Submitted by Amanda Carolina Paulino de Oliveira (amanda@posgrad.fcav.unesp.br) on 2018-07-30T23:45:45Z No. of bitstreams: 1 Dissertação final - ACPO - Versão online.pdf: 8789441 bytes, checksum: a845ac98a1435e414423acdc29c30ff5 (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2018-07-31T11:04:06Z (GMT) No. of bitstreams: 1 oliveira_acp_me_iabo.pdf: 8789441 bytes, checksum: a845ac98a1435e414423acdc29c30ff5 (MD5) / Made available in DSpace on 2018-07-31T11:04:06Z (GMT). No. of bitstreams: 1 oliveira_acp_me_iabo.pdf: 8789441 bytes, checksum: a845ac98a1435e414423acdc29c30ff5 (MD5) Previous issue date: 2018-07-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Xanthomonas citri subsp. citri 306 (XccA) é o agente causal do cancro cítrico (CC), doença endêmica que afeta a citricultura. Durante a interação patógeno-hospedeiro o sistema de secreção tipo três (SST3) codificado pela XccA age na translocação de efetores e no estabelecimento da doença. A montagem do aparato de SST3 depende da síntese, remodelagem e degradação da parede celular bacteriana, sendo este processo realizado pela ação enzimática de transglicosilases líticas de mureína (LTs). XccA codifica diversas LTs, porém, pouco é conhecido sobre a diversidade de famílias e relação com a virulência. Dentre as LTs com provável relação com a virulência, duas ORFs parálogas presentes no cromossomo e plasmídeo pXAC64, respectivamente; são genes passageiros do TnXax1, um transposon da família Tn3, relacionado a evolução e emergência da patogenicidade nas Xanthomonadales. Portanto, este estudo objetivou elucidar o provável papel e diversidade das LTs presentes no genoma de XccA, caracterizando funcionalmente as LTs presentes em TnXax1 pela técnica de mutação sítio dirigida. Foram identificadas no genoma de XccA 13 LTs, sendo 12 pertencentes às famílias: 1A, 1B, 1C, 1D, 1F, 1G, 3A, 3B (2 cópias), 5A e 6A, e uma não classificada. A LT não classificada, é exclusiva do gênero Xanthomonas e relacionada à família 3B, porém contém um domínio adicional relacionado ao metabolismo de carboidratos. As LTs classificadas em famílias apresentam provável função relacionada com a remodelagem da parede celular para inserção de sistemas de secreção tipo 3, 4 e 6, inserção de flagelo, divisão celular, reciclagem da parede celular e degradação e controle da produção do peptidoglicano. As LTs do TnXax1 pertencem a família 3B, não são essenciais para XccA e desenvolvimento do CC, porém estão relacionadas ao aumento da virulência, diminuição da formação de biofilme, agregação e aumento na produção de goma xantana, corroborando o papel do TnXax1 como agente propagador da patogenicidade e virulência em Xanthomonadales. Em resumo, os resultados lançam novos conhecimentos frente ao papel das LTs com o metabolismo do peptidoglicano e relação com os mecanismos de transferência lateral, virulência e patogenicidade de XccA. / Xanthomonas citri subsp. citri 306 (XccA) is a causal agent of type A citrus canker (CC), one of the most devastating citriculture diseases. Type 3 Secretion Systems (T3SS) play a fundamental role in XccA pathogenicity. T3SS components are embedded in the bacterial inner and outer membrane and act as a channel for injection of effector proteins directly into the plant host cell cytosol. T3SS assembly and installation relies on bacterial cell wall synthesis, remodeling and degradation. Murein lytic transglycosylases (LT) are important in this process and are responsible for peptidoglycan cleavage and its remodeling. Information about the XccA LT arsenal is scarce: little is known about family diversity, their exact role and their connection to virulence in this bacterium. Among the LTs with probable relation to virulence, two paralogue ORFs (one in chromosome, one in pXAC64 plasmid) are passenger genes of the Tn3 family transposon TnXax1, known to play a significant role for evolution and emergence of pathogenicity in Xanthomonadales. This study addresses LT diversity in the XccA genome and examines the role of plasmid and chromosomal TnXax1 LT passenger genes using site-directed deletion mutagenesis and functional characterization. We identified 13 XccA LTs: 12 belong to families 1A, 1B, 1C, 1D (2 copies), 1F, 1G, 3A, 3B (2 copies), 5A, 6A and one which is non-categorized. This noncategorized gene, is exclusive to the Xanthomonas genus and related to the 3B family but contains an additional domain linked to carbohydrate metabolism, whilst the other catalyzes peptidoglycan biosynthesis, and is widely distributed in gammaproteobacteria. The categorized LTs are probably involved in cell wall remodeling to allow insertion of type 3, 4 and 6 secretion systems, flagellum assembly, division and recycling of cell wall and degradation and control of peptidoglycan production. The TnXax1 passenger LTs (3B family) are not essential to XccA and CC development but are implicated in virulence, biofilm production and aggregation decrease and xanthan gum production increase, corroborating the role of TnXax1 transposon as a virulence and pathogenicity propagating agent in XccA. These findings also suggest that LTs acquisition by horizontal gene transfer mediated by TnXax1 improved bacterial fitness, bringing adaptive advantages to the plant-pathogen interaction process. / 33004102070P6

Dermatófitos são um grupo de fungos intimamente relacionados, que tem a capacidade de invadir tecidos queratinizados como pele, cabelos e unhas de homens e outros animais causando dermatofitoses. Os agentes envolvidos nessas infecções pertencem aos gêneros Trichophyton, Microsporum ou Epidermophyton e, de acordo com seu habitat natural, são classificados em espécies geofílicas, zoofílicas ou antropofílicas. A maior incidência de dermatofitoses é causada pelo gênero Trichophyton, sendo T. rubrum a espécie mais prevalente em infecções de pele e unhas em humanos. Devido à severidade e longevidade destas infecções, e à resistência ao tratamento, o estudo de fatores envolvidos na interação patógeno-hospedeiro, na resistência dos dermatófitos a agentes antifúngicos e na manutenção do processo infeccioso são de grande relevância. Por análises morfológicas, fisiológicas e de expressão gênica, comparamos cinco dermatófitos cujos genomas foram sequenciados por iniciativa do Broad Institute, Microsporum canis, Trichophyton equinum, Trichophyton interdigitale, Trichophyton rubrum e Trichophyton tonsurans. Cultivos em queratina, mimetizando o processo infeccioso, foram utilizados para analisar o envolvimento dos dermatófitos na interação patógeno-hospedeiro e manutenção do processo infeccioso. Também expusemos as espécies a concentrações subinibitórias de agentes terapêuticos, de modo a verificar a resposta destes fungos a diferentes drogas. Observamos que o acúmulo de transcritos dos genes relacionados à virulência em dermatófitos avaliados durante o crescimento em queratina sugere que a maquinaria metabólica com atividade de formação da parede celular do fungo, metabolização do substrato e adesão ao hospedeiro ativa nos períodos iniciais de infecção. Contudo, um padrão de expressão correlacionado à similaridade das sequências genômicas não foi observado nas condições testadas. Também não se observa correlação direta entre o nicho preferencial dos dermatófitos e os níveis transcricionais em resposta à queratina de origem animal. Analisamos três genes envolvidos na resistência a múltiplas drogas (MDR) durante crescimento na presença de drogas com atividade antifúngica. Nossos dados sugerem que os genes MDR atuam sinergicamente em dermatófitos, e podem atuar de forma compensatória quando em presença de drogas antifúngicas, o que pode ser uma importante causa de falhas no tratamento. Nossos resultados fornecem evidências de que a expressão dos genes analisados não se correlaciona com as relações filogenéticas entre estes dermatófitos, visto que apesar da íntima relação entre o conteúdo genético e organização do genoma, os níveis transcricionais destes genes são diferentes entre as espécies. Assim, diferenças na adaptação a nichos específicos e a progressão da doença entre os dermatófitos podem ser explicadas por diferentes perfis de transcrição do gene. / Dermatophytes are a group of closely related fungi, which have the ability to invade keratinized tissues, such as skin, hair, and nails of both human and animal hosts causing dermatophytosis. The agents involved in these infections belong to the genera Trichophyton, Microsporum or Epidermophyton and, according to their natural habitat, are classified as geophilic, zoophilic or anthropophilic species. The higher incidence of dermatophytosis is caused by the genera Trichophyton, being the specie T. rubrum the most prevalent causative of human skin and nail infections. Because of the severity and longevity of these infections and their resistance to treatment, the study of the factors involved in host-pathogen interaction, in resistance of dermatophytes to antifungal agents, and in maintenance of the infection is relevant. Through morphological, physiological and gene expression analysis we compared five dermatophytes, whose genomes were sequenced by initiative of the Broad Institute: Microsporum canis, Trichophyton equinum, Trichophyton interdigitale, T. rubrum and Trichophyton tonsurans. Growth in keratin, which mimetize the infectious process, was used to analyze the involvement of dermatophytes in host-pathogen interaction and maintenance of the infectious process. We also exposed the species to subinibitory concentrations of therapeutic agents to verify the response of these fungi to different drugs. We observed that the accumulation of transcripts of genes related to virulence in dermatophytes evaluated during growth in keratin, suggest that the metabolic machinery with activity on fungal cell wall formation, substrate metabolization, and host adhesion is activated in early stages of infection. However, an expression pattern correlating to genomic sequence similarity was not observed in the conditions tested. We also did not observe a direct correlation between the preferential niche of these dermatophytes and the transcriptional levels in response to the keratin from animal origin. We analyze three genes involved in multidrug resistance (MDR) during growth in the presence of drugs with antifungal activity. Our data suggest that MDR genes act synergistically in dermatophytes, and they may compensate for one another when challenged with antifungal drugs, which can be an important cause of therapeutic failure. We provide evidence that the expression of the analyzed genes does not correlate with the phylogeny of these dermatophytes since, in spite of the different species being highly related in gene content and genome organization, the transcription level of these genes is different among these species. Thus, differences in adaptation to a specific niche and disease progression among dermatophytes would be explained by different gene transcription profiles.

Les répertoires de gènes sont des objets extrêmement dynamiques : Des gènes sont dupliqués et perdus, transférés d’un génome à l’autre et des nouveaux gènes sont créés. L’étude de ces processus et de leur impact sur l’évolution des répertoires de gènes est fondamentale pour notre compréhension de l’énorme diversité de la vie sur terre. J’ai reconstruit les familles des gènes homologues chez les levures du clade Lachancea et je les ai classées en trois catégories selon leur présence chez les espèces en dehors du clade en: transmises verticalement (98.2 %), transmises horizontalement (0.15 %) et spécifiques aux Lachancea (1.63 %). Ensuite, j’ai reconstruit l’évolution de chaque famille de gènes le long de l’arbre phylogénétique des Lachancea en terme de gains et de pertes depuis l’origine du clade. Mes résultats suggèrent que les réarrangements chromosomiques balancés (translocations, inversions) peuvent interrompre, au niveau de leurs points de cassure, la séquence codante des gènes, et entraîner jusqu’à 14 % des pertes de gènes observées (rupture de gène). En outre, j’ai observé des corrélations entre le taux de divergence des séquences codant pour des protéines et les taux de duplication de gènes, de translocations et d’inversions, et de rupture de gène, suggérant l’existence d’une horloge génomique qui coordonnerait ces processus. Par la suite, je me suis focalisé sur l’émergence de nouveaux gènes de novo à partir de séquences non-codantes, dont l’impact global sur les génomes n’est pas encore connu. J’ai pour cela analysé les gènes taxonomiquement restreints aux levures des clades Lachancea et Saccharomyces sensu stricto et j’ai pu identifier un ensemble de 596 gènes ayant fort probablement émergé de novo. Le taux d’émergence de novo est constant chez les levures au sein du même clade mais varie d’un ordre de grandeur entre les 2 clades (2.8 gènes/ma chez les Saccharomyces et 0.27 gènes/ma chez les Lachancea). Ces nouveaux gènes sont distribués uniformément sur les chromosomes. Ils sont le plus souvent orientés de façon divergente par rapport à leur voisin en 5’, ce qui suggère que leur transcription pourrait être initiée au niveau de promoteurs divergents, favorisant ainsi la transition d’une séquence intergénique non transcrite à une séquence codante transcrite (puis traduite). Enfin, j’ai montré que dans certains cas, seul un petit nombre de mutations permettent la création d’un gène bien adapté à son environnement génomique, en comparaison avec des gènes plus «anciens». Cela signifie que sous certaines conditions la transition d’une séquence non-codante vers une séquence codante peut être relativement rapide. Globalement, mes résultats suggèrent que l’émergence de novo est un processus évolutif non négligeable, représentant une source importante de création de nouvelles protéines. / Gene repertoires are highly dynamic : Genes are duplicated, lost, transferred from one genome toanother and new genes are formed. Studying these processes and how they shape gene repertoireevolution is fundamental to our understanding of how the enormous diversity of life on earth came to be. I reconstructed the homologous gene families of the yeasts of the Lachancea genus and categorized them based on their conservation in species outside the genus into vertically inherited (98.2%), horizontally transferred (0.15%) and taxonomically restricted (1.63%). Then, I inferred the evolution of each family along the genus’ phylogeny and identified the gene gain and loss events that occurred since the genus’ origin. I found that balanced chromosomal rearrangements may be responsible for up to 14% of gene losses by disrupting the coding sequence at their breakpoints and detected 3 cases with clear traces of the disruption at the sequence level. Additionally, I found that correlations exist between the rate of protein-coding sequence divergence and the rates of gene duplication, chromosomal inversions and translocations, and gene disruptions by balanced rearrangements, suggesting the existence of a genomic clock coordinating these processes. Next, I focused on the emergence of new genes de novo from non-coding sequences, a process whose overall impact remains a matter of debate. I thus analyzed taxonomically restricted genes in the two model yeast genera Lachancea and Saccharomyces sensu stricto and identified a robust set of 596 genes that have likely emerged de novo. I found that de novo emergence rates are constant among yeasts of the same genus but differ by an order of magnitude between the two genera with 2.8 genes/my in the Saccharomyces and 0.27 genes/my in the Lachancea. De novo genes are uniformly distributed on yeast genomes and are found divergently oriented relative to their 5’ neighbors suggesting that divergent transcription might play a role in their transition from non-transcribed intergenic sequences to transcribed (and translated) coding sequences. Moreover, through specific examples I was able to show that a few enabling mutations are sufficient for a young de novo gene to emerge already well-adapted relative to older genes, indicating that the transition from non-coding to coding can happen rapidly. Overall, my results support de novo emergence as a ubiquitous evolutionary process and a potent source of novel proteins.

Hamiltonella defensa est un endosymbiote secondaire ayant établi deux associations très distinctes chez les insectes phloémophages. Chez les pucerons, la bactérie protège l'hôte contre les parasitoïdes. Elle infecte de nombreux tissus dans l'hôte, et notamment l'hémolymphe, ce qui favoriserait le contact avec les oeufs de parasitoïdes. Malgré ce phénotype protecteur, les coûts importants que sa présence inflige à son hôte empêchent sa fixation dans les populations. Chez l'aleurode Bemisia tabaci, on ne retrouve la bactérie que dans des cellules spécialisées dans l'hébergement des endosymbiotes, les bactériocytes. Elle s'y trouve entre autres en présence du symbiote primaire, Portiera aleyrodidarum, des conditions de vie propices aux échanges entre les deux symbiotes. Elle est fixée dans les populations d'insectes, ce qui suggère un rôle important pour le consortium, qui serait nutritif. Dans le cadre de cette thèse, nous nous sommes intéressés aux spécificités de chacun de ces systèmes. Nous nous sommes également attardés sur l'évolution génomique du genre Hamiltonella, en comparant des souches infectant B. tabaci à une souche de puceron. Pour finir, nous nous sommes intéressés aux phénomènes d'accélération des taux de mutations chez H. defensa, comparativement à son espèce-soeur Regiella insecticola, également endosymbiotique et protectrice du puceron. Après avoir éliminé l'hypothèse selon laquelle la transition vers la vie intracellulaire aurait eu lieu indépendamment dans les deux lignées, nous avons tenté d'établir un lien entre ces différentiels d'évolvabilité chez les endosymbiotes et leur contenu en gènes, notamment ceux impliqués dans l'écologie et la réparation de l'ADN. L'ensemble des résultats obtenus au cours de ce Doctorat ont permis de mieux comprendre l'évolution de l'espèce H. defensa, depuis le dernier ancêtre jusqu'aux espèces actuelles, en tâchant de faire le lien entre phénotype de la bactérie et évolution génomique / Hamiltonella defensa is a secondary endosymbiont that established two distinct associations with phloemophagous insects. In aphids, it protects the host against parasitoid attacks. Its ability to infect many host tissues, notably the hemolymph, could promote its contact with parasitoid eggs. Despite this protective phenotype, the high costs associated with its presence within the host prevent its fixation in the population. In the whitefly Bemisia tabaci however, this symbiont is found only in cells specialized in hosting endosymbionts, the bacteriocytes. In these cells, it cohabits with other symbiotic species, such as the primary symbiont Portiera aleyrodidarum, a proximity that favors potential exchanges between the two symbionts. It is fixed in populations of B. tabaci, which suggests an important role for the consortium, probably nutritious. As part of this PhD thesis, we studied the specificities of each of these systems. We also focused on the genomic evolution of the genus Hamiltonella, by comparing the strains infecting B. tabaci with a strain infecting the aphids. Finally, we studied the phenomenon of ‘accelerated mutation rate’ in H. defensa, compared to its sister species Regiella insecticola, which is also a clade of protective endosymbionts of aphids. After excluding the assumption that the transition to the intracellular life occurred independently in the two lineages, we tried to establish a link between these differences in terms of evolvability in the endosymbionts and of their gene contents, particularly for genes involved in ecology and DNA repair. All the results obtained during this PhD have provided insight into the evolution of the species H. defensa, since the last ancestor to the present species, by establishing a link between bacterial phenotype and genomic evolution

Scientific Workflows (SWFs) are widely used to model applications in e-Science. In this programming model, scientific applications are described as a set of tasks that have dependencies among them. During the last decades, the execution of scientific workflows has been successfully performed in the available computing infrastructures (supercomputers, clusters and grids) using software programs called Workflow Management Systems (WMSs), which orchestrate the workload on top of these computing infrastructures. However, because each computing infrastructure has its own architecture and each scientific applications exploits efficiently one of these infrastructures, it is necessary to organize the way in which they are executed. WMSs need to get the most out of all the available computing and storage resources. Traditionally, scientific workflow applications have been extensively deployed in high-performance computing infrastructures (such as supercomputers and clusters) and grids. But, in the last years, the advent of cloud computing infrastructures has opened the door of using on-demand infrastructures to complement or even replace local infrastructures. However, new issues have arisen, such as the integration of hybrid resources or the compromise between infrastructure reutilization and elasticity, everything on the basis of cost-efficiency. The main contribution of this thesis is an ad-hoc solution for managing workflows exploiting the capabilities of cloud computing orchestrators to deploy resources on demand according to the workload and to combine heterogeneous cloud providers (such as on-premise clouds and public clouds) and traditional infrastructures (supercomputers and clusters) to minimize costs and response time. The thesis does not propose yet another WMS, but demonstrates the benefits of the integration of cloud orchestration when running complex workflows. The thesis shows several configuration experiments and multiple heterogeneous backends from a realistic comparative genomics workflow called Orthosearch, to migrate memory-intensive workload to public infrastructures while keeping other blocks of the experiment running locally. The running time and cost of the experiments is computed and best practices are suggested. / Los flujos de trabajo científicos son comúnmente usados para modelar aplicaciones en e-Ciencia. En este modelo de programación, las aplicaciones científicas se describen como un conjunto de tareas que tienen dependencias entre ellas. Durante las últimas décadas, la ejecución de flujos de trabajo científicos se ha llevado a cabo con éxito en las infraestructuras de computación disponibles (supercomputadores, clústers y grids) haciendo uso de programas software llamados Gestores de Flujos de Trabajos, los cuales distribuyen la carga de trabajo en estas infraestructuras de computación. Sin embargo, debido a que cada infraestructura de computación posee su propia arquitectura y cada aplicación científica explota eficientemente una de estas infraestructuras, es necesario organizar la manera en que se ejecutan. Los Gestores de Flujos de Trabajo necesitan aprovechar el máximo todos los recursos de computación y almacenamiento disponibles. Habitualmente, las aplicaciones científicas de flujos de trabajos han sido ejecutadas en recursos de computación de altas prestaciones (tales como supercomputadores y clústers) y grids. Sin embargo, en los últimos años, la aparición de las infraestructuras de computación en la nube ha posibilitado el uso de infraestructuras bajo demanda para complementar o incluso reemplazar infraestructuras locales. No obstante, este hecho plantea nuevas cuestiones, tales como la integración de recursos híbridos o el compromiso entre la reutilización de la infraestructura y la elasticidad, todo ello teniendo en cuenta que sea eficiente en el coste. La principal contribución de esta tesis es una solución ad-hoc para gestionar flujos de trabajos explotando las capacidades de los orquestadores de recursos de computación en la nube para desplegar recursos bajo demando según la carga de trabajo y combinar proveedores de computación en la nube heterogéneos (privados y públicos) e infraestructuras tradicionales (supercomputadores y clústers) para minimizar el coste y el tiempo de respuesta. La tesis no propone otro gestor de flujos de trabajo más, sino que demuestra los beneficios de la integración de la orquestación de la computación en la nube cuando se ejecutan flujos de trabajo complejos. La tesis muestra experimentos con diferentes configuraciones y múltiples plataformas heterogéneas, haciendo uso de un flujo de trabajo real de genómica comparativa llamado Orthosearch, para traspasar cargas de trabajo intensivas de memoria a infraestructuras públicas mientras se mantienen otros bloques del experimento ejecutándose localmente. El tiempo de respuesta y el coste de los experimentos son calculados, además de sugerir buenas prácticas. / Els fluxos de treball científics són comunament usats per a modelar aplicacions en e-Ciència. En aquest model de programació, les aplicacions científiques es descriuen com un conjunt de tasques que tenen dependències entre elles. Durant les últimes dècades, l'execució de fluxos de treball científics s'ha dut a terme amb èxit en les infraestructures de computació disponibles (supercomputadors, clústers i grids) fent ús de programari anomenat Gestors de Fluxos de Treballs, els quals distribueixen la càrrega de treball en aquestes infraestructures de computació. No obstant açò, a causa que cada infraestructura de computació posseeix la seua pròpia arquitectura i cada aplicació científica explota eficientment una d'aquestes infraestructures, és necessari organitzar la manera en què s'executen. Els Gestors de Fluxos de Treball necessiten aprofitar el màxim tots els recursos de computació i emmagatzematge disponibles. Habitualment, les aplicacions científiques de fluxos de treballs han sigut executades en recursos de computació d'altes prestacions (tals com supercomputadors i clústers) i grids. No obstant açò, en els últims anys, l'aparició de les infraestructures de computació en el núvol ha possibilitat l'ús d'infraestructures sota demanda per a complementar o fins i tot reemplaçar infraestructures locals. No obstant açò, aquest fet planteja noves qüestions, tals com la integració de recursos híbrids o el compromís entre la reutilització de la infraestructura i l'elasticitat, tot açò tenint en compte que siga eficient en el cost. La principal contribució d'aquesta tesi és una solució ad-hoc per a gestionar fluxos de treballs explotant les capacitats dels orquestadors de recursos de computació en el núvol per a desplegar recursos baix demande segons la càrrega de treball i combinar proveïdors de computació en el núvol heterogenis (privats i públics) i infraestructures tradicionals (supercomputadors i clústers) per a minimitzar el cost i el temps de resposta. La tesi no proposa un gestor de fluxos de treball més, sinó que demostra els beneficis de la integració de l'orquestració de la computació en el núvol quan s'executen fluxos de treball complexos. La tesi mostra experiments amb diferents configuracions i múltiples plataformes heterogènies, fent ús d'un flux de treball real de genòmica comparativa anomenat Orthosearch, per a traspassar càrregues de treball intensives de memòria a infraestructures públiques mentre es mantenen altres blocs de l'experiment executant-se localment. El temps de resposta i el cost dels experiments són calculats, a més de suggerir bones pràctiques. / Carrión Collado, AA. (2017). Management of generic and multi-platform workflows for exploiting heterogeneous environments on e-Science [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/86179 / TESIS

Small nucleolar RNAs (snoRNAs) are one of the most abundant and evolutionary ancient group of small non-coding RNAs. Their main function is to target chemical modifications of ribosomal RNAs (rRNAs) and small nuclear (snRNAs). They fall into two classes, box C/D snoRNAs and box H/ACA snoRNAs, which are clearly distinguished by conserved sequence motifs and the type of modification that they govern. The box H/ACA snoRNAs are responsible for targeting pseudouridylation sites and the box C/D snoRNAs for directing 2’-O-methylation of ribonucleotides. A subclass that localize to the Cajal bodies, termed scaRNAs, are responsible for methylation and pseudouridylation of snRNAs. In addition an amazing diversity of non-canonical functions of individual snoRNAs arose. The modification patterns in rRNAs and snRNAs are retained during evolution making it even possible to project them from yeast onto human. The stringent conservation of modification sites and the slow evolution of rRNAs and snRNAs contradicts the rapid evolution of snoRNA sequences. Recent studies that incorporate high-throughput sequencing experiments still identify undetected snoRNAs even in well studied organisms as human. The snoRNAbase, which has been the standard database for human snoRNAs has not been updated ince 2006 and misses these new data. Along with the lack of a centralized data collection across species, which incorporates also snoRNA class specific characteristics the need to integrate distributed data from literature and databases into a comprehensive snoRNA set arose. Although several snoRNA studies included pro forma target predictions in individual species and more and more studies focus on non-canonical functions of subclasses a systematic survey on the guiding function and especially functional homologies of snoRNAs was not available. To establish a sound set of snoRNAs a computational snoRNA annotation pipeline, named snoStrip that identifies homologous snoRNAs in related species was employed. For large scale investigation of the snoRNA function, state-of-the-art target pedictions were performed with our software RNAsnoop and PLEXY. Further, a new measure the Interaction Conservation Index (ICI) was developed to evaluate the conservation of snoRNA function. The snoStrip pipeline was applied to vertebrate species, where the genome sequence has been available. In addition, it was used in several ncRNA annotation studies (48 avian, spotted gar) of newly assembled genomes to contribute the snoRNA genes. Detailed target analysis of the new vertebrate snoRNA set revealed that in general functions of homologous snoRNAs are evolutionarily stable, thus, members of the same snoRNA family guide equivalent modifications. The conservation of snoRNA sequences is high at target binding regions while the remaining sequence varies significantly. In addition to elucidating principles of correlated evolution it was possible, with the help of the ICI measure, to assign functions to previously orphan snoRNAs and to associate snoRNAs as partners to known but so far unexplained chemical modifications. As further pattern redundant guiding became apparent. For many modification sites more than one snoRNA encodes the appropriate antisense element (ASE), which could ensure constant modification through snoRNAs that have different expression patterns. Furthermore, predictions of snoRNA functions in conjunction with sequence conservation could identify distant homologies. Due to the high overall entropy of snoRNA sequences, such relationships are hard to detect by means of sequence homology search methods alone. The snoRNA interaction network was further expanded through novel snoRNAs that were detected in data from high-throughput experiments in human and mouse. Through subsequent target analysis the new snoRNAs could immediately explain known modifications that had no appropriate snoRNA guide assigned before. In a further study a full catalog of expressed snoRNAs in human was provided. Beside canonical snoRNAs also recent findings like AluACAs, sno-lncRNAs and extraordinary short SNORD-like transcripts were taken into account. Again the target analysis workflow identified undetected connections between snoRNA guides and modifications. Especially some species/clade specific interactions of SNORD-like genes emerged that seem to act as bona fide snoRNA guides for rRNA and snRNA modifications. For all high confident new snoRNA genes identified during this work official gene names were requested from the HUGO Gene Nomenclature Committee (HGNC) avoiding further naming confusion.

4 ABSTRACT (English) This thesis is based on three published research papers studying bacterial REP (repetitive extragenic palindrome) elements. REP elements are one of the best-characterized groups of bacterial DNA repeats, distributed mostly in gammaproteobacteria, including enterobacteria. They are present in noncoding parts of host genomes, usually occurring in hundreds of copies. REPs are typically aggregated in higher order repeats. In the Gram-negative model Escherichia coli, interactions of several proteins important for cell's physiology with REPs were described, indicating significant role for these elements for host cells. The first work (Nunvar et al. 2010) presents the discovery of a protein class, related to IS200/IS605 transposases. These proteins, termed RAYTs (REP-associated tyrosine transposases), contain characteristic motifs in their amino acid sequences, which are absent in canonical IS200/IS605 transposases. Another attribute of RAYTs is the arrangement of their encoding genes. These are single copy genes, always flanked at both termini by at least two REPs in inverted orientation. Based on the similarity between the REP-rayt-REP unit and insertion sequences of the IS200/IS605 family, between RAYTs and tyrosine transposases and between REPs and subterminal sequences of the IS200/IS605...

The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration. Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene. The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes. The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes. Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes. Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions. RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz. During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins. In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World.

Les levures oléagineuses ascomycètes font partie des productrices de lipides les plus connus de notre époque. Elles peuvent produire des lipides, des molécules chimiques dérivées et des acides organiques à partir de sucres simples ou complexes. Nous avons choisi les levures du genre Blastobotrys afin de définir un nouvel organisme modèle pour la production d'acides gras et de lipides, car ces levures sont capables de synthétiser et de stocker naturellement 15 à 25% de lipides dans leur biomasse sèche à partir de glucose et xylose, soit plus que Yarrowia. lipolytica dans les mêmes conditions. La plupart des études d'ingénierie métabolique connues ont utilisé les levures du genre Blastobotrys dans une logique de production de molécules différentes des lipides. Nous avons caractérisé les traits oléagineux de deux souches appartenant à deux espèces du genre Blastobotrys, en utilisant comme substrats du glucose, xylose, glycérol, fructose, cellobiose, saccharose, galactose avec un rapport C/N de 60. La plus forte production de lipides vient du cellobiose (35%) et du glucose (32%).Ensuite, afin de mieux comprendre le métabolisme des lipides des levures du genre Blastobotrys, nous avons exploré l'effet de la température sur leur physiologie, production de lipides et le profil lipidique en utilisant un milieu YNB contenant 30 g/L de glucose. Nous n'avons pas trouvé de différence marquée de transition de formes entre les formes hyphes et les levures en milieu YNB sous l’effet de quatre températures (28°C, 37°C, 42°C, 45°C), mais la production des lipides est favorisée à 28°C et le C18:1 est l'acide gras le plus abondant dans le profil lipidique. Nous avons transformé avec succès l’espèce B. raffinosifermentans grâce au système Xplor2. Nous avons pu augmenter l'accumulation de lipides en sur-exprimant deux diacylglycérol acyltransférase endogènes, DGA1 et DGA2. Le niveau d’expression élevé de DGA1 dans nos mutants n’est pas corrélé à une production élevée de lipides alors que celui de DGA2 l’est. Notre meilleure souche, dérivée de la souche parentale G1212, a produit 26,5% de lipides à partir de 30 g/L de glucose en culture en flasque. Ce travail représente l’une des premières ingénieries métaboliques de souches de Blastobotrys pour la production de lipides. Ce sont donc des levures oléagineuses comme Y. lipolytica avec un potentiel biotechnologique avéré. / Ascomycetous oleaginous yeasts are among the highest known producers of lipids of our era that may supply lipids compounds, derived chemicals and organic acids from simple or complex carbon sources. We chose oleaginous yeasts species of Blastobotrys genus for defining a new model organism for fatty acid production and lipids, because these oleaginous yeasts natively produce higher lipids rate than Yarrowia lipolytica in the same conditions and can metabolize glucose and xylose. Most of the metabolic engineering studies on these yeast species focused on other molecules compounds than lipids. We characterized the oleaginous traits of two strains belonging to two different species of genus Blastobotrys, using glucose, xylose, glycerol, fructose, cellobiose, sucrose, galactose, starch and oleic acid as substrates with a C/N ratio of 60. We found the higher lipid production (35%) on cellobiose and glucose (32%).Next, in order to further understand the lipid metabolism in Blastobotrys, we explored the effect of temperature on cell physiology, lipid production and lipid profile using YNB medium with 30 g/L glucose. No markedly transition were found from the hyphae to budding form or reversely on YNB medium under four temperatures (28°C, 37°C, 42°C, 45°C). The lipids production is favored at 28°C and C18:1 is the most abundant fatty acid in the lipid profile. We successfully transformed the yeast species B. raffinosifermentans using the Xplor2 system. We increased lipid accumulation by over-expressing two native diacylglycerol acyltransferase genes, DGA1 and DGA2. Our best strain, derived from the parental strain G1212, produced 26.5 g/L lipid from 30g/L glucose in shake-flask experiments. This strain also produced citric acid like Y. lipolytica. We didn’t find significant overall elevated expression in lipid synthesis pathway for DGA1 gene when lipid production was favored on contrary to DGA2 gene. This work represents one of the first metabolic engineering of B. adeninivorans for lipid production.

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