In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues. Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues. Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining. The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues. In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

No description available.

Lo sviluppo delle tecnologie di sequenziamento ha portato alla nascita di strumenti in grado di produrre gigabasi di dati di sequenziamento in una singola corsa. Queste tecnologie, comunemente indicate come Next Generation Sequencing o NGS, producono grandi e complessi dataset la cui analisi comporta diversi problemi a livello bioinformatico. L'analisi di questo tipo di dati richiede la messa a punto di pipelines computazionali il cui sviluppo richiede un lavoro di scripting necessario per concatenare i softwares già esistenti. Questa tesi tratta l'aspetto metodologico dell'analisi di dati NGS ottenuti con tecnologia Illumina. In particolare in essa sono state sviluppate tre pipelines bioinformatiche applicate ai seguenti casi studio: 1) uno studio di espressione genica mediante RNA-seq in "Olea europaea" finalizzato all’indagine dei meccanismi molecolari alla base dell’acclimatazione al freddo in questa specie; 2) uno studio mediante RNA-seq finalizzato all’identificazione dei polimorfismi di sequenza nel trascrittoma di due razze bovine mirato a produrre un ampio catalogo di marcatori di tipo SNPs; 3) il sequenziamento, l’assemblaggio e l’annotazione del genoma di un ceppo di Lactobacillus plantarum che mostrava potenziali proprietà probiotiche. / The advance in sequencing technologies has led to the birth of sequencing platforms able to produce gigabases of sequencing data in a single run. These technologies commonly referred to as Next Generation Sequencing or NGS produce millions of short sequences called “reads” generating large and complex datasets that pose several challenges for Bioinformatics. The analysis of large omics dataset require the development of bioinformatics pipelines that are the organization of the bioinformatics tools in computational chains in which the output of one analysis is the input of the subsequent analysis. A work of scripting is needed to chain together a group of existing software tools.This thesis deals with the methodological aspect of the data analysis in NGS sequencing performed with the Illumina technology. In this thesis three bioinformatics pipelines were developed.to the following cases of study: 1) a global transcriptome profiling of “Oleaeuropeae” during cold acclimation, aimed to unravel the molecular mechanisms of cold acclimation in this species; 2) a SNPs profiling in the transcriptome of two cattle breeds aimed to produce an extensive catalogue of SNPs; 3) the genome sequencing, the assembly and annotation of the genome of a Lactobacillus plantarum strain showing probiotic properties.

Les neuroblastomes olfactifs (NBOs) sont des tumeurs rares de la base du crâne. Les outils de classification de ces tumeurs sont insuffisants, il n’existe notamment aucune classification moléculaire des NBO. La biologie de ses tumeurs est mal connue. Nos travaux se sont appuyés sur l’analyse des exomes, du transcriptome, du méthylome et des caractéristiques histopathologiques et immunitaires d’une série de 59 NBOs bien annotés cliniquement. Nous avons mis en évidence l’existence de 2 sous-types de NBO présentant un profil d’expression et un profil anatomo-clinique différent. Le type neural, correspond à une tumeur bien différenciée, peu agressive, présentant des caractéristiques plutôt neuronales et partageant des caractéristiques phénotypiques avec les progéniteurs directs de neurones olfactifs. Le type basal correspond à une tumeur moins différenciée, plus agressive, ayant des caractéristiques plutôt embryonnaires, partageant des caractéristiques phénotypiques avec les cellules basales de renouvellement de l’épithélium olfactif. Nous avons mis en évidence que la charge mutationnelle était plus élevée dans le type basal, avec notamment des mutations IDH2 R172 récurrentes, associées à un phénotype CpG Island Methylator (CIMP). Nous avons également montré que les NBOs de type basal étaient infiltrés par un nombre plus important de lymphocytes T avec, dans certaines tumeurs, une expression plus marquée de checkpoints immunitaires et de facteurs immunosuppresseurs. Ce travail ouvre la perspective d’une classification moléculaire permettant de mieux stratifier les patients et ouvre également le champ de nouvelles stratégies thérapeutiques dans ces tumeurs rares. / Olfactory neuroblastomas (ONBs) are rare tumors arising in the skull base. Classification tools are poor, notably; no molecular classification of ONB has been reported. Literature data about their cell of origin, the existence of molecular therapeutic targets or their immune environment being scarce, the biology of these tumors is still poorly understood. Our work was based on exome, transcriptome and methylome analysis, but also on histopathological and immune characteristics of a series of 59 clinically well annotated ONBs. We highlighted 2 sub-types of ONB showing different expression and clinicopathologic patterns. The neural type is a well differentiated, poorly aggressive tumor which shows neurons characteristics and shares phenotypic similarities with olfactory neuron progenitors. The basal type is a less differentiated tumor, displaying an aggressive phenotype, with embryonic phenotypical characteristics, which shares similarities with basal renewing cells of the olfactory epithelium. We showed that the mutational load was higher in basal tumors, with notably recurrent IDH2 R172 mutations associated with a CpG Island Methylator Phenotype (CIMP). We also showed that basal type ONBs were infiltrated by a greater number of T cells with, in some cases, a higher expression of immune checkpoints and immunosuppressive factors. This work paves the way towards a new molecular classification which will allow a better stratification of patients and will open the field of new therapeutic strategies for this rare tumor.

Submitted by JULIANA JOICE PEREIRA LIMA null (julianajoicelima@yahoo.com.br) on 2016-06-28T20:01:19Z No. of bitstreams: 1 Final_version_UNESP com ficha.pdf: 2890371 bytes, checksum: 99c3a37a64a79be1ed5e60fb7720deb9 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-29T18:48:22Z (GMT) No. of bitstreams: 1 lima_jjp_dr_bot_par.pdf: 731237 bytes, checksum: 6b7a35a2397a27e5b7522bcee2cf6435 (MD5) / Made available in DSpace on 2016-06-29T18:48:22Z (GMT). No. of bitstreams: 1 lima_jjp_dr_bot_par.pdf: 731237 bytes, checksum: 6b7a35a2397a27e5b7522bcee2cf6435 (MD5) Previous issue date: 2016-04-29 / Item withdrawn by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-10-09T12:55:09Z Item was in collections: Teses - Agronomia (Agricultura) - FCA (ID: 320) No. of bitstreams: 3 lima_jjp_dr_bot_par.pdf.jpg: 3708 bytes, checksum: f4776b7545f49e0381732de6fe127643 (MD5) lima_jjp_dr_bot_par.pdf.txt: 70796 bytes, checksum: a8b790348c0cd59ac464aff1569d634a (MD5) lima_jjp_dr_botfca.pdf: 2807174 bytes, checksum: 00e8dea3c713cb320435c39d6d97bae6 (MD5) / Item reinstated by Vivian Rosa Storti null (vstorti@reitoria.unesp.br) on 2018-10-10T14:33:51Z Item was in collections: Teses - Agronomia (Agricultura) - FCA (ID: 320) No. of bitstreams: 3 lima_jjp_dr_bot_par.pdf.jpg: 3708 bytes, checksum: f4776b7545f49e0381732de6fe127643 (MD5) lima_jjp_dr_bot_par.pdf.txt: 70796 bytes, checksum: a8b790348c0cd59ac464aff1569d634a (MD5) lima_jjp_dr_botfca.pdf: 2807174 bytes, checksum: 00e8dea3c713cb320435c39d6d97bae6 (MD5) / Item reinstated by Vítor Silvério Rodrigues (vitor.s.rodrigues@unesp.br) on 2018-11-06T19:07:52Z Item was in collections: Teses - Agronomia (Agricultura) - FCA (ID: 320) No. of bitstreams: 5 lima_jjp_dr_bot_par.pdf.jpg: 3708 bytes, checksum: f4776b7545f49e0381732de6fe127643 (MD5) lima_jjp_dr_botfca.pdf.jpg: 4544 bytes, checksum: af481a0bb7992130d84da3a17c3b7c0d (MD5) lima_jjp_dr_bot_par.pdf.txt: 70796 bytes, checksum: a8b790348c0cd59ac464aff1569d634a (MD5) lima_jjp_dr_botfca.pdf.txt: 211012 bytes, checksum: 72d4f897af91998bbeca736aa4fc7e7f (MD5) lima_jjp_dr_botfca.pdf: 2807174 bytes, checksum: 00e8dea3c713cb320435c39d6d97bae6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Le soja est l'une des plus importantes espèces de cultures d'huile utilisée aussi bien en nourriture que dans diverses gammes d'applications industrielles. C'est pourquoi produire des graines vigoureuses est un levier essentiel pour augmenter efficacement de la production de la récolte. La qualité de graine physiologiquement faible, qui est plus à même de se produire sous un environnement tropical, mène à un pauvre établissement des plantes ainsi qu'à une diminution du rendement. La longévité d'une graine est la capacité de celle-ci à survivre à la sécheresse durant de longues périodes et représente une caractéristique importante sur la qualité d'une graine. Ici, l'objectif était d'obtenir une idée sur les mécanismes en régulant l'acquisition progressive de la longévité. En utilisant le séquençage à haut-débit, ARN a été séquencé en sept différentes étapes durant l'acquisition de longévité, générant entre 14 et 38 millions de reads. Ces reads ont été alignés sur les modeles de gene de Glycine max Wm82.a2.v1. Les transcripts différentiellement exprimés (DET) sont corrélée avec l'augmentation de la longévité de la graine. L’analise d’enrichissement via GO de ces DET ont révélé une importante surreprésentation des termes associés à la réponse au stress et traitement et modification de l'ARN. Le processu biologique Photosynthèse était liée à une faible longévité des semences. HSF (heat shock factor) et plusieurs facteurs de transcription associés à la défense biotique (WRKY 3 et NLFX1) sont des gènes candidats dont putative rôle dans la longévité des graines et méritent une caractérisation plus poussée. Nous avons également effectué la détermination de la teneur en sucres solubles non réducteurs, et nous avons observé que l'accumulation de sucres solubles non réducteurs sont liés à l'acquisition de la longévité, mais seulement l’accumulation d’eux ne suffit pas à expliquer la croissance de la longevité. / Soja é uma das mais culturas oleaginosas usadas para alimentação animal e humana bem como para uma larga aplicação industrial. Dada a sua capacidade de fixar nitrogênio atmsférico, é fundamental para o desenvolvimento de uma agricultura sustentável. Produzir sementes altamente vigorosas é a chave para aumentar a eficiência da produção da cultura. Longevidade de semente é a capacidade de sobreviver no estado seco por períodos prolongados e representa uma importante característica de qualidade da semente. Nesta pesquisa o objetivo foi obter insights sobre processos moleculares que regulam a aquisição de longevidade em sementes de soja. Com o sequenciamento de nova geração da Illumina, o RNA foi sequenciado a partir de sete estágios diferentes durante a aquisição de longevidade, gerando entre 14 e 38 milhões de reads. Estes reads foram alinhados com os modelos de genes de Glycine max Wm82.a2.v1 preditos no genoma de soja. Transcritos diferencialmente expressos (DET) foram correlacionados com o aumento da longevidade. Análise de enriquecimento da ontologia do gene daqueles DET revelaram uma siginificante sobre representação de termos associados com resposta a estresse e processamento e modificação de RNA. Processo biológico fotossíntese foi relacionado à baixa longevidade. Heat Shock Factors (HSF) e vários fatores de transcrição associados com resposta a estresse biótico (WRKY e NFXL1) são genes candidatos com possíveis papéis na longevidade de semente e merecem uma caracterização. Também foi determinado o conteúdo de açúcares solúveis não redutores. Foi observado que o acúmulo desses açúcares estão relacionados à aquisição da longevidade, porém somente eles não são suficientes para explicar o ganho da longevidade. / Soybean is one of the most important oil crop species used for food and feed as well as a range of industrial applications. However, producing highly vigorous seeds is a key lever to increase crop production efficiency. Low physiological seed quality, which is more prone to occur under tropical environment, leads to poor stand establishment and decreased in yields. Seed longevity is the ability to survive the dry state for prolonged periods of time and represents an important trait for seed quality. Here, the objective was to obtain insights into the mechanisms regulating the progressive acquisition of longevity. Using Illumina high-throughput sequencing, RNA was sequenced from seven different stages during the acquisition of longevity, generating between 14 and 38 million of reads. These reads were aligned to the Glycine max Wm82.a2.v1 gene model. Differentially expressed transcripts (DET) were correlated with the increase in seed longevity. Transcriptome and GO enrichment analyses of these DET revealed a significant over-representation of terms associated with response to stress and RNA processing and modification. Photosynthesis biological process was related to low seed longevity. HSF and several TF associated with biotic defense (WRKY3 and NLFX1) are candidate genes whose putative role in seed longevity deserve further characterization. We also performed the determination of the content of non-reducing soluble sugars, and we observed that the accumulation of non-reducing soluble sugars are related to acquisition of longevity but only the accumulation of them is not enough to explain the increase in longevity.

La transcription pervasive génère des milliers de lncRNA pouvant réguler l'expression des gènes et jouer un rôle important dans le développement et dans différentes maladies. Parmi eux, les asRNA restent mal caractérisés malgré l'importance de leurs rôles régulateurs. Durant ma thèse, nous avons utilisé des données de séquençage à haut débit directionnelles de diverses lignées cellulaires et tissus humains pour définir de nouveaux asRNA. L'analyse bioinformatique nous a permis de classer les nouveaux asRNA selon 3 critères: niveau d'expression, la présence d'EST épissée et de jonction prédite. Nous avons défini 4 catégories: la classe1 répond aux 3 critères (la plus robuste), la classe2a satisfait les critères d'expression et de jonction, la classe2b satisfait les critères d'expression et d'EST et la classe3 satisfait le critère d'expression. Le niveau d'expression de ces asRNA est très faible et comparable à celui des lncRNA. Ils sont exprimés de manière spécifique, enrichis dans le noyau, coiffés et polyadénylés. Deuxièmement, j'ai posé la question de savoir s'ils sont impliqués dans le cancer et dans l'EMT. Nous avons analysé l'expression des gènes dans les tumeurs de la prostate, du sein et d'angiosarcomes. Nous avons comparé le tissu tumoral au sain et avons obtenu une liste de gènes dérégulés incluant des asRNA. Cette analyse nous a permis de découvrir 3 nouveaux asRNA spécifiques aux tumeurs de la prostate pouvant être utilisés comme outil de diagnostic. L'analyse de l'EMT a permis de définir des ARN spécifiques au système étudié. Cette analyse a révélé l'importance de la transcription antisens chez l'Homme et leur rôle potentiel dans la régulation des gènes. / Pervasive transcription generates thousands of lncRNAs that can regulate gene expression and play an important role in development and in different diseases. Among them, asRNAs remain poorly characterized despite the importance of their regulatory roles in cells. During my PhD, we used high-throughput sequencing directional data from multiple human cell lines and tissues to define new asRNAs. Bioinformatics analysis allowed us to classify new asRNAs according to 3 criteria: level of expression, presence of spliced EST and predicted junction. We defined 4 categories: class1 responds to the 3 criteria (strongest class), class2a satisfies the criteria of expression and splice junction, class2b satisfies the criteria of expression and spliced EST and class3 responds to criterion of expression. The level of expression of these asRNAs is lower than mRNAs but is comparable to lncRNAs. They are specifically expressed in a tissue or cell line, enriched in the nucleus, capped and polyadenylated. Secondly, I posed the question of whether this asRNAs are expressed in cancer and in the EMT. We analyzed gene expression in prostate tumors, breast and angiosarcoma. We compared tumor tissue and healthy one and we obtained a list of genes differentially expressed, among them asRNAs. We defined for each tissue a specific transcriptome signature. This analysis allowed us to discover 3 new asRNAs specific to prostate tumors that can be used as a diagnostic tool. Analysis of the EMT helped to define specific markers in the studied system including mRNAs, lncRNAs and asRNAs. This analysis revealed the importance of antisense transcription in humans and their potential role in gene regulation.

No description available.

No description available.

Tesis por compendio / [ES] En las últimas décadas, se ha observado un incremento en la emergencia de nuevas virosis en los cultivos del sudeste español, principal región abastecedora de frutas y hortalizas al resto de Europa. Entre las virosis emergentes más recientes en esta área de cultivo, se encuentra el tomato leaf curl New Delhi virus (ToLCNDV), un begomovirus de genoma bipartito transmitido por moscas blancas (Bemisia tabaci). En 2012 se detectó por primera vez en las provincias de Murcia y Almería, y desde entonces se está propagando por otros países de la cuenca del Mediterráneo, generando graves daños en los cultivos de cucurbitáceas, y limitando la producción, principalmente, de calabacín (Cucurbita pepo) y melón (Cucumis melo). En esta tesis doctoral se ha desarrollado un programa de mejora genética enfocado a la obtención de variedades de cucurbitáceas resistentes al aislado español del ToLCNDV(-ES). Por primera vez, hemos identificado fuentes de resistencia al ToLCNDV en el género Cucurbita y en pepino (Cucumis sativus). Todas las accesiones resistentes que describimos pertenecen a tipos silvestres o variedades locales, la mayoría originarias de la India, donde se describió por primera vez el ToLCNDV. Es posible que en esta área se haya producido un fenómeno de co-evolución entre plantas silvestres de cucurbitáceas y este begomovirus. Dos accesiones de Cucurbita moschata han mostrado un elevado nivel de resistencia, y a pesar de su cruzabilidad intermedia con C. pepo, ha sido posible la obtención de descendencia interespecífica y la transferencia parcial de la resistencia. Las accesiones resistentes identificadas se han caracterizado para determinar el tipo de herencia que regula la resistencia al ToLCNDV. Mediante el desarrollo de poblaciones segregantes de mejora y el aprovechamiento de herramientas genéticas de mapeo y cartografía hemos identificado tres QTLs que controlan la resistencia al ToLCNDV en melón, uno de efecto mayor y herencia parcialmente dominante en el cromosoma 11 y dos que modifican su efecto mediante interacciones epistáticas en los cromosomas 2 y 12. Siguiendo esta estrategia, también hemos identificado un locus de resistencia recesiva a ToLCNDV en el cromosoma 8 de C. moschata, aunque la penetración incompleta cuando se intenta transferir a C. pepo pone de manifiesto la influencia del fondo genético en el carácter. Las regiones genómicas involucradas en la resistencia de ambas especies son sinténicas y agrupan un conjunto de genes comunes no descritos previamente en la resistencia a otros virus en cucurbitáceas. Los genotipados mediante colecciones de SNPs han permitido identificar marcadores moleculares ligados a la resistencia al ToLCNDV en melón, calabaza y calabacín. Estos marcadores suponen un valioso recurso en programas de mejora, ya que permiten transferir de manera asistida las resistencias identificadas a fondos genéticos comerciales. Para profundizar en el conocimiento de los mecanismos moleculares que dan lugar a la resistencia, hemos realizado un ensayo de RNA-seq, comparando los transcriptomas de un genotipo resistente y otro susceptible de melón durante la infección con ToLCNDV. Los resultados obtenidos al analizar la expresión diferencial son compatibles con el tipo de herencia cuantitativa de la resistencia y reflejan un complejo sistema de regulación transcripcional. La infección sistémica del ToLCNDV en melón se ve influenciado por la desregulación de genes implicados en la ruta de señalización hormonal del ácido jasmónico, transportadores transmembrana, fotosíntesis y factores de transcripción. Además, hemos observado cambios en la expresión de genes implicados en la metilación del DNA, tanto en el genotipo resistente como susceptible, lo que sugiere que el silenciamiento génico mediado por RNA puede estar involucrado en la inhibición de la transcripción del genoma viral, favoreciendo la resistencia. / [EN] Different factors promote the emergence of new viruses in crops of the Southeastern Spain, the main supplier region of vegetables and fruits to the rest of European countries. A new strain of tomato leaf curl New Delhi virus (ToLCNDV) is among the most recent emergent viral diseases in this farming area, a bipartite begomovirus naturally transmitted by whiteflies (Bemisia tabaci). This strain was first detected in 2012 in the provinces of Murcia and Almeria, from where it propagated to other Mediterranean countries. ToLCNDV generates severe damages in cucurbits, decreasing yields mainly in squash (Cucurbita pepo) and melon (Cucumis melo) crops. In this doctoral thesis, a breeding program has been developed to obtain cucurbit varieties with resistance to the Spanish strain of ToLCNDV(-ES). Although cucurbit germplasm with resistance genes to this virus is poor and localized in few accessions, in this work we have identified for the first-time resistance sources to ToLCNDV in Cucurbita spp. and cucumber (Cucumis sativus). All resistant accessions here described are landraces or wild types not domesticated or with low levels of genetic manipulation. Moreover, most of these genotypes are originals from India, where ToLCNDV was first described. Likely, the occurrence of co-evolution events took place between wild cucurbits plants and this begomovirus. Despite not identifying resistance sources in squash, two Cucurbita moschata accessions have shown high resistance levels. Both species present intermediate crossability, even so the obtention of interspecific offspring and the partial transference of the resistance was possible. The resistant accessions identified in the works of this thesis and those identified by our group in previous assays, have been characterized to determine the heritage controlling ToLCNDV resistance. The development of breeding segregating populations and exploiting getenic tools for mapping and cartography, we identifed three QTLs controlling resistance to ToLCNDV in melon, one partially dominant with mayor effect in chromosome 11, and two minor modifiers in chromosomes 2 and 12. Epistatic interactions between them have been detected. Following the same strategy, we have also identified a locus with recessive heritage in chromosome 8 of C. moschata. However, when we tried to transfer it to C. pepo incomplete penetrance occurrence was observed, reflecting the influence of the genetic background on the trait. The genomic regions identified in both species are synthenic and share a common cluster of genes not described previously in cucurbits conferring resistance to other viruses. The advances in next generation sequencing technologies have offered us a huge quantity of genomic and transcriptomic information. Genotyping by SNPs collections allowed the identification of molecular markers linked to ToLCNDV resistance in melon, pumpkin and squash. These markers suppose a valuable resource in breeding programs, since can be used to assist the transference of the identified resistances to commercial genetic backgrounds. To further understand the molecular mechanism regulating the resistance, we have performed an RNA sequencing assay (RNAseq), comparing transcriptomes between a resistant and a susceptible accession of C. melo in the time course of ToLCNDV infection. The results obtained from the analysis of differential expression levels are compatible whit a complex transcriptional regulation. Deregulation of genes involved in hormonal jasmonic signaling, transmembrane transport, photosynthesis and transcription factors are involved in the ToLCNDV systemic infection through melon plants. Furthermore, in both resistant and susceptible genotypes, we observed expression changes of genes described in DNA methylation pathway. This suggests that RNA silencing mechanisms might be responsible of inhibit viral genome transcription, enhancing the resistance response. / [CA] Durant les últimes dècades, s'ha observat un increment en l'emergència de noves virosis als cultius del sud-est espanyol, principal regió proveïdora de fruites i hortalisses a la resta d'Europa. Entre les virosis emergents més recents en aquesta àrea de cultiu, es troba un nou aïllat del tomato leaf curl New Delhi virus (ToLCNDV), un begomovirus de genoma bipartit transmés per mosca blanca (Bemisia tabaci). En 2012 es va detectar per primera vegada a les províncies de Múrcia i Almeria, i des d'aleshores s'està propagant per altres països de la conca del Mediterrani, ací genera greus danys en els cultius de cucurbitàcies, i hi limita la producció, principalment de carabasseta (Cucurbita pepo) i meló (Cucumis melo). En aquesta tesi doctoral s'ha desenvolupat un programa de millora genètica enfocat a l'obtenció de varietats de cucurbitàcies resistents a l'aïllat espanyol del ToLCNDV(-ES). Hem pogut identificar per primera vegada fonts de resistència al ToLCNDV en el gènere Cucurbita i en cogombre (Cucumis sativus). Totes les accesions resistents que descrivim pertanyen a tipus silvestres o varietats locals, la major part originaris de l'Índia, on es va descriure per primera vegada el ToLCNDV. És possible que en aquesta àrea s'haja produït un fenomen de coevolució entre plantes silvestres de cucurbitàcies i aquest begomovirus. Dos accesions de Cucurbita moschata han mostrat un elevat nivell de resistència, i tot i la compatibilitat intermèdia que presenten ambdues espècies, ha sigut possible l'obtenció de descendència interespecífica i la transferència parcial de la resistència. Les accessions resistents identificades s'han caracteritzat per tal de determinar el tipus d'herència que regula la resistència al ToLCNDV. Mitjançant el desenvolupament de poblacions segregants de millora i l'aprofitament de ferramentes genètiques de mapeig i cartografia, hem identificat tres QTLs que controlen la resistència al ToLCNDV en meló, un d'efecte major i herència parcialment dominant en el cromosoma 11 i dos que modifiquen el seu efecte mitjançant interaccions epistàtiques als cromosomes 2 i 12. Seguint aquesta estratègia, també hem identificat un locus de resistència recessiva a ToLCNDV en el cromosoma 8 de C. moschata, encara que la penetració incompleta quan s'intenta transferir a C. pepo evidència la influència del fons genètic en el caràcter. Les regions genòmiques involucrades en la resistència de les dos espècies són sintèniques i agrupen un conjunt de gens comuns no descrits prèviament en la resistència a altres virus en cucurbitàcies. Els genotipats utilitzant col·leccions de SNPs han permés identificar marcadors moleculars lligats a la resistència al ToLCNDV en meló, carabassa i carabasseta. Aquests marcadors suposen un valuós recurs en programes de millora, ja que permeten transferir de forma assistida les resistències identificades a fons genètics comercials. Per tal d'aprofundir en el coneixement dels mecanismes moleculars que confereixen la resistència, hem realitzat un assaig de seqüenciació del RNA (RNA-seq), i hem comparat els transcriptomes d'un genotip resistent i altre susceptible de meló durant la infecció amb ToLCNDV. Els resultats obtinguts en analitzar l'expressió diferencial són compatibles amb el tipus d'herència quantitativa de la resistència i reflecteixen un complex sistema de regulació transcripcional. La infecció sistèmica del ToLCNDV en meló es veu influenciada per la desregulació de gens implicats en la ruta de senyalització hormonal de l'àcid jasmònic, transportadors transmembrana, fotosíntesi i factors de transcripció. A més, hem observat canvis en l'expressió de gens implicats en la metilació del DNA, tant en el genotip resistent com susceptible, el que suggereix que el silenciament gènic mediat per RNA pot estar involucrat en la inhibició de la transcripció del genoma viral, afavorint la resistència. / La Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana) y el Fondo Social Europeo (FSECV 2014-2020) han cofinanciado la contratación de la doctoranda como personal investigador de carácter predoctoral (ACIF/2016/188) y dos estancias predoctorales fuera de la Comunitat Valenciana (BEPFI/2017/011 y BEFPI/2018/028). La realización de esta tesis doctoral también se ha realizado en el marco de dos proyectos de investigación del Ministerio de Ciencia, Innovación y Universidades, con cofinanciación de fondos FEDER [Proyectos AGL2017-85563-C2-1-R y RTA2017-00061-C03-03 (INIA)] y del proyecto PROMETEO para grupos de excelencia (2017/078) de la Conselleria d’Educació, Investigació, Cultura i Esports (Generalitat Valenciana). / Sáez Sánchez, C. (2020). Mejora Genética de la Resistencia al tomato leaf curl New Delhi virus en Cucurbitaceas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153801 / Compendio

The pathogen Francisella tularensis subsp. tularensis has been classified as a Center for Disease Control (CDC) select agent. However, little is still known of what makes the bacteria cause dis-ease, especially the highly virulent type A1 strains. The work in this dissertation focused on type A1 strains from the Inzana laboratory, including a wildtype virulent strain TI0902, an avirulent chemical mutant strain TIGB03 with a Single Nucleotide Polymorphism in the wbtK gene, and several complemented mutants, [wbtK+]TIGB03, with dramatic differences in virulence and growth rates. One of the complemented clones (Clone12 or avp-[wbtK+]TIGB03-C12) was aviru-lent, but protected mice against challenge of a lethal dose of TI0902 and was considered as a possible vaccine strain. Whole genome sequencing was performed to identify genetic differences between the virulent, avirulent and protective strains using both Roche/454 and Illumina next-generation sequencing technologies. Additionally, RNASeq analysis was performed to identify differentially expressed genes between the different strains. This comprehensive genomic analysis revealed the critical role of transposable elements in inducing genomic instability resulting in large du-plications and deletions in the genomes of the chemical mutant and the complemented clones that in turn affect gene dosage and expression of genes known to regulate virulence. For exam-ple, whole genome sequencing of the avirulent chemical mutant TIGB03 revealed a large 75.5 kb tandem duplication flanked by transposable elements, while the genome of a virulent Clone01 (vir-[wbtK+]TIGB03-C1) lost one copy of the 75.5 kb tandem duplicated region but gained a tandem duplication of another large 80kb region that contains a virulence associated transcription factor SspA. RNAseq data showed that the dosage effect of this extra region in Clone1 suppresses expression of MglA regulated genes. Since MglA regulates genes that are known to be crucial for virulence, including the well-studied Francisella Pathogenicity Island (FPI), these results suggest that gene dosage effects arising from large duplications can trigger unknown virulence mechanisms in F. tularensis subsp. tularensis. These results are important especially when designing live vaccine strains that have repeated insertion elements in their genomes. / Ph. D.