(Abstract) Some of heat shock proteins (Hsp), for example the inducible form Hsp70, are expressed on the surface of tumour cells. High Hsp expression is reflected in tumour cell features, such as ability to progression, to metastasize and resistance to apoptosis. The question is whether Hsp gene expression correlates with surface expression. The aim of this master thesis is to compare surface and gene expression of Hsp70 and observe the gene expression of some other Hsp proteins (Hsp27, Hsp60, Hsp90 and HspBP1) in leukaemia. The research was carried out on cell lines obtained from leukaemic blasts of patients with acute myeloid leukaemia: UoC-M1, HL-60, OCI/AML3, THP-1, HU-3 and TF-1 that had been cultivated in vitro. Hsp70 surface expression was detected using flow cytometry, and gene expression of each Hsp was studied using real-time RT-PCR. It was found out that high surface expression of Hsp70 did not correlate with gene expression in consequence of negative feedback applied in Hsp expression regulation. Hsp27 gene expression was increased compared to negative (healthy) control on all tumour cell lines, with the highest increase on the THP-1 line. Hsp60 gene expression was increased compared to negative (healthy) control on all tumour cell lines and there were not remarkable differences in...

Acute myeloid leukemia (AML) is a malignancy with a poor prognosis. The expansion of leukemic cells is associated with the impairment of normal hematopoiesis, which can lead to severe morbidity in affected patients. Recent studies suggested that leukemic cells can remodel the bone marrow (BM) microenvironment, the so-called BM niche, creating permissive conditions favoring leukemic stem cell expansion over normal hematopoietic stem and progenitor cells (HSPCs) [1]. BM niche is a complex three-dimensional (3D) structure that provides factors necessary for the maintenance, survival, and differentiation of healthy HSPCs [2]. Most of the BM niche studies were performed using murine models. Mouse BM niche is defined based on the anatomical localization of different cell types and includes mesenchymal stromal cells (MSCs), endothelial cells, macrophages, lymphocytes, and megakaryocytes [2]. In addition, sensory and sympathetic nerve fibers (SNF) penetrating the BM were described for the first time in animals more than 50 years ago [3]. Later, neuropeptide Y-expressing neurons and parasympathetic nerve fibers were detected in rat femurs [4]. The recent development of 3D imaging suggests that the sympathetic nervous system regulates balanced blood cell production [5], HSPC migration [6], and regeneration of hematopoiesis in response to genotoxic stresses [7] in murine models. Sensory signaling has been reported to modulate the functionality of MSCs and osteoclasts in murine BM [4]. In the case of AML, it was recently shown in a murine model generated by retroviral infection of HSPCs with MLL-AF9 that functional SNFs were disrupted around arterioles, leading to the expansion of MSCs and endothelial cells [8]. Therefore, it is reasonable to hypothesize that SNFs potentially can also regulate hematopoiesis in human BM.

Les leucémies aigues myéloïdes (LAM) dérivent de progéniteurs hématopoïétiques dans lesquels se sont accumulés des événements génétiques conduisant à leur transformation. En établissant la hiérarchie clonale de multiples lésions récurrentes, nous démontrons que les événements impliquant les régulateurs de l'épigénétique sont les premiers événements dans le clone. A l'inverse, les mutations régulant la prolifération se produisent tardivement. Les événements précoces sont quasi constamment détectables en rémission complète. Les clones qui persistent servent de réservoir pour les rechutes, avec une variation qui augmente avec la durée de la rémission. Après xénogreffe, les échantillons de patients porteurs de lésions épigénétiques sont capables de repopulation hématopoïétique, ce qui est la signature fonctionnelle des événements pré-leucémiques. Cette hiérarchie est observée chez la majorité des patients. Cependant dans 1/3 des cas, il n'est pas retrouvé d'événement épigénétique, et on observe notamment des prédispositions germinales aux LAM, ou des mutations acquises de TP53. Chez 3 patients avec des LAM de novo, les mutations de TP53 sont associées à des mutations de DNMT3A dans un même clone. Cela suggère que les mutations de DNMT3A et de TP53 sont complémentaires pour obtenir une dominance clonale pré leucémique. En conclusion, dans la majorité des LAM, des hiérarchie clonales récurrentes, initiées par des lésions pré-leucémiques variées vont promouvoir l'expansion ou la variation clonale, jusqu'à l'émergence de la maladie. Ces données sont essentielles pour le développement de nouvelles stratégies personnalisées de suivi de la maladie résiduelle. / Acute myeloid leukaemia (AML) emerge from haematopoietic stem/progenitor cells that acquiregenomic or chromosomal aberrations, some being considered as pre-leukemic lesions. Here, byestablishing the chronological hierarchy of multiple driver lesions in AML, we show that most eventsaffecting epigenetic regulators - DNMT3A, TET2, ASXL1 mutations, as well as MLL and chromosome20q rearrangements - are the first lesions in the clone. In contrast to late mutations involving signallingpathways such as FLT3 or RAS, these early lesions are frequently detectable in complete remissionsample. By studying late relapses, we show that persistent clones behave as long-term relapsereservoirs, and variegate increasingly with delay to relapse. Cells from patients with early epigeneticdefects can repopulate bone marrow of xenotransplanted NOD/SCID/IL-2Rgc-null (NSG) mice withleukemic or non leukemic engraftment, a functional signature of pre-leukemic events. This genetichierarchy is observed in most patients but in one third of them lesions in epigenetic regulators are notthe first events. Some of these patients have genetic predisposition, or somatic mutations in TP53. Inthree de novo AMLs, but not in three secondary or therapy-related AMLs, these TP53 mutations wereaccompanied by DNMT3A mutations in a unique clone. This suggests that DNMT3A mutations maycomplement TP53 mutations to generate a dominant pre-leukemic clone. We conclude that in mostAMLs recurrent genetic hierarchies, initiated by distinct pre-leukemic lesions, promote progressiveclonal expansion or variegation, leading to the development of a full blown disease. These data areessential for the development of new personalised strategies for MRD evaluation.

Student Number : 9807768F - MSc (Med) research report - Faculty of Health Sciences / Fanconi anaemia (FA) is an autosomal recessive disorder characterised by aplastic anaemia (AA) and a high risk of developing acute myeloid leukaemia (AML). It is unknown whether heterozygote carriers are also predisposed to developing these disorders. The black South African population group is ideal for FA mutation screening because the presence of a founder mutation, FANCG 637-643, makes screening relatively straight forward. Three individuals with AML (115 screened) and one with AA (78 screened) were found to be heterozygous for the black South African founder mutation. From our data it seems unlikely that this mutation places heterozygous carriers of the mutation at high risk of developing AML or AA. Three children with AA out of 26 screened, were homozygous for the mutation. This finding reiterates the importance of screening all children with AA for FA. The frequency of certain congenital abnormalities in black South African FA patients was compared to patients described by other research groups. The frequencies of the abnormalities were similar to other FANCG cohorts described but significant differences to a group of FA patients from unspecified complementation groups were found. This difference could be because different complementation groups are associated more or less strongly with specific abnormalities. It was found previously that particular congenital abnormalities in FA patients are associated with a poor haematological outcome. We concluded that black South African FANCG patients have a high risk of early development of AA even though they do not have a high frequency of congenital abnormalities.

Dans le monde, 350 000 leucémies sont diagnostiquées chaque année. La rechute reste un problème majeur des leucémies aiguës myéloïdes (LAM) et le métabolisme oxydatif pourrait jouer un rôle essentiel dans la réponse au traitement. Un faible niveau des espèces réactives de l’oxygène (ROS) est associé à des propriétés des cellules souches leucémiques et la quiescence alors qu’un niveau plus élevé caractérise les leucoblastes proliférants. L’homéostasie des ROS repose sur un équilibre entre les systèmes oxydants et antioxydants. Les antioxydants sont bien documentés dans les LAM alors que les connaissances sur l’activité oxydante sont encore limitées. Dans ce travail nous avons choisi d’étudier les sept complexes NADPH oxydases (NOX) dans 25 lignées issues de LAM humaines et des LAM primaires. L’analyse des ARNm et des protéines montre des profils d’expression variables entre les lignées avec une expression plus forte des sous-unités du complexe NOX2 dans les lignées correspondant à des stades de différenciation myéloïde plus avancés. L’activité enzymatique des NOX est cependant équivalente entre les lignées. Deux inhibiteurs, DPI et VAS3947, ont été utilisés pour connaître la contribution des NOX à la production des ROS cellulaires. Alors qu’ils ont inhibé l’activité, ils ont aussi généré un stress oxydatif majeur conduisant à une diminution de la prolifération cellulaire et une forte apoptose, le DPI en augmentant les ROS mitochondriaux et VAS3047 les ROS cytoplasmiques. Afin de connaitre les sous-unités impliquées et de mieux comprendre les mécanismes, les sous-unités NOX2 et p22phox ont été inhibée par ARN interférence. Celle-ci n’ont pas affecté la prolifération mais ont montré des effets compensatoires. Nos data montrent qu’inhiber les NOX pourrait s’avérer une stratégie thérapeutique en augmentant le stress oxydatif dans les cellules leucémiques. / 350,000 leukaemia are diagnosed each year worldwide. In acute myeloid leukaemia (AML), relapse remains a major problem and the oxidative metabolism might play a crucial role in the therapeutic response. Low level of reactive oxygen species (ROS) is associated with properties of leukemic stem cells and quiescence whereas higher level promotes leukoblasts proliferation. ROS homeostasis relies on a tightly regulated balance between the oxidant and antioxidant systems. Although the antioxidant system is extensively studied in AML, the oxidant system remains poorly documented. In this work we aimed to study the seven NADPH oxidases (NOX) complexes in 25 AML human cell lines and primary samples. NOX transcriptional and protein profiles are variable with a higher expression of NOX2 in cell lines belonging to mature differentiation stages. An equivalent level of enzymatic activity was observed across all the cell lines. To reveal the contribution of NOX to global ROS production in the cells, two NOX inhibitors, DPI and VAS3947, were then used. Although both inhibitors efficiently blocked NOX activity they unexpectedly triggered strong oxidative stress leading to reduced cell proliferation and strong apoptosis, DPI by increasing mitochondrial ROS while VAS3947 by increasing cytoplasmic ROS production. To highlight which of the subunits were involved and to understand the mechanisms, NOX2 and p22phox subunits were inhibited using shRNA strategy. These did not affect cell proliferation but revealed a compensation effect. Our data suggest that NOX inhibition might be potential therapeutic strategy by increasing oxidative stress in leukemic cells.

Acute myeloid leukemia (AML) is a malignancy of the hematopoietic system caused by somatic mutations that accumulate in hematopoietic stem and progenitor cells. The cells are thereby transformed into leukemic stem cells (LSCs), which cannot be efficiently eliminated with the standard chemotherapy treatment. Thus, LSCs pose a risk of relapse for AML patients. There- fore, identification and characterization of LSCs is a major challenge in the field of AML research. Through next generation sequencing approaches the mutational spectrum of AML cells has been established and a continuous effort is being made to resolve the order of mutation acquisition and their functional consequences. In the subgroup of AML patients that bear a mutation in Nucleophosmin 1 (NPM1 ), a mutation in DNA-methyltransferase 3 A (DNMT3A) has been often found as a co-occurring event. Evidence suggests that this mutation arises early in leukemogene- sis and marks leukemic progenitors and stem cells. However, the functional consequences of this mutation are far from being understood. In this thesis work, I set out to unravel novel functional dependencies of the DNMT3A-mutant AML cells that can be exploited for therapeutic purposes. To nominate genes that are essential for the survival of primary AML cells, I performed a func- tional RNA interference-mediated drop out screen in 38 DNMT3A- and NPM1-mutant AML patient lines. The patients in this cohort were divided into two groups, based on the treatment outcome: an early relapse (ER) group and a long term remission (LTR) group. To nominate can- didate genes in each group, I have selected 12 screens with the highest data quality and performed a differential bioinformatic analysis. The analysis yielded 7 potential candidates, from which I initially validated three: Glucocorticoid modulatory element binding protein 1 (GMEB1), Mouse double minute 4 (MDM4) – both shared between the ER and the LTR groups – and Thioredoxin domain containing protein 9 (TXNDC9 ), which scored only in the ER group. Additional rounds of validation nominated MDM4 as the strongest candidate. To investigate the role of MDM4 in LSCs, I knocked it down in three patient samples and performed the long-term culture-initiating cell assay. However, the number of progenitor colonies that formed by the end of the assay was not enough for a statistical evaluation, probably due to the low frequency of long-term culture- initiating cells in the samples. Therefore, no conclusion regarding the functional dependency of LSCs on MDM4 could be made. However, a recent study suggested that loss of MDM4 causes cell cycle arrest and induces apoptosis in leukemic cell lines and primary cells, including progenitor populations, confirming the findings of this thesis. Nevertheless, the question about the role of MDM4 in NPM1 -mutant AML cells remains open. The NPM1 involvement in the p14Arf-MDM2- p53 pathway and the deregulation of this pathway caused by the NPM1 mutation indicate that MDM4 might poses special functions in NPM1 -mutant AML. Therefore, it should be investigated if MDM4 is a particularly suitable therapeutic target in AML with NPM1 mutation.

Measurable residual disease (MRD) detected by multiparametric flow cytometry (MFC) is associated with unfavorable outcome in patients with AML. A simple, broadly applicable eight-color panel was implemented and analyzed utilizing a hierarchical gating strategy with fixed gates to develop a clear-cut LAIP-based DfN approach. In total, 32 subpopulations with aberrant phenotypes with/without expression of markers of immaturity were monitored in 246 AML patients after completion of induction chemotherapy. Reference values were established utilizing 90 leukemia-free controls. Overall, 73% of patients achieved a response by cytomorphology. In responders, the overall survival was shorter for MRDpos patients (HR 3.8, p = 0.006). Overall survival of MRDneg non-responders was comparable to MRDneg responders. The inter-rater-reliability for MRD detection was high with a Krippendorffs α of 0.860. The mean time requirement for MRD analyses at follow-up was very short with 04:31 minutes. The proposed one-tube MFC approach for detection of MRD allows a high level of standardization leading to a promising inter-observer-reliability with a fast turnover. MRD defined by this strategy provides relevant prognostic information and establishes aberrancies outside of cell populations with markers of immaturity as an independent risk feature. Our results imply that this strategy may provide the base for multicentric immunophenotypic MRD assessment.

[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