Les progrès en biotechno]ogie ont permis le développement d’outils pour le transfert de gène intégratif en transgénèse, bioproduction et thérapie génique. Cependant, trois challenges majeurs doivent être relevés pour garantir un système sécurisé : l’innocuité et l’efficacité du transfert, l’intégration ciblée et contrôlée dans le génome, le niveau et la durée d’expression du transgène au cours du temps. Dans ce but, mes travaux de thèse ont consisté à tester des solutions pour améliorer la biosécurité du transposon piggyBac qui nécessite un plasmide porteur du gène d’intérêt à insérer dans le génome et une source de transposase catalysant la réaction d’intégration du transgène. Une des stratégies de ma thèse repose sur l’apport de la source de transposase sous forme d’ARN messager au lieu d’ADN afin d’améliorer la stabilité de l’intégration et de réduire les effets génotoxiques en limitant la transposase dans les cellules. Pour la première fois, la biodisponibilité de l’ARNm de la transposase et les conditions optimales d’utilisation en cellules humaines ont été déterminées pour augmenter la biosêcurité du système. Le second objectif de mes travaux consiste à améliorer l’expression du transgène en ajoutant des insulateurs connus pour s’opposer à l’extinction de l’expression des gênes. En termes de biosécurité, cette stratégie permet de réduire le nombre de copies du transgène nécessaires pour obtenir une expression suffisante. Deux candidats ont été identifiés pour améliorer l’expression du transgène. La combinaison des approches ARNrn et insulateurs est prometteuse pour sécuriser le transfert de gène médié par piggyBac et pour maintenir l’expression du gène d’intérêt. / Advances in biotechnology have enabled the development of tools for gene transfer applicable to transgenesis, bioproduction and gene therapy. But, 3 major challenges must be met to ensure a secure system: the safety and effectiveness of the transfer. the targeted and controlled integration into the genome. and the level of transgene expression over time. In this aim, my thesis project was to validate solutions to improve the biosafety of the piggyBac transposon, which requires a plasmid carrying the gene of interest to be inserted in the genome, and a source of transposase which catalyzes the transgene integration. One approach of my thesis work is to deliver the source of piggyBac transposase as an mRNA molecule instead of DNA. This strategy aims to improve the stability of the integration and reduce the genotoxic effects by limiting the transposase in the cells. For the 1st time, the bioavailability of the transposase rnRNA and the optimal conditions for its use in human cells were determined to increase the biosafety of the transposon system. The 2nd objective ofmy project is to improve the expression of the transgene by adding insulators known to counteract the transgene silencing. This strategy reduces the number of integrations required ta get a sufficient expression of the transgene and thus, improve biosecurity. Two candidates have been identified to improve transgene expression. The combination of the mRNA and insulator strategies is promising to secure the piggyBac-mediated gene transfer and to maintain the expression of the gene of interest

O objetivo deste projeto foi entender a organização genômica e o conteúdo de genes de dois fitopatógenos relacionados geneticamente, mas que infectam diferentes hospedeiros: Leifsonia xyli subsp. xyli (Lxx), um patógeno de cana-de-açúcar; e Leifonia xyli subsp. cynodontis (Lxc), um patógeno de gramíneas do gênero Cynodon. Os resultados do seqüenciamento parcial do genoma de Lxc são descritos, incluindo as seqüências comuns ao genoma de Lxx e os fragmentos de organização distinta e genes específicos de Lxc. Para alcançar o objetivo, bibliotecas genômicas do genoma de Lxc foram construídas. A estratégia revelou seqüências específicas, algumas provavelmente adquiridas por transferência horizontal, e regiões não sintênicas do genoma de Lxc, quando comparadas com Lxx. Regiões específicas somaram 311.353 pb e foram anotadas. Devido a associação de elementos genéticos móveis com reorganização cromossômica e transferência horizontal de genes, um estudo detalhado dos transposons do tipo IS, presentes em ambos os genomas, foi realizado. A análise revelou um número variável de elementos para cada genoma atuando na diversificação dos mesmos. / This study aimed to understand genome organization and gene content of two closely related plant pathogens that infect different hosts: Leifsonia xyli subsp. xyli (Lxx), a sugarcane pathogen; and Leifonia xyli subsp. cynodontis (Lxc), a Cynodon grass pathogen. We describe the results of a partial genome sequence of Lxc, assessing the similarity to Lxx completely sequenced genome, describing differences in genome organization and uncovering genes specific to Lxc genome. To accomplish the objective, genomic libraries were constructed. The strategy uncovered specific sequences, some probably acquired by horizontal transfer and non-syntenic regions of Lxc genome compared to Lxx. Specific regions of Lxc genome accounted for 311,353 bp and were annotated. Because mobile genetic elements are often associated with rearrangements and horizontal gene transfer, a detailed study of all insertion sequence (IS) elements presented in both genomes were realized. The analysis revealed a variable number of transposable elements acting upon genomic diversity.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Die Ergebnisse dieser Arbeit zeigen erstmals die weite Verbreitung des IS100 innerhalb der Spezies E. coli und große Ähnlichkeiten bezüglich der chromosomalen Lokalisationen einzelner Kopien in einem heterogenen Kollektiv von E. coli-Stämmen. / The results of the present study attest to the wide distribution of the IS100 element among bacteria of the E. coli species and reveal significant similarities with regard to the chromosomal localisation of numerous single copies within a heterogenous E. coli strain collection.

Human intestinal microbiomes exhibit a large degree of interindividual compositional variation. Animal models, such as the zebrafish, facilitate the design of controlled and highly replicated studies that allow us to understand the normal variation in vertebrate intestinal composition and to study the rules guiding normal assembly of these complex communities. The smaller intestinal size and high fecundity of the zebrafish allow us to fully sample the intestinal contents of many animals, while the optical transparency allows direct in vivo observation of fluorescently labeled bacterial species within the intestine. The studies in this dissertation utilize these advantages to investigate the composition, colonization dynamics and functional requirements for colonization in the vertebrate intestine. We first describe the taxonomic composition and diversity of the zebrafish intestinal microbiota from wild-caught and domesticated zebrafish. In the process, we identify a set of core bacterial genera that are consistently present in zebrafish intestines. We then use species from two of these genera in subsequent studies to gain a detailed understanding of the colonization dynamics and genetic requirements of the two species. We initially describe the application of light sheet microscopy to imaging the zebrafish intestine and associated colonizing bacteria. We find that a single species, Aeromonas veronii, does not occupy the entire intestinal space and that competition within the same species appears to prevent further colonization. These results are extended to a zebrafish isolated Vibrio species as well as A. veronii by tagging bacteria with transposon insertions and tracking the changes in colonizing population sizes. These insertion libraries also identify genes in each bacterial species that are important in the process of colonization, highlighting the key role for motility and chemotaxis in this process. The descriptions and methods discussed in this dissertation advance the use of this important model organism towards the understanding of vertebrate host-microbial interactions. This dissertation includes previously published co-authored material as well as unpublished co-authored material. / 10000-01-01

Transposable elements (TEs) are utilized for the creation of a wide range of transgenic organisms. However, in some systems, this technique is not very efficient due to low transposition frequencies and integration into unstable or transcriptionally inactive genomic regions. One approach to ameliorate this problem is to increase knowledge of how transposons move and where they integrate into target genomes. Most transposons do not insert randomly into their host genome, with class II TEs utilizing target sequences of between 2 – 8 bp in length, which are duplicated upon insertion. Furthermore, amongst insertion sites, certain sites are preferred for insertion and hence are classified as hot spots, while others not targeted by TEs are referred to as cold spots. The hypothesis tested in this analysis is that in addition to the primary consensus target sequence, secondary and tertiary DNA structures have a significant influence on TE target site preference. Bioinformatics was used to predict and analyze the structure of the flanking DNA around known insertion sites and cold spots for various TEs, to understand why insertion sites are used preferentially to cold spots for element integration. Hidden Markov Models were modeled and trained to analyze datasets of insertions of the P element in the Drosophila melanogaster genome, the Tc1 element in the Caenorhabditis elegans genome, and insertions of the Mos1, piggyBac and Hermes transposons into the target plasmid pGDV1. Analysis of the DNA structural profiles of the insertion sites for the P element and Hermes transposons revealed that both transposons targeted regions of DNA with a relatively high degree of bendability/flexibility at the insertion site. However, similar trends were not observed for the Tc1, Mos1 or piggyBac transposons. Hence, it is believed that the secondary structural features of DNA can contribute to target site preference for some, but not all transposable elements.

Estudos realizados no Laboratório de Drosophila da UFRGS tem caracterizado linhagens de Drosophila willistoni quanto à presença de Elementos Transponíveis (TEs) e à existência do fenômeno de Disgenesia Híbrida nesta espécie. Como conseqüência destes estudos, o presente trabalho se propôs a ampliar o conhecimento sobre o papel destes elementos na geração de variabilidade nesta espécie e abordou os TEs P, hobo, gypsy e 412, anteriormente identificados no genoma de D. willistoni, quanto à sua atividade transcricional. Em nosso trabalho, verificamos a presença de transcritos de P, gypsy e 412 em adultos, sugerindo uma regulação pós-transcricional destes elementos, como já sugerido para o elemento P, considerando que as linhagens utilizadas não se caracterizam pela hipermutabilidade. Devido à descrição prévia da Síndrome da Disgenesia do Híbrido na prole do cruzamento entre as linhagens 17A2 e Wip de D. willistoni, foi estabelecido o padrão de expressão do elemento P durante o desenvolvimento embrionário das duas linhagens. O padrão de expressão embrionário em D. melanogaster também foi estabelecido, para fins comparativos, uma vez que se aceita a ocorrência de um evento de transferência horizontal de P de D. willistoni para D. melanogaster. A similaridade entre os padrões de expressão nas duas espécies, sugere que o elemento P é regulado pelo seu próprio promotor e que não é dependente de promotores de genes vizinhos aos seus sítios de inserção. Foi estabelecida a presença de transcritos potenciais da transposase e de um repressor da transposição de P nos embriões analisados. Além do transcrito correspondente ao repressor, que é gerado por processamento alternativo do transcrito da transposase, obteve-se indícios da presença de transcritos antisenso do próprio elemento nos embriões, sugerindo a regulação por interferência de RNA (RNAi) neste estágio do desenvolvimento de Drosophila. Diferenças transcricionais do elemento P entre D. willistoni e D. melanogaster, estão relacionadas ao número de transcritos deletados de P que são expressos em maior número em D. melanogaster do que em D. willistoni, corroborando a idéia da invasão recente do genoma da primeira por este elemento. A expressão dos TEs descrita neste trabalho relata a regulação complexa destes elementos, evidenciando a importância da continuidade deste estudo. / Studies accomplished in the Laboratory of Drosophila of UFRGS have been characterizing strains of Drosophila willistoni in respect to the presence of Transposable Elements (TEs) and to the occurrence of the Hybrid Dysgenesis phenomenon in this species. As a consequence of these studies, the present work aimed to broaden the knowledge about the role of these elements in the genesis of variability in this species, by approaching the transcriptional expression of P, hobo, gypsy and 412 TEs, already described in the D. willistoni genome. In our work, we verified the presence of P, gypsy, and 412 transcripts in adults, suggesting post-transcriptional regulation, like already described for P element in D. melanogaster, considering that the strains studied were not characterized by hypermutability. Due to the previous description of the Hybrid Dysgenesis Syndrome in the offspring resulting of crosses between 17A2 and Wip D. willistoni strains, their P element expression pattern during the embryonic development was established. The embryonic P element expression pattern in D. melanogaster was also established, for comparative purpose, since the occurrence of a horizontal transfer event of this element from D. willistoni to D. melanogaster is accepted. The similarity among these expression patterns in both species suggests that P element is regulated by its own promoter and that it’s not dependent of the insertion sites neighboring genes promoters. The presence of putative P element transcripts of transposase and of the transposase repressor was established in the analyzed embryos. Besides the transposase repressor transcript, that is result of an alternative splicing of the transposase transcript, it was obtained indication of the presence of antisense transcripts of P element in the embryos, suggesting the regulation by RNA interference (RNAi) in this stage of development of Drosophila. Transcriptional differences of the P element between D. willistoni and D. melanogaster, are related to the number of deleted transcripts of P that are expressed in larger number in D. melanogaster than in D. willistoni, corroborating the idea of the recent invasion of the genome of the first species by this element. The expression of TEs described in this work suggests a complex regulation of these elements, evidencing the importance of the continuity of this study.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T11:34:47Z No. of bitstreams: 1 resumo.pdf: 15826 bytes, checksum: 80e9778f9db86d01b847c607230b3867 (MD5) / Made available in DSpace on 2017-06-13T11:34:47Z (GMT). No. of bitstreams: 1 resumo.pdf: 15826 bytes, checksum: 80e9778f9db86d01b847c607230b3867 (MD5) Previous issue date: 2002-04-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O elemento transponível impala, isolado do fungo fitopatogênico Fusarium oxysporum, é capaz de sofrer transposição em Penicillium griseoroseum, porém, com uma baixa frequência. Em trabalho anterior, condições de estresse, testadas com o objetivo de aumentar a transposição de impala em P. griseoroseum, foram eficientes, sendo isoladas 691 colônias revertentes, em que o elemento impala havia sofrido transposição. Neste trabalho, foram feitas a caracterização molecular das linhagens revertentes e a avaliação da produção de enzimas pectinolíticas, com o objetivo de testar a eficiência de impala como ferramenta para a etiquetagem e isolamento de genes que codificam pectinases. As análises por hibridização das linhagens revertentes, utilizando como sonda o elemento impala e o gene niaD de A. nidulans, demonstraram que o tratamento por irradiação foi mais eficiente do que o choque térmico para a ativação da transposição em P. griseoroseum. Nas linhagens revertentes obtidas após a irradiação, um maior número de sítios de reinserção de impala foi observado. Entre as linhagens revertentes provenientes do choque térmico, uma apresentou perda total do elemento impala. Das 650 linhagens revertentes analisadas quanto à produção de pectinases, uma linhagem obtida após choque térmico apresentou atividade significativamente inferior de pectina liase quando comparada à linhagem selvagem. A região flanqueadora do novo sítio de inserção do elemento impala foi amplificada por PCR invertido. O fragmento amplificado de 600pb foi clonado e será utilizado como sonda para o isolamento do gene completo em um banco genômico da linhagem selvagem de P. griseoroseum. Este trabalho demonstra, pela primeira vez, que o elemento impala pode ser usado para a etiquetagem de genes no fungo Penicillium griseoroseum. / The tranposable element impala, isolated from the phytopathogenic fungus Fusarium oxysporum, suffers transposition in Penicillium griseoroseum, albeit at low frequency. In a previous work, stress conditions tested to increase impala transposition in P. griseoroseum have been show to be very efficient, resulting in the production of 691 revertant colonies in which impala had suffered transposition. In this work, a molecular characterization of revertant colonies and the analysis of pectinolytic enzyme were done to test impala efficiency as a tool for tagging and isolating pectinase gene. Hybridization analysis of revertant colonies using impala and the niaD gene of Aspergillus nidulans as probes demonstrated that UV treatment was more efficient than thermal shock for the activation of impala transposition in P. griseoroseum. In the revertant colonies obtained after UV irradiation, a larger number of reinsertion sites was observed. Among the revertants produced by thermal shock, one strain showed total loss of impala. Among 650 revertant colonies analyzed for pectinase production, one isolate obtained after thermal shock treatment presented significantly lower pectin lyase activity than the wild type strain. The flanking region of the new insertion site of impala was amplified by inverted PCR. A 600-pb amplified fragment was cloned and will be used as probe for the isolation of the complete gene from a library of the type strain of P. griseoroseum. ixThis work demonstrates for the first time that the element impala can be used for efficient gene tagging in P. griseoroseum.