas inducen la producción de 2-HG, la mutación R172 produce mayor cantidad de oncometabolito, presenta menos concurrencias con otras alteraciones genéticas y se asocia a una peor respuesta a la quimioterapia y un mayor riesgo de recaída. Los modelos de investigación han permitido conocer el papel de las mutaciones genéticas en el desarrollo de la enfermedad. A pesar de ello, es necesario desarrollar nuevos modelos que expresen de forma endógena estas mutaciones para estudiar en profundidad las vías moleculares afectadas. Por todo ello, en esta Tesis se han desarrollado nuevas estrategias de edición génica mediante el sistema CRISPR/Cas9 con el objetivo de desarrollar nuevos modelos in vitro e in vivo de mutaciones implicadas en LMA. Debido a la baja eficiencia de transfección de los plásmidos CRISPR en las líneas celulares leucémicas, el método más empleado para introducir los elementos CRISPR han sido principalmente vectores lentivirales. Para evitar los inconvenientes de este tipo de vectores, en esta Tesis se ha desarrollado una estrategia alternativa para la introducción de la nucleasa Cas9 y los guías CRISPR. El gen codificante de la Cas9 se introdujo en el genoma de células NB4 mediante transducción con lentivirus, generando una línea celular con expresión constitutiva de la nucleasa. Por otro lado, se desarrolló un sistema sencillo de producción de los guías CRISPR mediante PCR con los elementos esenciales para su expresión y la expresión del reportero GFP de forma opcional. Con el objetivo de optimizar la técnica y probar su eficiencia en distintas dianas se modificaron dos genes implicados en LMA. Estos fueron el gen IDH2, en el cuál se buscó introducir la mutación R172, y el gen MYBL2. Finalmente, las eficiencias de edición obtenidas se compararon con el uso de complejos de ribonucleoproteínas CRISPR, muy utilizados por su alta eficiencia. Mientras que los complejos de ribonucleoproteínas presentaron una mayor eficiencia de corte, la eficiencia de edición de la mutación R172 fue similar en ambas estrategias. Mediante secuenciación masiva se confirmó y caracterizó esta edición y se comprobó que la maquinaria de edición no había producido cortes inespecíficos en regiones similares del genoma. Por tanto, la nueva metodología desarrollada permitió editar de forma precisa líneas celulares leucémicas con eficiencias similares a otras técnicas CRISPR más extendidas y sin producir efectos inespecíficos no deseados. Por otro lado, gracias a la gran conservación evolutiva del gen IDH2, los residuos R140 y R172 se encuentran conservados en la proteína idh-2 de Caenorhabditis elegans. Se empleó la estrategia co-CRISPR para desarrollar y seleccionar cepas mutantes con las mutaciones ortólogas a R140 y R172, y una cepa con ambas mutaciones. A pesar de la conservación, no se observó el aumento del oncometabolito 2-HG esperado en las cepas mutantes en comparación con la cepa salvaje control N2. Un estudio exhaustivo de las vías implicadas nos serviría para desarrollar modelos de investigación con las alteraciones moleculares observadas en los pacientes. Para concluir, la estrategia desarrollada de introducción de elementos CRISPR en líneas celulares, junto a los modelos producidos en C. elegans, permitirán en futuros estudios investigar en detalle los efectos moleculares de mutaciones detectadas en pacientes de LMA, su implicación en el desarrollo y pronóstico de la LMA y comprender su papel en la estratificación de los pacientes. / [CA] La leucèmia mieloide aguda (LMA) es tracta d'un grup heterogeni de desordres hematològics produïts per alteracions genètiques en les cèl·lules precursores mieloides. Les mutacions en l'enzim Isocitrato deshidrogenasa 2 (IDH2) son d'aquestes alteracions. Les mutacions més freqüents en aquesta proteïna afecten a les posicions R1240 i R172, produint un guany de funció amb la producció de l'oncometabolit D-2-hidroxiglutarat (2-HG). A pesar que ambdues indueixen la producció de 2-HG, la mutació R172 produeix mes quantitat de oncometabolit, presenta menys co ocurrències con altres alteracions genètiques i s'associa a una pitjor resposta a la quimioteràpia i un major risc de recaiguda. Els models d'investigació han permés conéixer el paper de les mutacions genètiques en el desenvolupament de la malaltia. Malgrat això, és necessari desenvolupar nous models que expressen de manera endògena aquestes mutacions per a estudiar en profunditat les vies moleculars afectades. Per tot això, en aquesta Tesis s'han desenvolupat noves estratègies d'edició gènica mitjançant el sistema CRISPR/Cas9 amb l'objectiu de desenvolupar nous models in vitro i in vivo de les mutacions implicades en la LMA. Degut a la baixa eficiència de transfecció dels plasmids CRISPR en les línies cel·lulars leucèmiques, el mètode més emprat per a introduir els elements CRISPR han sigut principalment vectors lentivirals. Per a evitar els inconvenients d'aquesta mena de vectors, en aquesta Tesis s'ha desenvolupat una estratègia alternativa per a la introducció de la nucleasa Cas9 i els guies CRISPR. El gen codificant de la Cas9 es va introduir al genoma de cèl·lules NB4 mitjançant transducció amb lentivirus, generant una línia cel·lular amb expressió constitutiva de la nucleasa. D'altra banda, es va desenvolupar un sistema fàcil de producció dels guies CRISPR mitjançant PCR amb els elements essencials d'expressió i amb l'expressió del reporter GFP de manera opcional. Amb l'objectiu d'optimitzar la tècnica i provar la seua eficiència en diferents dianes es van modificar dos gens implicats en LMA. Aquests van ser el gen IDH2, en el qual es va buscar introduir la mutació R172, i el gen MYBL2. Finalment, les eficiències d'edició obtingudes amb la nova estratègia es van comparar amb l'ús de complexos ribonucleotproteïnes CRISPR, molt utilitzats per la seua alta eficiència. Mentre que els complexos de ribonucleoproteïnes van presentar una major eficiència de tall, l'eficiència d'edició de la mutació R172 va ser similar en les dues estratègies. Mitjançant seqüenciació massiva es va confirmar i caracteritzar aquesta edició i es va comprovar que la maquinària d'edició no havia produït talls inespecífics en regions similars del genoma. D'aquesta manera, la nova metodologia desenvolupada permet editar de manera precisa línies cel·lulars leucèmiques amb eficiències similars a altres tècniques CRISPR més esteses i sense produir efectes inespecífics no desitjats. D'altra banda, gràcies a la gran conservació evolutiva del gen IDH2, els residus R140 i R172 es troben conservats en la proteïna idh-2 de Caenorhabditis elegans. Es va utilitzar l'estratègia co-CRISPR per a desenvolupar i seleccionar ceps mutants amb les mutacions ortòlogues a R140 i R172, i un cep amb dues mutacions. Malgrat l'alta conservació, no es va observar l'augment del oncometabolit 2-HG esperat en els ceps mutants en comparació amb el cep salvatge control N2. Un estudi exhaustiu de les vies implicades ens serviria per a desenvolupar models d'investigació amb les alteracions moleculars observades en els pacients. Per a concloure, l'estratègia desenvolupada d'introducció d'elements CRISPR en línies cel·lulars, al costat dels models produïts en C. elegans permetran en estudis futurs investigar detalladament els efectes moleculars de mutacions detectades en pacients, la seua implicació en el desenvolupament i prognosi de la LMA i comprendre el seu paper en l'estratificació dels pacients. / [EN] Acute Myeloid Leukaemia (AML) is a heterogeneous group of haematological disorders caused by genetic alterations in myeloid precursors. Mutations in the Isocitrate dehydrogenase enzyme are among these alterations. The most frequent mutations in this protein affect R140 and R172 positions, leading to a gain of function with the production of the oncometabolite D-2-hydroxyglutarate (2-HG). Although both induce the 2-HG production, the R172 mutation generates greater amount of oncometabolite, has fewer co-occurrences with other genetic alterations and is associated with worse chemotherapy response and higher relapse risk. Research models have made possible to study the role of genetic mutations in disease development. Despite this progress, new models with endogenous expression of these mutations are needed to study in depth the molecular pathways involved. Therefore, in this Thesis we have developed new gene editing strategies using the CRISPR/Cas9 system with the aim of developing new in vitro and in vivo models of mutations involved in AML. Regarding in vitro model, due to the low transfection efficiency of CRISPR plasmids in leukemic cell lines, the most commonly method used for introducing CRISPR elements have been mainly lentiviral vectors. To avoid the disadvantages of this type of vectors, in this Thesis we have developed an alternative strategy for introducing Cas9 nuclease and CRISPR guides. The gene encoding the Cas9 was introduced into NB4 genome by lentiviral transduction producing a stable cell line that constitutively express the nuclease. On the other hand, a simple system for the production of CRISPR guides by PCR with essential elements of expression was developed and with GFP reporter expression optionally. In order to optimise the technique and test its efficiency in different targets, two genes involved in AML were modified. These were IDH2 gene, in which R172 mutation was introduced, and MYBL2 gene. Finally, editing efficiencies obtained with the new strategy were compared with CRISPR ribonucleoproteins methodology, widely used for its high efficiency. Whereas ribonucleoprotein complexes showed higher cut efficiencies, the efficiency of edition of R172 mutation efficiency was similar in both strategies. These results were validated and characterized by means of next generation sequencing, and no off-target effects were found. Therefore, the new developed methodology allows precise gene editing in leukemic cell lines with similar efficiencies with other popular CRISPR techniques and without off-target effects. On the other hand, thanks to the high evolutive conservation of IDH2 gene R140 and R172 residues are conserved in Caenorhabditis elegans idh-2 protein. The co-CRISPR strategy was used to produce and select mutant strains with ortholog mutations to R140, R172 and one strain with both mutations. Despite the high conservation, the expected increase in oncometabolite 2-HG concentration was not detected in mutant strains compared to the N2 wild type strain. A comprehensive study of the pathways involved would help us to develop a research model with molecular alterations noticed in patients. In conclusion, the new developed strategy for CRISPR elements introduction in cell lines, together with C. elegans models, will allow an in-depth research of molecular effect of mutations detected in patients, its implication in AML progression and prognosis and understand their role in patient stratification. / González Romero, E. (2022). Investigación de la Leucemia Mieloide Aguda mediante el desarrollo de modelos in vitro e in vivo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181891 / TESIS