Dissertations / Theses on the topic 'NGS'

As craniossinostoses são malformações craniofaciais caracterizadas pelo fechamento precoce de uma ou mais suturas cranianas. Elas são doenças congênitas e são causadas por mutações em diversos genes devido ao grande número de vias envolvidas na formação e manutenção das suturas cranianas. Embora mutações em 53 genes já tenham sido descritas o conhecimento da genética e da patofisiologia das craniossinostoses ainda é incompleto. Nesse trabalho tivemos como objetivo a identificação de novas mutações associadas às craniossinostoses bem como o aprofundamento do conhecimento sobre a atuação dessas mutações em células humanas por meio de estudos funcionais. Para identificarmos novas mutações utilizamos metodologias de sequenciamento em larga escala conhecidas como sequenciamento de noiva geração (NGS). Identificamos a mutação causal em uma paciente proveniente de um casamento consanguíneo portadora da síndrome de Raine (p.P496L em FAM20C). Também delimitamos a poucas mutações candidatas outros onze casos atípicos de craniossinostose. Por fim estudamos os efeitos de diferentes FGFs sobre o comportamento de células com a mutação mais comum causadora da S. de Apert, p.S252W em FGFR2. Descobrimos que os FGFs10 e 19 têm ações distintas sobre o perfil transcricional e sobre a taxa de proliferação de células mutantes. Também descobrimos que as células tronco mesenquimais e as células fibroblastóides têm comportamentos distintos ao serem tratadas com FGF19. Os resultados aqui apresentados serão de grande serventia para o melhor delineamento da biologia das suturas cranianas e da patofisiologia das craniossinostoses
Craniosynostosis are craniofacial malformations defined by early closure of the cranial sutures. They are congenital diseases caused by mutations in several genes due to the diversity of pathways involved in the development and maintenance of the cranial sutures. Even though 53 genes have already been linked to various forms of craniosynostosis, the knowledge about the genetics and pathophysiology is incomplete. In this work we aimed to identify new mutations associated with craniosynostosis as well as to further the knowledge of how those mutations act in human cells. To identify new variants associated with craniosynostosis we used large scale sequencing techniques known as next generation sequencing (NGS). We were able to identify the causal mutation in one patient from a consanguineous marriage with Raine syndrome (p.P496L in FAM20C). We also were able to elect candidate mutations in other eleven cases of atypical craniosynostosis. Lastly, we studied the effects of different FGFs over the behavior of human cells harboring the most common Apert syndrome mutation, p.S252W in FGFR2. We discovered that FGFs 10 and 19 have different effects over the transcriptional profile and proliferation rate of mutant cells. We also found that FGF19 have opposite effects in mesenchymal stem cells and fibroblastoid cells osteogenic differentiation. The results shown here will be of great service to better understand the biology of cranial suture and the pathophysiology of craniosynostosis

Les cellules NK, membres de l’immunité innée, sont impliquées dans le contrôle des infections virales et notamment l’infection à cytomégalovirus (CMV). Généralement bien tolérée chez l’individu immunocompétent, l’infection à CMV demeure associée à une forte morbidité chez les individus dont le système immunitaire est compromis (patients transplantés, coinfectés par le VIH) ou immature (fœtus et nouveaux nés). Lors de l’infection par le CMV, une relation étroite s’établit entre le système immunitaire et le virus. Celle-ci génère en effet une forte mobilisation, associée à un profond remodelage, de différents compartiments immuns. Au cours de ce travail de thèse, nous nous sommes intéressées à l’équilibre qui s’instaure entre le virus et le système immunitaire. Dans une première approche menée in vitro, nous avons exploré le rôle de différentes sous-populations NK dont les cellules NK NKG2C+, caractéristiques de l’infection à CMV, en réponse à des cellules endothéliales, isolées à partir de donneurs de rein, infectées par le CMV. Ensuite, une cohorte rare d’adultes immunocompétents souffrant d’une primo-infection symptomatique à CMV nous a permis d’étudier la course naturelle de l’infection à CMV. Cette approche ex vivo a constitué d’une part en l’analyse du polymorphisme de la réponse de l’hôte au virus, par l’étude phénotypique et transcriptomique non seulement de la réponse NK mais aussi d’autres effecteurs lymphocytaires. D’autre part, nous avons exploré l’impact du polymorphisme génétique viral, par le séquençage par NGS d’isolats cliniques du CMV, sur le pouvoir pathogène du virus. L’ensemble de ces travaux devrait contribuer à la meilleure compréhension du rôle des cellules NK dans le contrôle de l’infection à CMV
NK cells are innate lymphocyte effectors involved in the control of viral infections and particularly cytomegalovirus (CMV) infection. Usually well tolerated in immunocompetent individuals, CMV infection remains life life-threatening in immunosuppressed patients, as transplant recipients or HIV-infected patients, or for fetuses in case of congenital infection. Upon primary infection, CMV establishes a close relationship with the immune system. CMV infection is known to drive an important immune response and to deeply imprint several immune compartments. In this present work, we focused on the host-virus balance that takes place upon infection. Through a first in vitro approach, we investigated the role of different NK cell subpopulations, including NKG2C+ NK cells which represent one of the hallmarks of CMV infection, in response to CMVinfected endothelial cells isolated from kidney donors. Then, an ex vivo approach was conducted in a cohort of immunocompetent adults diagnosed with symptomatic primary CMV infection. On one hand, our aim was to explore the host immune response polymorphism, through phenotypic and transcriptomic analyses of lymphocyte responses. On the other hand, we investigated the viral genome polymorphism, through NGS sequencing of clinical CMV isolates, which could modulate the viral pathogenicity. Taken together, these findings should contribute to the better understanding of the role of NK cells during the course of CMV infection

In this PhD I have used NGS technologies in different organisms and scenarios such as in ENCODE, comparing the conservation and evolution of long non-coding RNA sequences between human and mouse, using experimental evidences from genome, transcriptome and chromatin. A similar approach was followed in other organisms such as the mesoamerican common bean and in chicken. Other analysis carried with NGS data involved the well known parasite, Leishmania Donovani, the causative agent of Leishmaniasis. I used NGS data obtained from genome and transcriptome to study the fate of its genome in survival strategies for adaptation and long term evolution. All this work was approached while working in tools and strategies to efficiently design and implement the bioinformatics analysis also known as pipelines or workflows, in order to make them easy to use, easily deployable, accessible and highly performing. This work has provided several strategies in order to avoid lack of reproducibility and inconsistency in scientific research with real biological applications towards sequence analysis and genome evolution.
En aquest doctorat he utilitzat tecnologies NGS en diferents organismes i projectes com l'ENCODE, comparant la conservació i evolució de seqüències de RNA llargs no codificant entre el ratolí i l'humà, utilitzant evidències experimentals del genoma, transcriptoma i cromatina. He seguit una estratègia similar en altres organismes com són la mongeta mesoamericana i el pollastre. En altres anàlisis he hagut d'utilitzar dades NGS en l'estudi del conegut paràsit leishmània Donovani, l'agent causatiu de la malaltia Leishmaniosis. Utilitzant dades NGS obtingudes del genoma i transcriptoma he estudiat les conseqüències del genoma en estratègies d'adaptació i evolució a llarg termini. Aquest treball es va realitzar mentre treballava en eines i estratègies per dissenyar eficientment i implementar els anàlisis bioinformàtics coneguts com a diagrames de treball, per tal de fer-los fàcils d'utilitzar, fàcilment realitzables, accessibles i amb un alt rendiment. Aquest treball present diverses estratègies per tal d'evitar la falta de reproductibilitat i consistència en la investigació científica amb aplicacions reals a la biologia de l'anàlisi de seqüències i evolució de genomes.

The main theme of this work is the detection of overlaps in NGS data. The work contains an overview of NGS sequencing technologies that are the source of NGS data. In the thesis, the problem of overlapping detection is generally defined. Next, an overview of the available algorithms and approaches for detecting overlaps in NGS data is created. Principles of these algorithms are described herein. In the second part of this work a suitable tool for detecting approximate overlaps in NGS data is designed and its implementation is described herein. In conclusion, the experiments performed with this tool and the conclusions that follow are summarized and described.

[EN] The work done for this doctorate thesis focuses on error correction of Next Generation Sequencing (NGS) data in the context of High Performance Computing (HPC). Due to the reduction in sequencing cost, the increasing output of the sequencers and the advancements in the biological and medical sciences, the amount of NGS data has increased tremendously. Humans alone are not able to keep pace with this explosion of information, therefore computers must assist them to ease the handle of the deluge of information generated by the sequencing machines. Since NGS is no longer just a research topic (used in clinical routine to detect cancer mutations, for instance), requirements in performance and accuracy are more stringent. For sequencing to be useful outside research, the analysis software must work accurately and fast. This is where HPC comes into play. NGS processing tools should leverage the full potential of multi-core and even distributed computing, as those platforms are extensively available. Moreover, as the performance of the individual core has hit a barrier, current computing tendencies focus on adding more cores and explicitly split the computation to take advantage of them. This thesis starts with a deep analysis of all these problems in a general and comprehensive way (to reach out to a very wide audience), in the form of an exhaustive and objective review of the NGS error correction field. We dedicate a chapter to this topic to introduce the reader gradually and gently into the world of sequencing. It presents real problems and applications of NGS that demonstrate the impact this technology has on science. The review results in the following conclusions: the need of understanding of the specificities of NGS data samples (given the high variety of technologies and features) and the need of flexible, efficient and accurate tools for error correction as a preliminary step of any NGS postprocessing. As a result of the explosion of NGS data, we introduce MuffinInfo. It is a piece of software capable of extracting information from the raw data produced by the sequencer to help the user understand the data. MuffinInfo uses HTML5, therefore it runs in almost any software and hardware environment. It supports custom statistics to mould itself to specific requirements. MuffinInfo can reload the results of a run which are stored in JSON format for easier integration with third party applications. Finally, our application uses threads to perform the calculations, to load the data from the disk and to handle the UI. In continuation to our research and as a result of the single core performance limitation, we leverage the power of multi-core computers to develop a new error correction tool. The error correction of the NGS data is normally the first step of any analysis targeting NGS. As we conclude from the review performed within the frame of this thesis, many projects in different real-life applications have opted for this step before further analysis. In this sense, we propose MuffinEC, a multi-technology (Illumina, Roche 454, Ion Torrent and PacBio -experimental), any-type-of-error handling (mismatches, deletions insertions and unknown values) corrector. It surpasses other similar software by providing higher accuracy (demonstrated by three type of tests) and using less computational resources. It follows a multi-steps approach that starts by grouping all the reads using a k-mers based metric. Next, it employs the powerful Smith-Waterman algorithm to refine the groups and generate Multiple Sequence Alignments (MSAs). These MSAs are corrected by taking each column and looking for the correct base, determined by a user-adjustable percentage. This manuscript is structured in chapters based on material that has been previously published in prestigious journals indexed by the Journal of Citation Reports (on outstanding positions) and relevant congresses.
[ES] El trabajo realizado en el marco de esta tesis doctoral se centra en la corrección de errores en datos provenientes de técnicas NGS utilizando técnicas de computación intensiva. Debido a la reducción de costes y el incremento en las prestaciones de los secuenciadores, la cantidad de datos disponibles en NGS se ha incrementado notablemente. La utilización de computadores en el análisis de estas muestras se hace imprescindible para poder dar respuesta a la avalancha de información generada por estas técnicas. El uso de NGS transciende la investigación con numerosos ejemplos de uso clínico y agronómico, por lo que aparecen nuevas necesidades en cuanto al tiempo de proceso y la fiabilidad de los resultados. Para maximizar su aplicabilidad clínica, las técnicas de proceso de datos de NGS deben acelerarse y producir datos más precisos. En este contexto es en el que las técnicas de comptuación intensiva juegan un papel relevante. En la actualidad, es común disponer de computadores con varios núcleos de proceso e incluso utilizar múltiples computadores mediante técnicas de computación paralela distribuida. Las tendencias actuales hacia arquitecturas con un mayor número de núcleos ponen de manifiesto que es ésta una aproximación relevante. Esta tesis comienza con un análisis de los problemas fundamentales del proceso de datos en NGS de forma general y adaptado para su comprensión por una amplia audiencia, a través de una exhaustiva revisión del estado del arte en la corrección de datos de NGS. Esta revisión introduce gradualmente al lector en las técnicas de secuenciación masiva, presentando problemas y aplicaciones reales de las técnicas de NGS, destacando el impacto de esta tecnología en ciencia. De este estudio se concluyen dos ideas principales: La necesidad de analizar de forma adecuada las características de los datos de NGS, atendiendo a la enorme variedad intrínseca que tienen las diferentes técnicas de NGS; y la necesidad de disponer de una herramienta versátil, eficiente y precisa para la corrección de errores. En el contexto del análisis de datos, la tesis presenta MuffinInfo. La herramienta MuffinInfo es una aplicación software implementada mediante HTML5. MuffinInfo obtiene información relevante de datos crudos de NGS para favorecer el entendimiento de sus características y la aplicación de técnicas de corrección de errores, soportando además la extensión mediante funciones que implementen estadísticos definidos por el usuario. MuffinInfo almacena los resultados del proceso en ficheros JSON. Al usar HTML5, MuffinInfo puede funcionar en casi cualquier entorno hardware y software. La herramienta está implementada aprovechando múltiples hilos de ejecución por la gestión del interfaz. La segunda conclusión del análisis del estado del arte nos lleva a la oportunidad de aplicar de forma extensiva técnicas de computación de altas prestaciones en la corrección de errores para desarrollar una herramienta que soporte múltiples tecnologías (Illumina, Roche 454, Ion Torrent y experimentalmente PacBio). La herramienta propuesta (MuffinEC), soporta diferentes tipos de errores (sustituciones, indels y valores desconocidos). MuffinEC supera los resultados obtenidos por las herramientas existentes en este ámbito. Ofrece una mejor tasa de corrección, en un tiempo muy inferior y utilizando menos recursos, lo que facilita además su aplicación en muestras de mayor tamaño en computadores convencionales. MuffinEC utiliza una aproximación basada en etapas multiples. Primero agrupa todas las secuencias utilizando la métrica de los k-mers. En segundo lugar realiza un refinamiento de los grupos mediante el alineamiento con Smith-Waterman, generando contigs. Estos contigs resultan de la corrección por columnas de atendiendo a la frecuencia individual de cada base. La tesis se estructura por capítulos cuya base ha sido previamente publicada en revistas indexadas en posiciones dest
[CAT] El treball realitzat en el marc d'aquesta tesi doctoral se centra en la correcció d'errors en dades provinents de tècniques de NGS utilitzant tècniques de computació intensiva. A causa de la reducció de costos i l'increment en les prestacions dels seqüenciadors, la quantitat de dades disponibles a NGS s'ha incrementat notablement. La utilització de computadors en l'anàlisi d'aquestes mostres es fa imprescindible per poder donar resposta a l'allau d'informació generada per aquestes tècniques. L'ús de NGS transcendeix la investigació amb nombrosos exemples d'ús clínic i agronòmic, per la qual cosa apareixen noves necessitats quant al temps de procés i la fiabilitat dels resultats. Per a maximitzar la seua aplicabilitat clínica, les tècniques de procés de dades de NGS han d'accelerar-se i produir dades més precises. En este context és en el que les tècniques de comptuación intensiva juguen un paper rellevant. En l'actualitat, és comú disposar de computadors amb diversos nuclis de procés i inclús utilitzar múltiples computadors per mitjà de tècniques de computació paral·lela distribuïda. Les tendències actuals cap a arquitectures amb un nombre més gran de nuclis posen de manifest que és esta una aproximació rellevant. Aquesta tesi comença amb una anàlisi dels problemes fonamentals del procés de dades en NGS de forma general i adaptat per a la seua comprensió per una àmplia audiència, a través d'una exhaustiva revisió de l'estat de l'art en la correcció de dades de NGS. Esta revisió introduïx gradualment al lector en les tècniques de seqüenciació massiva, presentant problemes i aplicacions reals de les tècniques de NGS, destacant l'impacte d'esta tecnologia en ciència. D'este estudi es conclouen dos idees principals: La necessitat d'analitzar de forma adequada les característiques de les dades de NGS, atenent a l'enorme varietat intrínseca que tenen les diferents tècniques de NGS; i la necessitat de disposar d'una ferramenta versàtil, eficient i precisa per a la correcció d'errors. En el context de l'anàlisi de dades, la tesi presenta MuffinInfo. La ferramenta MuffinInfo és una aplicació programari implementada per mitjà de HTML5. MuffinInfo obté informació rellevant de dades crues de NGS per a afavorir l'enteniment de les seues característiques i l'aplicació de tècniques de correcció d'errors, suportant a més l'extensió per mitjà de funcions que implementen estadístics definits per l'usuari. MuffinInfo emmagatzema els resultats del procés en fitxers JSON. A l'usar HTML5, MuffinInfo pot funcionar en gairebé qualsevol entorn maquinari i programari. La ferramenta està implementada aprofitant múltiples fils d'execució per la gestió de l'interfície. La segona conclusió de l'anàlisi de l'estat de l'art ens porta a l'oportunitat d'aplicar de forma extensiva tècniques de computació d'altes prestacions en la correcció d'errors per a desenrotllar una ferramenta que suport múltiples tecnologies (Illumina, Roche 454, Ió Torrent i experimentalment PacBio). La ferramenta proposada (MuffinEC), suporta diferents tipus d'errors (substitucions, indels i valors desconeguts). MuffinEC supera els resultats obtinguts per les ferramentes existents en este àmbit. Oferix una millor taxa de correcció, en un temps molt inferior i utilitzant menys recursos, la qual cosa facilita a més la seua aplicació en mostres més gran en computadors convencionals. MuffinEC utilitza una aproximació basada en etapes multiples. Primer agrupa totes les seqüències utilitzant la mètrica dels k-mers. En segon lloc realitza un refinament dels grups per mitjà de l'alineament amb Smith-Waterman, generant contigs. Estos contigs resulten de la correcció per columnes d'atenent a la freqüència individual de cada base. La tesi s'estructura per capítols la base de la qual ha sigut prèviament publicada en revistes indexades en posicions destacades de l'índex del Journal of Citation Repor
Alic, AS. (2016). Improved Error Correction of NGS Data [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/67630
TESIS

Hemolytic disease of fetus or newborn (HDFN) är en komplikation där foster eller nyföddas erytrocyter förstörs för tidigt. HDFN uppstår när det föreligger blodgruppsinkompatibilitet mellan moder och barnet. Komplikationerna/ symptomen kan variera allt från mildare symptom till fosterdöd. HDFN orsakas framförallt av antikropp D (RhD-immunisering) och på grund av detta utförs det typning av fetalt RhD i maternell plasma. Utöver RhD-immuniseringar kan svåra fall av HDFN ibland orsakas av andra blodgruppssystem som c (Rh) och K (Kell). Fetal RhD-typning görs idag som screening på alla RhD-negativa gravida mödrar och utförs i Stockholm och Lund. Metoden som används är baserad på realtids-PCR och har använts sedan 2009. Fetal typning av c och K görs när höga titrar av anti-c respektive anti-K påvisas i mammans plasma. För fetal typning av c och K skickas prover från Huddinge till Lund respektive Amsterdam. Motsvarande immunisering mot erytrocytantigen kan även bildas mot trombocytantigen främst Human Platelet Antigene (HPA-1a) som uttrycks hos fostret och leda till fetal neonatal alloimmun trombocytopeni (FNAIT). En next generation sequencing (NGS) baserad metod för genomisktypning av alla kliniskt relevanta blodgruppssystem finns idag på Karolinska universitetssjukhuset. Denna metod är dock inte anpassad för fetal blodgruppstypning där cell-fritt foster- DNA (cffDNA) analyseras. Syftet med det här projektet var att optimera designade oligonukleotider (oligos) anpassade för fetal blodgruppstypning med NGS-metodik. Design av oligos utfördes innan examensarbetet påbörjades. Dessa inkluderade primer- par som amplifierar upp alla blodgruppssystem som kan orsaka HDFN och FNAIT. Utöver det inkluderade designade oligos markörer som amelogenin-XY (Amel-XY) som är könsspecifika markörer och insertion/deletion markörer (Indel-markörer). Med Amel-XY markörerna kan könsspecifika Y-genen påvisas i provet, vilket kommer att fungera som en kontroll på att foster-DNA har analyserats (fungerar endast vid manligt foster). Designade Indel-markörer består av 2 - 3 bp insertioner/deletioner där olika markörerna används till att skilja DNA från foster och modern i framtiden. De designade oligonukleotiderna undersöktes för om de ger tillräckligt bra amplifiering av sökta sekvenser med NGS genom att analysera två slumpmässigt valda försöksprover (genomisk DNA 2 ng/ μL) från en man och en kvinna. Resultatet visade att tillräckligt bra amplifieringar/signaler erhålls från samtliga markörer som ingår i studien. Den totala signalen för samtliga markörer blev 114234 läsningar (”reads”) vilket gav ett medelvärde på 3939 läsningar. För att mäta amplifieringsförmågan hos varje enskild markör beräknades procent av medelvärdet på 3939 läsningar. En signal på 20 – 180 % av medelvärdet ansågs vara godkänt för varje markör. Alla undersökta primer-markörer uppvisade en signal på minst 45 % av medelvärdet vilket var godkänt (godkänt intervall 20 – 180 %). Efter optimering av oligos som ger tillräckligt bra amplifiering genotypades 32 slumpmässigt valda DNA prover från 21 män och 11 kvinnor med hjälp av NGS. Efter NGS-körning och analys av fastQ-filer med hjälp av mjukvaruprogrammet R erhölls resultat i form av signaler. Signaler för respektive markör över 50 läsningar ansågs vara en positiv signal det vill säga att den sökta genen har påvisats. Signal-värde under 50 innebar att det förekom bakgrundssignal då önskat signal-värde vid avsaknad av sökt gensekvens är 0 (åsatt gränsvärde <1 % av högst uppmätta positiva signalen för varje markör). Samtliga undersökta markörer visade låg bakgrundssignal (<1 % ). Bakgrundssignalen beräknades för att veta vilken tillförlitlig nivå varje markör ska ligga på för att kunna detektera känsligt foster-DNA i framtiden. Resultaten i denna studie visade att de optimerade oligos kan användas till genotypning av cffDNA i framtiden eftersom markörerna uppvisade låga bakgrundssignaler vilket indikerar att metoden är tillräckligt effektiv för att kunna detektera känsligt foster-DNA i framtiden.

Cette étude a pour objet la mise en évidence de traces d'ADN bactérien pathogène dans des échantillons animaux et humains anciens, et ainsi améliorer les connaissances sur l'évolution des maladies au cours du temps. En parallèle, nous avons étudié les phénomènes de dégradation de l'ADN dans le sol sur des cadavres de souris enterrées après avoir été contaminées par des bactéries non pathogènes. Cette étude des processus taphonomiques s'est étalée sur trois ans et a permis de montrer une disparition rapide des bactéries simulantes, remplacé par l'ADN des bactéries du sol, qui colonisent rapidement la dépouille et dégradent tant l'ADN endogène (murin) qu'exogène (bactérien). Cette disparition rapide explique la grande difficulté à mettre en évidence des pathogènes dans des échantillons anciens, à de rares exceptions près. Notre étude n'a pas permis de détecter d'agents pathogènes particuliers dans les échantillons que nous avons étudié, mais nous avons mis en évidence l'intérêt d'analyser certains types de restes pour accéder à une information génétique préservée. Le tartre dentaire indique est un bon indicateur de la flore buccale de l'hôte et les kystes calcifiés assurent une bonne préservation de l'ADN endogène, moins soumis à contamination et digestion par les bactéries de l'environnement. Les kystes présentent en règle générale une teneur en ADN endogène supérieure à tous les autres tissus étudiés
The aim of this study was the identification of pathogenic bacterial DNA traces in ancient animal and human samples, and thus improve knowledge of past diseases that affect humankind over time. In parallel, we studied the DNA degradation phenomena in the soil on the buried corpses of mice after being contaminated by non-pathogenic bacteria. This study of taphonomic processes was spread over three years and has shown a rapid disappearance of simulant bacteria, replaced with the DNA of soil bacteria that colonize the body quickly after burial and degrade both the endogenous DNA (murine) that exogenous (bacteria). This quick degradation can explain the high difficulty to detect and identify bacterial pathogens in old samples, with very few exceptions. Despite the fact in our study we were not able to detect specific pathogens in the samples we have studied, we have shown the interest to analyze certain types of remnants to access preserved and informative genetic data. Dental calculus is a good indicator of the oral flora of the host and calcified cysts ensure good preservation of the endogenous DNA, less subject to contamination and digestion by bacteria from the environment. Cysts generally have an endogenous DNA content higher than all other tissues examined

L’émergence et l’évolution des réseaux de nouvelles génération (NGN) a soulevé plusieurs défis surtout en termes d’hétérogénéité, de mobilité et de sécurité. En effet, l’utilisateur est capable, dans un tel environnement, d’avoir accès à plusieurs réseaux, à travers différents terminaux, avec un choix vaste de services fournis par différents fournisseurs. De plus, les utilisateurs finaux demandent à être constamment connectés n’importe où, n’importe quand et n’importe comment. Ils désirent également avoir un accès sécurisé à leurs services à travers une session dynamique, seamless et continue selon leurs préférences et la QoS demandée. Dans ce contexte, la sécurité représente une composante majeure. Face à cette session user-centric sécurisée, plusieurs défis se posent. L’environnement est de plus en plus ouvert, de multiples services ne sont pas connus d’avance et nous avons une diversité de communications entre les services et les utilisateurs. L’hétérogénéité des ressources (terminaux, réseaux et services) impliquées dans la session de l’utilisateur accentue la complexité des tâches de sécurité. Les différentes déclinaisons de mobilité (mobilité de l’utilisateur, mobilité du terminal, mobilité du réseau et mobilité du service) modifient la session user-centric que l’on veut unique, sécurisée et seamless avec la délivrance d’un service continu
The emergence and evolution of Next Generation Networks (NGN) have raised several challenges mainly in terms of heterogeneity, mobility and security. In fact, the user is able, in such environment, to have access to many networks, via multiple devices, with a vast choice of services offered by different providers. Furthermore, end-users claim to be constantly connected anywhere, anytime and anyhow. Besides, they want to have a secure access to their services through a dynamic, seamless and continuous session according to their preferences and the desired QoS. In this context, security represents an important concern. In fact, this user-centric session should obviously be secured. However, many challenges arise. In such environment, system boundaries, which were well delimited, become increasingly open. Indeed, there are multiple services which are unknown in advance and multiple communications between services and with users. Besides, heterogeneity of involved resources (terminals, networks and services) in the user session increases the complexity of security tasks. In addition, the different types of mobility (user, terminal, network and service mobility) affect the user-centric session that should be unique, secure and seamless and ensure continuity of services

Regeneration is a relatively widespread phenomenon in nature, although different organisms exhibit different abilities to reconstitute missing structures. Due to the diversity in the extent of damage the organisms can repair it has been debated for a long time whether those abilities are evolutionary traits that arose independently in multiple organisms or whether they represent a by-product of more basic processes. To date, due to constant increase in the amount of available genomic information this question can be approached by means of comparative genomics by comparing several taxa that have different regenerative capabilities. Two relatively closely related salamander species, newt, Notophthalmus viridescens, and the Mexican axolotl, Ambystoma mexicanum, offer a unique opportunity to compare two organisms with well-known regenerative capabilities. Despite their importance for regeneration research, relatively little sequence information was available until recently, owing mainly to the large sizes of the respective genomes. In this work I aimed to create a comprehensive transcriptome assembly of the axolotl by sequencing and then assembling the sequence data from a number of tissues and developmental stages. I also incorporated available sequence information that mostly comes from cDNA libraries sequenced previously. I assessed the completeness of the transcriptome by comparing it to a set of available axolotl sequences and found that 96% of those have homologs in the assembly. Additionally, I found that 7,568 of 7,695 protein families common to vertebrates are also represented in the transcriptome. In order to turn the assembly from a merely collection of sequences into a valuable and useful resource for the entire research community I first annotated the sequences, predicted the open reading frames and protein domains and additionally put together multiple bits of information available for each sequence including but not limited to time-course and tissue- specific expression data and in situ hybridization results. The assembly was thereafter made available for the entire axolotl research community through a web portal I developed. Not only does the web portal provide access to the transcriptome data, it is also equipped with an engine for automated data retrieval, which could facilitate automated cross-species bioinformatics analyses. The study crossed the boundary between pure bioinformatics and biology as the transcriptome allowed for computational comparison of the axolotl and the newt in order to identify salamander-specific genes possibly implicated in regeneration and subsequent functional analysis thereof in the lab. Since regeneration closely resembles embryonic development in terms of genes involved in both processes, I first identified approximately 200 homologous contigs in axolotl and newt, which had a predicted open reading frame, but did not have homologs in non-regenerating species. The expression profile of one of those candidate genes suggested that it had a role in regeneration. I studied the molecular function of that gene using CRISPR/Cas system to confirm that it was protein-coding and to create knock-out animals to study the effect of gene knock-down and knock-out. Knock-out animals exhibited significant delays in both, limb development and tail regeneration. The exact mechanism causing this delay is currently being investigated.

Les études préexistantes identifient quatre taxons de base (C. reticulata les mandariniers, C. maxima les pamplemoussiers, C. medica les cédratiers et C. micrantha) à l'origine de l'ensemble des formes cultivées suite à des événements de réticulations. Il en résulte des structures génotypiques complexes, généralement fixées par l'apomixie, fortement hétérozygotes et formées d'une mosaïque de grands fragments chromosomiques d'origines phylogénétiques différentes. La structuration de la variabilité phénotypique suggère que la différenciation initiale des taxons ancestraux est à l'origine d'une part importante de la variabilité utile des agrumes. La connaissance de l'origine des formes cultivées et de leurs structures phylogénomiques est donc indispensable à la bonne gestion des collections et à l'optimisation des programmes d'amélioration génétique. A cette fin, cette thèse explore différentes approches d'analyse de la diversité des génomes. Elle a bénéficié de l'évolution rapide des NGS et propose une utilisation raisonnée des outils disponibles en fonction des questions de recherches. Une analyse plus poussée a été conduite sur les limettiers et citronniers. Le pyroséquençage 454 (Roche) d'amplicons a été utilisé pour décrypter la structure en mosaïque interspécifique du chromosome 2 de 50 variétés à partir d'une information haplotypique multiloci et pour identifier des marqueurs diagnostiques des taxons ancestraux. Ces marqueurs ont permis, en association avec des SSR et indels, d'apporter un nouvel éclairage sur l'origine des limettiers et citronniers, par un génotypage exhaustif des collections Inra/Cirad et Ivia. Enfin, les données de re-séquençage complet Illumina de sept variétés de limettiers et de citronniers comparées à celles de représentants des taxons ancestraux nous ont permis de reconstituer la structure interspécifique de leurs génomes et de schématiser leurs caryotypes phylogénomiques. Les différentes approches ont conduit à des conclusions convergentes. Nos résultats confirment les hypothèses concernant la séquence évolutive à l'origine des bigaradiers (C. aurantium), des orangers (C. sinensis) et des pomelos (C. paradisi) à partir des pools géniques de C. maxima et C. reticulata. Ils mettent en évidence de fréquentes introgressions de C. maxima dans le génome de mandariniers considérées comme représentatifs de C. reticulata. Les contributions relatives de ces deux taxons ancestraux aux génomes de nombreuses variétés de petits agrumes (mandariniers, tangors et tangelos) ont pu être estimées. Les limettiers et citronniers résultent de multiples évènements de réticulation et C. medica est identifié comme parent mâle de la majorité des variétés diploïdes. Deux grands groupes de citronniers, sont différenciés, ceux issus d'hybridations directes C. reticulata × C. medica et ceux impliquant trois taxons ancestraux (C. maxima, C. reticulata et C. medica). Le bigaradier serait le parent femelle à l'origine des citronniers type Lisbonne (C. limon). Les limettiers de type Mexicain (C. aurantifolia) seraient issus d'une hybridation directe C. micrantha × C. medica. Enfin, les limes à gros fruits, triploïdes, ont deux origines. Les types Tahiti résulteraient probablement de la fécondation d'un ovule de citronnier type Lisbonne par un gamète diploïde de limettier type Mexicain. L'autre grand type serait issu d'un backcross entre C. aurantifolia (gamète diploïde) et C. medica. Ces connaissances sur la structure génomique des espèces secondaires permettent d'envisager une reconstruction d'idéotypes à partir du germplasm des taxons ancestraux. Elles ouvrent également la voie à des études de génétique d'association s'appuyant sur la phylogénomique des gènes impliqués dans l'élaboration des caractères de qualité, de résistance et d'adaptation. Enfin, les marqueurs diagnostiques d'espèces développés trouveront de nombreuses applications pour la caractérisation des collections et diverses études de génétiques
Citrus fruit, the most important fruit crop in the world, show a wide phenotypic diversity. Previous studies (molecular markers) identified four ancestral taxa (Citrus reticulata Blanco, mandarins; C. maxima (Burm.) Merr., pummelos; C. medica L., citrons; C. micrantha Wester, papedas) as the ancestors of all cultivated Citrus after reticulate evolutions. As a result, modern citrus varieties have complex and highly heterozygous genotypic structures, generally fixed by apomixis, and formed by a mosaic of large chromosomal fragments of different phylogenetic origins. Furthermore, the structuration of the phenotypic variability suggests that the initial differentiation of the basic taxa is the main source of most of the variability of the useful citrus phenotypic diversity. A thorough knowledge of the origin of cultivated citrus and their phylogenomic structure are essential for the management of biological resources and breeding program optimization. This thesis explores different approaches for analyzing genome diversity in order to identify the phylogenetic origins of the various horticultural citrus groups and to decipher their phylogenomic genome's structures. We focused on limes and lemons. This thesis takes advantage of the rapid evolution of NGS and proposes a rational use of available tools, based on research questions. Roche 454 parallel sequencing of amplicons provides multi-loci haplotype information on 500 base fragments. It was used to decipher the interspecific mosaic structure of chromosome 2 for fifty varieties and to identify ancestral taxa diagnostic SNP markers. The genotyping of all limes and lemons of the Inra/Cirad and Ivia germplasms with these markers, in association with SSR and indel markers, allowed to propose new hypothesis on the origins of limes and lemons. Data from Illumina whole genome re-sequencing of 7 varieties of limes and lemons, compared to those of representatives of the ancestral taxa, allowed to infer the interspecific structure of their genomes and to map out, for the first time, their phylogenomic karyotypes. The different approaches led to similar conclusions. Our results confirm previous hypothesis about the evolutionary steps at the origin of sour orange (C. aurantium), sweet orange (C. sinensis) and grapefruit (C. paradisi) involving C. maxima and C. reticulata gene pools. They highlight frequent introgressions of C. maxima in the genome of mandarin varieties despite the fact they were considered as representative of C. reticulata. We were also able to quantify the relative proportions of these two ancestral taxa in the genome of many varieties of small citrus fruit (mandarin hybrids, tangors and tangelos). Our work on limes and lemons demonstrate that C. medica is the male parent of this varietal group at the diploid level. Two groups of lemons are clearly differentiated: one from direct hybridizations between C. reticulata and C. medica, and one from crosses between hybrids (C. maxima × C. reticulata) and C. medica. Sour orange seems to be the female parent of ‘Eureka' type lemons (C. limon). The ‘Mexican' limes (C. aurantifolia) seems to come from a direct hybridization C. micrantha × C. medica. Finally, triploid big fruit limes have two major origins. The ‘Tahiti' type probably results from an ‘Eureka' type lemon (C. limon) ovule fecundated by a diploid gamete of a ‘Mexican' type lime (C. aurantifolia), while the other type would come from a back-cross between C. aurantifolia (diploid gamete) and C. medica. This new insights in genomic structure of secondary species makes to consider possible a reconstruction of these ideotypes from ancestral taxa germplasm. They also open new ways for association genetic studies based on phylogenomics of genes involved in the development of quality, resistance and adaptation traits. Finally, developed specific taxa diagnostic markers will find many applications for the characterization of collections and further genetic studies

Les facteurs de transcription ETS sont divisés en sous-familles en fonction de leurs similitudes en matière de séquence protéique, de séquences de liaison à l'ADN et d’interactions avec différents cofacteurs. Ils sont régulés par des signaux extracellulaires et contribuent à divers processus cellulaires, dont la prolifération cellulaire et la transformation tumorale. Les gènes de la famille ETS sont fréquemment ciblés par des processus oncogéniques que ce soit des translocations chromosomiques ou des gains du nombre de leurs copies. Le gène PU.1/SPI1 est également ciblé par des mutations ponctuelles inactivatrices dans les hémopathies myéloïdes humaines. Nous avons étudié une mutation somatique récurrente du gène PU.1/SPI1 (c.676C>G, p.Q226E), identifiée chez environ 6% des patients atteints d’une macroglobulinémie de Waldenström (MW), un syndrome lymphoprolifératif B chronique rare. La mutation modifie les caractéristiques de liaison à l'ADN de la protéine mutante, passant des séquences classiques reconnues par SPI1 à des séquences reconnues par d’autres protéines ETS comme ETS1, et d’une liaison à des régions enhancer à une liaison à des régions promotrices. La liaison accrue du mutant de SPI1 aux régions promotrices active des programmes transcriptionnels impliquant des voies de signalisation intracellulaire généralement favorisées par d'autres membres de la famille ETS. Les conséquences fonctionnelles de cette mutation sont une augmentation de la prolifération cellulaire et une diminution de la différenciation lymphoïde B terminale dans une lignée cellulaire modèle et des échantillons primaires de MW. Nous décrivons ici un mécanisme de subversion oncogénique de la fonction d’un facteur de transcription suite à la modification subtile de la spécificité de liaison à l'ADN de la protéine mutante, menant à un arrêt de différenciation. La démonstration qu'une mutation somatique ponctuelle peut modifier l'équilibre de liaison d’un facteur de transcription à l’échelle du génome fournit un paradigme mécanistique sur la façon dont les mutations faux sens dans les gènes codant pour des facteurs de transcription pourraient être oncogéniques dans les tumeurs humaines
The ETS-domain transcription factors are divided into subfamilies based on protein similarities, DNA binding sequences and interaction with cofactors. They are regulated by extracellular clues and contribute to a variety of cellular processes, including proliferation and transformation. ETS genes are targeted by oncogenic processes through chromosomal translocations and copy number gains. The PU.1/SPI1 gene is also targeted by inactivating point mutations in human myeloid malignancies. We investigated a recurrent somatic missense mutation (Q226E) of the PU.1/SPI1 gene in Waldenström macroglobulinemia, a human B-cell lymphoproliferative disorder. The mutation changes DNA binding of the mutant protein from classical SPI1 to ETS1-like sequences, shifting the balance from binding to promoter regions from enhancers. Increased binding by mutant SPI1 at promoters activates gene expression of intracellular signaling pathways typically promoted by other ETS factor family members. The functional consequences are decreased terminal B-cell differentiation in a model cell line and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration DNA binding specificity leading to differentiation arrest. The demonstration that a somatic point mutation subtly changes the balance of genome binding provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors

Les hautes montagnes des Andes du Nord abritent un écosystème de type tropical alpin connu localement sous le nom de páramo. En dépit de conditions climatiques stressantes, ces habitats totalisent 10 à 20% de la richesse de la flore Andine et contiennent les radiations de plantes alpines les plus rapides au monde. Les Espeletiinae (Asteraceae; Heliantheae), endémique de ces habitats et figurant parmi ces exemples majeurs de diversification andine, ont su profiter des avantages écologiques fournit par la formation des páramos, en développant une remarquable diversité morphologique et écologique en moins de 3 Ma pour faire face aux nombreux stress abiotiques présents dans ces écosystèmes. Ce complexe de +/- 135 espèces inclut des formes de vie arborescentes ramifiées, des rosettes acaules naines, et des rosettes caulescentes aussi bien sessiles que géantes, constituant une adaptation emblématiques des milieux tropicaux alpins. Ces plantes sont également caractérisées par une distribution d'espèces diversifiée au niveau du páramo dans des milieux humides, des prairies alpines ouvertes ou des pentes rocailleuses à partir de la limite supérieur des forets andines vers 2500m d'altitude jusqu'aux bords des glaciers à environ 4600m. Cette forte diversité morphologique et écologique observée, associée à de forts taux de sympatrie entre ces espèces, a conduit à l’hypothèse d’une radiation adaptative des Espeletiinae dans les páramos. Or les reconstructions phylogénétiques préalablement effectuées, permettant de tester cette origine évolutive, s’avèrent peu résolues en raison de l’évolution très récente du complexe couplées à l’utilisation de marqueurs phylogénétiques traditionnels ou encore en raison de nombreux événements d’hybridation observés pouvant biaiser les reconstructions phylogénétiques. Aujourd’hui, l’avènement des nouvelles technologies de séquençage offre de nouvelles perspectives en phylogénomique. Si bien qu’au travers de ce travail de thèse, l’utilisation de fragments génomiques aléatoires (par méthode de shotgun-sequencing) et ciblés (par méthode de ddRAD-sequencing) a permis de reconstruire pour la première fois des phylogénies robustes et d’étudier les événements d’hybridations à l’intérieur du complexe, apportant ainsi de nouvelles réponses quant à l’évolution de ces taxons dans les páramos
The high-elevations ecosystems in the Northern Andes, known as paramos, exhibit an exceptional diversity of species accounting for 10 to 20% of Andean flora's richness and contain the fastest alpine plant radiation in the world. The Espeletiinae (Asteraceae, Heliantheae), endemic to these habitats and among these major examples of Andean diversification, took advantage of the ecological benefits provided by the uplift of the páramos, by developing a remarkable morphological and ecological diversity in less than 3 Ma to cope with the abiotic stresses of these ecosystems. This complex of +/- 135 species includes branched tree life forms, dwarf rosettes, and caulescent as well sessile as giant rosettes, constituting an emblematic adaptation of tropical alpine environments. These plants are also diversified in páramo, in wetlands, open alpine meadows or rocky slopes from the upper limit of the Andean forests at 2500m altitude to the edges of the glaciers at 4600m. Such morphological and ecological diversity, in association with high sympatric rates between these species, led to the hypothesis of an adaptive radiation of Espeletiinae. However, the first phylogenetic reconstructions, testing this evolutionary origin, failed to depict any relationships between these species because of the very recent evolution of the complex, the use of traditional phylogenetic markers and/or the widely hybridization events, which could skew the phylogenetic signal. Today, the advent of new sequencing technologies offers new perspectives in phylogenomics. As a result of this thesis work, the use of random (shotgun-sequencing) and targeted genomic fragments (by ddRAD-sequencing method) made it possible to reconstruct for the first time robust phylogenies and to study the hybridization events inside this complex, bringing new answers to the evolution of these plants in the páramos

Estudos filogenômicos usando Sequenciamento de Próxima Geração (do inglês, Next Generation Sequencing - NGS) estão se tornando cada vez mais comuns. O uso de marcadores oriundos do sequenciamento de DNA de uma biblioteca genômica reduzida, neste caso ddRADSeq (do inglês, Double Digestion Restriction Site Associated DNA Sequencing), para este fim é promissor, pelo menos considerando sua relação custo-benefício em grandes conjuntos de dados de grupos não-modelo, bem como a representação genômica recuperada. Aqui usamos ddRADSeq para inferir a filogenia em nível de espécie do gênero Cereus (Cactaceae). Esse gênero compreende em cerca de 25 espécies reconhecidas predominantemente sul-americanas distribuídas em quatro subgêneros. Nossa amostra inclui representantes de Cereus, além de espécies dos gêneros próximos, Cipocereus e Praecereus, além de grupos externos. A biblioteca ddRADSeq foi preparada utilizando as enzimas EcoRI e HPAII. Após o controle de qualidade (tamanho e quantificação dos fragmentos), a biblioteca foi sequenciada no Illumina HiSeq 2500. O processamento de bioinformática a partir de arquivos FASTQ incluiu o controle da presença de adaptadores, filtragem por qualidade (softwares FastQC, MultiQC e SeqyClean) e chamada de SNPs (software iPyRAD). Três cenários de permissividade a dados faltantes foram realizados no iPyRAD, recuperando conjuntos de dados com 333 (até 40% de dados perdidos), 1440 (até 60% de dados perdidos) e 6141 (até 80% de dados faltantes) loci. Para cada conjunto de dados, árvores de Máxima Verossimilhança (MV) foram geradas usando duas supermatrizes: SNPs ligados e Loci. Em geral, observamos algumas inconsistências entre as árvores ML geradas em softwares distintos (IQTree e RaxML) ou baseadas no tipo de matriz distinta (SNPs ligados e Loci). Por outro lado, a precisão e a resolução, foram melhoradas usando o maior conjunto de dados (até 80% de dados perdidos). Em geral, apresentamos uma filogenia com resolução inédita para o gênero Cereus, que foi resolvido como um provável grupo monofilético, composto por quatro clados principais e com alto suporte em suas relações internas. Além disso, nossos dados contribuem para agregar informações sobre o debate sobre o aumento de dados faltantes para conduzir a análise filogenética com loci RAD.
Phylogenomics studies using Next Generation Sequencing (NGS) are becoming increasingly common. The use of Double Digest Restriction Site Associated DNA Sequencing (ddRADSeq) markers to this end is promising, at least considering its cost-effectiveness in large datasets of non-model groups as well as the genome-wide representation recovered in the data. Here we used ddRADSeq to infer the species level phylogeny of genus Cereus (Cactaceae). This genus comprises about 25 species recognized predominantly South American species distributed into four subgenera. Our sample includes representatives of Cereus, in addition to species from the closely allied genera Cipocereus and Praecereus, besides outgroups. The ddRADSeq library was prepared using EcoRI and HPAII enzymes. After the quality control (fragments size and quantification) the library was sequenced in Illumina HiSeq 2500. The bioinformatic processing on raw FASTQ files included adapter trimming, quality filtering (FastQC, MultiQC and SeqyClean softwares) and SNPs calling (iPyRAD software). Three scenarios of permissiveness to missing data were carry out in iPyRAD, recovering datasets with 333 (up tp 40% missing data), 1440 (up to 60% missing data) and 6141 (up to 80% missing data) loci. For each dataset, Maximum Likelihood (ML) trees were generated using two supermatrices: SNPs linked and Loci. In general, we observe few inconsistences between ML trees generated in distinct softwares (IQTree and RaxML) or based in distinctive matrix type (SNP linked and Loci). On the other hand, the accuracy and resolution were improved using the larger dataset (up to 80% missing data). Overall, we present a phylogeny with unprecedent resolution for genus Cereus, which was resolved as a likely monophyletic group, composed by four main clades and with high support in their internal relationships. Further, our data contributes to aggregate information on the debate about to increasing missing data to conduct phylogenetic analysis with RAD loci.

Il termine Next generation sequencing (NGS) si riferisce ad un gruppo di moderne tecnologie di sequenziamento di acidi nucleici che rende possibile l’analisi di un elevato numero di sequenze di DNA o RNA sia per scopi diagnostici che di ricerca. Rispetto al sequenziamento tradizionale secondo Sanger, l’NGS permette di analizzare allo stesso tempo molti geni responsabili di patologie umane o di individuare nuovi geni ancora ignoti. Nel primo caso, vengono utilizzati in diagnostica pannelli genici dedicati, con costi relativamente contenuti e notevole risparmio di tempo rispetto al sequenziamento tradizionale. I disturbi del movimento sono un gruppo eterogeneo di disordini neurologici che possono manifestarsi in tutte le età della vita con un’anormale produzione del movimento stesso. Esistono molti disturbi del movimento su base genetica, specialmente nella popolazione pediatrica e l’NGS sta contribuendo in maniera significativa ad ampliare le conoscenze relative a queste patologie. Presso l’Istituto Carlo Besta vengono seguiti molti pazienti affetti da disturbi del movimento, inclusa un’ampia percentuale di casi ad esordio pediatrico. Durante il mio Dottorato di Ricerca, abbiamo analizzato tramite NGS pazienti selezionati, famiglie o gruppi omogenei di pazienti con lo stesso disturbo del movimento (es. distonia, corea) sia a fini diagnostici sia come parte di collaborazioni scientifiche con istituti esteri, focalizzandoci in particolar modo sui pazienti pediatrici. Grazie a ciò, abbiamo individuato nuovi pazienti con mutazioni in geni di recente scoperta (ADCY5, PDE10A) ed ampliato lo spettro fenotipico di geni già noti (GNAL, PSEN1). Inoltre, abbiamo partecipato a progetti di ricerca internazionali che hanno portato alla scoperta di nuovi geni responsabili di disturbi del movimento (KCTD17, PDE10A) con esordio prevalentemente pediatrico. Nonostante permangano alcuni limiti tecnici che necessitano di qualche cautela, l’utilizzo dell’NGS sta profondamente cambiando il modo in cui i clinici impostano l’iter diagnostico nei disturbi del movimento e sta contribuendo a ridurre progressivamente la percentuale di pazienti affetti da malattie rare ancora non caratterizzate geneticamente.
Next generation sequencing (NGS) refers to a group of innovative sequencing techniques that allow analyzing a high number of DNA or RNA sequences both for diagnostic and research purposes. As compared to traditional Sanger sequencing, NGS allows analyzing at the same time a large number of genes causally linked to human pathology or to search for new genes. In the first case, customized gene panels are used, with relatively low costs and substantial advantages in terms of time. Movement disorders are a heterogeneous group of neurological diseases that can manifest at any age with an abnormal movement production. There are several genetic causes of movement disorders, especially in the pediatric population, and NGS is contributing to significantly widen our knowledge of the genetic bases of several of these rare disorders. At Carlo Besta Neurological Institute, a large cohort of patients with rare movement disorders are followed, including a substantial proportion of pediatric cases. During my PhD course, we applied targeted resequencing and Whole Exome Sequencing to selected patients, kindreds or homogeneous subgroup of patients affected by specific movement disorders (e.g. chorea, dystonia) as part of routine diagnostic workup or in collaboration with foreign Institutes, with a particular focus on pediatric patients. We were able to identify carriers of recently discovered genes (ADCY5, PDE10A) and to widen the phenotypic spectrum previously associated with known genes (GNAL, PSEN1). Moreover, we contributed to international projects that allowed the discovery of new genes responsible for movement disorders (KCTD17, PDE10A) mainly with onset in childhood. Despite some technical limitations that still need some caution, the advent of NGS is profoundly changing the way clinicians diagnose movement disorders and is contributing to progressively decrease the proportion of patients with genetically undefined rare disease.

Orientador: Cesar Martins
Abstract: One of the biggest challenges in chromosome biology is to understand the occurrence and complex genetics of extra, non-essential karyotype elements, commonly known as supernumerary B chromosomes (Bs). Bs are present in diverse species of eukaryotes and their molecular characterization remains elusive for years. A distinguished feature that makes them different from the normal chromosomes (called A chromosomes) is their way of inheritance in irregular fashion. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. The non-Mendelian inheritance and unpairing/non-recombining abilities make the B chromosomes immensely interesting for genomics studies, thus arising different questions about their genetic composition, survival, maintenance and role inside the cell. This study aims to uncover these phenomena in different species. Here, we sequenced the genomes of three model organisms including fish species Astyanax mexicanus and Astyanax correntinus, and grasshopper Abracris flavolineata with (B+) and without Bs (B-) to identify the B-localized sequences, called B chromosome blocks (“B-blocks”). We established approaches for this analysis that comprised of steps such as comparative genomics analysis and annotation of B chromosomal genes and DNA repeat types. The next generation sequencing (NGS) analyses identified thousands of genes fragments as well as... (Complete abstract click electronic access below)
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As doenças do lenho são das mais importantes doenças associadas à videira. Este trabalho teve como objetivo principal estudar as diferenças da diversidade dos fungos endofíticos e patogénicos e a suscetibilidade de duas cultivares distintas: Trincadeira e Aragonez, em dois locais (A e B). Situadas na Região do Alentejo todas testadas em material lenhoso. Os fungos endofíticos mais encontrados nas amostras de planta foi o pertencente ao género Alternaria, tanto para os dois locais como para as duas cultivares. Nos fungos patogénicos, os mais encontrados nas amostras das plantas foram os fungos das espécies Diplodia seriata, Botrytis cinerea e Rhizopus spp. Verificou-se que a cultivar Aragonez, no local A é menos suscetível às doenças do lenho, ao contrário do que acontece no local B, neste local a cultivar menos suscetível às doenças do lenho é a Trincadeira. As espécies Botryosphaeria dothidea e Diplodia seriata são causadores das doenças do lenho; Evaluation of endophytic fungal communities in two grapevine cultivars from Alentejo Region showing different susceptibility to trunk diseases Abstract: Trunk diseases is one of the most important diseases of the grapevine. The main objective of this work was to study the differences in the diversity of endophytic and pathogenic fungi and the susceptibility of two cultivars: Trincadeira and Aragonez in two sites (A and B) located in Alentejo Region and all tested in woody material. The most endophytic fungi found in the plant samples was, the one belonging to the genus Alternaria, for both sites and cultivars. For pathogenic fungi, the most found in the plant samples were the species Diplodia seriata, Botrytis cinerea and Rhizopus spp. It was also found, that the cultivar Aragonez in the organic production mode is less susceptible to trunk diseases, unlike what happens in the conventional production mode. In this mode the cultivar Trincadeira is less susceptible to these diseases. Wood diseases are caused by the species: Botryosphaeria dothidea and Diplodia seriata.

L’Italia ha rappresentato a lungo un crocevia del Mediterraneo, dove popoli e culture diverse hanno trovato facile approdo nel corso del tempo, giungendo ad un apice durante l’età dei Metalli. Attualmente le conoscenze genetiche dei popoli che hanno concorso al moderno modello di variabilità genetica italiana sono piuttosto scarse. Inoltre, la quasi totalità dei dati disponibili si basa sull’analisi del DNA mitocondriale, in grado di spiegare solo in parte il complesso quadro migratorio. L’indagine genetica delle caratteristiche del cromosoma Y (Y-chr) nelle popolazioni antiche risulta pertanto essenziale per contestualizzare gli eventi migratori passati. Esso presenta proprietà che hanno influenze sulla struttura genetica, sui processi mutazionali e sulla diversità intra- ed inter-popolazionistica. Inoltre è un marker d'elezione in genetica di popolazioni grazie a qualità come l’aploidia, la trasmissione uniparentale e la non ricombinazione per la maggior parte della sua lunghezza. In questo lavoro sono stati isolati un totale di 79565 SNPs localizzati nella regione X-degenerata del Y-chr e considerati essenziali per produrre inferenze relative alle linee di discendenza per via paterna, ed idonei anche per l’identificazione di individui imparentati. Sono stati condotti due differenti disegni sperimentali per la costruzione di sonde a RNA su un subset delle posizioni totali, ma in grado di fornire le informazioni ricercate con un ottimo livello di risoluzione. L’efficienza, specificità e resa delle sonde prodotte sono state confrontate mediante l’applicazione di 4 diversi protocolli per l’arricchimento delle regioni selezionate; in particolare sono stati adottati i protocolli suggeriti dalle aziende produttrici delle sonde, oltre a due modificati per fornire maggior resa in caso di substrati degradati. I test comparativi, eseguiti su un set di 4 campioni moderni e 4 antichi, hanno mostrato che è possibile effettuare analisi di determinazione degli aplogruppi (Hg) estremamente dettagliate. I risultati ottenuti mostrano che entrambi gli assetti valutati presentano una buona resa, ma l’utilizzo di disegni sperimentali e protocolli ad hoc per substrati degradati aumenta significativamente l’efficienza delle sonde e le informazioni deducibili. Con questo approccio ottimizzato è possibile giungere alla lettura di un elevato numero di posizioni del Y-chr, rendendo la metodologia innovativa ed applicabile ad una varietà di contesti. È stato successivamente eseguito uno screening per la determinazione genetica del sesso, su 101 campioni associati al periodo dell’età del Ferro, recuperati da 8 siti del Centro e Sud Italia. Le analisi condotte sul Y-chr di 42 campioni selezionati tra i maschi disponibili, hanno messo in luce la differenziazione genetica della popolazione in 4 principali Hg (R1b, I2, G2a e J) tipici dell’attuale variabilità della popolazione italiana maschile. Inoltre, se si esclude la componente maggioritaria ottenuta con l’Hg R1b, omogeneamente diffuso in tutta Europa, è possibile ipotizzare un cline di variabilità genetica distinto tra Nord e Sud Italia, già proposto in lavori precedenti. La comparazione delle frequenze degli Hg del Y-chr della popolazione in analisi, con quelle di popolazioni antiche e moderne recuperate in letteratura, ha permesso di mostrare una vicinanza genetica della popolazione italiana dell’età del Ferro con quelle dello stesso periodo recuperate da diversi contesti italiani, e con le popolazioni dell’età dei metalli provenienti dalle steppe pontico-caspiche. È stata inoltre riscontrata una particolare affinità con individui italiani del successivo periodo romano. Per poter chiarire in maggior dettaglio la complessa situazione italiana dell’età del Ferro sarà necessario genotipizzare un numero maggiore di individui dello stesso arco temporale e consolidare i dati ad oggi ottenuti mediante datazioni al radiocarbonio.
Italy has long been a crossroads of the Mediterranean sea, where different peoples and cultures have come into contact over time, and in particular during the Metal Age, due to the intensification of the commercial maritime routes. However, the genetic knowledge of the peoples who have contributed to the modern model of Italian genetic variability is rather scarce. In addition, almost all the data currently available are based on the analysis of mitochondrial DNA, which can explain only part of the complex migratory framework. Therefore, together with the matrilinear marker, the genetic investigation of the Y chromosome (Y-chr) variation in ancient population is essential to better contextualize past migratory events. Indeed the Y-chr displays properties such as the haploidy, the uniparental transmission and the non-recombination for most of its length, that have significant influences on the genetic structure, mutational processes and diversity between and within populations. In this work, a total of 79565 SNPs located in the X-degenerate region of the Y-chr have been isolated. The selected positions are considered crucial to provide inferences related to the male genealogy but are also suitable for the identification of individuals who share the same paternal ancestry. Two different experimental design were carried out for the production of RNA probes aiming to enrich a subset of the total positions previously identified but which still exhibit an excellent level of resolution. The efficiency, specificity and the total yield of the probes produced were tested through 4 different enrichment protocols of the selected regions. In particular, experimental analysis were carried out both through the protocols suggested by the probes’ manufacturer, as well as through two protocols specifically developed for degraded material. The analysis were conducted on 4 modern and 4 ancient (previously successfully analyzed) samples, and showed that with both the set of probes, is possible to perform a detailed haplogroups (Hg) assignation. In addition, the performed analysis showed that the use of experimental designs and protocols specifically developed for damaged substrates , significantly increases the specificity of the probes and the deducible information. After the development of the best method to provide maximum performance in the target enrichment phases, 101 samples recovered in 8 Italian necropolis associated with the Iron Age were screened for the genetic sex. The analysis conducted on the Y-chr variation among 42 samples (selected from the total amount of male available) highlighted the population distribution into four main Hg (R1b, I2, G2a and J); these Hg are typical of the modern genetic makeup of the Italian males. Furthermore, although the marked presence of the Hg R1b (which is homogeneously distributed among Europe), it is possible to hypothesize a well-established genetic variability cline that would differentiate the variability of the Italian Y-chr in two latitudinal regions, the north and the south, as proposed in previous works. The Y-chr Hg variation of the analyzed population were compared with those of ancient and modern European populations recovered in the literature. The results obtained showed a genetic closeness of the Iron Age Italian peoples with those of the same period recovered by different Italian contexts, and with the Metal Age populations from the Pontic-Caspian steppes. A particular affinity with Italian individuals of the subsequent Roman period was also found. It will be necessary to both broaden the Y-chr analysis on a larger set of samples of the same periods, as well as radiocarbon dating in order to better clarify the historical context and the genetic situation during the Iron Age.

Il collagene VI è una proteina della matrice extracellulare che forma un reticolo di microfilamenti nel muscolo scheletrico ed in altri organi. Le mutazioni dei geni del collagene VI causano, nell'uomo, principalmente due malattie genetiche: la miopatia di Bethlem e la distrofia muscolare congenita di Ullrich. Come centro di riferimento nazionale per le neuropatie muscolari, in 12 anni, abbiamo raccolto una casistica di 245 pazienti, indagati a livello genomico e trascrittomico, al fine di catalogare e caratterizzare le mutazioni nei tre geni del collagene VI. La casistica a livello nazionale si è dimostrata sovrapponibile a quella presente in letteratura sia per frequenza di mutazione e fenotipo, sia per tipo di mutazioni riscontrate. Considerata l’importanza di indagare il trascritto in generale nelle patologie neuromuscolari e, nello specifico, nei disordini del collagene VI, in questo lavoro abbiamo paragonato due differenti metodiche: RNA sequencing (RNAseq) e FluiCol6 micro-fluidic exome array. Il sequenziamento dell’RNA si è rivelato il miglior strumento da utilizzare, in parallelo con l’analisi genomica, nella diagnosi delle patologie del collagene VI. Al contrario, la micro-fluidic card FluiCol6 ha fornito diversi falsi positivi e si è dimostrata inefficace nell’identificare la mutazione di splicing del controllo positivo. Ad oggi manca un modello cellulare che riproduca fedelmente e in modo completo il fenotipo patologico, indotto dal deficit di collagene VI. A tale scopo si è scelto di studiare e validare le cellule staminali di origine urinaria, rivelatesi un buon modello in vitro per lo studio della Distrofia muscolare di Duchenne. I primi risultati ottenuti hanno fornito buone aspettative; le cellule staminali urinarie infatti mostrano lo stesso pattern di espressione, per i geni del collagene VI e relative isoforme, dei fibroblasti. Inoltre, i primi esperimenti di immunoistochimica rivelano la presenza della proteina nella matrice extracellulare anche se, la completa caratterizzazione proteica non è ancora conclusa. I modelli cellulari per il collagene VI, utilizzati solitamente in diagnostica e ricerca sono i fibroblasti e i mioblasti isolati da biopsie cutanee e muscolari, rispettivamente. Ottenere questi due tipi cellulari richiede l’utilizzo di metodiche invasive perciò, se le USCs si rivelassero un modello cellulare alternativo agli attuali sistemi si avrebbe la possibilità di ottenere linee cellulari in modo semplice, poco costoso, ripetibile e ben accettato dai pazienti, soprattutto da quelli in età pediatrica.
Collagen VI-related myopathies are a group of rare inherited genetic disorders with varying degree of clinical severity, caused by mutations in the Collagen type VI genes: COL6A1, COL6A2, and COL6A3. As a national reference center for diagnosis of neuromuscular disorders, 245 patients were analyzed in the Medical Genetics Unit over a 12-year period (2006-2018). The aim of this thesis is to provide a nationwide study of patients with COLVI phenotype and an overview of COL6 genes variants. The detection of mutations in collagen VI genes remains the gold standard for diagnosis, but traditional diagnostic tools do not completely achieve this goal. Next generation sequencing panel offers the ability to efficiently and cost-effectively screening all exons of the three COL6A genes. However, it is known the importance of studying transcriptome to enhance the diagnostic rate and to study the mutation functional consequence, therefore confirming their pathogenicity. In our results, the RNA-seq was shown to be the innovative strategy to explore RNA profile of COL6 genes in patients. To date, a disease cellular model for COL6-RD with the capacity to completely recapitulate the pathological phenotype in humans is yet to be established. Having this in mind, the use of Urine Stem cells (USCs) as a collagen VI cellular model was in this study developed and validated at the RNA level. Despite being a preliminary study defining whether USCs could be a good collagen VI cellular model, the data presented herein hold good promise. In contrast to skin and muscle biopsies, USCs can easily and non invasively be retrieved from urine samples. Hence, the collagen VI cellular model employing USCs with the ability to function equally well to the existing using fibroblasts could have many advantages. We propose these cells as COLVI disease in vitro model for functional studies, drug screening and validation.

Dans la première partie j'ai analysé des jeux de données de RNA-seq de transcriptome de petits ARNs disponibles dans les bases de données publiques. J'y ai observé 2 points intrigants : - une grande partie des lectures (bien que courtes) ne peux pas être alignée sur le génome de référence sans discordance et cette fraction non-alignable est parfois majoritaire. - de nombreuses lectures ont des tailles autours de 15-18nt qui ne correspondent à aucun type de petits ARNs connues, cette fraction est également majoritaires dans certains cas. Ces expériences sont souvent conçues pour la détection des miRNAs et l'analyse bioinformatique de ces données passent toujours par un alignement sur le génome de référence ou sur des séquences connues pour donner des petits ARNs. J'ai donc simplement éliminé la contrainte d'alignement dans l'analyse de ces données et effectué un regroupement des lectures par similarité (à la manière des ESTs). Ce regroupement donne une vision différente des données dans laquelle la notion de position génomique n'est plus centrale et ouvre la possibilité d'y découvrir des phénomènes non-standard. La deuxième partie est tirée d'une collaboration avec le laboratoire U675 INSERM. J'ai fait l'analyse bioinformatique des gènes dérégulés par la répression par RNAi du gène REST dans une lignée de neuroblastome de souris (N18). Ce gène est un facteur de transcription qui réprime les gènes neuronaux dans les cellules non neuronales. Ce répertoire de gènes dérégulés est potentiellement constitué de gènes clefs dans la biologie des neurones
In first part of this thesis I have analysed small RNA-seq transcriptome data. I have noticed : - a large fraction of reads can't be aligned perfectly on reference genome - lot of reads are very short (15-18 nt) and don't match on previously known functionnal small RNAs. These experiments are designed for miRNA discovery and bioinformatics analysis of these data use alignments on genome or on known small RNA precursors sequences. I have eliminated the alignment and I have clustered these sequences. This clustering let me to observe these data with a new view in wich the genomic location is not central and open the gate to discover unconventional events. The second part is the analysis of deregulate genes by the silencing of the gene REST/NRSF in mouse N18 cell line. This gene is a transcription factor and it works as a repressor of neuronal genes in non neuronal cells. This deregulate genes repertoire potentially contains key genes in neuron biology. We found in this repertoire a network of genes centered on SWI/SNF complex including SMARCA2. This gene was associated to schizophrenia (SZ) in association studies and structural variation studies. In this network we found another genes associated to SZ. We show that these genes exhibit positive evolution in primate compare to rodents

Les accidents nucléaires des centrales de Tchernobyl et de Fukushima rendent primordial la compréhension des transferts de la contamination radioactive dans l'environnement et de ses conséquences écologiques. Bien que certaines études aient été réalisées sur les organismes supérieurs, trop peu ont étudié les communautés bactériennes telluriques, qui jouent pourtant un rôle essentiel dans la mobilité des contaminants dans les sols en diminuant ou en améliorant leur transfert vers d'autres compartiments (eau, végétaux, animaux). Cependant, les radionucléides (RNs) peuvent avoir des effets toxiques sur les bactéries, entrainant une inhibition de leur rôle dans ce transfert. Les objectifs de cette étude étaient (1) d'évaluer l'impact d’une contamination radioactive sur les communautés bactériennes d’un sol de la zone d’exclusion de Tchernobyl (sol de la tranchée n°22) et (2) d’étudier les interactions bactérie-uranium pour une souche résistante, isolée à partir de ce sol
The nuclear power plants accidents of Chernobyl and Fukushima demonstrate the importance of the understanding of the transfers of the radioactive contamination in the environment and their ecological consequences. Although certain studies have been realized on superior organisms of the food chain, studies on telluric bacterial communities are scarce. The later play nevertheless an essential role in the mobility of contaminants in soils by decreasing or by improving their transfer towards other compartments (water, vegetables, and animals). Moreover, radionuclides (RNs) can have toxic effects on bacteria, leading to an inhibition of their participation in such transfer. The objectives of this study were (1) to estimate the impact of radioactive contamination on bacterial communities belonging to a soil of a Chernobyl exclusion zone (trench n°22) and (2) to study the uranium-bacteria interactions of a resistant strain, isolated from this soil

La vinificación es un proceso complejo que involucra varias etapas hasta el embotellado y comercialización del vino. Durante este proceso, la cantidad limitada de nutrientes provoca la competencia microbiana, que resulta en la producción de metabolitos que modulan el producto final del vino. Esta actividad microbiana puede conferir características organolépticas beneficiosas o indeseables a la calidad del vino. En los últimos años, el enfoque principal se ha centrado en la detección y el seguimiento de microorganismos determinados, que supuestamente estropean el vino, y la aplicación de metodologías empíricas para la prevención del crecimiento microbiano indeseable. Sin embargo, los hallazgos de las investigaciones han mostrado una base multifactorial del deterioro del vino, y han subrayado la necesidad de una estrategia innovadora que permita el estudio de la diversidad microbiana en su totalidad. La secuenciación de última generación parece un enfoque adecuado y prometedor para este propósito, ya que parece capaz de superar las limitaciones de las metodologías convencionales
evaluación de los resultados derivados en función de su alineación con hallazgos anteriores y su capacidad para proporcionar nuevos conocimientos. En general, el trabajo actual ha logrado corroborar estudios previos, sugerir mejoras sobre las implementaciones relacionadas con la bioinformática y la estadística y ampliar nuestro conocimiento sobre varios factores que influyen en la vinificación. Winemaking is a intricate process, involving various stages until the wine bottling and commercialization. During this process, the limited amount of nutrients leads to microbial competition, which in turn results in the production of metabolites that modulate the final wine product. This microbial activity may confer beneficial or undesirable organoleptic characteristics to the wine quality. The past years, the main focus has been given to the detection and monitoring of specific putative wine-spoiling microorganisms and the application of empirical methodologies for the prevention of unwanted microbial growth. Nevertheless, research findings have shown a multifactorial basis of the wine spoilage and underlined the need for an innovative strategy that will allow the study of the microbial diversity in its entirety. Next-generation-sequencing appears a suitable and promising approach for this purpose, as it seems able to overcome the limitations of conventional methodologies. In this work, various aspects associated to the NGS-based metataxonomic analysis have been studied, in relation to the performance of the NGS technology against conventional applications, and the establishment of a bioinformatic and statistical framework for the analysis of metataxonomic data.

L'épigénomique pourrait nous aider à mieux comprendre pourquoi différents types cellulaires montrent différents comportements. Puisque, dans le cadre d'études épigénétiques, il peut êtrenécessaire de comparer plusieurs profils de séquençage, il y a un besoin urgent en nouvelles approches et nouveaux outils pour pallier aux variabilités techniques sous-jacentes. Nous avons développé NGS-QC, un système de contrôle qualité qui détermine la qualité de données et Epimetheus, un outil de normalisation d'expériences de modifications d'histones basé sur les quartiles afin de corriger les variations techniques entre les expériences. Enfin, nous avons intégré ces outils dans un pipeline d'analyse allèle-spécifique afin de comprendre le statut épigénétique de XCI dans le cancer du sein où la perte du Xi est fréquent. Notre analyse a dévoilé des perturbations dans le paysage épigénétique du X et des réactivations géniques aberrantes dans le Xi, dont celles associées au développement du cancer
Epigenomics would help us understand why various cells types exhibit different behaviours. Aberrant changes in reversible epigenetic modifications observed in cancer raised focus towards epigenetic targeted therapy. As epigenetic studies may involve comparing multi-profile sequencing data, thereis an imminent need for novel approaches and tools to address underlying technical variabilities. Wehave developed NGS-QC, a QC system to infer the experimental quality of the data and Epimetheus, a quantile-based multi-profile normalization tool for histone modification datasets to correct technical variation among samples. Further, we have employed these developed tools in an allele-specific analysis to understand the epigenetic status of X chromosome inactivation in breast cancer cells where disappearance of Xi is frequent. Our analysis has revealed perturbation in epigenetic landscape of X and aberrant gene reactivation in Xi including the ones that are associated with cancer promotion

Next-generation sequencing (NGS) has rapidly become the current standard in genetic related analysis. This switch from microarray to NGS required new statistical strategies to address the research questions inherent to the considered phenomena. First and foremost, NGS dataset usually consist of discrete observations characterized by overdispersion - that is, discrepancy between expected and observed variability - and an abundance of zeros, measured across a huge number of regions of the genome. With respect to chromatin immunoprecipitation sequencing (ChIP-Seq), a class of NGS data, it is of primary focus to discover the underlying (unobserved) pattern of `enrichment': more particularly, there is interest in the interactions between genes (or broader regions of the genome) and proteins, as they describe the mechanism of regulation under different conditions such as healthy or damaged tissue. Another interesting research question involves the clustering of these observations into groups that have practical relevance and interpretability, considering in particular that a single unit could potentially be allocated into more than one of these clusters, as it is reasonable to assume that its participation is not exclusive to one and only biological function and/or mechanism. Many of these complex processes, indeed, could also be described by sets of ordinary differential equations (ODE's), which are mathematical representations of the changes of a system through time, following a dynamic that is governed by some parameters we are interested in. In this thesis, we address the aforementioned tasks and research questions employing different statistical strategies, such as model-based clustering, graphical models, penalized smoothing and regression. We propose extensions of the existing approaches to better fit the problem at hand and we elaborate the methodology in a Bayesian environment, with the focus on incorporating the structural dependencies - both spatial and temporal - of the data at our disposal.

Next-generation sequencing (NGS) has rapidly become the current standard in genetic related analysis. This switch from microarray to NGS required new statistical strategies to address the research questions inherent to the considered phenomena. First and foremost, NGS dataset usually consist of discrete observations characterized by overdispersion - that is, discrepancy between expected and observed variability - and an abundance of zeros, measured across a huge number of regions of the genome. With respect to chromatin immunoprecipitation sequencing (ChIP-Seq), a class of NGS data, it is of primary focus to discover the underlying (unobserved) pattern of `enrichment': more particularly, there is interest in the interactions between genes (or broader regions of the genome) and proteins, as they describe the mechanism of regulation under different conditions such as healthy or damaged tissue. Another interesting research question involves the clustering of these observations into groups that have practical relevance and interpretability, considering in particular that a single unit could potentially be allocated into more than one of these clusters, as it is reasonable to assume that its participation is not exclusive to one and only biological function and/or mechanism. Many of these complex processes, indeed, could also be described by sets of ordinary differential equations (ODE's), which are mathematical representations of the changes of a system through time, following a dynamic that is governed by some parameters we are interested in. In this thesis, we address the aforementioned tasks and research questions employing different statistical strategies, such as model-based clustering, graphical models, penalized smoothing and regression. We propose extensions of the existing approaches to better fit the problem at hand and we elaborate the methodology in a Bayesian environment, with the focus on incorporating the structural dependencies - both spatial and temporal - of the data at our disposal.

Single Nucleotide Polymorphisms (SNPs) represent the most abundant type of genetic variation and they are a valuable tool for several biological applications like linkage mapping, integration of genetic and physical maps, population genetics as well as evolutionary and protein structure-function studies. SNP genotyping by mapping DNA reads produced via Next generation sequencing (NGS) technologies on a reference genome is a very common and convenient approach in our days, but still prone to a significant error rate. The need of defining in silico true genetic variants in genomic and transcriptomic sequences is prompted by the high costs of the experimental validation through re-sequencing or SNP arrays, not only in terms of money but also time and sample availability. Several open-source tools have been recently developed to identify small variants in whole-genome data, but still the candidate variants, provided in the VCF output format, present a high false positive calling rate. Goal of this thesis work is the development of a bioinformatic method that classifies variant calling outputs in order to reduce the number of false positive calls. With the aim to dissect the molecular bases of grape acidity (Vitis vinifera L.), this tool has been then used to select SNPs in two grapevine varieties, which show very different content of organic acids in the berry. The VCF parameters have been used to train a Support Vector Machine (SVM) that classifies the VCF records in true and false positive variants, cleaning the output from the most likely false positive results. The SVM approach has been implemented in a new software, called VerySNP, and applied to model and non-model organisms. In both cases, the machine learning method efficiently recognized true positive from false positive variants in both genomic and transcriptomic sequences. In the second part of the thesis, VerySNP was applied to identify true SNPs in RNA-seq data of the grapevine variety Gora Chirine, characterized by low acidity, and Sultanine, a normal acidity variety closely related to Gora. The comparative transcriptomic analysis crossed with the SNP information lead to discover non-synonymous polymorphisms inside coding regions and, thus, provided a list of candidate genes potentially affecting acidity in grapevine.

Single Nucleotide Polymorphisms (SNPs) represent the most abundant type of genetic variation and they are a valuable tool for several biological applications like linkage mapping, integration of genetic and physical maps, population genetics as well as evolutionary and protein structure-function studies. SNP genotyping by mapping DNA reads produced via Next generation sequencing (NGS) technologies on a reference genome is a very common and convenient approach in our days, but still prone to a significant error rate. The need of defining in silico true genetic variants in genomic and transcriptomic sequences is prompted by the high costs of the experimental validation through re-sequencing or SNP arrays, not only in terms of money but also time and sample availability. Several open-source tools have been recently developed to identify small variants in whole-genome data, but still the candidate variants, provided in the VCF output format, present a high false positive calling rate. Goal of this thesis work is the development of a bioinformatic method that classifies variant calling outputs in order to reduce the number of false positive calls. With the aim to dissect the molecular bases of grape acidity (Vitis vinifera L.), this tool has been then used to select SNPs in two grapevine varieties, which show very different content of organic acids in the berry. The VCF parameters have been used to train a Support Vector Machine (SVM) that classifies the VCF records in true and false positive variants, cleaning the output from the most likely false positive results. The SVM approach has been implemented in a new software, called VerySNP, and applied to model and non-model organisms. In both cases, the machine learning method efficiently recognized true positive from false positive variants in both genomic and transcriptomic sequences. In the second part of the thesis, VerySNP was applied to identify true SNPs in RNA-seq data of the grapevine variety Gora Chirine, characterized by low acidity, and Sultanine, a normal acidity variety closely related to Gora. The comparative transcriptomic analysis crossed with the SNP information lead to discover non-synonymous polymorphisms inside coding regions and, thus, provided a list of candidate genes potentially affecting acidity in grapevine

Single Nucleotide Polymorphisms (SNPs) represent the most abundant type of genetic variation and they are a valuable tool for several biological applications like linkage mapping, integration of genetic and physical maps, population genetics as well as evolutionary and protein structure-function studies. SNP genotyping by mapping DNA reads produced via Next generation sequencing (NGS) technologies on a reference genome is a very common and convenient approach in our days, but still prone to a significant error rate. The need of defining in silico true genetic variants in genomic and transcriptomic sequences is prompted by the high costs of the experimental validation through re-sequencing or SNP arrays, not only in terms of money but also time and sample availability. Several open-source tools have been recently developed to identify small variants in whole-genome data, but still the candidate variants, provided in the VCF output format, present a high false positive calling rate. Goal of this thesis work is the development of a bioinformatic method that classifies variant calling outputs in order to reduce the number of false positive calls. With the aim to dissect the molecular bases of grape acidity (Vitis vinifera L.), this tool has been then used to select SNPs in two grapevine varieties, which show very different content of organic acids in the berry. The VCF parameters have been used to train a Support Vector Machine (SVM) that classifies the VCF records in true and false positive variants, cleaning the output from the most likely false positive results. The SVM approach has been implemented in a new software, called VerySNP, and applied to model and non-model organisms. In both cases, the machine learning method efficiently recognized true positive from false positive variants in both genomic and transcriptomic sequences. In the second part of the thesis, VerySNP was applied to identify true SNPs in RNA-seq data of the grapevine variety Gora Chirine, characterized by low acidity, and Sultanine, a normal acidity variety closely related to Gora. The comparative transcriptomic analysis crossed with the SNP information lead to discover non-synonymous polymorphisms inside coding regions and, thus, provided a list of candidate genes potentially affecting acidity in grapevine.

Introduction/aims. Currently, there are no straightforward guidelines for the clinical and diagnostic management of neuromuscular disorders. Therefore, I have aimed to describe the diagnostic workflow which is used in my neuromuscular clinic for evaluating patients with this condition. The neuromuscular clinic is situated in IRCCS Policlinico San Martino in Genova and is a neuromuscular university centre in Northwest Italy. Methods. I describe our diagnostic approach to two frequent neuromuscular disorders: hyperCKemia and CMT neuropathy. The first work is an Italian multicentre study evaluating our diagnostic workflow for isolated hyperCKemia, which is based on electrodiagnostic data, biochemical screening and first-line genetic investigations, followed by successive targeted sequencing panels. Using this approach, we established a definitive diagnosis in one third of the patients. The detection rate was higher in patients with severe hyperCKemia and abnormal electromyographic findings. The second work includes patients affected by CMT with regular follow-ups in our CMT clinic. I describe the genetic distribution of CMT subtypes in our cohort and report a peculiar phenotype. Moreover, I define our diagnostic experiences as a multidisciplinary outpatient clinic, combining a gene-by-gene approach or targeted gene panels based on clinical presentation. Discussion/conclusion. Taking as a model our experience, I generalise the genetic approach to neuromuscular disorders: the diagnosis strategy should be flexible and tuned to the clinical features of the patient in order to select the best molecular approach for each patient.

O número crescente de descobertas de substâncias bioativas produzidas pelo metabolismo secundário de cianobactérias tem despertado o interesse de grupos de pesquisa no mundo todo com o objetivo comum de descrever e explorar estas moléculas e entender a sua biossíntese. No Brasil, as pesquisas sobre moléculas bioativas produzidas por linhagens de cianobactérias nativas são escassas. Neste trabalho, utilizando iniciadores específicos da PCR e sequenciamento, a presença de genes envolvidos na biossíntese da neurotoxina saxitoxina (STX) foi confirmada em representantes dos gêneros Dolichospermum, Sphaerospermopsis, Cuspidothrix e Cylindrospermopsis, enquanto que genes da citotoxina cilindrospermopsina (CYN) foram detectados somente em representantes de Cylindrospermopsis. Genes envolvidos com a produção dos inibidores enzimáticos, microviridina (MDN) e a cianobactina microciclamida (MCA) foram sequenciados em isolados do gênero Microcystis. Os genomas das linhagens de Cylindrospermopsis raciborskii CENA302 e CENA303 foram sequenciados usando a plataforma HiScan SQ (Ilumina) com biblioteca pareada 2 x 100 pb. O genoma da Sphaerospermopsis torques-reginae ITEP-024 foi sequenciado utilizando a plataforma Ion Torrent (Life Technologies) com tamanhos de fragmentos de até 200 pb. As tentativas de montagem ab initio dos genomas foram realizadas e o agrupamento gênico da saxitoxina (28 kb) da linhagem C. raciborskii CENA302 foi identificado e caracterizado. As análises filogenéticas das sequências de aminoácidos envolvidos com a biossíntese das moléculas bioativas avaliadas demonstraram que os isolados brasileiros de cianobactérias formam clados com elevado valor de reamostragem com sequências homólogas de cianobactérias conhecidas como produtoras dessas moléculas. Neste estudo é relatada pela primeira vez a presença de genes cyr em linhagens da América do Sul de C. raciborskii e a presença simultânea de genes cyr e sxt em uma única linhagem de C. raciborskii. Além disso, este é o primeiro estudo que relata a presença de genes envolvidos na biossíntese de MDN e MCA nas espécies de cianobactérias M. protocystis, M. panniformis e M. wesenbergii. Análises por espectrometria de massas acoplada a cromatografia líquida (LC-MS) e imunoensaio enzimático (ELISA) foram utilizadas a fim de detectar e identificar variantes estruturais das moléculas bioativas das cianobactérias que tiveram os genes biossintéticos sequenciados. A análise de LC-MS mostrou a produção das variantes GTX2, GTX3, STX e dc-STX pela linhagem C. raciborskii CENA302, enquanto que a linhagem C. raciborskii CENA305 apresentou as variantes NEO, C1 e dcGTX3. As quatro novas variantes de MCY, [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR e [D-Phe1]MC-LR, foram encontradas nas espécies M. panniformis SPC702 e M. protocystis SPC697. Este é o primeiro relato da produção de MCY por essas duas espécies de Microcystis. Dezesseis linhagens que ainda não possuíam as sequências do gene de RNAr 16S foram sequenciadas. O resultado da análise filogenética das sequências do gene de RNAr 16S foi coerente com as descrições morfológicas, sendo que todas as linhagens foram caracterizadas em nível de espécie. As informações geradas neste estudo contribuem para o aumento do conhecimento da diversidade metabólica dos isolados brasileiros de cianobactérias e trazem nova visão sobre a evolução dessas moléculas produzidas pelo metabolismo secundário
The growing numbers of discoveries of bioactive substances produced by cyanobacterial secondary metabolism has attracted the interest of research groups around the world with the common goal of describing and exploring these molecules and understanding their biosynthesis. In Brazil, researches on bioactive molecules produced by native cyanobacterial strains are scarce. In this work, using specific PCR primers and sequencing, the presence of genes involved in the biosynthesis of the neurotoxin saxitoxin (STX) was confirmed in representatives of the genera Dolichospermum, Sphaerospermopsis, Cuspidothrix and Cylindrospermopsis, while genes of the cytotoxin cylindrospermopsin (CYN) were detected only in representatives of Cylindrospermopsis. Genes involved in the production of protease inhibitors, microviridin (MDN) and the cianobactin microciclamide (MCA), were sequenced in isolates of the genus Microcystis. The genomes of Cylindrospermopsis raciborskii strains CENA302 and CENA303 were sequenced using the high-throughput platform HiScan SQ (Illumina) as paired-ends 2 x 100 bp. The Sphaerospermopsis torques-reginae ITEP-024 genome was sequenced using the high-throughput platform Ion Torrent (Life Technologies) with fragment sizes up to 200 bp. Attempts of ab initio genomes assembly were performed and the 28 kb saxitoxin gene cluster of C. raciborskii strains CENA302 was identified and characterized. Phylogenetic analyses of amino acid sequences involved in the biosynthesis of the bioactive molecules evaluated showed that the Brazilian cyanobacterial isolates formed clades with high bootstrap values with homologous sequences of known cyanobacterial producers of these molecules. In this study is reported for the first time the presence of cyr genes in South America strains of C. raciborskii and the simultaneous presence of cyr and sxt genes in a single C. raciborskii strain. Furthermore, this is the first study reporting the presence of genes involved in the biosynthesis of MDN and MCA in the cyanobacterial species M. protocystis, M. panniformis e M. wesenbergii. Analyses by mass spectrometry coupled to liquid chromatography (LC-MS) and enzyme immunoassay (ELISA) were used to detect and identify structural variants of bioactive molecules of the cyanobacteria that had the biosynthetic genes sequenced. Analysis of LC-MS showed the production of the variants GTX2, GTX3, STX and dc-STX by the C. raciborskii strain CENA302, whereas the strain C. raciborskii CENA305 presented the variants NEO, C1 and dcGTX3. The new four MCY variants [D-Val1]MC-RR, [D-Leu/Ile1]MC-RR, [D-Leu/Ile1]MC-YR and [D-Phe1]MC-LR were found in the species M. panniformis SPC702 and M. protocystis SPC697. This is the first report of the MCY production by these two species of Microcystis. Sixteen strains that still lacked the 16S rRNA gene sequences were sequenced. The result of the phylogenetic analysis of 16S rRNA gene sequences was consistent with the morphological descriptions, and all strains were characterized to species level. The informations generated in this study contribute to the increase of knowledge on metabolic diversity of Brazilian cyanobacterial strains and bring new insight into the evolution of these molecules produced by secondary metabolism

Background & aims: Up to 15% inflammatory bowel diseases (IBD) rising before the age of 6 years, defined as Very-Early-Onset IBD (VEO-IBD), may have a monogenic disease. More rarely monogenic defects are found in later onset IBD. Monogenic IBD are associated with high morbidity and mortality and timely genetic diagnosis is essential for adequate treatment. Due to the wide phenotypic and genetic heterogeneity of these conditions, it is often difficult to reach a genetic diagnosis and the best diagnostic approach is still debated. Next generation sequencing (NGS) techniques have been proposed as a screening tool especially in patients with poorly defined phenotypes. In a cohort study that included patients with VEO-IBD and Early-onset IBD with severe/atypical phenotypes (EO-IBD s/a) we aimed to: describe the genetic diagnoses and their therapeutic implications, define the clinical characteristics associated with monogenicity, suggest a diagnostic approach to monogenic IBD. Methods: Clinical information of patients with VEO-IBD and EO-IBD s/a referred to 3 Italian Centers for a genetic work-up over 10 years (2008-2017) were collected. From 2015 newly diagnosed patients and patients without a previous genetic diagnosis were screened using NGS, except patients with disease specific features in whom candidate gene analysis was chosen. Results: 93 patients were collected and 14 (15%) reached a genetic diagnosis. Selective sequencing was performed in 47 patients (50%), NGS in 84 patients (90%). Causative defects were revealed by NGS in 5 patients (NOD2, TTC37, DKC1, XIAP, FERMT3) and candidate sequencing in 8 patients (2WAS, CYBA, CYBB, FOXP3, 2CD40L, XIAP). In 8 of 9 patients diagnosed with candidate sequencing, the analysis was guided by the presence of disease specific features. One patient, with unspecific presentation, underwent sequential sequencing of multiple genes over 15 months before reaching the diagnosis (XIAP). NGS identified a new NOD2 mutation previously missed with single gene approach. One patient with WAS, in whom Sanger sequencing had not revealed mutations, was diagnosed externally through WGS which revealed a large genomic inversion. Genetic diagnosis impacted patient management in 10 patients (71%): 6 underwent bone marrow transplant (2XIAP, 2WAS, 2CD40L, FOXP3), 1 gene therapy, 2 anti-infective prophylaxis and 1 introduced danazole (DKC1). Patients with monogenic IBD more frequently had a history of infections (71%vs30%; p<.001), thrombocytopenia (21% vs 3%; p.003), hemophagocytosis (21% vs 3%; p.02), and disease onset ≤ 1 month of life (36% vs 1%; p<.001) when compared to the non-monogenic group. Conclusion: We suggest using NGS in all patients presenting with non-specific clinical profiles and selective gene sequencing when clinical characteristics suggestive of specific monogenic conditions are present.

Les récents progrès technologiques en termes de séquençage de données génomiques ont entraîné une forte croissance des données disponibles et l'apparition de nouveaux besoins. Initialement limitée à l'analyse de petite quantité de données, la bioinformatique a dû s'adapter à ce nouveau contexte technologique et scientifique afin de répondre aux nouveaux challenges proposés. Par l'intermédiaire de différents projets réalisés dans des contextes différents, cette thèse s'intègre dans ce changement contextuel où la bioinfomatique n'est plus limitée à l'utilisation successive d'outils à objectifs unitaire entrecoupée d'étapes humaine dépendantes. Focalisés sur le développement de stratégies d'analyse complexes pour le développement ou la mise à disposition d'outils entièrement automatisés et la production de données à haute valeur ajoutée, ces travaux permettent de comprendre le rôle important de la bioinformatique 2.0. Ainsi nous montrerons comment elle doit être à même de répondre à des objectifs précis par l'intermédiaire de stratégies intégrant les concepts de la biologie, les outils bioinformatiques existants et l'expertise humaine associée au domaine. En conclusion nous discuterons du nouveau rôle et de l'impact futur de la bioinformatique 2.0 qui requiert une expertise tant sur le plan biologique qu'informatique adaptée aux données NGS
Recent technological advances in terms of genomic sequencing data led to a strong growth of available data and the emergence of new needs. Initially limited to the analysis of simple sequence or limited amount of data, bioinformatics has to adapt to this new technological and scientific context to meet the new challenges offered. Through different projects in different genomic era, this thesis fits into this contexts change where bioinfomatics is no longer limited to the use of tool with unitary goal and human dependent steps. Focused on the development of complex analysis strategies for the development or the availability of fully automated tools and high-value data, this work introduce the important role of bioinformatics version 2.0. We will show how it is able to answer to precise biological question through specific strategy that integrate all the biological concepts, existing bioinformatics tools and human expertise related to the domain. To conclude, we discuss about the role and the impact of the bioinformatics 2.0 that requires a expert vision at biological and computers level adapted to NGS data

Introduction : Les shunts artérioveineux cérébraux provoquent céphalées, épilepsie et déficits neurologiques. Nous avons étudié la physiopathologie de ces affections en IRM de perfusion cérébrale à 1. 5 T, afin d'essayer de corréler les symptômes cliniques avec les anomalies hémodynamiques. Matériels et Méthodes : 39 patients et de 10 volontaires sains, ont été imagés en IRM de perfusion à 1. 5 T, avec injection pulsée de Gadolinium. Les séries d'images IRM ont été analysées en utilisant la méthode de dilution des indicateurs. Les paramètres CBV et CBF ont été mesurés dans deux zones cérébrales spécifiques chez tous les sujets, en estimant quatre fonctions d'entrée artérielle (AIF) : « Locale », « Régionale », « Régionale Proportionnelle », et « Globale ». La meilleure méthode d'estimation de l'AIF a été déterminée en utilisant la méthode statistique « NGS » (No Gold Standard). Nous avons ensuite analysé les valeurs de CBV et CBF chez les témoins et les patients afin de relier signe clinique et anomalies de perfusion, en utilisant des tests statistiques de Fisher, V de Cramer, du Chi deux et Phi. Résultats : Les CBV et CBF des patients étaient différents de ceux des témoins quelle que soit la méthode d’AIF. Trois types d'anomalies de perfusion étaient présentes à distance de la zone de shunt (hyperperfusion, hypoperfusion et congestion veineuse) chez 91 % des patients. La meilleure méthode d'estimation selon NGS était la « Régionale Proportionnelle », permettant seule d'identifier une relation significative entre hyperperfusion et épilepsie. Conclusion : Nous avons observé une relation liant l'hyperperfusion et l'épilepsie, et proposé des hypothèses physiopathologiques
Introduction: Brain Arteriovenous shunts are diseases that induce seizures, headaches and focal deficits. We studied their pathophysiology using 1. 5 T Perfusion MR trying to relate hemodynamics abnormalities and symptoms. Material and Methods: 39 patients and 10 healthy subjects were included. We performed perfusion MRI on a 1. 5 T scanner with Gd bolus injection. MRI series were processed using indicator dilution theory CBV and CBF parameters were estimated in two specific brain areas using four arterial input function estimations: "Local", "Regional", "Regional Scaled", and 'Global". Best of these AIF estimates was assessed using the NGS statistical method. We then compared patients and volunteers CBV and CBF results trying to relate symptoms and perfusion abnormalities using Fisher, Cramer, Chi square and Phi tests. Results: Patients CBV and CBF values were different to those estimated in volunteers in all AIF estimates. Three perfusion abnormalities were observed: hypo, hyperperfusion and venous congestion in 91% of patients. The best AIF estimate assessed using NGS was "Regional Scaled", with significant relation between hyperperfusion and seizures. Conclusion: We observed a significant relation between hyperperfusion and seizures in the AVS patients

Orientador: Eduardo Bagagli
Resumo: O reino fungi é constituído por amplos grupos de organismos com reconhecida importância ecológica, industrial ou médica. O solo é um importante reservatório para microrganismos, especialmente fungos, que atuam na decomposição e reciclagem de nutrientes ou se associam com animais e plantas em relações simbióticas ou causando patogenias. Ainda são poucos os estudos relacionados a estrutura do micobioma, sendo rasa a compreensão da diversidade e ecologia de fungos. A compreensão dos padrões de distribuição da diversidade fúngica é essencial para mensurar as mudanças naturais e antrópicas relacionadas a estas comunidades, tal como entender a ecologia e epidemiologia dos fungos considerados patogênicos. Neste trabalho caracterizamos a diversidade fúngica das amostras de solo em diferentes ambientes no município de Botucatu (Brasil), descrevendo a estrutura da comunidade fúngica e mensurando a prevalência dos fungos patogênicos, mediante a técnica de Sequenciamento de Nova Geração (NGS). No sequenciamento mediante plataforma Illumina, analisamos 44 amostras provenientes de 8 ambientes diferentes, em dois períodos (outono e primavera), incluindo tocas de tatus, corujas e outros ambientes relacionados. A riqueza e diversidade variaram entre os ambientes, sendo favorecido nas amostras ambientais da floresta sazonal semidecidual e das tocas de tatus, os quais possuem uma maior cobertura vegetal. Ainda, ambientes mais antropizados, com menores efeitos de cobertura vegetal, como edifício... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Pneumocystis species are obligate fungi capable of causing opportunistic infection called Pneumocystis Pneumonia (PCP) in those who are immunocompromised, often resulting in serious disease and a high mortality rate. Due to host specificity and inability to conduct in vitro studies, molecular methods have stepped in to undertake the bulk of epidemiological studies. Many unknows regarding the fungus still exist, and there is little concordance between researchers. This thesis aimed to improve and standardize the identification and genotyping techniques currently used to enable the global research community to bridge gaps collaboratively. Chapter 1 outlines Pneumocystis epidemiology, the pitfalls facing researchers and how they have been addressed. As a major influencing factor in determining sequencing success is the quality of the DNA obtained, Chapter 2 investigates and compares several DNA extraction kits and methods for an effective protocol. The gold standard for P. jirovecii currently remains to be multilocus sequence typing (MLST), therefore Chapter 3 optimizes primers and PCR protocols of the current MLST scheme used by the International Society for Human & Animal Mycology. It identified pitfalls within the scheme and addresses them by reviewing all genotyping loci and schemes used till now to determine the most effective scheme to be used in the future. This resulted in the proposal of a new global consensus MLST scheme, which was then successfully applied to a clinical cohort in Chapter 4. Chapter 5 examines the epidemiology of globally collected clinical samples using the scheme, uncovering unique alleles that formed 49 sequence types, and demonstrated the importance of a collaborative database. The importance of the genetic locus DHPS to monitor global resistance is discussed in Chapter 6. Finally, long-read next-generation sequencing using the MinION sequencer is applied to detect PCP in clinical samples to determine its potential for early diagnostics of PCP in Chapter 7.

The rust fungi are obligate biotrophic pathogens of a wide range of plants. The plant-pathogen interactions follow a gene for gene manner: a resistance (R) gene from plants can recognize a corresponding avirulence (Avr) gene from rust fungi. This thesis focused on searches for Avr gene in two rust fungi, the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt), and the barley leaf rust pathogen Puccinia hordei (Ph), with Next-generation sequencing (NGS) technologies. In Chapter 2, two Pgt isolates, one wildtype and one mutant derivative that differed in virulence to host R gene Sr50, were sequenced to identify the corresponding Avr gene AvrSr50. Genome comparison of the two isolates revealed amino acid-changing variations in 18 genes encoding haustorially-expressed secreted proteins. One of these genes was validated to encode the AvrSr50 protein, showing for the first time the effectiveness of Avr gene search via comparative genomics in rust fungi. In Chapter 3, a de novo genome assembly was performed for a Ph isolate Ph612, producing 15,913 scaffolds amounting to 127Mbp. A total of 16,354 genes were predicted, including 1,072 secreted protein-encoding genes. In Chapter 4, four additional Ph isolates derived from a same clonal lineage as Ph612 were studied. The five isolates differed in virulence for three barley R genes Rph3, Rph13 and Rph19. To identify the corresponding Avr genes AvrRph3, AvrRph13, and AvrRph19, the isolates were sequenced for genome comparisons, which revealed in 114, 99 and 120 candidates for the three Avr genes, respectively. The results and logical framework presented in this thesis have contributed to the knowledge pool of rust virulence, which will assist in development of novel resistance to these pathogens in both wheat and barley.

Orientador: Renate Krause Sakate
Resumo: A espécie Arachis pintoi, conhecida como amendoim forrageiro, é uma leguminosa nativa do Brasil. Muitas são as utilidades atribuídas ao amendoim forrageiro, sendo seu uso mais comum como espécie forrageira, fornecendo alimento em grande quantidade e qualidade aos animais, em plantios puros ou consórcio com gramíneas. A ocorrência de sintomas de viroses em genótipos e cultivos de amendoim forrageiro tem sido observada por pesquisadores em diferentes estados brasileiros. No Brasil, apenas duas espécies virais já foram relatadas: o Peanut mottle virus- PeMoV e o Cowpea mild mottle virus - CpMMV. O objetivo deste trabalho foi estudar os vírus de plantas de amendoim forrageiro do Banco de Germoplasma da Embrapa Acre e detectar viroses em plantas de pimenta Cumari-do-Pará. Para detectar possíveis vírus nesses genótipos, uma análise de sequenciamento de nova geração foi realizada. A extração total de RNA dos 22 acessos foi realizada com o kit RNA Viral PureLink (Invitrogen) seguida de preparação da biblioteca e sequenciamento do transcriptoma utilizando a plataforma Illumina HiSeq2500. A montagem de novo das leituras de 24.659.442 foi realizada usando o software CLC Genomics Workbench v7.0.3. Os 9.709 contigs obtidos foram submetidos a uma pesquisa BLASTn usando o software Geneious v.9.1.5. A análise metagenômica permitiu a identificação de sete espécies de vírus: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus sub-grupo IB (Cucumovirus), Cowpea chlorotic mottle virus... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The species Arachis pintoi, known as forage peanuts is a native legume from Brazil. There are many uses attributed to forage peanut, being its most common use as a forage species, providing food in large quantity and quality to the animals, in single crops or consortium with grasses. The occurrence of virus symptoms in forage peanut genotypes and crops has been observed by researchers in different Brazilian states. In Brazil, only two viral species have been reported: Peanut mottle virus-PeMoV and Cowpea mild mottle virus-CpMMV. The objective of this work was to study the forage peanut plant viruses of the Embrapa Acre Germplasm Bank and to detect viruses in Cumari-do-Pará pepper plants. To detect possible viruses in these genotypes, a new generation sequencing analysis was performed. Total RNA extraction from the twenty-two accessions was performed with the PureLink Viral RNA Kit (Invitrogen) followed by library preparation and transcriptome sequencing using the Illumina HiSeq2500 platform. Re-assembly of the 24,659,442 readings was performed using the CLC Genomics Workbench v7.0.3 software. The 9,709 contigs obtained were subjected to a BLASTn search using the Geneious v.9.1.5 software. Metagenomic analysis allowed the identification of seven virus species: Peanut mottle virus - PeMoV (Potyvirus), Cucumber mosaic virus subgroup IB (Cucumovirus), Cowpea chlorotic mottle virus - CCMV (Bromovirus) and a probable new member of the Potyviridae family, and also new species of the... (Complete abstract click electronic access below)
Doutor

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Made available in DSpace on 2016-12-01T12:56:42Z (GMT). No. of bitstreams: 1 Evelyn Anly Ishikawa Hayashi.pdf: 995457 bytes, checksum: 6e8e0ab5195fade5b6ad359231618e69 (MD5) Previous issue date: 2016-02-29
Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The yam (Dioscorea spp.) has an important socio-economic role in tropical and subtropical regions of Asia, Africa and the Americas including the Caribbean. In Brazil, it is a significant source of income and food for the local populations and family agriculture, especially in the Northeast region of the country. The crop yield is very affected by both abiotic factors and biotic agents, including fungi, nematodes and viruses. Diseases caused by viruses are important because the vegetative propagation of yam provides the accumulation and spread of these pathogens on successive crops. To date, the reported viruses in this crop belong to nine genera: Aureusvirus, Badnavirus, Carlavirus, Comovirus, Cucumovirus, Fabavirus, Macluravirus, Potexvirus and Potyvirus. The objective of the present work was to analyze, through the Next Generation Sequencing (NGS), different viral species that infect the yam in fields located in the states of Pernambuco and Paraíba and in the Federal District. Leaf tissue samples of D. rotundata and D. alata were subjected to partial virus purification, the total RNA was extracted and submitted to NGS. The nucleotide reads obtained were assembled using CLC Genomics Workbench 6.5 program and the contigs using Geneious program. Based on the data obtained from NGS it was possible to detect three new virus species reported in this work. It was sequenced the complete genome of two new species, one belonging to the family Secoviridae, with the proposed name Dioscorea virus S (DVS), and another to Foveavirus genus of the family Betaflexiviridae, called Dioscorea virus F (DVF). For the third species described, belonging to the family Closteroviridae, it was done only the viral detection in the collected samples and proposed the name Dioscorea virus C (DVC).
O inhame (Dioscorea spp.) apresenta importante papel socioeconômico nas regiões tropicais e subtropicais da Ásia, África e Américas incluindo o Caribe. No Brasil, se constitui uma expressiva fonte de renda e alimento para as populações locais e agricultura familiar, principalmente na região Nordeste do país. A produtividade da cultura é bastante afetada, tanto por fatores abióticos, como por agentes bióticos, entre os quais fungos, nematoides e vírus. Doenças causadas por vírus são importantes, pois a propagação vegetativa do inhame proporciona o acúmulo e disseminação desses patógenos em cultivos sucessivos. Até o momento, os vírus relatados nesta cultura pertencem a nove gêneros: Aureusvirus, Badnavirus, Carlavirus, Comovirus, Cucumovirus, Fabavirus, Macluravirus, Potexvirus e Potyvirus. No presente trabalho objetivou-se analisar, através do Sequenciamento de Nova Geração (Next Generation Sequencing - NGS), as diferentes espécies virais que infetam o inhame em plantios localizados nos estados de Pernambuco e Paraíba e no Distrito Federal. Amostras de tecido foliar de D. rotundata e D. alata foram submetidas a purificação viral parcial, o RNA total foi extraído e submetido ao NGS. As leituras nucleotídicas obtidas foram montadas utilizando o pragrama CLC Genomics Workbench 6.5 e os contigs utilizando o programa Geneious. Por meio dos dados obtidos por NGS foi possível a detecção de três espécies virais novas relatadas neste trabalho. Foi sequenciado o genoma completo de duas espécies, uma pertencente à família Secoviridae, que recebeu o nome Dioscorea virus S (DVS), e outra ao gênero Foveavirus da família Betaflexiviridae, denominada de Dioscorea virus F (DVF). Para a terceira espécie descrita, pertencente à família Closteroviridae, foi feita apenas a detecção viral nas amostras coletadas e a proposição do nome Dioscorea virus C (DVC).

Complex diseases are caused by a complex interaction among genetic and environmental factors. The Identified common variants occount for only a small fraction of the genetic component of these diseases. Therefore, low (1X<5%) and rare (MAR<15) frequency varlants may help fill in some of the heritability Eap. We have focused on two different neurological diseases, Multiple Sclerosis (MS) and Epilepsy. The general aim of the study was on ole side to dentify new low and fare frequency genetic variants associated to the susceptibility to MS in the Italian continental population, and, on the other side associated to Epilepsy. From the analysis of Multiplex MS families, our study allowed us to identify 10 geres harboring rare coding variants and other 11 genes herboring rare non-coding variants. The extension of these analyses to other cohorts of non-familial MS patients, MS multiple families and Controls:{HC) is ongoing to further assess the role in MS Susceptibility of the identified rare variants. From the research of rare functional variants in MS associated loci, our study allowed us to identify one gene with the most promising result especially for disruptive variants (stop-gain, stop-loss and splicing), Identifying a novel low frequency functional variant associated with MS susceptibility. Moreover, regarding a project on pediatric MS, our preliminary wGRSs analyses on already avallable pediatric MS patients suggested that pediatric MS have a higher risk score compared to adult-onset MS and HC, conferred by common non-HLA MS associated variants and more slightly by the analyzed 5 HLA markers: From the screening of epileptic patients, for diagnostic and research purposes, our study identified four rare pathogenic variants causative of the epilepsy forms in four different patients: Moreover, our analyses showed that epileptic patients can have an increased burden of rare variants in genes associated to epilepsy.

The Ph.D. research work is an integral part of a Horizon 2020 project, called HERCULES project (CompreHEnsive chaRacterisation and effeCtive combinatorial targeting of high-grade seroUs ovarian cancer via singLE-cell analysiS). The topics of the Horizon 2020 project are to comprehensively characterize high grade serous ovarian cancer (HGS-OvCa) by integrating and modeling clinical and biological data (e.g., genetics, transcriptomics, protein binding, drug screens) from primary, metastatic and relapsed tumors from various anatomical sites of HGS-OvCa patients, and establish combinatorial treatment modalities that effectively kill HGS-OvCa tumor cell subpopulations. The role of the Company in HERCULES project is to develop and validate a marketable prototype biomarker kit for predicting HGS-OvCa patients response to combinatorial therapeutic modalities. In detail, the kit hypothesized for this application is a NGS gene panel, based on an Illumina platform and technology, capable to predict the outcome of a pharmacological therapy using high grade serous ovarian cancer subpopulation genetic biomarkers. A further aim of the Ph.D. project is more generally to study the processes required for developing an in vitro diagnostic (IVD) workflow finalized to a next generation sequencing analysis for oncology applications, not only focused on high grade serous ovarian cancer, having diagnostic, prognostic and predictive purposes. In order to achieve this goal, the following activities were carried out: 1. Evaluation of the “state of art” regarding the presence of patents relevant for the HERCULES project. 2. Definition of a gene panel design based on several genes involved in HGS-OvCa. 3. Identification and selection of FFPE DNA and RNA extraction kits having features, in terms of nucleic acid yields, quality and purity, that are compatible with the NGS downstream analysis and an IVD workflow. 4. Study of the processes necessary to sequence 12 human FFPE samples, having both KRAS wild type and mutated, on the Illumina platform using amplicon technology. As a result of this work three commercial column-based DNA extraction kits have been identified as the most effective when included in a NGS workflow based on Illumina technology. The study and the development of the NGS workflow were done employing available clinical FFPE sections of human colorectal cancer with already determined KRAS mutational status using established IVD assays. The results obtained with the NGS analysis have also demonstrated that the gene panel design for the library preparation kit, which uses both amplicon and UMI (unique molecular identifier) technologies, provides high UMI incorporation, high coverage depth of the regions of interest. This are fundamental aspects required for the identification of false-positive and mutations expressed at very low levels. These results will be used for developing clinical custom genes panel, intended to be used by pathologists and oncologists, capable to provide: -Prediction cancer onset. -Characterization of different tumor types. - Precise information about the exact therapeutic treatment for different cancer types (HGS-OvCa included). - Prognosis.

Els dos subtipus més freqüents de càncer d’endometri (CE) són el carcinoma endometrioide (CEE) i el carcinoma serós (CSE). El diagnòstic diferencial entre aquestes dues tipologies no sempre és fàcil, hi ha casos que presenten característiques histològiques molt dubtoses i ambigües que dificulten el correcte diagnòstic final. Aquests dos tipus tumorals presenten diferents perfils moleculars i un pronòstic diferencial, fent que el CSE sigui el subtipus més agressiu i amb el pronòstic més desfavorable. Actualment s’està posant en coneixement la importància que pot jugar l’heterogeneïtat intratumoral (HIT) en els comportaments més agressius dels tumors i en la resistència als tractaments, com és el cas del CSE. En els últims anys, la irrupció de les tecnologies d’alt rendiment com la NGS (Next Generation Sequencing) i la MLPA (Multiple Ligase-dependent Probe Amplification) han ofert l’opció d’ampliar el coneixement molecular del càncer d’una manera més precisa i resolutiva. Per aquests motius, els dos primers objectius d’aquesta tesi s’han centrat en analitzar, en els dos tipus més freqüents de càncer d’endometri, diverses alteracions moleculars que ens permetin obtenir uns perfils genètics específics, i classificar d’una manera més precisa i objectiva aquests dos tipus tumorals. Per realitzar les anàlisis de les alteracions genètiques s’han utilitzat, l’aplicació de targeted sequencing (NGS) amb un panell personalitzat de 40 gens per determinar els gens alterats respecte a variants somàtiques, i la tècnica MLPA per determinar les alteracions somàtiques en el número de còpies (ASNC) de 106 gens. Finalment, el tercer objectiu s’ha focalitzat en conèixer el paper que juga l’heterogeneïtat intratumoral en el CSE. L’efecte de l’HIT ha estat estudiat a dos nivells d’alteració molecular, a nivell de l’alteració dels gens determinant variants somàtiques i de l’alteració en el número de còpies dels gens, mitjançant les dues tècniques anteriors. Els resultats del primer objectiu d’aquesta tesi han demostrat la idoneïtat del nostre estudi personalitzat de NGS com a eina molecular addicional per confirmar la classificació histològica del CE. Aquesta estratègia sembla interessant com una eina per classificar tumors amb troballes microscòpiques inusuals i ambigües. Amb els resultats del segon objectiu s’ha descrit que de manera general el CSE presenta moltes més ASNC que el CEE. Tot i això, també s’ha mostrat que dins el CSE hi ha un percentatge de casos (42%) que presenten unes característiques genètiques que no es corresponen amb el fenotip, i que per tant, en aquests casos pot ser de gran ajuda l’ús del perfil genètic d’ASNC per realitzar una classificació més correcta. A més a més, s’ha suggerit l’ús de la combinació de la determinació de p53 per immunohistoquímica i del número de còpies de la CCNE1 per classificar els casos dins el grup serous-like. Per finalitzar, els resultats del tercer objectiu han mostrat que el CSE, per una banda, presenta unes alteracions moleculars clonals com les variants somàtiques al gen TP53 i guanys en els gens CCNE1 i PIK3CA, i per altra banda, que la seva heterogeneïtat intratumoral està caracteritzada principalment per les ASNC dels gens. A més a més, s’ha remarcat que un dels gens principalment afectats per aquesta heterogeneïtat és el gen ERBB2, que és una diana terapèutica àmpliament utilitzada.
Los dos subtipos más frecuentes de cáncer de endometrio (CE) son el carcinoma endometrioide (CEE) y el carcinoma seroso (CSE). El diagnóstico diferencial entre estas dos tipologías no siempre es fácil, hay casos que presentan características histológicas muy dudosas y ambiguas que dificultan su correcto diagnóstico final. Estos dos tipos tumorales presentan perfiles moleculares y pronósticos diferenciales, haciendo que el CSE sea el subtipo más agresivo y con el pronóstico más desfavorable. Actualmente se está poniendo en conocimiento la importancia que puede jugar la heterogeneidad intratumoral (HIT) en los comportamientos más agresivos de los tumores y en la resistencia a los tratamientos, como es el caso del CSE. En los últimos años, la irrupción de las tecnologías de alto rendimiento como la NGS (Next Generation Sequencing) y la MLPA (Multiple Ligase-dependiente Probe Amplification) han ofrecido la opción de ampliar el conocimiento molecular del cáncer de una manera más precisa y resolutiva. Por estos motivos, los dos primeros objetivos de esta tesis se han centrado en analizar en los dos tipos más frecuentes de cáncer de endometrio varias alteraciones moleculares que nos permitan obtener unos perfiles genéticos específicos, y clasificar de una manera más precisa y objetiva estos dos tipos tumorales. Para realizar los análisis de las alteraciones genéticas se han utilizado, la aplicación de targeted sequencing (NGS) con un panel personalizado de 40 genes para determinar los genes alterados respecto a variantes somáticas, y la técnica MLPA para determinar las alteraciones somáticas en el número de copias (ASNC) de 106 genes. Finalmente, el tercer objetivo se ha focalizado en conocer el papel que juega la heterogeneidad intratumoral en el CSE. El efecto de la HIT ha sido estudiado a dos niveles de alteración molecular, a nivel de la alteración de los genes analizando las variantes somáticas y la alteración en el número de copias de los genes, mediante las dos técnicas anteriores. Los resultados del primer objetivo de esta tesis han demostrado la idoneidad de nuestro estudio personalizado de NGS como herramienta molecular adicional para confirmar la clasificación histológica del CE. Esta estrategia parece interesante como una herramienta para clasificar tumores con hallazgos microscópicos inusuales y ambiguos. Con los resultados del segundo objetivo se ha descrito que de manera general el CSE presenta muchas más ASNC que el CEE. Sin embargo, también se ha mostrado que dentro el CSE hay un porcentaje de casos (42%) que presentan unas características genéticas que no corresponden con el fenotipo, por lo que en estos casos puede ser de gran ayuda el uso del perfil genético de ASNC para realizar una clasificación más correcta. Además, se ha sugerido el uso de la combinación de la determinación de p53 por inmunohistoquímica y del número de copias de la CCNE1 para clasificar los casos dentro del grupo serous-like. Para finalizar, los resultados del tercer objetivo han mostrado que el CSE, por una parte, presenta unas alteraciones moleculares clonales como las variantes somáticas en el gen TP53 y ganancias en los genes CCNE1 y PIK3CA, y por otra parte, que su heterogeneidad intratumoral está caracterizada principalmente por las ASNC de los genes. Además, se ha remarcado que uno de los genes principalmente afectados por esta heterogeneidad es el gen ERBB2, que es una diana terapéutica ampliamente utilizada.
The two most common subtypes of endometrial cancer (CE) are endometrioid carcinoma (CEE) and serous carcinoma (CSE). Differential diagnosis between these two types is not always easy, there are cases with very doubtful and ambiguous histological features that make it difficult their final diagnostic. These two tumor types have different molecular profiles and differential prognosis, making the CSE the most aggressive subtype and with the most unfavorable prognosis. At present, the importance of intratumoral heterogeneity (HIT) can be played out in the most aggressive behaviors of tumors and in resistance to treatment, as in the case of CSE. In recent years, the advent of high-performance technologies such as NGS (Next Generation Sequencing) and MLPA (Multiple Ligase-dependent Probe Amplification) have offered the option of extending molecular knowledge of cancer in a way more accurate and resolute. For these reasons, the first two objectives of this dissertation have been to analyze in the two most common types of endometrial cancer, various molecular alterations that allow us to obtain specific genetic profiles, and to classify more precisely and objective these tumor types. To preform genetic alteration analyzes have been used the application of targeted sequencing (NGS) with a personalized panel of 40 genes to determine the altered genes with respect to somatic variants, and the MLPA technique to determine somatic alterations in the number of copies (ASNC) of 106 genes. Finally, the third objective was focused on understanding the role that intratumoral heterogeneity plays in CSE. The effect of HIT has been studied at two levels of molecular alteration, at the level of the genes determining somatic variants and the number of copies of the genes, using the two previous techniques. The results of the first objective of this thesis have shown the suitability of our personalized NGS study as an additional molecular tool to confirm the histological classification of CE. This strategy seems interesting as a tool for classifying tumors with unusual and ambiguous microscopic findings. The results of the second objective have described that in general the CSE has many more ASNCs than the EEC. However, it has also been shown that in the CSE there is a percentage of cases (42%) that have genetic characteristics that do not correspond to the phenotype, and therefore in these cases the use may be very helpful of the ASNC gene profile for more accurate classification. In addition, the combination of p53 determination by immunohistochemistry and the number of CCNE1 copies has been suggested to classify cases within the group serous-like. Finally, the results of the third objective have shown that CSE, on the one hand, has clonal molecular alterations such as the somatic variants in the TP53 gene and gains in the CCNE1 and PIK3CA genes, and on the other hand, that its intratumoral heterogeneity is characterized mainly by the ASNC of the genes. It has also been emphasized that one of the genes that is most affected by this heterogeneity is the ERBB2 gene, which is a widely used therapeutic target.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Vice Direção de Ensino, Informação e Comunicação. Rio de Janeiro, RJ, Brasil.
As tecnologias NGS (Next-Generation Sequencing), desenvolvidas para reduzir o custo e o tempo do processo de sequenciamento, geram uma grande massa de dados, a um custo relativamente baixo e com grande acurácia. No entanto, as leituras curtas, por elas produzidas, dificultam sobremaneira o processo de montagem de genomas, originando novos problemas computacionais. Para tentar suplantar esses desafios, várias ferramentas de software estão disponíveis e continuam a ser desenvolvidas. Cada um desses pacotes possui vantagens e desvantagens e, na maioria das vezes, se apresenta como uma solução individual, não estando integrado a outros. Além disso, tipicamente é exigido um conhecimento mais avançado de informática para a sua correta instalação, configuração e operação; o que, nem sempre, é a realidade do usuário final. Neste contexto, o projeto nomeado LASZLO (Linkage of Assembly Scripts Zero-costed and with License Opened) @ GALAXY propõe combinar diferentes ferramentas de tratamento de dados de NGS de uso livre, na forma de um protótipo básico de serviço de montagem de genomas, buscando facilitar o trabalho do usuário através da disponibilização de uma interface Web, sugestões de parametrização e de fluxos de trabalho para esse tipo de análise. Tomando por base o framework Galaxy, foram agregados fluxos de trabalho para montagens de dados de sequenciamento reais de diferentes organismos e provenientes das tecnologias Illumina, SOLiD™ e 454. O caráter aplicado do projeto originou soluções pontuais para atender a necessidades específicas, as quais foram reunidas sob o módulo NGS: LASZLO's Sandbox, uma "caixa de ferramentas" especialmente designada às abordagens de montagem do tipo de novo e com auxílio de genoma de referência. Durante a pesquisa, o protótipo LASZLO @ GALAXY processou, por exemplo, dados de sequenciamento de Leishmania amazonensis, contribuindo para um primeiro processo de avaliação do genoma do referido organismo. Atualmente, observa-se que a produção de dados não é o mais o "gargalo" em projetos de sequenciamento, mas sim o fluxo de análise subsequente sobre o material obtido. Muitas vezes, tais dados não se traduzem imediatamente em expansão do conhecimento biológico, devido às dificuldades encontradas pelo biólogo experimental em lidar, não somente com a miríade de ferramentas disponíveis, mas também com fatores como a inerente necessidade de integração entre elas e a implementação de infra-estrutura adequada para a sua operação. Os resultados obtidos no projeto indicam que o sistema proposto, vislumbrado como um eventual serviço institucional ou mesmo de menor âmbito, pode se tornar um aliado do usuário final quanto à manipulação dos dados de NGS.
The NGS (Next-Generation Sequencing) technologies, designed to reduce sequencing process costs and time, generate a huge amount of data, at a relatively low cost and with great accuracy. However, the produced short reads strongly difficult the genome assembly process, originating new computational issues. To overcome those challenges, there are several software tools available and continuously being developed. Each of these tools presents advantages and disadvantages and most of them are isolated, not integrated solutions. Moreover, typically it is required a higher level of computer-literacy for their proper installation, configuration and usage, which, not always, is the end-user reality. In this context, the project named LASZLO (Linkage of Assembly Scripts Zero-costed and with License Opened) @ GALAXY suggests to combine different open source tools for NGS data handling, as a basic prototype service for genome assembly, aiming at simplifying the end-user task by providing a Web interface, suggestions of parametrization and workflows for this kind of analysis. Based on the Galaxy framework, some workflows for the assembly of real sequencing data from different organisms and produced by the Illumina, SOLiD™ and 454 technologies were aggregated. Also, due to the applied characteristic of the project, a few punctual solutions were generated to address specific needs. Those solutions were encapsulated in the NGS: LASZLO's Sandbox module, a "toolbox" especially tailored for the de novo and reference-guided assembly approaches. During the research, the LASZLO @ GALAXY prototype processed, for instance, sequencing data of the Leishmania amazonensis organism, contributing for a first evaluating process of its genome. Presently, it's noticed that the data generation is no longer the "bottleneck" of the sequencing projects, but the downstream data analysis. Frequently, the acquired data is not immediately translated into biological knowledge expansion, due to the obstacles met by the experimental biologist when dealing, not only with the myriad of available tools, but also with factors like the inherent need of their integration and the deployment of the adequate infrastructure for their operation. The results achieved during project execution indicate that the proposed system, glimpsed as an eventual institutional service or even as one of smaller scope, might become an end-user's ally in the NGS data manipulation.

Le syndrome de Usher (USH) est une maladie transmise selon le mode autosomique récessif caractérisée par l’association d’une surdité congénitale (HL) et d’une rétinite pigmentaire (RP), et dans certains cas, d’une aréflexie vestibulaire. Une hétérogénéité clinique et génétique est reconnue. Environ 10 % des cas USH restent non résolus après analyse moléculaire exhaustive des différents gènes. Ces cas incluent les patients qui ne portent aucune mutation dans un des gènes USH connus ainsi que les patients porteurs d’une seule mutation dans un gène USH. Au cours de cette thèse, nous nous sommes intéressés à l’étude des patients porteurs d’une seule mutation dans les gènes USH2A et PCDH15.Dans la première partie de la thèse, nous avons analysé une cohorte de patients avec un phénotype USH2A bien défini : 5 patients pour lesquels une seule mutation à l’état hétérozygote avait été identifiée dans le gène USH2A et un patient porteur d’un variant silencieux en trans d’une mutation non-sens.Pour les 5 patients, nous avons émis l’hypothèse que la seconde mutation, restant à être identifiée, pourrait se trouver dans des régions introniques profondes. Pour cela, nous avons développé une approche de séquençage à haut débit (NGS) de l’ADN pour identifier les variants introniques profonds dans le gène USH2A et évaluer leurs conséquences sur l’épissage. Comme preuve de concept et pour valider l’approche, y compris le pipeline bio-informatique et l’évaluation des outils de prédiction de l’épissage, nous avons analysé un patient porteur d’un pseudoexon (PE) connu dans le gène USH2A. Ensuite, les 5 patients ont été étudiés en utilisant le pipeline défini, ce qui a conduit à l’identification de 3 nouveaux variants introniques profonds chez 4 d’entre eux. Tous les variants ont été prédits comme pouvant avoir un impact sur l’épissage et aboutir à l’insertion de PE. Ces prédictions ont été validées par les essais minigènes. Grâce à cette étude, nous présentons une stratégie innovante pour identifier les mutations introniques profondes, lorsque l’analyse des transcrits n’est pas possible. Par ailleurs, le pipeline bio-informatique développé fonctionne indépendamment de la taille du gène analysé, ce qui permet l’application possible de cette approche à n’importe quel gène. Par ailleurs, un oligonucléotide antisens de type morpholino (AMO) a été évalué in vitro afin de rétablir l’altération d’épissage induite par une des mutations identifiées. Les résultats ont montré un taux d’exclusion élevé du transcrit aberrant et suggèrent une application possible en thérapie moléculaire. Nous avons ensuite effectué des études sur le variant USH2A c.1377T>A, un variant silencieux afin d’évaluer son effet sur l’épissage. L’analyse de l’ARN issu de cellules nasales du patient a montré que ce variant conduit au saut de l’exon 8 dans les transcrits USH2A. Ceci a été confirmé par un essai minigène. En outre, des études préliminaires ont été réalisées en utilisant des outils de prédictions et des essais minigènes pour évaluer l’implication des éléments cis-régulateurs dans le défaut d’épissage observé chez le patient. Dans la deuxième partie de la thèse, nous avons analysé une patiente USH1, pour laquelle une seule mutation avait été identifiée dans le gène PCDH15. Dans ce cas, nous avons combiné la culture des cellules épithéliales nasales avec l’analyse des transcrits PCDH15. Celle-ci a été réalisée par séquençage de cinq RT-PCR chevauchantes. Grâce à cette analyse, nous avons réussi à délimiter une région d’intérêt dans le transcrit, dont l’amplification a échoué exclusivement pour l’allèle porteur de la mutation non identifiée. D’autres analyses ont été effectuées dans la région génomique correspondante par capture ciblée couplée au séquençage NGS et LongRange PCR suivi de séquençage Sanger. Cependant, aucun variant candidat n’a été identifié à ce jour. Nous suggérons l’implication de mécanismes moléculaires complexes qui restent à être caractérisés
Usher syndrome (USH) is an autosomal recessive disorder characterized by the association of sensorineural hearing loss (HL) and retinitis pigmentosa (RP), and in some cases, vestibular areflexia. Clinical and genetic heterogeneity are recognised. Indeed, three clinical types can be caused by mutations in one of the 10 known genes and USH2A represents the most frequently involved gene.Approximately 10 % of the USH cases remain genetically unsolved after extensive molecular analysis of the different genes, which includes sequencing of the exons and their intronic boundaries, combined to large rearrangements screening by array CGH. These unsolved cases include patients who do not carry any mutation in any of the known USH genes and patients who carry a single USH mutation. During this thesis we focalised on the study of patients carrying a single mutation in USH2A and PCDH15 gene.First, we have analysed a cohort of well-defined USH2A patients: five patients, for whom a single USH2A heterozygous mutation had been identified and one patient carrying a silent variant in trans to a nonsense mutation. For the 5 patients, we supposed that the second mutation remaining to be found could be localised deep in the introns. Indeed, a deep intronic mutation resulting in the inclusion of a pseudoexon (PE 40) in USH2A transcripts had been identified, following RNA analysis from nasal cells. Unfortunately, analysing USH2A transcripts still represent a challenging approach in a diagnostic settings and it is not always possible. To circumvent this issue, we have developed a DNA-Next Generation Sequencing (NGS) approach to identify deep intronic variants in USH2A and evaluate their consequences on splicing. As a proof of concept and to validate this approach, including the bioinformatics pipeline and the assessment of splicing predictor tools, the patient carrying the PE 40 was analysed at first. Then, the 5 patients were studied using the defined pipeline, which led to the identification of 3 distinct novel deep intronic variants in 4 of them. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. Through this study, we present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. In addition, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Moreover, an antisense morpholino oligonucleotide (AMO) tested in vitro for its ability to restore the splicing alterations caused by one of the identified mutation provided high inhibition rates. These results are indicative of a potential application for molecular therapy.In the second case, we have performed studies on the USH2A c.1377T>A silent variant to investigate its effect on splicing. Analysis of RNA from nasal cells of patients showed that this variant led to the skipping of exon 8 in USH2A transcripts. This was confirmed by minigene assay. Moreover, preliminary studies have been performed using prediction tools and minigene assays to assess the involvement of cis-acting elements in causing the aberrant splicing.In the second part of the thesis, we have analysed an USH1 patient, for whom only one mutation had been identified in the PCDH15 gene. In this case, we combined nasal epithelial cells culture with the analysis of the PCDH15 transcripts. This was performed by sequencing five overlapping RT-PCRs. Through this analysis, we were able to delimit a region within the transcript, which failed to be amplified exclusively in the allele carrying the unidentified mutation. Further analyses have been performed in the corresponding genomic region by NGS-target capture and LongRange PCR associated with Sanger sequencing. However, no evident mutation has been identified so far. Therefore, we suggest the involvement of complex molecular mechanisms that remain to be characterised

The liquid biopsy is a new emerging and repeatable low risky approach able to detect drive mutations that characterize the tumor, to monitor cancer evolution over time, and to overcome the standard tissue biopsy limits. The biomarker par excellence is the circulating cell-free DNA (cfDNA) that was the principal leading actor of this study. The scope of this study was to perform different liquid biopsy analysis both in metastatic cancer and in vascular malformations patients to detect, from a precision medicine perspective, the sniper clone responsible for the tumor evolution or the vascular malformations. The cfDNA was extracted from plasma coming from peripheral and/or efferent vein of vascular malformation. The obtained cfDNA was used to perform the libraries using two different genes panel of 52 and 77 cancer-driver genes, respectively the Oncomine™ Pan-Cancer Cell-Free Assay and AVENIO ctDNA Expanded Kit. The most frequent mutations that we found in metastatic patients were the SNV in TP53, follow by PIK3CA, KRAS, and CNV in FGFR3. In the majority of cases, the mutations found at first liquid biopsy were confirmed by an increased allele frequency at the second one. In vascular anomalies affected patients, the PIK3CA, MET, and KRAS mutated genes were found in Klippel-Trenaunay syndrome, in lymphovenous malformations, and in artero-venous malformations respectively, with a very low allele frequency percentage. In conclusion, repeated analysis of liquid biopsy lead to the identification of key cancer genes and the following of clonal evolution over time. Moreover, the liquid biopsy is suitable not only for cancer patients but also for the diagnosis of vascular malformation. Our data prove that in the new era of precision medicine, this novel approach, based on the combination of NGS and liquid biopsy from the efferent vein at the vascular malformation site, allows to detect even low-grade somatic mosaicism responsible for the vascular phenotype. This approach let to bypassing the need for a highly risky tissue biopsy and lead to a tailored personalized treatment.

Le tecnologie NGS hanno rivoluzionato il mondo della genetica e della medicina, influenzando fortemente la diagnosi delle malattie ereditarie. Il grande numero di applicazioni, sia di diagnostica che di ricerca, ha generato la necessità di adattare l’analisi dei dati prodotti da queste tecnologie per ottimizzare la risposta ai problemi specifici. Il processo di analisi è implementato tramite trasformazioni consecutive dei dati genetici (pipeline) utilizzando un grande numero di tool e software bioinformatici. Spesso le performance dei diversi tool dipendono dal tipo dei dati in ingresso e l’integrazione dei software adatti ai diversi tipi di dati è diventato un passaggio critico per la qualità delle informazioni prodotte. Inoltre, l’utilizzo dei tool, la loro configurazione, la progettazione di pipeline robuste e lo sviluppo di nuove soluzioni di analisi, sono processi complessi che richiedono competenze di coding e la conoscenza dell’esteso panorama bioinformatico. I laboratori che non dispongono di personale specializzato in applicazioni bioinformatiche, possono incontrare difficoltà nell’ottimizzazione del workflow analitico, che spesso viene affidato a software commerciali che applicano uguali regole e sistemi a tutti i geni indistintamente. Durante il percorso di dottorato di ricerca effettuato presso il Centro malattie genetiche cardiovascolari dell’ospedale San Matteo di Pavia, è stata sviluppata la piattaforma Helper. Helper è nata per la progettazione e l’adattamento semplificato delle pipeline bioinformatiche dedicate all’analisi di dati NGS derivati da applicazioni di targeted sequencing. Helper è dotato di una semplice interfaccia grafica mirata a facilitare l’esperienza di sviluppo dei processi analitici bioinformatici anche per chi non possiede particolari conoscenze di sviluppo di codice. Tramite Helper è possibile scegliere quali step effettuare nel workflow di analisi e quali evitare, quali tools e software utilizzare in ogni step selezionato, e con quali argomenti settare i tool utilizzati. Helper permette inoltre di utilizzare le pipeline, progettate ed effettuare l’analisi dei dati NGS, ed è modificabile in base all’esperimento di sequenziamento dal quale derivano i campioni e in base al tipo e all’organizzazione dei campioni. Helper può essere utilizzato sia su workstation, sia su un comune PC, dimostrandosi compatibile con i tempi di analisi dei laboratori di genetica anche in presenza di soluzioni a bassa capacità computazionale. Nel workflow di analisi genetica, Helper è dedicato a quella che è definita come analisi secondaria, che trasforma i dati NGS grezzi in un set di varianti utili all’interpretazione del test genetico. Il lavoro di tesi si è proposto inoltre di introdurre due questioni fondamentali per la diagnosi genetica. La prima è rappresentata dal problema della classificazione patogenica delle varianti identificate dall’analisi bioinformatica. La classificazione delle varianti è un processo delicato a causa della difficoltà esistenti nel trovare regole uniformi e robuste da applicare a tutti i difetti genici. In questa tesi viene proposto un sistema di classificazione per le varianti del gene DES, che prende in considerazione le caratteristiche specifiche del gene che codifica per la proteina di Desmina. Il secondo è l’identificazione dei geni responsabili di un determinato fenotipo, necessaria per l’ottimizzazione del test diagnostico e per la gestione dei pazienti. In questo contesto viene approfondito il problema dei tumori ereditari della mammella e dell’ovaio, tramite lo studio dei risultati di analisi del database genetico sviluppato presso il San Matteo per il monitoraggio delle cause genetiche delle patologie oncologiche.
Next generation sequencing (NGS) technologies have revolutionized the world of genetics and medicine, strongly influencing the diagnosis of hereditary diseases. The large number of applications, both diagnosis, and research, has generated the need to adapt the analysis of the data produced by these technologies to optimize the clinical path of many human diseases. The analysis process is implemented through consecutive modifications of the genetic data (pipeline) using bioinformatics tools and software. Often, the performance of the different tools depends on the type of input data; the integration of software suitable for different types of data is a critical step for the quality of the information produced. Furthermore, the use of bioinformatics tools, their configuration, the design of robust pipelines, and the development of new analysis solutions is a complex process that requires coding skills and knowledge of the wide range of existing tools. In this context, bioinformaticians achieved a key role within genetics laboratories, thanks to the skills of developing computer systems combined with the integration of knowledge on target biology systems and related applications; these “in house” tailored activities favor the adaptation of the analyses to each specific questions/objectives. Laboratories using outsourcing analysis tools or entrusting to commercial software that apply the same rules and systems to all genes, without distinction, often face difficult optimization of the analytical workflow. Hence, the growing need for simple and fast tools that can support professionals with limited computer skills in the design of customized pipelines and their use to analyze NGS data. During the PhD course carried out at the Center for Cardiovascular Genetic Diseases of the San Matteo Hospital in Pavia, the Helper platform was developed. Helper was born for the design and simplified adaptation of bioinformatics pipelines for the analysis of NGS data derived from targeted sequencing applications. Helper is equipped with a simple graphic interface aimed at facilitating the development experience of bioinformatics analytical processes even for professionals who do not have coding knowledge. Helper allows the selection of: the steps to carry out (or to avoid) in the analysis workflow; the tools and software to use in each selected step; the arguments to set the tools employed in each application. Helper further allows the use of the pipelines, the design and carrying out of the analysis of NGS data; it can be modified based on the sequencing experiment from which the samples are derived, and on the basis of the organization of the samples. Helper can be used both on a workstation and on a common PC, proving to be compatible with the analysis times of the genetics laboratories even in the presence of solutions with low computational capacity. In the genetic analysis workflow, Helper is part of the process of translating raw NGS data into a set of variants useful for the interpretation of the genetic test. The thesis finally aimed at addressing two fundamental questions for genetic diagnosis. The first question addresses the complex issue of the variant classification as identified by bioinformatics analysis. The classification of genetic variants is a process that reflects difficulties in finding uniform and robust rules shared by all genes. In this thesis, a classification system is proposed for the variants of the DES gene, which takes into consideration the specific characteristics of the gene encoding the Desmin protein. The second question addressed the identification of the genes responsible for a specific phenotype, necessary for the optimization of the diagnostic test and for patient management. In this context, hereditary breast and ovarian tumors is investigated through the study of the results of the analysis of the genetic database developed at San Matteo for monitoring the genetic basis of oncological diseases.

Le développement des méthodes de séquençage de nouvelle génération a permis la production de grandes quantités de données à moindre coût. Cependant, les fragments obtenus, appelés reads, possèdent des longueurs plus courtes et des taux d’erreurs plus élevés que ceux obtenus avec les premières méthodes de séquençage. Cela a créé de nouveaux défis pour l’assemblage de génomes. Même si de nombreux assembleurs sont publiés chaque année et que les algorithmes sont de plus en plus élaborés, la reconstruction d’un génome entier de novo, en l’absence de génome de référence, reste un problème difficile. Une des principales causes est la présence des répétitions dans les génomes. Cette thèse décrit des algorithmes visant à améliorer l’assemblage de novo de répétitions. Nous présentons d’abord nos solutions axées sur les répétitions en tandem. L’algorithme appelé DExTaR a été conçu pour améliorer la détection de répétitions en tandem exactes suite à un assemblage de novo global basé sur l’approche de de Bruijn. Le second algorithme, appelé MixTaR, effectue seulement des assemblages locaux afin de détecter des répétitions en tandem exactes et approximatives. En utilisant deux types de reads, courts et longs, MixTaR ne requiert pas un assemblage global préalable. Nous roposons ensuite plusieurs algorithmes pour simplifier le problème d’assemblage basé sur une nouvelle structure de données, le graphe de de Bruijn pairé. Ce graphe inclut les informations des reads pairés dès le début du processus d’assemblage afin d’améliorer la détection de répétitions et la qualité de l’assemblage
The development of the next-generation sequencing methods has allowed the generation of vast amounts of data at a lower cost and time. However, the fragments obtained, called reads, have shorter lengths and higher error rates that the ones obtained with the first sequencing methods. This new type of data created new challenges in genome assembly. Even though many assembly software are published every year and algorithms are becoming more and more complex, reconstructing a whole genome de novo, in the absence of a reference genome, remains a difficult problem. One of the main causes is represented by the presence of repetitive regions in the genomes. This thesis describes algorithms designed to improve the de novo assembly of repeats. We first present our solutions focused on tandem repeats. The algorithm called DExTaR aims at extending the work done by a de novo assembly in the detection of exact tandem repeats. Based on a de Bruijn graph constructed by an assembler, our approach assembles new exact tandem repeats by analysing the parts of the graph left unresolved. The second algorithm, called MixTaR, performs only local assemblies in order to detect exact and approximate tandem repeats. Using the two types of reads obtained by the new sequencing methods, short and long reads, MixTaR does not require a global de novo assembly. We then propose several algorithms for simplifying the assembly problem based on a new data structure, the paired de Bruijn graph. This graph uses the paired-end information from the beginning of the assembly process as a solution to a better repeat detection and higher quality results

Le virus de l'hépatite C (VHC) est une menace majeure avec plus de 130 millions de personnes infectées chaque année. Il constitue la principale cause de cancer du foie. Le VHC est un virus transmis par le sang soit au cours de consommation de drogue par voie intraveineuse soit lors de transfusions sanguines. Il s'adapte à l'environnement de l'hôte grâce à un taux de mutation élevé qui amoindrit l’efficacité des traitements. Le virus se multiplie rapidement dans l'hôte et crée ainsi une population de virus génétiquement hétérogènes, appelée quasi-espèces, qui peut ainsi répondre aux pressions sélectives liées au traitement. Les traitement antiviraux existants sont des tri-thérapies contenant des peg-interféron, de la ribavirine et des inhibiteurs de la protéine (PI). Les inhibiteurs comme la telaprevir ou la bocéprévir ciblent la région NS3 du génome en bloquant le mécanisme de réplication. Cependant, en raison de la nature dynamique des quasi-espèces, les séquences cibles sont variables et les inhibiteurs conçus pour se lier à une région génomique particulière sont rendus inefficaces.Nous analysons ces populations virales en utilisant les techniques modernes de séquençage et le pyroséquencage profond qui permet l’analyse à grande échelle des données génétiques. La technique “Amplicon Sequencing” permet de cibler des régions particulières du génome viral, comme les régions NS3 ou NS5B qui participent au mécanisme de réplication et qui sont des cibles pour les thérapies antivirales. Par rapport au séquençage Sanger, notre pipeline NGS permet d’appréhender l’hétérogénéité de la population virale au sein d’un hôte. Pour analyser les données NGS, nous avons implémenté un pipeline d’analyse bioinformatique qui a été automatisé avec eHive.Nous étudions des échantillons de VHC de 40 patients traités par trithérapie. Deux sources de cellules virales sont utilisées pour le séquençage: les cellules du plasma et les cellules mononuclées du sang périphérique. L'objectif est de vérifier si une analyse des mutations de la région génomique NS3 peut aider à prédire le résultat du traitement. Nous constatons que des mutations de résistance aux antiviraux se trouvent à la fois chez les individus qui ont répondu et qui n’ont pas répondu au traitement. Nous avons donc recherché d'autres signatures génétiques de l'échec du traitement. Nous constatons que l'hétérogénéité génétique est plus faible chez les individus qui répondent de manière favorable au traitement. Notre conclusion est que l'hétérogénéité virale est un facteur indépendant pour prédire la réponse à un traitement, en plus de la présence de mutations spécifiques dans les régions ciblées par le traitement.Les techniques NGS permettent également d’étudier l'évolution virale au sein d'un seul hôte. En utilisant de multiples temps d'échantillonnage, nous pouvons mesurer les caractéristiques de l'évolution de la population virale. Pour trois patients avec des échantillons viraux couvrant une période de 13 ans, nous avons utilisé la technique “Amplicon Sequencing“ pour les régions NS3 et NS5B. Des infections mixtes comprenant de multiples génotypes sont retrouvées chez deux patients. Nous avons montré qu’il existe de la structure de populations et des lignées divergentes de VHC au sein de chaque patient. Au cours du traitement, l'hétérogénéité génétique et la taille efficace de la population dans la région NS5B augmente fortement après le début du traitement. Ces résultats mettent en évidence un processus de sélection diversifiante suite au traitement qui augmente l'hétérogénéité génétique virale. Nous mettons ainsi en évidence un processus dit de balayage sélectif doux qui est observé pour la première fois chez des patients infectées par des génotypes multiples du virus VHC.Notre analyse NGS montre que l'hétérogénéité génétique du VHC est liée à l'échec ou à la réussite du traitement et que son évolution permet de mieux comprendre la façon dont les virus s'adaptent au traitement
Hepatitis C virus (HCV) is a major threat to global health, with over 130 million annual infections. HCV is a blood borne virus transmitted primarily via intravenous drug use or hospital transfusions. It infects the liver cells and is the leading cause of liver cancer. It adapts to the host environment with a high mutation rate and can make efficient treatment very difficult. Due to poor replication proofreading, the virus multiplies rapidly in the host and creates a population of viruses which is genetically heterogeneous enough to escape selective pressures. This HCV population called quasispecies is found within and between infected hosts. Current antiviral treatment consists of a triple therapy of peg-Interferon, ribavirin and protein inhibitors (PI). PIs such as telaprevir, boceprevir target the NS3 region of the genome, blocking the replication mechanism. However due to the highly dynamic nature of the quasispecies, the target sequences are variable and PIs designed to bind to a particular genomic region are therefore rendered ineffective.We analyse viral populations of HCV using Next generation Sequencing (NGS) technologies and ultradeep pyrosequencing, which allow for rapid and large scale analysis of genetic data. Amplicon sequencing allows for targeting particular regions of the viral genome, such as the NS3 or NS5B which form a part of the replication mechanism and hence are targets for antiviral therapy. Compared to Sanger sequencing, our NGS pipeline ascertains viral population heterogeneity within a host. We implemented the bioinformatics workflow manually and in eHive as an automated pipeline.We study HCV samples from 40 patients treated with triple therapy. Two sources of the virus, plasma and peripheral blood mononuclear cells are used for sequencing. The main aim is to check if a baseline analysis of the NS3 genomic region can help to predict the outcome of the treatment. We find that antiviral resistance mutations are found in both responders and non-responders to the treatment. Since no correlation exists between observed mutations and failure of tri-therapy, we look for other genetic signatures of treatment failure. We find that genetic heterogeneity, calculated using Shannon’s entropy, is lower in responders. We conclude that the viral heterogeneity can be used as an independent factor to predict response to treatment, more than presence of specific mutations at baseline.NGS also enables large-scale studies of viral evolution within a single host. Using multiple sampling time points, we gain insights about viral evolutionary characteristics of HCV and responses to selective pressures during infection. For three patients with viral samples covering a period of 13 years, we perform amplicon sequencing on the NS3 and NS5B regions. Mixed infections comprising of multiple genotypes are found in two patients. We find considerable population structure and diverging HCV lineages within each patient. Over the course of treatment, genetic heterogeneity and effective population size in the NS5B regions increases sharply after treatment initiation compared to baseline. These results provide evidence of diversifying selection occurring post-treatment, acting on standing genetic variation resulting in high genetic heterogeneity. These are characteristics of a soft selective sweep, which is observed for the first time in chronic HCV patients infected with multiple genotypes.Our NGS analysis show that genetic heterogeneity in HCV is related to treatment failure and that its evolution provides insights about how viruses adapt to treatment

Orientador: Fausto Foresti
Resumo: Characidium é um grupo de peixes amplamente distribuídos pela região Neotropical, embora seja considerado o mais especioso dentro de Crenuchidae, do ponto de vista citogenético o número de espécies investigadas ainda é baixo, o que dificulta a caracterização quanto a organização cromossômica do gênero. Em relação ao número diploide, as espécies de Characidium conservaram um cariótipo com 2n = 50 cromossomos, do tipo metacêntricos e submetacêntricos (com exceções), o que resulta em uma macroestrutura homogênea para o grupo. Porém, investigações utilizando sequências repetitivas têm contribuído para ilustrar que a organização microestrutural cromossômica pode diferir entre as espécies, refletindo o hábito destes peixes constituírem populações pequenas e isoladas em cabeceiras de riachos. Adicionalmente, algumas espécies de Characidium também foram descritas portando cromossomos B em seus cariótipos, e a utilização de ferramentas citomoleculares têm contribuído para explorar quanto a origem e evolução destes componentes cariotípicos. Neste sentido, o objetivo do presente estudo foi agregar técnicas citomoleculares com resultados de sequenciamento massivo, para tentar compreender a ocorrência de cromossomos B no genoma de Characidium sp. aff. C. vidali. Os resultados obtidos mostraram que i) o mapeamento físico de diferentes sondas de DNA repetitivo contribuíram não apenas para caracterizar o cariótipo da espécie em estudo, como também adicionaram mais informações quanto a organi... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Characidium is a group of fish widely distributed in the Neotropical region, although it is considered the most specious within Crenuchidae, from the cytogenetic point of view the number of species investigated is still low, which makes it difficult to characterize the chromosomal organization of the genus. In relation to the diploid number, Characidium species retained a karyotype with 2n = 50 chromosomes, metacentric and submetacentric (with exceptions), resulting in a homogeneous macrostructure for the group. However, investigations using repetitive sequences have contributed to illustrate that the chromosomal microstructural organization may differ between species, reflecting the habit of these fish constituting small and isolated populations in headwaters of streams. In addition, some species of Characidium have also been described carrying B chromosomes in their karyotypes, and the use of cyto-molecular tools has contributed to explore the origin and evolution of these karyotype components. In this sense, the objective of the present study was to aggregate cyto-molecular techniques with massive sequencing results to try to understand the occurrence of B chromosomes in the genome of Characidium sp. aff. C. vidali. The results showed that i) the physical mapping of different repetitive DNA probes contributed not only to characterize the karyotype of the species under study, but also added more information about the organization and evolution of the chromosomal microstruct... (Complete abstract click electronic access below)
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Several hypotheses have been formulated to explain Amazonian biodiversity patterns, whose biotic diversification has been seen as a result of historically complex scenarios covering a wide range of temporal and spatial scales. Anurofauna has the potential to enhance our understanding of the biogeographic patterns of diversification and processes of speciation, since it serves as a model for inferring historical events. However, the challenge for those seeking to elucidate the processes of diversification of Amazonian frogs is that large portion of its diversity is cryptic, which result in an inaccuracy of limits and distributions of species, which drastically alters our perception of structuring of biodiversity and obscures biogeographic patterns. One of the components of the Amazon anurofauna is the species Hypsiboas cinerascens, which was used as a model to investigate and contribute to the knowledge of the patterns of genetic distribution of anurofauna in the Amazon. Given its wide geographic distribution, some authors have indicated the existence of a species complex with molecular data (mitochondrial gene sequences and genomic data) providing of important tools for the delimitation of evolutionary lineages and their distributional limits, thus clarifying current taxonomy, and for the identification of cryptic species, and thus biogeographic patterns. Given the above, we used sequences of mitochondrial genes 16S RNA and cytochrome b together with the new8 generation sequences (ddRAD-tags) to study the distribution patterns of genetic diversity of H. cinerascens in the Amazon and so testing for cryptic lineages, and inferring biogeographic patterns of the lineages found. Through genetic distance analyses and formation of biological groups with 16S rRNA, concatenated phylogeny of the mitochondrial 16S rRNA and Cytochrome B genes, phylogenomic analyses of the ddRAD-tags, and estimating the time of divergence of both genomes, we identified the possible existence of nine evolutionary lineages in H. cinerascens that originated in the Miocene to the Pliocene: Japurá-Peru, Manaus-Juruti-Guyana, Matupiri-Purus, Santarém-Alta Floresta, Tefé-Jutaí, Morrinho-Rondônia, Uacari, French Guiana and Negro-Trombetas. The filogenomic analyzes confirmed the lineages found in the mtDNA, but with some discrepancies between the topologies. Due to the robustness of the dating of gDNA, we use it to infer the biogeographical history of the group. We suggested that the transcontinental formation of the Amazon River in the last 10 Ma may have been the precursor event for the diversification of the lineages, but due to the complexity of the relationships between groups and the lack of sampling throughout the complete distribution of the H. cinerascens species complex, possibly different historical and ecological factors events influenced their distributions, which can not be identified accurately with our data. The Negro-Trombetas lineage has a distinct biogeographic history of the other lineages of the complex, which may be associated with open forest environments in the region of Guyana. In the future a taxonomic revision of the group should be carried out to verify the existence of new species.
Diversas hipóteses foram formuladas para explicar os padrões de biodiversidade amazônica, cuja diversificação biótica tem sido vista como um produto que envolve cenários historicamente complexos e que abrangem uma ampla gama de escalas temporais e espaciais. A anurofauna possui o potencial de aprimorar o entendimento dos processos biogeográficos nos padrões de especiação e diversificação, já que serve como modelo para inferir eventos históricos. Porém, o desafio para quem busca elucidar o processo de diversificação de anuros amazônicos é que grande parcela de sua diversidade é críptica, que tem por consequência uma imprecisão de limites e distribuições das espécies, o que altera drasticamente a nossa percepção da estrutura da biodiversidade e oculta padrões biogeográficos. Um dos componentes da anurofauna amazônica é a espécie Hypsiboas cinerascens, que foi utilizada como modelo para investigar e contribuir com o conhecimento sobre os padrões de distribuição genética da anurofauna na Amazônia. Considerando sua ampla distribuição geográfica, alguns autores indicam a existência de um complexo de espécies e os dados moleculares (sequências de genes mitocondriais e dados genômicos) são importantes ferramentas para a delimitação de linhagens evolutivas e seus limites de distribuição, de forma a clarificar a taxonomia vigente, bem como para a identificação de espécies crípticas, e consequentemente a mostrar padrões biogeográficos. Conforme o exposto, utilizamos o sequenciamento dos genes mitocondriais 16S rRNA e Citocromo b juntamente com o sequenciamento de nova geração (ddRAD-tags), para estudar os padrões de distribuição da diversidade genética de H. cinerascens na Amazônia e assim testar a presença de linhagens crípticas, inferindo padrões biogeográficos sobre as linhagens encontradas. Por meio de análises de distâncias genéticas e formação de grupos biológicos com o 16S rRNA, filogenia concatenada dos genes mitocondriais 16S rRNA e Citocromo B, filogenômica dos ddrad-tags, e estimação do tempo de divergência de ambos genomas, definimos a possível existência de 9 linhagens evolutivas em H.. cinerascens que se originaram do Mioceno ao Plioceno: Japurá- Peru, Manaus-Juruti-Guiana, Matupiri-Purus, Santarém-Alta Floresta, Tefé-Jutaí, Morrinho- Rondônia, Uacari, Guiana Francesa e Negro-Trombetas. As análises filogenômicas confirmaram as linhagens encontradas com o mtDNA, porém com algumas discordâncias entre as topologias. Devido a maior robustez da datação do gDNA, a utilizamos para inferir a história biogegráfica do grupo. Sugerimos que a formação transcontinental do Rio Amazonas nos últimos 10 Ma pode ter sido o evento precursor da diversificação de linhagens, porém devido a complexidade das relações entre os grupos e a falta de amostragem da completa distribuição da espécie, possivelmente diferentes eventos históricos e fatores ecológicos influenciaram as suas distribuições, nos quais não podem ser definidos com exatidão com os nossos dados. A linhagem Negro-Trombetas possui uma história biogeográfica distinta das outras linhagens do complexo, que pode estar associada a ambientes florestais mais abertos na região das Guianas. Futuramente deve ser realizada a revisão taxonômica do grupo para verificar a existência de novas espécies.

Décrypter les données omiques : importance du contrôle qualité. Application au cancer de l’ovaire Au cours des dix dernières années, la taille et la complexité des données biologiques ont littéralement explosé, et une attention particulière doit être portée au contrôle qualité. En effet, certaines données omiques (données génomiques et post-génomiques obtenues à haut débit) sont très incomplètes et/ou contiennent de nombreux biais et erreurs qu’il est facile de confondre avec de l’information biologiquement intéressante. Dans cette thèse, nous montrons que les interactions protéine-protéine issues de curation de la littérature et les interactions identifiées à haut débit sont beaucoup plus corrélées que ce qui est communément admis. Nous examinons l’interactome de la levure d’un point de vue original, en prenant en compte le degré d’étude des protéines par la communauté scientifique et nos résultats indiquent que cette corrélation s’estompe lorsqu’on se restreint aux protéines très étudiées. Ces observations nous permettent de proposer une méthode simple et fiable pour estimer la taille d’un interactome. Notre méthode conduit à une estimation d’au moins 37 600 interactions physiques directes chez S. cerevisiae, et montre que les évaluations précédentes sont trop faibles. Par ailleurs, nous étudions des données de séquençage nouvelle génération de l’ADN. Par une analyse des biais existant entre les short-reads alignés sur un brin ou sur l’autre du génome, nous mettons en évidence de nombreuses erreurs systématiques. De plus, nous observons de multiples positions présentant entre 20 et 40% de short-reads portant l’allèle variant : celles-ci ne peuvent pas être génotypées correctement. Nous proposons une méthode fiable pour appeler les génotypes à partir des données NGS qui permet de s’affranchir de ses difficultés. Enfin, nous appliquons cette méthode sur des données massives de séquençage d’exome de cellules saines et tumorales de 520 patientes atteintes du cancer de l’ovaire, produites par le consortium TCGA. Nous détectons en moyenne 30 632 variants germinaux par patiente. Parmi ces variants, nous identifions ceux les plus enclins à conférer un risque accru de développer la maladie : nous nous restreignons notamment aux variants induisant une perte de fonction de la protéine encodée et significativement plus présents chez les patientes que dans la population générale. Cela conduit à 44 SNVs par patiente en moyenne, répartis sur 334 gènes dans l’ensemble de la cohorte. Parmi ces 334 gènes, 42 ont été reportés comme impliqués dans la cancerogénèse, confirmant que la liste de candidats identifiés est fortement enrichie en gènes de susceptibilité au cancer de l’ovaire. En particulier, nos travaux confirment le rôle de suppresseur de tumeur de la protéine MAP3K8, très récemment proposée comme jouant un rôle clé dans d’autres cancers
Deciphering omics data : on the importance of quality control. Application to ovarian cancer. Over the past 10 years, the size and complexity of biological data have exploded, and quality control is critical to interpret them correctly. Indeed, omics data (high- hroughput genomic and post-genomic data) are often incomplete and contain bias and errors that can easily be misinterpreted as biologically interesting findings. In this work, we show that literature-curated and high-throughput protein-protein interaction data, usually considered independent, are in fact significantly correlated. We examine the yeast interactome from a new perspective by taking into account how thoroughly proteins have been studied, and our results show that this bias can be corrected for by focusing on well- studied proteins. We thus propose a simple and reliable method to estimate the size of an interactome, combining literature-curated data involving well-studied proteins with high- hroughput data. It yields an estimate of at least 37,600 direct physical protein-protein interactions in S.cerevisiae, a significant increase over previous estimates. We then focus on next-generation DNA sequencing data. An analysis of the bias existing between short- eads aligned on each strand of the genome allows us to highlight numerous systematic errors. Furthermore, we observe many positions that exhibit between 20 and 40% of reads carrying the variant allele : these cannot be genotyped correctly.We then propose a method to overcome these biases and reliably call genotypes from NGS data. Finally, we apply our method to exome-seq data produced by the TCGA for tumor and matched normal samples from 520 ovarian cancer patients. We detect on average 30,632 germline variants per patient. Though an integrative approach, we then identify those which are likely to increase cancer risk : in particular, we focused on variants inducing a loss of function of the encoded protein, and selected those that are significantly more present in the patients than in the general population. We find 44 SNVs per patient on average, impacting 334 genes overall in the cohort. Among these genes, 42 have been previously reported as involved in carcinogenesis, confirming that our list is highly enriched in ovarian cancer susceptibility genes. In particular, our results confirm the tumor suppressor role of the MAP3K8 protein, recently identified in other types of cancer