<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>

Les éléments transposables sont des séquences génomiques capables de se répliquer et de se déplacer dans les génomes. Leur capacité à s'insérer près des gènes et à produire des réarrangements chromosomiques par recombinaison entre copies, font des éléments transposables des agents mutagènes. Les éléments transposables sont de plus capables de modifier l'expression des gènes voisins grâce aux régions promotrices qu'ils possèdent. Les éléments transposables ont été trouvés dans la plupart des génomes dans lesquels ils ont été recherchés. Ils forment ainsi 45 % du génome de l'homme et peuvent représenter jusqu'à 90 % du génome de certaines plantes. Dans la première partie de ma thèse, je me suis penché sur les facteurs qui déterminent la distribution de ces éléments. Je me suis intéressé à un facteur particulier, qui est la fonction des gènes dans le voisinage des insertions d'éléments transposables. Dans la deuxième partie, j'ai essayé de déterminer l'impact de l'altération des modifications épigénétiques (modifications d'histones plus précisément) associées aux différents composants géniques, dont les éléments transposables, sur la variation de l'expression des gènes en condition tumorale.

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.

We did functional characterisation of Drosophila melanogaster linker histone, dH1. In the mutant state for this protein, we observed structural changes in polytene chromosomes chromocenter and nucleoli of mutant larvae. In addition, we performed a microarray analysis in H1 mutant background in order to determine contribution of dH1 to gene expression regulation. We determined effects of dH1 loss in different types of chromatin and we identified groups of differentially expressed (DE) genes, groups in sense of physical clusters of genes and genomic elements rather than groups of functionally related genes. We found that dH1 affects in greater extent expression of heterochromatin genes compared to its effect on euchromatin genes; that dH1 regulates transcription in a regional manner, since the genes physically nearest to the most DE genes tend to be upregulated as well; and that dH1 is negatively regulating expression of transposable elements and members of certain gene families. In addition, we found that dH1 is necessary for preserving genome stability. Among DE transposable elements we detected R1 and R2 retrotransposons, elements that are integrating specifically in rRNA locus. We showed that activation of their transcription is also upregulating expression of aberrant, transposon-inserted, rDNA units of the locus. In this regard we observed an accumulation of extra-chromosomal rDNA circles, increased γ-H2Av content, stop in cell proliferation and activation of apoptosis. Altogether, these results are revealing so far unknown role of histone H1 in preserving genome stability and its effects on cell proliferation.

Transposable elements are DNA sequences that can change their location within the genome and can be found in almost all organisms. The mariner transposable element belongs to Class II, Tc1-mariner superfamily. An inactive insertion of this element in the gene white (w+) located on the X chromosome, causes a phenotypic change in color's eyes becoming them a peach color in the place of the wild coloration, being for this reason called whitepeach (wpch). Crossing of mutant females with males obtained from nature, an offspring with eyes the color of peach or variegated with spots of wild color is formed enabling estimate transposicional activity in natural populations. From the use of this system, several studies have been performed to understand if the number of copies and the activity of the mariner element are involved with environmental factors, historical and/or populational. In this study, the existence of intrapopulation variability of D. simulans collected in two places at Santa Maria (RS) city was investigated by crossing with a line of D. simulans wpch. Moreover, data on another species, D. melanogaster, were also obtained and analyzed: interspecific crosses generated progeny with variegation. The isolines that showed these properties were studied using of molecular research tools, such as PCR, cloning and sequencing, it was also necessary. The results suggest the existence of intrapopulation variability in D. simulans and indicate the discovery of sequences very similar to the element mariner in the genome of natural populations of D. melanogaster. / Os elementos transponíveis são seqüências de DNA capazes de alterar sua localização dentro do genoma e podem ser encontrados em praticamente todos os organismos. O elemento transponível mariner pertence à classe II, superfamília Tc1-mariner. Uma inserção inativa deste elemento no gene white (w+) localizado no cromossomo X de Drosophila simulans, causa uma alteração fenotípica na cor dos olhos tornando-os cor de pêssego no lugar da coloração selvagem, sendo por este motivo denominada white-peach (wpch). Do cruzamento de fêmeas mutantes com machos coletados na natureza, uma prole com olhos cor de pêssego ou variegados com pontos de coloração selvagem é formada, possibilitando estimar a atividade transposicional em populações naturais. A partir do uso deste sistema, diversos estudos têm sido realizados para compreender se o número de cópias e a atividade do elemento mariner estão envolvidos com fatores ambientais, históricos e/ou populacionais. Neste trabalho, a existência de variabilidade intrapopulacional de D. simulans coletadas em dois pontos da cidade de Santa Maria RS foi investigada através de cruzamentos com uma linhagem de D. simulans wpch. Além disso, dados sobre uma outra espécie, D. melanogaster, também foram obtidos e analisados: cruzamentos interespecíficos que geraram descendência com variegação fez com que o uso de ferramentas de pesquisa molecular, como PCR, clonagem e seqüenciamento, também fossem necessárias. Os resultados obtidos sugerem a existência de variabilidade intrapopulacional em D. simulans e indicam a descoberta de seqüências semelhantes ao elemento mariner no genoma de populações naturais de D. melanogaster.

Made available in DSpace on 2014-06-11T19:26:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-27Bitstream added on 2014-06-13T19:12:52Z : No. of bitstreams: 1 ricci_j_me_sjrp.pdf: 1040646 bytes, checksum: cec6211f3f5843239b67ee910f199d37 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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... / 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)

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.

Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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. / Orientador: Cláudia Márcia Aparecida Carareto / Coorientador: Marie Anne Van Sluys / Banca: Hermione Elly Melara de Campos Bicudo / Banca: Maria Magdalena Rossi / Banca: Galina Ananina / Banca: Elgion da Silva Loreto / Doutor