Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-18Bitstream added on 2014-06-13T20:14:43Z : No. of bitstreams: 1 barbosa_acdr_me_jabo_prot.pdf: 1040447 bytes, checksum: 48ed6a565f670b0e8919f670ff1f64ba (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A seca é um dos principais fatores abióticos que afetam a produtividade das plantas. Para detectar os genes expressos sob condições de deficiência hídrica desenvolveu-se uma análise da expressão gênica das plantas de cana-de-açúcar de uma cultivar tolerante (cv. SP83-2847) e outra sensível (cv. SP90-1638) à seca, utilizando macroarranjos de DNA para monitorar a expressão gênica de 3.575 clones de folhas de cana-de-açúcar e, a partir dos resultados obtidos foram selecionados dois ESTs (SCJFLR2036B11 e SCBFLR1005E12) que se apresentaram como diferencialmente expressos nas duas cultivares. Os clones foram seqüenciados e identificados e correspondem ao gene que codifica para a Nitrilase e para elementos genéticos móveis (Retrotransposon). Os dados de expressão gênica foram validados por meio de RT-PCR semiquantitativo e Southem blotting e a seqüência de nucleotídeos obtida foi utilizada nas buscas em bancos de dados e na comparação de seqüência o que contribuiu para a atribuição de função biológica. O gene similar ao da Nitrilase apresentou-se induzido tanto na situação de deficiência hídrica no cultivar sensível (SP90-1638) como no cultivar tolerante (SP83-2847) nas plantas não expostas ao déficit hídrico. O gene que codifica um elemento genético móvel (Retrotransposon) apresentou-se induzido no cultivar tolerante na situação controle em detrimento da situação estressada e invariável para cultivá-Io sensível nas duas situações. / Drought is one of the main abiotic stresses which aftect plant productivity. To detect genes expressed under drought conditions, a gene expression study of sugarcane plants was performed, with drought tolerant (cv. SP83-2847) and sensitive (cv. SP90-1638) cultivars, using DNA macroarrays to monitor gene expression of 3.575 sugarcane leaf clones, and from the obtained results two ESTs (SCJFLR2036811 and SC8FLR1005E12) identified as difterentially expressed in both cultivars. were selected. The clones were sequenced and identified as the gene which codifies Nitrilase and mobile genetic elements (Retrotransposon). Gene expression data were confirmed by Semiquantitative RT-PCR and Southern blotting analysis, and the obtained nucleotide sequence used in database searches and in sequence comparisons, which contributed for the biological function attribution. The gene similar to Nitrilase appeared to be induced under water deficiency in the sensitive cultivar (SP90¬ 1638) as well as in the tolerant cultivar (SP83-2847) in plants not submitted to water deficit. The gene which codifies a mobile genetic element (Retrotransposon) appeared to be induced in the tolerant cultivar under the controlled condition in detriment of the stress, and unchanged for the sensitive cultivar under both conditions.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24749号 / 生博第490号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 石川 冬木, 教授 高田 穣, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Inbred lines for hybrid crop production have been a mainstay of plant breeding. Biotechnological approaches to hasten the process are available including anther culture to halve the genome and protoplast fusion to create hybrids between incompatible partners. We applied these techniques to potato to evaluate their potential for breeding highly heterozygous, cross-pollinating species. Four families of monoploids (2n=1x=12), developed from diploid hybrids with diverse genomic constitutions but heavily favoring Solanum phureja, a primitive cultivated potato, were used in electrofusion experiments to create intermonoploid somatic hybrids (SH). The "monoploid sieve" results in the survival of only those gametes free of lethal and deleterious genes but generates sterile sporophytes, necessitating protoplast fusion for SH development. From six intermonoploid electrofusion combinations, 276 plants were regenerated over 6-9 months. Fusion conditions were optimized. Ploidy was determined by flow-cytometry and SH confirmed by microsatellite analysis. Field evaluations over three years revealed that intermonoploid SH were inferior to cultivars. Dihaploids derived by anther culture of a tetraploid intermonoploid SH were reduced in vigor with an increase in homozygosity, while 2x X 2x sexually derived populations had better yield than the SH, suggesting that producing SH introduced or eliminated factors required for productivity. Molecular analysis of the SH was conducted to examine genomic stability through protoplast isolation and plant regeneration. Sequence specific amplified polymorphism (S-SAP) represents a hybrid system incorporating amplified fragment length polymorphism (AFLP) technology in conjunction with the use of a defined genomic sequence, e.g., retrotransposon display (RD) when the defined sequence is anchored into a consensus sequence of a retrotransposon such as the long terminal repeat (LTR) sequence of Tst1. Parental monoploids, SH and various Solanaceae were evaluated by RD. Fluorescently-labeled retrotransposon-based primers were used in the ALFexpress automated fragment analyzer system. Eleven probes from RD were created for Southern blot analysis and used to verify taxonomic relationships between selected Solanaceae. Blots of intermonoploid somatic hybrids confirmed hybridity and occasional loss of genomic fragments. No activation or replication of retrotransposons was detected. Sequencing of inter-retrotransposon amplified polymorphism (IRAP) and S-SAP fragments revealed that all fragments had the expected Tst1 retroelement and/or the AFLP adaptor sequence. BLAST analysis identified 4 of the 17 fragments sequenced as part of the chloroplast genome, a tobacco anther-specific gene, repetitive DNA, and the phytochrome F gene. / Ph. D.

Schistosomen verursachen die Tropenparasitose Schistosomiasis mit 250-300 Millionen infizierten Menschen weltweit. Zur Analyse von Genfunktionen, durch die wirksame Medikamente entwickelt werden könnten, wird ein System zur Herstellung transgener Schistosomen benötigt. In dieser Arbeit wurde daher versucht, ein System zur Transfektion von Schistosoma mansoni von der transienten Expression von Reportergenen ausgehend zu entwickeln. Die Funktionalität der hergestellten Plasmide zur transienten Transfektion wurde durch die Expression der Reporter Enhanced Green Fluorescent Protein (EGFP) und beta-Galatosidase unter der Kontrolle der schistosomalen Promotoren für das Calreticulin, die 28 kDa-Glutathion-S-Transferase und das 70 kDa-Hitzeschockprotein in cos7-Zellen nachgewiesen. Für den Gentransfer in S. mansoni wurden Mikroinjektion, Elektroporation und Mikroprojektilbeschuss eingesetzt. Der Mikroprojektilbeschuss erwies sich als geeignete Methode, Plasmid-DNA zur transienten Expression von EGFP und beta-Galaktosidase in adulte und larvale Stadien zu transferieren. Da die beobachtete Reportergenexpression schwach war, wurde durch den Einsatz von Discosoma Red Fluorescent Protein (DsRed) als Reportergen versucht, das Transfektionsssytem zu optimieren. Außerdem wurden die potentiellen Promotoren für das Cathepsin D und für ein Aktin durch inverse PCR aus genomischer DNA isoliert. Die Funktionalität dieser Promotoren wurde in cos7- bzw. HeLa-Zellen nachgewiesen, eine verbesserte Expression in S. mansoni dagegen wurde mit diesen zwei Promotoren und unter Einsatz von DsRed nicht erreicht. Da S. mansoni sich in vitro nicht kultivieren lässt, muss für die Etablierung eines Transfektionssystems ein in seinen Keimzellen transgenes Stadium in den Lebenszyklus eingeschleust werden. Durch Mikroprojektilbeschuss transfizierte Mirazidien (freilebende Stadien des Parasiten) infizierten Zwischenwirtsschnecken und entwickelten sich zu transgenen Sporozysten, was durch die Transkription von EGFP nachgewiesen wurde. Für eine stabile Integration in das Genom werden unter anderem mobile genetische Elemente eingesetzt. Boudicca ist ein endogenes long terminal repeat (LTR) Retroelement. In dieser Arbeit wurde gezeigt, dass es in adulten Würmern, Sporozysten und Zerkarien transkribiert wird. Das LTR kann in vitro als Promotor zur Expression von EGFP eingesetzt werden. Außerdem wurde eine transkribierte Kopie kloniert und analysiert. Die in dieser Arbeit gewonnen Ergebnisse weisen darauf hin, dass Boudicca als Vektor zur stabilen Transfektion von S. mansoni eingesetzt werden kann. / Schistosomiasis caused by schistosomes affects about 250-300 million people world wide. The establishment of a transgenesis system for schistosomes is a pre-requisite to the identification of new drug targets and to the analysis of gene regulation and will add to our knowledge of parasite physiology and development. In this study, plasmids expressing Enhanced Green Fluorescent Protein (EGFP) and beta-galactosidase driven by the schistosomal promoters of the calreticulin, the 28kDa-glutathione-S-transferase and the 70kDa-heatshock protein were cloned and successfully tested in cos7-cells. Microinjection, electroporation and particle bombardment were used to introduce plasmid DNA into Schistosoma mansoni. Adult and larval stages of S. mansoni subjected to particle bombardment transiently expressed the reporter genes, although the observed transfection rates were very low. To optimize the transfection system Discosoma Red Fluorescent Protein (DsRed) was used as a reporter gene. Furthermore, the schistosomal promoters of the aspartic protease cathepsin D and a cytoplasmatic actin gene were isolated from genomic DNA using inverse PCR. The isolated promoters were able to drive EGFP and DsRed expression in cos7- or HeLa-cells, but improved expression could not be observed in S. mansoni. Due to the complexity of the life cycle S. mansoni cannot be cultured in vitro. In order to develop a transgenesis system for schistosomes, the life cycle must consequently be completed by genetically modified parasites. In this study it was shown that the free-living and mobile miracidium that infects the intermediate host snail could be transformed by particle bombardment and reintroduced in the life cycle using the natural path of infection. Development into transgenic sporocysts was shown through the isolation of EGFP transcripts from intermediate host snails. Mobile genetic elements are used as molecular tools to achieve stable integration of a foreign gene into a genome. Boudicca is an endogeneous long-terminal repeat (LTR) transposon in the schistosomal genome. In this study it was shown that it is transcribed in adult worms, sporocysts and cercariae. The LTR drives EGFP expression in cell cultures. Furthermore, a Boudicca transcript was cloned and analyzed by sequencing. The results suggest that Boudicca can be used as a molecular tool for stable integration of transgenes into the schistosomal genome.

Orientador: Claudia Marcia Aparecida Carareto / Banca: Cristina Vieira / Banca: Douglas Silva Domingues / Resumo: Três modelos têm sido propostos para explicar o modo de expansão dos elementos de transposição nos genomas - master gene, transposon e intermediário. O modelo master gene aplica-se à situação em que existe apenas uma única sequência ativa, de uma subfamília particular, que dá origem às demais sequências, as quais não apresentam capacidade de mobilização, enquanto o modelo transposon assume que todas as sequências de uma subfamília são ativas. Por outro lado, o modelo intermediário, considera que algumas cópias apresentam capacidade de mobilização e outras permanecem inativas. Os modelos propostos podem ser relacionados aos mecanismos de transposição, copy-and-paste e cut-and-paste, característicos das classes de elementos transponíveis I e II, respectivamente. Contudo, os estudos de dispersão intragenômica se concentram em elementos da classe I sem LTRs. Com o objetivo de ampliar o entendimento da dinâmica de dispersão genômica dos elementos de transposição e buscando verificar se os modelos propostos para a dispersão dos retroposons ajustam-se aos transposons e aos retrotransposons, foram analisados o transposon Bari e o retrotransposon 412 no genoma das espécies do grupo melanogaster de Drosophila e em populações naturais de D. melanogaster e D. simulans. Assim, buscas por seqüência similares a ambos os elementos selecionados foram realizadas nos genomas seqüenciados de seis espécies do grupo melanogaster; bem como, foram amplificadas, clonadas e sequenciadas cópias presentes nas populações naturais de ambas as espécies. Foram reconstruídas as relações evolutivas entre as sequências dos genomas, como também entre aquelas das populações naturais por meio do programa Network, utilizando o algoritmo Median Joining. Nossos resultados indicam a ausência de um modelo único de dispersão para ambos os elementos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Three models have been proposed to explain the expansion of transposable elements within genomes - master gene, transposon and intermediate. The master gene model applies to situations in which there is only one active sequence of a particular subfamily, which gives rise to other sequences which have no capacity for mobilization, while the transposon model assumes that all sequences of a subfamily are active. On the other hand, the intermediate model considers that some copies have the capacity for mobilization and others remain inactive. The proposed models can be related to mechanisms of transposition, copy-and-paste and cut-and-paste, characteristic of class I and II of transposable elements, respectively. However, studies of intragenomic dispersion concentrate on elements of the class I without LTRs. Aiming at enhancing the understanding of the transposable elements genomic dynamics of dispersion and trying to verify whether the proposed models for dispersion of retroposons fit to transposons and retrotransposons, we analyzed the retrotransposon 412 and Bari transposon in the genome of the species melanogaster group of Drosophila and in natural populations of D. melanogaster and D. simulans. Thus, searches for sequences similar to both elements were done in the sequenced genomes of six species of the melanogaster group; as well as copies present in natural populations of both species were amplified, cloned and sequenced. We reconstructed the evolutionary relationships between the sequences of the genomes, as well as those amplified from samples of wild populations through the Network program, using the Median Joining algorithm. Our results indicate the absence of a single model of dispersion for both transposable elements, Bari and 412, in the different species analyzed, as well as in the populations of both species... (Complete abstract click electronic access below) / Mestre

L’élément L1 (Long Interspersed Nuclear Element 1 ou L1) est le seul rétrotransposon autonome et actif connu dans notre génome, représentant 17% de celui-ci. Capable de se répliquer grâce à un intermédiaire à ARN et un mécanisme de transcription inverse initiée au site d’intégration, il encode deux protéines ORF1p et ORF2p, qui s’associent à l’ARN L1 pour former une particule ribonucléoprotéique (RNP). L’élément L1 rétrotranspose préférentiellement en cis : un L1 défectif est complémenté en trans par un élément fonctionnel de façon inefficace. Ce travail s'intéresse à deux étapes clefs du cycle réplicatif du L1 : l'assemblage de la RNP L1 en cis ou en trans afin d’explorer le mécanisme de la cis-préférence et la spécificité de l’initiation de la reverse transcription initiée. Nous avons d’abord comparé deux méthodes d’analyse de l’activité RT. Puis, nous avons montré l’importance de la complémentarité entre queue poly(A) de l’ARN L1 et site d’intégration durant l’initiation de la RT, ainsi que l’impact de mésappariements terminaux éventuels. Enfin, nous avons étudié les bases biochimiques de la cis-préférence, à travers la coexpression et la purification de deux éléments distincts étiquetés, ce qui nous a permis de suivre l'assemblage et l'activité de leurs RNPs respectives. Nos données suggèrent que ORF1p et ORF2p peuvent lier en trans l’ARN L1 de façon efficace et que la cis-préférence pourrait nécessiter des quantités limitantes de L1. / The Long Interspersed Nuclear Element 1 (LINE-1 ou L1) is the only known active and autonomous retrotransposon in the human genome and constitutes around 17% of our genomic DNA. The L1 element is able to replicate through an RNA intermediate by a mechanism called target-primed reverse transcription and encodes two proteins ORF1p and ORF2p, which associate with the L1 RNA to form a ribonucleoprotein particle (RNP). L1 preferentially retrotranspose in cis: a defective L1 can only be rescued in trans at low levels by a replication-competent copy. During this work, we focused on two essential steps of the L1 replication cycle: the assembly of the L1 RNP in cis or in trans to explore the mechanism of the cis-preference and the specificity of L1 reverse transcription priming. First, we compared two different methods to detect L1 RT activity. Then, we showed the importance of base-pairing between the poly(A) tail of the L1 RNA and the integration site to prime reverse transcription and the impact of potential mismatches. Finally, we investigated the biochemical basis of the cis-preference through the coexpression and purification of two different tagged L1 elements, which allowed us to follow the assembly and activity of their RNP. Our data suggest that binding of ORF1p and ORF2p in trans is efficient and that the cis-preference might requires limiting L1 levels.

Durant ces dernières décennies, la fertilité masculine est en constante diminution à l’échelle mondiale. Même si les facteurs environnementaux ont une part de responsabilité indéniable, il n’en reste pas moins que les altérations génétiques mais également épigénétiques semblent aussi largement impliquées. La compréhension des mécanismes épigénétiques qui régulent la fertilité masculine est récente mais essentielle pour le développement de nouvelles approches thérapeutiques. Dans ce contexte, l’objectif de mes travaux de thèse s’est focalisé sur l’étude du rôle du variant d’histone H3.3 dans la spermatogenèse. H3.3 possède la capacité de remplacer l’histone canonique H3 dans la chromatine modifiant ainsi les propriétés épigénétiques de cette dernière. H3.3 est nécessaire à la spermatogenèse mais son rôle reste à élucider. Grace à plusieurs modèles murins, mes travaux de thèse ont montré que la forme H3.3B est essentielle à la reproduction masculine notamment pour la transition méiose/post-méiose. Lors de cette transition, on observe une forte régulation des piARN, des rétrotransposons et des chromosomes sexuels. Nos expériences révèlent pour la première fois que la perte de H3.3B provoque une chute de l’expression des piARN. À l’inverse, l’absence de H3.3B est aussi associée à une augmentation de l’expression de l’ensemble des gènes des chromosomes sexuels comme des rétrotransposons RLTR10B et RLTR10B2. Ces changements d’expression se traduisent par une spermatogenèse altérée et une infertilité. Par des expériences de ChIP-seq, nous avons observé que H3.3 est fortement enrichie sur les piRNA, les rétrotransposons RLTR10B et RLTR10B2 et l'ensemble des chromosomes sexuels. Toutes ces expériences ont permis de mieux caractériser la fonction régulatrice de l’histone H3.3B au cours de la spermatogenèse. En particulier, elles démontrent que H3.3B, en fonction de sa localisation sur la chromatine, intervient dans la régulation positive ou négative de l'expression de régions chromatiniennes définies. Ces résultats montrent l’importance des contrôles épigénétiques au cours de la spermatogenèse et ouvrent de nouvelles pistes dans la compréhension des causes d’infertilité masculine. / In last decades, male fertility has been steadily declining worldwide. Even if environmental factors have an undeniable responsibility, the fact remains that both genetic and epigenetic alterations also seem to be widely implicated. The understanding of the epigenetic mechanisms that regulate male fertility is recent but essential to develop a new therapeutic approaches. In this context, the objective of my thesis work focused on the study of the role of histone variant H3.3 in spermatogenesis. H3.3 has the ability to replace the H3 canonical histone in chromatin thus modifying the epigenetic properties of chromatin. H3.3 is necessary for spermatogenesis but its role remains unclear. Used to several mouse models, my thesis work has shown that the H3.3B form is essential for male reproduction and especially for the meiosis/post-meiosis transition. During this transition, there is a strong regulation of piRNAs, retrotransposons and sex chromosomes. Our experiments reveal at the first time that the loss of H3.3B resulted in down-regulation of the expression of piRNA. In contrast, the absence of H3.3B is also associated with increased expression of all sex chromosom genes as well as of both RLTR10B and RLTR10B2 retrotransposons. These expression changes result in altered spermatogenesis and infertility. By ChIP-seq experiments, we observed that H3.3 is markedly enriched on the piRNA clusters, RLTR10B and RLTR10B2 retrotransposons and the whole sexual chromosomes. All these experiments allowed bettering characterizing the regulatory function of histone H3.3B during spermatogenesis. In particular, he demonstrates that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined chromatin regions. These results show the importance of epigenetic controls during spermatogenesis and open new tracks for understanding the causes of male infertility.

Considerable variation exists both within and between species in nuclear DNA content. Despite there being no obvious functional role for much of this DNA, many studies have reported phenotypic correlations with genome size at various taxonomic levels. This suggests that DNA plays a functional role beyond the traditionally understood mechanisms. One such example of a phenotypic correlation with DNA content is present in the genus Silene, where a negative correlation between DNA content and flower size exists within and between species. This relationship is consistent with the direction of sexual dimorphism in DNA content (caused by heteromorphic sex-chromosomes) and flower size in the most studied species in the genus: S. latifolia. This thesis takes a comparative approach between two closely related species in the genus (S. latifolia and S. marizii), which differ markedly in their nuclear DNA content, in order to investigate the nature and phenotypic impacts of variation in DNA content. A phenotypic survey from a number of S. marizii populations reveals that the pattern of DNA content variation in this species is very different to that in S. latifolia. In particular, phenotypic correlations with DNA content appear be much weaker, whilst sexual dimorphism in DNA content, when present, appears to occur in either direction. A survey of interspecific hybrids suggests that this may be due to an enlarged S. marizii X-chromosome and that DNA content in hybrids may be biased with regard to their parents. Repetitive elements may be significant constituents of plant genomes. A study of Ty1-copia class retrotransposons in the two species reveals that they are present as a large and highly heterogeneous population. Phylogenetic analysis of these elements suggests a substantial degree of genetic isolation between the two species. Finally, an assessment of the flow-cytometric method, used to estimate DNA content, reveals substantial error associated with the method, but only limited evidence for stoichiometric effects.

Le maintien de l'intégrité génomique est crucial pour la préservation du potentiel des cellules souches hématopoïétiques (CSH). Les lésions de l'ADN dans les CSH sont associées à une capacité réduite à reconstituer l'hématopoïèse, à altérer le potentiel de différentiation et à accroître le risque de développer des tumeurs myéloïdes. Les éléments rétrotransposables (ER), se propageant dans le génome à travers un ARN intermédiaire, ont été associés à la perte d'auto-renouvellement, au vieillissement et aux dommages à l'ADN. Cependant, leur rôle dans les CSH n'avait pas été abordé. Dans cette étude, nous avons constaté que les CSH expriment des niveaux élevés d'ARNm de plusieurs ER comprenant des rétrovirus endogènes (ERV) et des L1 (LINE-1: Long Interspersed Nuclear Elements 1). Leur expression augmente avec l'irradiation. En utilisant des souris transgéniques L1-EGFP, on a montré que la rétrotransposition de L1 se produit dans les CSH in vivo. En outre, les inhibiteurs de la transcriptase inverse Efavirenz et ddC sauve à la fois les CSH des dommages persistants à l'ADN induit par l’irradiation et de la perte de prolifération in vitro. Ceci démontre que la rétrotransposition endogène joue un rôle important dans l'instabilité génomique de CSH induite par l’irradiation et dans leur perte de fonction. Nous avons précédemment montré que la thrombopoïétine (TPO), un facteur d'auto-renouvellement critique pour le CSH, limite les lésions de l'ADN induites par l’irradiation en améliorant la réparation de l'ADN. Nous avons découvert que le traitement par TPO empêche également l'expression et la mobilisation d’ER induite par l’irradiation. Nous avons aussi constaté que l’expression et la retrotransposition de L1 augmente dans les CSH provenant de souirs Mpl-/- et L1-EGFPxMpl-/-. Cela montre que la signalisation TPO in vivo est nécessaire pour restreindre l’expression et la retrotransposition d’ER dans les CSH au niveau basal et dans des conditions de stress. L'analyse transcriptomique a révélé que la TPO induit une réponse d'expression génique antivirale d'interféron (IFN) de type I dans les CSH. En utilisant des souris déficientes en STAT1/STAT2, nous démontrons que cette réponse est dépendante à la fois de STAT1 et de STAT2 et est requise pour l'inhibition de l'expression d’ER. En conclusion, cette étude montre que les ER représentent une importante source d’instabilité génomique dans les CSH. Les CSH sont capables de monter une réponse antivirale en réponse à la TPO comme un nouveau mécanisme pour limiter les dommages à l'ADN. Bien que la sécrétion constitutive d'IFN-I se produise chez des souris saines, les IFN sont produits abondamment principalement pendant les infections. Ainsi, la réponse d'expression de gène d'IFN induite par la TPO peut représenter un signal constitutif important et CSH-dédié; permettant à ces cellules de résister aux lésions de l'ADN induites par ER, tout en préservant leur capacité d'auto-renouvellement. / Maintenance of genomic integrity is crucial for the preservation of hematopoietic stem cell (HSC) potential. DNA damage in HSCs is associated with reduced ability to reconstitute hematopoiesis, altered lineage potential and accrued risk of developing myeloid malignancies. Retrotransposable elements (RE), spreading in the genome through an RNA intermediate, have been associated with loss of self-renewal, aging and DNA damage. However, their role in HSCs has not been addressed. In this study, we found that HSCs express high mRNA levels of several REs, including evolutionary recent long interspersed element-1 (L1) and endogenous retroviruses (ERV). Their expression further increases upon total body irradiation (TBI). Using L1EGFP transgenic reporter mice, we show that productive L1 retransposition occurs in HSCs in vivo. Furthermore, the reverse transcriptase inhibitors Efavirenz and ddC rescue TBI-induced both persistent DNA damage and HSC loss of proliferation in vitro. This demonstrates that endogenous retrotransposition plays an important role in TBI-induced HSC genomic instability and their loss of function. We have previously shown that thrombopoietin (TPO), a critical HSC self-renewal factor limits TBI-induced HSC DNA damage by improving DNA repair. We found that TPO treatment also prevents TBI-induced RE expression and mobilization. In addition, L1 expression and retrotransposition are increased in Mpl-/- and L1-EGFPxMpl-/- HSCs, showing that TPO signaling in vivo is required to restrain RE in HSCs, under both steady state and stress conditions. Transcriptomic analysis revealed that TPO induces an anti-viral, interferon (IFN) type-I like, gene expression response in HSCs. Using STAT1/STAT2-deficient mice, we demonstrate that this response is dependent on both STAT1 and STAT2 and is required for TPO-mediated RE expression inhibition in HSCs. Overall, this study shows that REs represent an important HSC intrinsic source of genomic instability and uncovers the ability of HSCs to mount an anti-viral innate immune state in response to TPO as a novel mechanism to minimize DNA damage. Although constitutive IFN-I secretion occurs in healthy mice, IFNs are produced abundantly mainly during infections. Thus, TPO-induced IFN gene expression response may represent an important constitutive, and HSC-dedicated, signal allowing HSCs to resist RE-induced DNA damage while preserving their self-renewal ability.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-06Bitstream added on 2014-06-13T19:42:42Z : No. of bitstreams: 1 setta_n_dr_sjrp.pdf: 1404480 bytes, checksum: e8ca57a6c89dd8308471f12f028ecfe8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O gênero Zaprionus tem sido eleito como um bom modelo biológico para estudos genéticocomparativos com as espécies do subgrupo melanogaster do gênero Drosophila, embora seu posicionamento filogenético dentro da família Drosophilidae ainda seja controverso. Na presente Tese foi investigada a presença de 10 elementos de transposição (TEs) em Zaprionus indianus e Drosophila malerkotliana, bem como a distribuição, a atividade transcricional e as relações evolutivas de três retrotransposons (copia, gypsy e micropia) em sete espécies do gênero Zaprionus. Para isso, foram empregadas as técnicas de Dot blot, PCR, RT-PCR e seqüenciamento. As seqüências obtidas foram comparadas às dos respectivos elementos das demais espécies de drosofilídeos disponíveis nas bases de dados genômicas. Os resultados indicam que Z. indianus e D. malerkotliana apresentam em seus genomas todos os TEs de D. melanogaster investigados. O retrotransposon copia foi seqüenciado e está transcricionalmente ativo nas sete espécies do gênero Zaprionus e constitui uma nova subfamília relacionada aos elementos do subgrupo melanogaster, que foi denominada subfamília GBFDouble-gap. Por outro lado, os retrotransposons gypsy e micropia foram identificados nas espécies do subgênero Zaprionus, onde também estão transcricionalmente ativos, e pertencem às subfamílias já descritas para as espécies do subgrupo melanogaster. As análises evolutivas sugeriram que esses três retrotransposons devem ter participado de eventos de transferência horizontal com as espécies do subgrupo melanogaster e com pelo menos um doador desconhecido, no caso do retrotransposon micropia. Além disso, o cálculo dos tempos de divergência dos elementos sugere que eles passaram por ondas de transferências horizontais, mais antigas para o retrotransposon copia, e mais recentes para gypsy e micropia. Esses resultados... / The Zaprionus genus has been elected as a good biological model for comparative analyses with the melanogaster subgroup of Drosophila genus, though its phylogenetic positioning within the Drosophilidae family is still controversial. This study aiming at investigating the occurrence of 10 transposable elements (TEs) in Zaprionus indianus and Drosophila malerkotliana species, as well the distribution, transcriptional activity and evolutionary relationships of three retrotransposons (copy, gypsy and micropia) in seven species of Zaprionus genus. To do so, Dot blot, PCR, RT-PCR and sequencing methods were employed. The Zaprionus sequences obtained were compared with the drosophilid sequences available in genomic databases. The results indicated that Z. indianus and D. malerkotliana harbor all D. melanogaster TEs investigated. The copia retrotransposon is present and transcriptionally active in seven species of the Zaprionus genus and represents a new subfamily related to that of the melanogaster subgroup, named as GBFDouble-gap subfamily. Additionally, gypsy and micropia retrotransposons were identified in the Zaprionus species subgenus, which are transcriptionally active and belong to the melanogaster subgroup subfamilies. The evolutionary analysis showed the three retrotransposons could have been involved in horizontal transfer events with species of the melanogaster subgroup for the three retrotransposons and at least one unknown donor regarding to micropia retrotransposon. Moreover, the time of divergence seems to indicate that the retrotransposons experienced horizontal transfer waves, the oldest involving the copia element followed by the gypsy and micropia retrotransposons in more recent times. These results suggest that the horizontal transfer phenomenon has happened repeatedly during the Zaprionus genus and melanogaster subgroup evolution in the Afrotropical region.