Ce document est dédié à la modélisation dynamique des interactions hôtes-parasites. Il porte sur deux modèles biologiques très différents, mais étudiés à l’aide de modèles épidémiologiques standards construits à partir de systèmes dynamiques à compartiments. La première contribution est l’implémentation d’un modèle ‘micro-parasites’ pour étudier la transmission du parasite protozoaire Trypanosoma cruzi, agent étiologique de la trypanosomiase américaine (ou ‘maladie de Chagas’), au sein d’une communauté d’hôtes synanthropiques et domestiques. L’analyse du modèle mathématique montre pour la première fois dans ce système biologique un effet de dilution associé aux hôtes aviaires, ainsi que la possibilité de réduire la transmission à l’homme par modification de la composition de la communauté d’hôtes domestiques. La seconde contribution porte sur la dynamique des ‘parasites génomiques’ que sont les éléments transposables. En utilisant les analogies entre concepts de génomique et d’écologie proposées par l’approche d’ « Écologie du génome », il a été possible d’adapter des modèles développés pour les ‘macro-parasites’ à la dynamique d’éléments transposables de classe 1, les retro-transposons. L’analyse de cesmodèles permet de formuler des hypothèses sur l’importance relative de la démographie des hôtes, de la distribution du nombre de copies entre les individus et des mécanismes moléculaires de silencing de ces éléments, sur leurpersistance au sein de population d’hôtes se reproduisant de façon asexuée. / This document is dedicated to the dynamic modeling of host-parasite interactions. It is about two distant biological models, who are studied using standard epidemiological models built from dynamic compartmental models. The first contribution is the implementation of a 'micro-parasites' model to study the transmission of the protozoan parasite Trypanosoma cruzi, the etiological agent of American trypanosomiasis (or 'Chagas' disease), within a host community of synanthropic and domestic animals. The analysis of the mathematical model shows for the first time in this biological system a dilution effect associated with avian hosts, as well as the possibility of reducing the transmission to humans by modifying the composition of the domestic host communities. The second contribution deals with the dynamics of the "genomic parasites" that are the transposable elements. Using the analogies between genomics and ecology concepts proposed by the "Genome Ecology" approach, it was possible to adapt models developed for 'macro-parasites' to the dynamics of transposable elements of class 1, retro-transposons. The analysis of these models makes it possible toformulate hypotheses on the relative importance of the host demography, the distribution of the number of copies between individuals and the molecular mechanisms of silencing of these elements, on their persistence within the population of hosts reproducing asexually.

Orientador: Claudia Marcia Aparecida Carareto / Banca: Ricardo De Marco / Banca: André Luís Laforga Vanzela / Resumo: A resistência aos inseticidas é um modelo de processo evolutivo onde o inseticida atua como agente seletivo e, como resposta à seleção, ocorre a evolução da resistência nas populações de insetos. As enzimas citocromo P450 monooxigenases (CYP) formam uma família responsável pela resistência aos inseticidas. Tem sido proposto que a inserção de elementos transponíveis (TEs) em regiões reguladoras ou codificadoras dos genes da família Cyp pode alterar a expressão gênica e induzir a resistência aos inseticidas. No presente estudo foram realizadas análises in silico que permitiram identificar a ocorrência de inserções de fragmentos de TEs em 35 genes Cyps com diferentes funções, e em seus genes flanqueadores, em Drosophila melanogaster e D. simulans, além de 13 genes Cyps de seis espécies do grupo melanogaster de Drosophila. As inserções de TEs ocorreram principalmente nas regiões flanqueadoras 5' dos Cyps associados à resistência aos inseticidas e à função monooxigenase geral. Os resultados não indicaram qualquer relação entre a distância em relação ao gene e o número de inserções. As análises mostraram que a maioria das inserções pertence à classe de transposons de DNA, sendo o transposon DNAREP1_DM o que apresentou o maior número de cópias. O fato de essas seqüências apresentarem putativos sítios de ligação de fatores de transcrição sugere que possam desempenhar algum papel na regulação dos genes Cyps. Também foi analisada a ocorrência de polimorfismos de inserção de TEs em regiões flanqueadoras de genes da família Cyp, em diferentes linhagens geográficas resistentes e suscetíveis, de D. melanogaster e D. simulans. Análises evidenciaram a presença de polimorfismo interpopulacional de tamanho das regiões flanqueadoras dos genes Cyp6w1, Cyp6a2 e Cyp12d1, porém, não indicaram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Insecticide resistance is a model of evolutionary process where the insecticide acts as the selective agent and resistance in the insect populations evolves as an answer to selection. Cytochrome monooxygenases (CYP) is family of enzymes responsible for the insecticide resistance. It has been proposed that insertion of transposable elements (TEs) in regulatory or coding regions of the Cyp genes can alter gene expression and induce insecticide resistance. In the present study in silico analyses allowed identifying the insertion of TE fragments in 35 Cyp genes with different functions, in Drosophila melanogaster and D. simulans, as well as in 13 Cyps of six species of the melanogaster group of Drosophila. The TE insertions occurred mainly in the 5' flanking regions of Cyp genes associated to resistance and to those with a general monooxygenase function. The results did not indicate any relationship between the number of insertions and the distance in relation to the gene. The analyses showed that most of the insertions belong to the DNA transposon class, being DNAREP1_DM the most numerous. Since this element carry putative biding sites of transcription factors it can be suggested they play same role in gene regulation. The polymorphism of TE insertions in the flanking regions of Cyp6w1, Cyp6a2 and Cyp12d1, genes associated to resistance, found in resistant and as well as in susceptible geographical strains of D. melanogaster and D. simulans, does not indicate any relationship between the presence of TEs in those regions and the insecticide resistance. The results also showed that the insertions of TEs in the proximities of the Cyps associated to resistance is differential among six species of the melanogaster group, not following the genomic proportion of TEs in each species. These results also suggest that TEs inserted in the Cyp flanking regions can carry out an adaptive... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2017-07-21T19:59:45Z (GMT). No. of bitstreams: 1 Cleberson Cezario Primo.pdf: 3495062 bytes, checksum: 39e96203835221786c05ef8ab3b75523 (MD5) Previous issue date: 2015-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Loricariidae family (Actinopterygii: Siluriformes) is morphologically diverse, has a number close to 900 valid species, distributed in seven subfamilies (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae and Hypostominae). However, cytogenetic studies in species of the family show evolutionary trends of karyotype diversification well defined for each of the subfamilies and the diploid number (2n) of 54 chromosomes is considered basal. Among the representatives of the subfamily Loricariinae, the variation of 2n is 36 to 74 chromosomes. Given these data, the Robertsonian rearrangements are the main mechanisms to explain the chromosome number variation in the subfamily. Rineloricaria is the most specious genus of Loricariinae, porting species with 2n = 36 to 2n = 70 chromosomes. However, little is known about what types of repetitive DNAs originate fission and fusion chromosome events. In this study, species of Rineloricaria from different rivers of the Paraná drainage were studied: Rineloricaria latirostris (Laranjinha river, Cinzas basin and Barra Grande river, Ivaí basin); Rineloricaria pentamaculata (Barra Grande and Juruba rivers, Tibagi basin); and, Rineloricaria stellata and Rineloricaria capitonia (Upper Uruguai river). The aim of this study was to characterize the karyotypes of populations/species of Rineloricaria and to check what types of repetitive DNAs may be related to Robertsonian events in the genus. In R. latirostris was detected 2n = 46 chromosomes for both populations, as well as for a triploid specimen from Laranjinha river. Rineloricaria pentamaculata had 2n = 56 chromosomes to populations from Barra Grande and Juruba rivers and a karyomorph in Barra Grande river with 2n = 54 chromosomes. Rineloricaria stellata had 2n = 54 chromosomes, while R. capitonia presented 2n = 64 chromosomes, both from the Uruguai river. The results using the chromosomal markers of 18S rDNA, 5S rDNA and TTAGGGn telomeric probe showed that these repetitive DNAs participated in end to end fusions of the st/a chromosomes in the karyotype diversification of R. latirostris. Vestiges of interstitial telomeric sites (ITS) were also detected in R. pentamaculata, karyomorph of 54 chromosomes from the Barra Grande river, suggesting chromosomal fusion to the diversification of this karyotype. The wide range of 2n between R. stellata and R. capitonia is compatible to the reproductive isolation of syntopic species and the diversification of R. capitonia can be explained by centric fusions. In addition to Robertsonian rearrangements, the pericentric inversions also assisted in the diversification of karyotypic formulas among the species/populations. In situ localization analysis using the transposable element Tc1-Mariner Like probe showed no evidence of the participation of transposon in chromosomal rearrangements and dispersion of multiple sites of 5S rDNA in Rineloricaria. Furthermore, analyzes of the Tc1-Mariner Like sequences showed intense molecular degeneration, especially in transposase domains. These results indicate the absence of activity of these sequences, which must be inert or serve to other genomic functions in the genus. Thus, this study discusses the telomeric instability, repetitive DNAs and the participation of rDNA gene families in karyotype diversification events in Rineloricaria. / A família Loricariidae (Actinopterygii: Siluriformes) é extremamente diversificada morfologicamente, conta com um número próximo a 900 espécies válidas, distribuídas em sete subfamílias (Lithogeneinae, Delturinae, Neoplecostominae, Hypoptopomatinae, Loricariinae, Ancistrinae e Hypostominae). Não obstante, os estudos citogenéticos em representantes da família mostram tendências evolutivas da diversificação cariotípica bem definidas para cada uma das subfamílias, sendo considerado basal o número diploide (2n) de 54 cromossomos. Entre os representantes da subfamília Loricariinae a variação do 2n é de 36 a 74 cromossomos. Diante destes dados, os rearranjos Robertsonianos são os principais mecanismos para explicar a variação cromossômica numérica na subfamília. Rineloricaria é o gênero mais especioso de Loricariinae, com espécies apresentando 2n = 36 até 2n = 70 cromossomos. Contudo, pouco se sabe sobre quais os tipos de DNAs repetitivos originam os eventos de fissão e fusão cromossômica. Neste estudo, foram avaliadas espécies de Rineloricaria de diferentes rios do sistema hidrográfico do Paraná: Rineloricaria latirostris (rio Laranjinha, bacia do rio das Cinzas e rio Barra Grande, bacia do rio Ivaí); Rineloricaria pentamaculata (rio Barra Grande e rio Juruba, bacia do rio Tibagi); e, Rineloricaria stellata e Rineloricaria capitonia (Alto Rio Uruguai). O objetivo foi de caracterizar cariotipicamente as populações/espécies de Rineloricaria estudadas, além de verificar quais os tipos de DNAs repetitivos podem estar relacionados aos eventos Robertsonianos no gênero. Em R. latirostris foi detectado 2n = 46 cromossomos para ambas populações, além de um exemplar triploide para o rio Laranjinha. Rineloricaria pentamaculata apresentou 2n = 56 cromossomos para as populações dos rios Barra Grande e Juruba e um cariomorfo 2n = 54 cromossomos no rio Barra Grande. Rineloricaria stellata apresentou 2n = 54 cromossomos, enquanto R. capitonia detém 2n = 64 cromossomos, ambas do rio Uruguai. Os resultados com marcadores cromossômicos de rDNA 18S, rDNA 5S e sonda TTAGGGn evidenciaram que estes DNAs repetitivos participaram dos eventos de fusão terminal para terminal (end to end fusions) de cromossomos st/a na diversificação cariotípica de R. latirostris. Vestígios de sítios teloméricos intersticiais (ITS) foram evidenciados também em R. pentamaculata, cariomorfo de 54 cromossomos do rio Barra Grande, sugerindo fusão cromossômica para a diversificação deste cariótipo. A ampla variação de 2n entre R. stellata e R. capitonia é compatível para o isolamento reprodutivo das espécies sintópicas e pode ser explicado por fissões cêntricas na diversificação de R. capitonia. Além dos rearranjos Robertsonianos, as inversões pericêntricas também auxiliaram na diversificação de fórmulas cariotípicas entre as espécies/populações. A análise de localização in situ do elemento transponível Tc1-Mariner Like não mostrou evidências da participação deste transposon nos rearranjos cromossômicos e na dispersão dos sítios múltiplos de rDNA 5S em Rineloricaria. Ainda, as análises das sequências Tc1-Mariner Like evidenciaram intensa degeneração molecular, principalmente nos domínios transposase. Estes resultados indicam a ausência de atividade destas sequências, as quais devem ser inertes ou servir para outras funções genômicas no gênero. Desta forma, este estudo discute a instabilidade telomérica, DNAs repetitivos e a participação das famílias gênicas de rDNA nos eventos de diversificação cariotípica em Rineloricaria.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2017-10-19T19:03:18Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Lucas Rosolen de Almeida Mello.pdf: 2555328 bytes, checksum: 3d7ce3cf485cd835d38b843b7b692900 (MD5) / Made available in DSpace on 2017-10-19T19:03:18Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Lucas Rosolen de Almeida Mello.pdf: 2555328 bytes, checksum: 3d7ce3cf485cd835d38b843b7b692900 (MD5) Previous issue date: 2015-02-27 / A ordem Coleoptera é a mais diversificada entre todos os seres vivos, existindo ampla possibilidades de estudos no que diz respeito à diversidade cariotípica e aos mecanismos de diferenciação. As espécies da subtribo Oedionychina (Alticinae; Chrysomelidae) são interessantes para estudos evolutivos, pois possuem cromossomos sexuais gigantes e assinápticos durante a meiose, podendo ser considerados altamente derivados. Assim, o objetivo do presente estudo foi propor os mecanismos moleculares envolvidos no processo de diferenciação e evolução dos cromossomos sexuais em espécies do gênero Omophoita. A análise de mapeamento, utilizando sondas de DNA C0t-1 total (cinética de reassociação de DNA altamente e moderadamente repetitivo) mostrou marcações distribuídas em todos os cromossomos, especialmente nos cromossomos sexuais. A hibridação cruzada entre as espécies produziu um padrão de localização muito semelhante, evidenciando que a maior parte do genoma é compartilhada entre as espécies de Omophoita. Análise em conjunto dos resultados obtidos com bandas C, fluorocromos e C0t-1 mostram que a heterocromatina das espécies em grande parte é composta de DNA repetitivo distribuída ao longo dos cromossomos sexuais e autossomos. O mapeamento cromossômico com sondas de microssatélites (SSRs) mostrou marcações conservadas para os autossomos e diversificadas para os cromossomos sexuais, evidenciando uma diferença de composição de SSRs dos cromossomos sexuais entre as espécies. Os resultados de hibridação com clones de elementos de transposição mostraram alguns padrões semelhantes aos obtidos com SSRs, podendo indicar que ao longo do processo evolutivo das espécies esses elementos estiveram presentes no processo de diferenciação. Considerando todos os resultados, pode se propor uma diferença de constituição nos cromossomos sexuais das espécies e, desta forma, inferir que os DNAs repetitivos tiveram um papel evolutivo na diferenciação desses cromossomos na subtribo. / The order Coleoptera is the most diverse of all living beings, with a wide possibilities of studies with regards to the karyotype diversity and the mechanisms of differentiation. The species of the subtribe Oedionychina (Alticinae; Chrysomelidae) are interesting for evolutionary studies due to the giant sex chromosomes and asynaptic during meiosis, can be considered highly derivate. The objective of this study was to propose the molecular mechanisms involved in the differentiation process and evolution of sex chromosomes in the Omophoita genus. The Mapping analysis using DNA C0t-1 total (reassociation kinetics highly and moderately repetitive DNA) showed marks distributed in all chromosomes, especially in the sex chromosomes. The cross-hybridization among species produced a very similar location pattern, indicating that most of the genome is shared among species Omophoita. Analysis of the results obtained in conjunct with C-bands, fluorochromes and C0t-1 together show that the heterochromatin of the species is largely composed of repetitive DNA distributed throughout the autosomes and sex chromosomes. Chromosome mapping with microsatellite (SSRs) probes showed conserved patterns for autosomes, but diversified to sex chromosomes, showing difference in SSRs composition in the sex chromosomes, of the species. The results of hybridization with transposition element clones showed some similarities patterns to the SSRs markers, which may indicate that throughout the evolutive process of species these elements were present. Considering all results we can propose differences in the constitution of sex chromosomes of the species studied, thus, we can infer an evolutionary role of repetitive DNA in the differentiation of chromosomes in the subtribe.

Une grande partie du génome des eucaryotes est constituée d’éléments transposables(ET). Ces séquences d’ADN répétées ont la capacité de se déplacer d’un site chromosomiqueà un autre et de multiplier le nombre de leurs copies, pouvant ainsi être la cause d’uneinstabilité génétique. Face à ce potentiel de mutagénèse, un certain nombre de systèmes ontété sélectionnés dans les génomes eucaryotes qui conduisent à une réduction de l’activité desET. Notamment, chez la drosophile, on a récemment mis en évidence des mécanismes derégulation impliquant les modifications d’histones, et une nouvelle classe de petits ARN,appelés piARN, qui contrôlent spécifiquement les éléments transposables dans les tissusreproducteurs.tirant est un rétrotransposon à LTR de la drosophile, de type Gypsy, isolé au sein dulaboratoire dans les populations naturelles de D. simulans, où le nombre de ses copies estvariable entre populations. Cet ET possède la même structure génomique que les rétrovirus.Dans la première partie de cette thèse, j’ai caractérisé un élément tirant actif dans lespopulations naturelles de D. simulans. Je me suis intéressé en particulier au gène de laprotéine d’enveloppe (env), qui confère le caractère infectieux du rétrovirus. La comparaisondes transcrits et de la protéine du gène env entre populations de D. simulans a montré quetirant est actif dans une population, et cette activation est associée à sa mobilisation, alors quedans les autres populations tirant est présent, mais régulé.Dans la deuxième partie de mon travail, je me suis intéressé à l’étude de l’influence detirant sur la structure de la chromatine au niveau de son site d’insertion et à son influence surl’expression des gènes voisins. J’ai étudié trois modifications d’histones dans troispopulations naturelles, dont une où tirant est inséré dans un intron du gène tkv. Les résultatsobtenus montrent que tirant est capable de modifier la structure de la chromatine au niveau deson site d’insertion, mais aussi en amont, par l’hétérochromatinisation d'un promoteur du gènetkv, en affectant ainsi son taux de transcription.Enfin, je me suis intéressé à la régulation post-transcriptionnelle de tirant par lespiARN. Par l’analyse de croisements intraspécifiques entre des souches contenant ou non descopies de tirant dans l’euchromatine, j’ai montré qu’une régulation post-transcriptionnelle parles piARN germinaux qui contrôle tirant dans les cellules folliculaires de l’ovaire. J’ai aussipu montrer une expression variable entre populations des gènes de la voie piARN. / Eukaryotic genomes harbor a wide variety of repeated sequences, such as transposableelements (TE). These sequences are able to move from one chromosomal site to another, tomultiply their number of copies, and can be the cause of a genetic instability. Sophisticatedgenomic defenses have evolved to restrict their activity. In Drosophila, epigeneticmodification such as post-translational histone modifications and RNAi interference areinvolved in TE silencing in reproductive tissues. The silencing of an LTR like element, tirant,has been deeply analyzed in this work. Tirant is a Gypsy like element, isolated in ourlaboratory in natural populations of D. simulans, in which a high level of copy numbervariability is observed between strains.Here, I first describe an active tirant element in natural populations of D. simulans. Ihave focused on the envelope protein gene (env), which confers the infectious behavior to theretrovirus. By comparison of tirant transcripts level and protein localization between naturalpopulations of D.simulans, I showed that tirant is active in one population, and this activationis correlated with its mobilization.I then focused on the effects of TE insertions on chromatin structure and in its influenceon the expression of the nearby genes. I studied three histone modification marks in threenatural populations, in the locus in which tirant was inserted. I show that tirant is associatedwith repressive marks and active marks, which explains the activity of the element. We alsoshowed that tirant modifies the structure of the chromatin at the level of its site of insertion,but also upstream, by the heterochromatinization of the promoter of tkv gene, interfering withthe level of transcription of the gene.Finally, I was interested in the post-transcriptional regulation of tirant involving thepiRNA pathway. By crossing D.simulans strains which contains different copy numbers ofthe tirant element, I showed that tirant is regulated in the follicular cells by the germ linepiRNA pathway. I was also able to show a variable expression between populations of theproteins of the piRNA pathway.

De nos jours, la quantité de données génétiques séquencées augmente de manière exponentielle sous l'impulsion d'outils de séquençage de plus en plus performants, tels que les outils de séquençage haut débit en particulier. De plus, ces données sont de plus en plus facilement accessibles grâce aux bases de données en ligne. Cette plus grande disponibilité des données ouvre de nouveaux sujets d'étude qui nécessitent de la part des statisticiens et bio-informaticiens de développer des outils adaptés. Par ailleurs, les progrès constants de la statistique, dans des domaines tels que le clustering, la réduction de dimension, ou les régressions entre autres, nécessitent d'être régulièrement adaptés au contexte de la bio-informatique. L’objectif de cette thèse est l’application de techniques avancées de statistiques à des problématiques de bio-informatique. Dans ce manuscrit, nous présentons les résultats de nos travaux concernant le clustering de séquences génétiques via Laplacian eigenmaps et modèle de mélange gaussien, l'étude de la propagation des éléments transposables dans le génome via un processus de branchement, l'analyse de données métagénomiques en écologie via des courbes ROC ou encore la régression polytomique ordonnée pénalisée par la norme l1. / Nowadays, the quantity of sequenced genetic data is increasing exponentially under the impetus of increasingly powerful sequencing tools, such as high-throughput sequencing tools in particular. In addition, these data are increasingly accessible through online databases. This greater availability of data opens up new areas of study that require statisticians and bioinformaticians to develop appropriate tools. In addition, constant statistical progress in areas such as clustering, dimensionality reduction, regressions and others needs to be regularly adapted to the context of bioinformatics. The objective of this thesis is the application of advanced statistical techniques to bioinformatics issues. In this manuscript we present the results of our works concerning the clustering of genetic sequences via Laplacian eigenmaps and Gaussian mixture model, the study of the propagation of transposable elements in the genome via a branching process, the analysis of metagenomic data in ecology via ROC curves or the ordinal polytomous regression penalized by the l1-norm.

<p>The <i>Murine Pneumotropic Virus </i>(MPtV), in contrast to the other <i>MurinePolyomavirus</i> (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. </p><p>The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. </p><p>MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in <i>Escherichia coli</i> DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.</p>

The Murine Pneumotropic Virus (MPtV), in contrast to the other MurinePolyomavirus (MPyV), appears to be non-tumourigenic in its natural host. Instead, MPtV causes acute pneumonia and can serve as a model in studies of polyomavirus-induced disease. In initial experiments, MPtV large T-antigen (LT) was expressed in a heterologous system. LT was characterized with regard to its metabolic stability and cell immortalizing activity and, after purification, to its specific DNA binding. The absence of permissive cell culture system for MPtV has hampered its study. We made attempts to widen the host range of the virus by modifying the regulatory and late regions of the genome. The enhancer substitution mutant (KVm1), having a transcriptional enhancer substituted with a corresponding DNA segment from MPyV, was able to replicate in mouse 3T3 cells and form virus particles that were infectious in mice. However, efficient infection of cells in vitro was not achieved with this mutant virus, possibly due to the absence of virus-specific receptors on the cells. The capsid protein substitution mutants, having capsid protein genes of MPyV, for which receptors are present on a variety of cell types, showed also no cytopathic effect, despite an enhanced viral DNA replication and assembly of virus particles. MPtV-DNA extracted from virus in lung tissue of infected mice had a heterogeneous enhancer segment. A majority of the DNA molecules had a structure differing from the standard-type. A 220 base-pair insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was a prominent variation. Other genome variants showed complete or partial deletions of the insertion and surrounding sequences in the viral enhancer. In relation to the standard-type, all variant genomes showed differences in the activities of transcriptional promoters and the origin DNA replication. Analysis by DNA reassociation showed that a large number of nucleotide sequences related to the 220 base-pair insert in the MPtV genome were present in mouse and human DNA, but not in Escherichia coli DNA. Together, the data suggest that the 220 base-pair insertion is related to a transposable element of a novel type.

The retrotransposon Ylt1 was described previously from the genome of the dimorphic fungus Yarrowia lipolytica. Remarkably, Ylt1 is currently the largest LTR retrotransposon reported from fungal genomes. However, little was known about its biology and its interactions with host genome. So, the aim of this work was the characterization of properties of Ylt1.Analysis of proteins encoded by Ylt1 (Gag protein and integrase) was carried out during this work. To enable their detection, both proteins were tagged with HA epitopes. The sizes of Gag protein and putative precursors of Gag protein and integrase were estimated, and a model for the proteolytic processing of the polyprotein of Ylt1 was proposed. It was shown that Gag protein of Ylt1 is about 2-fold larger than Gag proteins of other studied yeast retrotransposons. An analysis of Ylt1 expression was also performed. Production of the Ylt1 Gag protein under different conditions was analyzed by Western blotting. Expression of Ylt1 occurred on all tested carbon sources. The amount of Ylt1 decreased rapidly upon transition to stationary growth phase, in the presence of copper sulfate and under heat shock conditions. It is suggested that Ylt1 is expressed in actively growing cells, whereas stress conditions have a negative impact on its expression. Such expression pattern was not previously reported for other yeast retrotransposons. Activity of Ylt1 in vivo was characterized using an Ylt1 elements tagged with SUC2 gene of Saccharomyces cerevisiae. Mobilization of the marked Ylt1 element and its transposition from autonomous plasmid into host genome was observed in performed experiments. Obtained results strongly support the idea that Ylt1 is transpositionally active. Formation of tandem repeats by newly inserted Ylt1 elements was observed in several cases. It is suggested that integrase function was affected in this case, and that the integration was mediated by homologous recombination instead. Analysis of the Ylt1 insertion specificity and of the Ylt1 distribution in the genome of Y. lipolytica E150 was done. The remarkable sequence specificity of Ylt1 insertions, which is unusual for LTR retrotransposons, was revealed during this analysis. Also, it was shown that Ylt1 insertions are found mainly in intergenic regions, often at a significant distance (&amp;gt;500 bp) from the next reading frame. No association of Ylt1 insertions with tRNA genes was observed. Searches for Ylt1-related elements in the Y. lipolytica genome database were performed. The novel Ty3/gypsy element Tyl6 was found in the genome of Y. lipolytica E150. The sequence analysis of this element was carried out. It was shown that structural properties of Tyl6 resemble the properties of the Ty3 element of S. cerevisiae. However, two reading frames of Tyl6 (gag and pol) are separated by -1 frame-shift, which was not previously reported for retrotransposons of hemiascomycetous yeasts. Phylogenetic analysis placed Tyl6 within chromoviruses, and the Tse3 element of S. exiguus was shown to be the closest relative of Tyl6. The distribution of Tyl6 among Y. lipolytica strains was analyzed. Interestingly, the novel element was found only in strains derived from the strain YB423-12. The strains of independent origin included in the analysis were shown to be Tyl6-free. The same distribution was previously reported for the retrotransposon Ylt1 and for the DNA transposon Mutyl. Two models of the evolution of transposable elements in Y. lipolytica genome were proposed based on these results.

Eukaryotic genomes are packed into the confines of the nucleus through a nucleoproteic structure called chromatin. Chromatin is a dynamic structure that can respond to developmental or environmental cues to regulate and orchestrate the functions of the genome. The fundamental unit of chromatin, the nucleosome, consists of a protein octamer, which contains two molecules of each of the core histone proteins (H2A, H2B, H3, H4), around which 147 bp of DNA is wrapped. The post-translational modifications (PTMs) of histones and methylation of the cytosine residues in DNA (DNA methylation) constitute primary epigenomic markers that dynamically alter the interaction of DNA with nucleosomes and participate in the regulation and control access to the underlying DNA. The main objective of my thesis was to understand the spatial and temporal dynamics of chromatin states in Arabidopsis by investigating on a genome-wide scale, patterns of DNA methylation and a set of well-characterized histone post-translational modifications. DNA methylation, a hallmark of epigenetic inactivation and heterochromatin in both plants and mammals, is largely confined to transposable elements and other repeat sequences. I show in this thesis that in Arabidopsis, methylated TE sequences having no or few matching siRNAs, and therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery, acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, I propose that this spreading of DNA methylation through promoter regions can explain, at least in part, the negative impact of siRNA-targeted TE sequences on neighbouring gene expression. In a second part, I have contributed to integrative analysis of DNA methylation and eleven histone PTMs. I have shown through combinatorial and cluster analysis that the Arabidopsis epigenome shows simple principles of organisation and can be distinguished into four primary types of chromatin that preferentially index active genes, repressed genes, TEs, and intergenic regions. Finally, in a third part, I integrated epigenomics with transcriptome data at three different time points in a developmental window to investigate the temporal dynamics of chromatin states in response to an external stimulus. This used the light-induced transcriptional response as a paradigm to assess the impact of histone H2B monoubiquitination (H2Bub), and showed that this PTM is associated with active transcription and implicated in the selective fine-tuning of gene expression. Taken together, the work presented here contributes significantly to our understanding of the spatial organisation of chromatin states in plants, its dynamic nature and how it can contribute to allow plants to respond to a signal from the environment.