Galileo foi identificado em D. buzzatii e classificado como transposon do tipo foldback devido às suas longas IRs (repetições invertidas), que capacitam o elemento a formar uma estrutura secundária que leva a quebras e rearranjos cromossômicos. Evidências indicam que Galileo foi responsável pela geração de três inversões naturais segregantes em populações de D. buzzatii. A descoberta de Galileo em seis das 12 espécies de Drosophila sequenciadas, incluindo a D. willistoni, através de um estudo in silico, foi promissora para a busca de agentes causadores de quebras e rearranjos cromossômicos nesta espécie, que é altamente polimórfica, e em espécies a ela relacionadas. Nesse contexto, o presente estudo foi o primeiro a caracterizar o transposon Galileo em populações naturais de espécies do grupo willistoni de Drosophila. Para a investigação da presença de Galileo foram construídos primers com base na sequência de Galileo encontrada no genoma da linhagem sequenciada (GdH4) de Drosophila willistoni, para amplificar um fragmento do domínio central do elemento, assim como da inferida transposase e de seu domínio THAP. Os fragmentos polimórficos foram clonados e sequenciados para posterior caracterização molecular. O alinhamento das sequências nucleotídicas e de aminoácidos foi obtido no software Muscle com posterior edição no Bio Edit e o programa Gene Doc foi utilizado na tradução das sequências. Foram investigadas as espécies Drosophila willistoni, D. tropicalis, D. equinoxialis, D. insularis e quatro das semiespécies de D. paulistorum (Amazônica, Andino-Brasileira, Interior e Orinocana) do subgrupo willistoni, e D. nebulosa, D. capricorni e D. fumipennis, subgrupo bocainensis, do grupo willistoni, pelas técnicas de PCR e Dot blot. Foi encontrada variabilidade intra e interespecífica no tamanho dos fragmentos amplificados de Galileo, sendo que todas as sequências analisadas são indicativas de cópias defectivas. Foram caracterizadas 22 variantes a partir das sequências obtidas, permitindo a construção de um dendrograma de acordo com a presença ou ausência das variantes no genoma de todas as espécies estudadas por meio do software NTSYSpc 2.1. Os dados resultaram em dois clados bem-definidos: (1) formado pelas quatro semiespécies de D. paulistorum e (2) pelas espécies crípticas D. tropicalis, D. insularis e D. equinoxialis. Drosophila willistoni do subgrupo willistoni, D. sucinea, D. nebulosa e D. capricorni do subgrupo bocainensis, apresentaram variantes exclusivas e, assim, formaram ramos independentes aos dois agrupamentos. Entre todas as espécies crípticas do subgrupo willistoni somente D. willistoni não compartilha qualquer uma das suas quatro variantes. O quadro que emerge de nossos achados é sugestivo de mais de um evento evolutivo, implicando tanto a manutenção por ancestralidade de certas variantes quanto a ocorrência de perdas estocásticas. Além disso, o fenômeno de introgressão, já bem-documentado dentro do subgrupo willistoni, pode ter sido o mecanismo pelo qual tenham sido transferidas sequências de Galileo entre alguns táxons estudados, principalmente entre algumas semiespécies de D. paulistorum. A singularidade de D. willistoni entre as crípticas quanto às diferentes sequências de Galileo em seu genoma, pode ser consequência da presença de elementos quiméricos, uma vez que foi observada a inserção secundária de outros elementos em Galileo de D. buzzatii. Considerando a peculiaridade de Galileo, como gerador de inversões cromossômicas em populações naturais de D. buzzatii, nossos resultados sugerem que Galileo também pode ter desempenhado um papel importante na evolução genômica no grupo willistoni de Drosophila, uma vez que D. willistoni e D. paulistorum apresentam muitos arranjos cromossômicos segregando em suas populações naturais. / Galileo was identified in Drosophila buzzatii and classified as a foldback-like transposon due to its long IRs (inverted repeats), which allow the element to form a secondary structure that leads to breaks and chromosomal rearrangements. Several evidences indicate that Galileo was responsible for the generation of three natural segregating inversions in populations of D. buzzatii. The finding of Galileo in six of the 12 species of Drosophila that were sequenced, including D. willistoni, through an in silico study, is promising for the search of agents that cause chromosomal breaks and rearrangements in this species, that is highly polymorphic, and in related species. Based on this context, this was the first work to characterize the Galileo transposon in natural populations of the willistoni group of Drosophila. To detect the presence of Galileo, we used primers based on the sequence of Galileo found in the genome of the sequenced lineage (GdH4) of Drosophila willistoni, so that a fragment of the central domain of the element is amplified, as well as the putative transposase and its THAP domains. The polymorphic fragments were cloned and sequenced for later molecular characterization. The alignment of the nucleotide sequences and amino acids was obtained in the Muscle software with later edition in the Bio Edit, and the Gene Doc program was used to translate the sequences. We investigated, by PCR and Dot blot, the following species: D. willistoni, D. tropicalis, D. equinoxialis, D. insularis and four semispecies of D. paulistorum (Amazonian, Andean-Brazilian, Interior and Orinocan) of the subgroup willistoni, besides D. nebulosa, D. capricorni and D. fumipennis, subgroup bocainensis, group willistoni. The size variability of the Galileo amplified fragments was intra and interspecific; all the analyzed sequences indicate defective copies. Twenty two variants were identified in the obtained sequences, allowing the construction of a dendrogram based in the presence or absence of the variants in the genomes of all the species studied through the NTSYSpc 2.1 software. The data produced two well-defined clades: (1) formed by the four semispecies of D. paulistorum and (2) by the sibling species D. tropicalis, D. insularis and D. equinoxialis. D. willistoni (subgroup willistoni), D. sucinea, D. nebulosa and D. capricorni (subgroup bocainensis) have exclusive variants of Galileo, forming two independent branches of the two clades. Among all the sibling species of the subgroup willistoni, only D. willistoni does not share any of its four variants. The results of our observations suggest more than one evolutionary event, indicating both the maintenance by ancestrality of some variants and the occurrence of stochastic loss. Besides, the introgression phenomenon, well-documented in the willistoni subgroup, may have been the mechanism through which the Galileo sequences were transferred between some taxons that were studied, specially between some semispecies of D. paulistorum. The uniqueness of D. willistoni among siblings, regarding the different Galileo sequences in its genome, can be a consequence of the presence of quimeric elements, since a secondary insertion of other elements in Galileo of D. buzzatii was observed. Taking into account the Galileo element singularities, as a generator of chromosomal inversions in natural populations of D. buzzati, our findings suggest that Galileo could also have played an important role in the genomic evolution of the Drosophila willistoni group, since D. willistoni and D. paulistorum have many chromosomal rearrangements segregating in their natural populations.

Galileo foi identificado em D. buzzatii e classificado como transposon do tipo foldback devido às suas longas IRs (repetições invertidas), que capacitam o elemento a formar uma estrutura secundária que leva a quebras e rearranjos cromossômicos. Evidências indicam que Galileo foi responsável pela geração de três inversões naturais segregantes em populações de D. buzzatii. A descoberta de Galileo em seis das 12 espécies de Drosophila sequenciadas, incluindo a D. willistoni, através de um estudo in silico, foi promissora para a busca de agentes causadores de quebras e rearranjos cromossômicos nesta espécie, que é altamente polimórfica, e em espécies a ela relacionadas. Nesse contexto, o presente estudo foi o primeiro a caracterizar o transposon Galileo em populações naturais de espécies do grupo willistoni de Drosophila. Para a investigação da presença de Galileo foram construídos primers com base na sequência de Galileo encontrada no genoma da linhagem sequenciada (GdH4) de Drosophila willistoni, para amplificar um fragmento do domínio central do elemento, assim como da inferida transposase e de seu domínio THAP. Os fragmentos polimórficos foram clonados e sequenciados para posterior caracterização molecular. O alinhamento das sequências nucleotídicas e de aminoácidos foi obtido no software Muscle com posterior edição no Bio Edit e o programa Gene Doc foi utilizado na tradução das sequências. Foram investigadas as espécies Drosophila willistoni, D. tropicalis, D. equinoxialis, D. insularis e quatro das semiespécies de D. paulistorum (Amazônica, Andino-Brasileira, Interior e Orinocana) do subgrupo willistoni, e D. nebulosa, D. capricorni e D. fumipennis, subgrupo bocainensis, do grupo willistoni, pelas técnicas de PCR e Dot blot. Foi encontrada variabilidade intra e interespecífica no tamanho dos fragmentos amplificados de Galileo, sendo que todas as sequências analisadas são indicativas de cópias defectivas. Foram caracterizadas 22 variantes a partir das sequências obtidas, permitindo a construção de um dendrograma de acordo com a presença ou ausência das variantes no genoma de todas as espécies estudadas por meio do software NTSYSpc 2.1. Os dados resultaram em dois clados bem-definidos: (1) formado pelas quatro semiespécies de D. paulistorum e (2) pelas espécies crípticas D. tropicalis, D. insularis e D. equinoxialis. Drosophila willistoni do subgrupo willistoni, D. sucinea, D. nebulosa e D. capricorni do subgrupo bocainensis, apresentaram variantes exclusivas e, assim, formaram ramos independentes aos dois agrupamentos. Entre todas as espécies crípticas do subgrupo willistoni somente D. willistoni não compartilha qualquer uma das suas quatro variantes. O quadro que emerge de nossos achados é sugestivo de mais de um evento evolutivo, implicando tanto a manutenção por ancestralidade de certas variantes quanto a ocorrência de perdas estocásticas. Além disso, o fenômeno de introgressão, já bem-documentado dentro do subgrupo willistoni, pode ter sido o mecanismo pelo qual tenham sido transferidas sequências de Galileo entre alguns táxons estudados, principalmente entre algumas semiespécies de D. paulistorum. A singularidade de D. willistoni entre as crípticas quanto às diferentes sequências de Galileo em seu genoma, pode ser consequência da presença de elementos quiméricos, uma vez que foi observada a inserção secundária de outros elementos em Galileo de D. buzzatii. Considerando a peculiaridade de Galileo, como gerador de inversões cromossômicas em populações naturais de D. buzzatii, nossos resultados sugerem que Galileo também pode ter desempenhado um papel importante na evolução genômica no grupo willistoni de Drosophila, uma vez que D. willistoni e D. paulistorum apresentam muitos arranjos cromossômicos segregando em suas populações naturais. / Galileo was identified in Drosophila buzzatii and classified as a foldback-like transposon due to its long IRs (inverted repeats), which allow the element to form a secondary structure that leads to breaks and chromosomal rearrangements. Several evidences indicate that Galileo was responsible for the generation of three natural segregating inversions in populations of D. buzzatii. The finding of Galileo in six of the 12 species of Drosophila that were sequenced, including D. willistoni, through an in silico study, is promising for the search of agents that cause chromosomal breaks and rearrangements in this species, that is highly polymorphic, and in related species. Based on this context, this was the first work to characterize the Galileo transposon in natural populations of the willistoni group of Drosophila. To detect the presence of Galileo, we used primers based on the sequence of Galileo found in the genome of the sequenced lineage (GdH4) of Drosophila willistoni, so that a fragment of the central domain of the element is amplified, as well as the putative transposase and its THAP domains. The polymorphic fragments were cloned and sequenced for later molecular characterization. The alignment of the nucleotide sequences and amino acids was obtained in the Muscle software with later edition in the Bio Edit, and the Gene Doc program was used to translate the sequences. We investigated, by PCR and Dot blot, the following species: D. willistoni, D. tropicalis, D. equinoxialis, D. insularis and four semispecies of D. paulistorum (Amazonian, Andean-Brazilian, Interior and Orinocan) of the subgroup willistoni, besides D. nebulosa, D. capricorni and D. fumipennis, subgroup bocainensis, group willistoni. The size variability of the Galileo amplified fragments was intra and interspecific; all the analyzed sequences indicate defective copies. Twenty two variants were identified in the obtained sequences, allowing the construction of a dendrogram based in the presence or absence of the variants in the genomes of all the species studied through the NTSYSpc 2.1 software. The data produced two well-defined clades: (1) formed by the four semispecies of D. paulistorum and (2) by the sibling species D. tropicalis, D. insularis and D. equinoxialis. D. willistoni (subgroup willistoni), D. sucinea, D. nebulosa and D. capricorni (subgroup bocainensis) have exclusive variants of Galileo, forming two independent branches of the two clades. Among all the sibling species of the subgroup willistoni, only D. willistoni does not share any of its four variants. The results of our observations suggest more than one evolutionary event, indicating both the maintenance by ancestrality of some variants and the occurrence of stochastic loss. Besides, the introgression phenomenon, well-documented in the willistoni subgroup, may have been the mechanism through which the Galileo sequences were transferred between some taxons that were studied, specially between some semispecies of D. paulistorum. The uniqueness of D. willistoni among siblings, regarding the different Galileo sequences in its genome, can be a consequence of the presence of quimeric elements, since a secondary insertion of other elements in Galileo of D. buzzatii was observed. Taking into account the Galileo element singularities, as a generator of chromosomal inversions in natural populations of D. buzzati, our findings suggest that Galileo could also have played an important role in the genomic evolution of the Drosophila willistoni group, since D. willistoni and D. paulistorum have many chromosomal rearrangements segregating in their natural populations.

The purpose of this work is to determine the life cycle of different species of Drosophila of different subgenera and coming from different environments.

As our knowledge of the genome expands, so does our understanding of the characteristics of what we define as genes. Small Open Reading Frame (smORF) genes have eluded gene annotation until very recently, and evidence is mounting that these very short nucleotide sequences encode functional peptides that are ≤100 amino acids in size. From work conducted in the fruit fly, our lab has successfully characterised three Drosophila smORFs, of which two have been shown to have a function in higher vertebrates, including humans. The functional characterisation of one of these conserved smORF encoded peptides (SEPs), Hemotin, is presented in this thesis. Though the overall number is still low compared to the abundance of potential smORF-encoding genes in Drosophila, the information gathered here allows us to speculate on the wider role of smORF peptides through cell-based imaging studies conducted on Drosophila cells. Here, I will discuss the various techniques that can and should be employed in order to study the functions of SEPs. Chapter III describes the various phenotypic studies conducted on the Hemotin smORF which is expressed in Drosophila haemocytes, and are integral to the fruit fly immune system. This study showed that connecting subcellular localisation of an SEP to a direct functional assay in cells can reveal functional characteristics of the peptide for further study. Chapter IV details the results from a tagging-transfection assay, which began initially as a way to independently corroborate the translation of smORF mRNAs that were assessed as such by Ribosome Profiling. This experiment resulted in the discovery of several mitochondrial-localised SEPs in Drosophila S2 cells, opening the door for the direct functional assay described in Chapter V. The results from a small-scale RNAi screen conducted on the mitochondria of S2 cells provided a reliable read-out for functionality of a large proportion of the smORFs that were screened. This assay can potentially be used as a phenotypic read-out of mitochondrial-SEP function in any cell or tissue type. Elucidation of smORFs and the functions of the peptides that they encode will help us to expand the Drosophila proteome, along with providing evidence of their functionality across every organism in which they are found. Considering that characterised SEPs play very important roles in physiology and health, it is time for smORFs to be acknowledged as the important genomic elements that they are.

Despite the benefits of sexual reproduction, sharing a genome can put constraints on the evolvability of a species. This is due to sexual conflict, where the interests of each sex is in direct opposition to one another, and the benefit of one sex can be the cost of the other sex (i.e. sexual antagonism). Sex chromosomes have been the focus of much of the research done on sexual conflict due to their unique nature and are particularly interesting in the context of sexually antagonistic variance. In the first experiment (Chapter 2), I used experimental evolution to investigate the standing sexually antagonistic variation on the X-chromosome of the common vinegar fly, Drosophila melanogaster. Unlike most other experimental evolution experiments where selection has been limited to males, I limited the inheritance of the X-chromosome to females only. I used a non-recombining Xchromosome balancer to control the inheritance of the female-limited X-chromosome. Throughout the evolution experiment, I tested different phenotypic traits that have previously been shown to be sexual antagonistic, as well as investigating how the transcriptome changed through female-limited selection (Chapter 3). The results were mixed but indicated that limiting selection of the X-chromosome to females could, to some extent, change the antagonistic variation and move traits towards the female optimum. In the second experiment (Chapter 4), I exchanged sex chromosomes between populations with divergent geographic origins. I used flies with special genetic constructs (e.g. autosomal balancers, fused-X chromosomes) to control the population crosses, so that sex chromosomes were introduced into a new background without any prior interaction. I found that introducing a novel sex chromosome increased male reproductive fitness through improved sperm competition at the cost of offspring viability. 25 generations after introducing the novel sex chromosome (Chapter 5), the increase in male fitness was undetectable and their fitness was again the same as the wild types. Collectively, this indicates an antagonistic coevolution between the sex chromosomes. Together, these two experiments shed new light on sexual conflict and the antagonistic coevolution between the sexes at the genetic level, both between and within the sex chromosomes. These novel insights could help further the understanding of how sex chromosomes may affect speciation.

A metilação do DNA genômico é um dos principais mecanismos de regulação epigenética nos organismos. Dentre as diferentes classes de DNA MTase, as m5C-MTase são as que se distribuem amplamente de procariotos a eucariotos. Em vertebrados existem três diferentes famílias: DNMT1, DNMT2 e DNMT3a e 3b. A DNMT1 possui atividade junto ao DNA hemimetilado. As DNMT3a e 3b são responsáveis pela metilação de novo. Já a subfamília DNMT2 possui seus sítios catalíticos altamente conservados, desde procariotos até eucariotos, possuindo propriedades que permitem executar funções tanto de metilação de novo, assim como de manutenção de metilação. Além disso, as enzimas da família DNMT2 podem atuar metilando citosinas genômicas ou de tRNAs. Em mamíferos, invertebrados e plantas a DNMT2 é classificada, prioritariamente in vivo como uma tRNA MTase. Entretanto, já foi descrita atividade de DNA MTase por parte dessas enzimas, mesmo que em baixos níveis. O que se discute são as atividades preferenciais da DNMT2 e os mecanismos que modulam sua atividade, pois se reconhece que em organismos que não possuem as MTases canônicas (DNMT1 e DNMT3), mas apresentam metilação em seu genoma, é a DNMT2 que atua como MTase em ambos os substratos. Espécies de Drosophila são conhecidas como de Dnmt2-only, justamente por possuírem apenas a DNMT2 na função de metilação de citosinas. A importância de seu papel no âmbito ecológico e evolutivo nesse grupo de espécies se reflete na presença de fenômenos peculiares, como a metilação sexo-específica presente em espécies do subgrupo willistoni de Drosophila, descrito por nosso grupo de pesquisa. No presente trabalho realizou-se a modelagem das enzimas DNMT2 de D. melanogaster, D. willistoni e Mus musculus com diferentes metodologias. A partir desses modelos realizaram-se análises comparativas com as estruturas cristalográficas de DNMT2 depositadas no banco de dados PDB, com o objetivo de estabelecer as relações evolutivas e funcionais entre as diferentes enzimas. Adicionalmente, de posse de modelos de DNMT2 - de validada qualidade - de duas espécies pertencentes a diferentes grupos evolutivos de drosofilídeos, realizaram-se estudos de caracterização evolutiva e estrutural das 22 espécies que tiveram seus genomas sequenciados e depositados no bando de dados Flybase, somando-se a essas a sequência de DNMT2 de Drosophila tropicalis (subgrupo willistoni), sequenciado por nosso grupo de pesquisa. Os resultados das análises evolutivas e estruturais sugerem propriedades diferenciais entre as DNMT2 de espécies do subgrupo willistoni em relação às demais. Estes resultados indicam que mesmo em espécies que possuem relações evolutivas próximas possam ocorrer mecanismos adaptativos que estabeleçam gradações na afinidade das DNMT2 por diferentes substratos, sem que para isso ocorram drásticas mudanças na arquitetura da enzima. / The methylation of genomic DNA is a major mechanism of epigenetic regulation in organisms. Among the different classes of DNA MTase the M5C-MTase are as widely distributed in prokaryotes to eukaryotes. In vertebrates there are three different families: DNMT1, DNMT2 and DNMT3a and 3b. The DNMT1 has activity with the hemimethylated DNA. The DNMT3a and 3b are responsible for de novo methylation. While the DNMT2 subfamily has its catalytic sites highly conserved from prokaryotes to eukaryotes, having properties that allow performing functions of both de novo and maintenance methylation. Furthermore, the DNMT2 family can act methylating cytokines, tRNAs or DNA. In mammals, invertebrates and plants, DNMT2 is classified primarily in vivo as a tRNA MTase. However,DNA-MTase activity by these enzymes has been described, even at low levels. The question is the preferred activities of DNMT2 and mechanisms that modulate its activity, because in organisms that do not have the canonical DNA-MTases (DNMT1 and DNMT3), but have cytokines methylated in its genome, the DNMT2 acts as MTase on both substrates. Drosophila species are known as Dnmt2-only. The importance of DNMT2’s role in ecological and evolutionary context in this species group is reflected by presence of a peculiar phenomenon: the sex-specific methylation described by our research group, presents in willistoni subgroup of Drosophila. In this study, DNMT2 of D. melanogaster, D. willistoni and Mus musculus were modeled by different methodologies. comparative analyzes with the crystallographic DNMT2 structures deposited in the PDB database were performed from these models, in order to establish the evolutionary and functional relationships between the different enzymes. Additionally, evolutionary and structural characterization studies of the 22 species, with the validated DNMT2 models of two species belonging to different evolutionary drosophilids groups, were conducted that have had their genomes sequenced and deposited in FlyBase data pack, adding the DNMT2 sequence of Drosophila tropicalis (willistoni subgroup) sequenced by our research group. The results of evolutionary and structural analyzes suggest differences between the DNMT2 properties of subgroup willistoni species from the other drosophilids. These results indicate that even species that have close evolutionary relationships may have adaptive mechanisms that establish gradations of DNMT2 affinity for different substrates, without the need of drastic changes in enzyme architecture.

A metilação do DNA genômico é um dos principais mecanismos de regulação epigenética nos organismos. Dentre as diferentes classes de DNA MTase, as m5C-MTase são as que se distribuem amplamente de procariotos a eucariotos. Em vertebrados existem três diferentes famílias: DNMT1, DNMT2 e DNMT3a e 3b. A DNMT1 possui atividade junto ao DNA hemimetilado. As DNMT3a e 3b são responsáveis pela metilação de novo. Já a subfamília DNMT2 possui seus sítios catalíticos altamente conservados, desde procariotos até eucariotos, possuindo propriedades que permitem executar funções tanto de metilação de novo, assim como de manutenção de metilação. Além disso, as enzimas da família DNMT2 podem atuar metilando citosinas genômicas ou de tRNAs. Em mamíferos, invertebrados e plantas a DNMT2 é classificada, prioritariamente in vivo como uma tRNA MTase. Entretanto, já foi descrita atividade de DNA MTase por parte dessas enzimas, mesmo que em baixos níveis. O que se discute são as atividades preferenciais da DNMT2 e os mecanismos que modulam sua atividade, pois se reconhece que em organismos que não possuem as MTases canônicas (DNMT1 e DNMT3), mas apresentam metilação em seu genoma, é a DNMT2 que atua como MTase em ambos os substratos. Espécies de Drosophila são conhecidas como de Dnmt2-only, justamente por possuírem apenas a DNMT2 na função de metilação de citosinas. A importância de seu papel no âmbito ecológico e evolutivo nesse grupo de espécies se reflete na presença de fenômenos peculiares, como a metilação sexo-específica presente em espécies do subgrupo willistoni de Drosophila, descrito por nosso grupo de pesquisa. No presente trabalho realizou-se a modelagem das enzimas DNMT2 de D. melanogaster, D. willistoni e Mus musculus com diferentes metodologias. A partir desses modelos realizaram-se análises comparativas com as estruturas cristalográficas de DNMT2 depositadas no banco de dados PDB, com o objetivo de estabelecer as relações evolutivas e funcionais entre as diferentes enzimas. Adicionalmente, de posse de modelos de DNMT2 - de validada qualidade - de duas espécies pertencentes a diferentes grupos evolutivos de drosofilídeos, realizaram-se estudos de caracterização evolutiva e estrutural das 22 espécies que tiveram seus genomas sequenciados e depositados no bando de dados Flybase, somando-se a essas a sequência de DNMT2 de Drosophila tropicalis (subgrupo willistoni), sequenciado por nosso grupo de pesquisa. Os resultados das análises evolutivas e estruturais sugerem propriedades diferenciais entre as DNMT2 de espécies do subgrupo willistoni em relação às demais. Estes resultados indicam que mesmo em espécies que possuem relações evolutivas próximas possam ocorrer mecanismos adaptativos que estabeleçam gradações na afinidade das DNMT2 por diferentes substratos, sem que para isso ocorram drásticas mudanças na arquitetura da enzima. / The methylation of genomic DNA is a major mechanism of epigenetic regulation in organisms. Among the different classes of DNA MTase the M5C-MTase are as widely distributed in prokaryotes to eukaryotes. In vertebrates there are three different families: DNMT1, DNMT2 and DNMT3a and 3b. The DNMT1 has activity with the hemimethylated DNA. The DNMT3a and 3b are responsible for de novo methylation. While the DNMT2 subfamily has its catalytic sites highly conserved from prokaryotes to eukaryotes, having properties that allow performing functions of both de novo and maintenance methylation. Furthermore, the DNMT2 family can act methylating cytokines, tRNAs or DNA. In mammals, invertebrates and plants, DNMT2 is classified primarily in vivo as a tRNA MTase. However,DNA-MTase activity by these enzymes has been described, even at low levels. The question is the preferred activities of DNMT2 and mechanisms that modulate its activity, because in organisms that do not have the canonical DNA-MTases (DNMT1 and DNMT3), but have cytokines methylated in its genome, the DNMT2 acts as MTase on both substrates. Drosophila species are known as Dnmt2-only. The importance of DNMT2’s role in ecological and evolutionary context in this species group is reflected by presence of a peculiar phenomenon: the sex-specific methylation described by our research group, presents in willistoni subgroup of Drosophila. In this study, DNMT2 of D. melanogaster, D. willistoni and Mus musculus were modeled by different methodologies. comparative analyzes with the crystallographic DNMT2 structures deposited in the PDB database were performed from these models, in order to establish the evolutionary and functional relationships between the different enzymes. Additionally, evolutionary and structural characterization studies of the 22 species, with the validated DNMT2 models of two species belonging to different evolutionary drosophilids groups, were conducted that have had their genomes sequenced and deposited in FlyBase data pack, adding the DNMT2 sequence of Drosophila tropicalis (willistoni subgroup) sequenced by our research group. The results of evolutionary and structural analyzes suggest differences between the DNMT2 properties of subgroup willistoni species from the other drosophilids. These results indicate that even species that have close evolutionary relationships may have adaptive mechanisms that establish gradations of DNMT2 affinity for different substrates, without the need of drastic changes in enzyme architecture.

A metilação do DNA genômico é um dos principais mecanismos de regulação epigenética nos organismos. Dentre as diferentes classes de DNA MTase, as m5C-MTase são as que se distribuem amplamente de procariotos a eucariotos. Em vertebrados existem três diferentes famílias: DNMT1, DNMT2 e DNMT3a e 3b. A DNMT1 possui atividade junto ao DNA hemimetilado. As DNMT3a e 3b são responsáveis pela metilação de novo. Já a subfamília DNMT2 possui seus sítios catalíticos altamente conservados, desde procariotos até eucariotos, possuindo propriedades que permitem executar funções tanto de metilação de novo, assim como de manutenção de metilação. Além disso, as enzimas da família DNMT2 podem atuar metilando citosinas genômicas ou de tRNAs. Em mamíferos, invertebrados e plantas a DNMT2 é classificada, prioritariamente in vivo como uma tRNA MTase. Entretanto, já foi descrita atividade de DNA MTase por parte dessas enzimas, mesmo que em baixos níveis. O que se discute são as atividades preferenciais da DNMT2 e os mecanismos que modulam sua atividade, pois se reconhece que em organismos que não possuem as MTases canônicas (DNMT1 e DNMT3), mas apresentam metilação em seu genoma, é a DNMT2 que atua como MTase em ambos os substratos. Espécies de Drosophila são conhecidas como de Dnmt2-only, justamente por possuírem apenas a DNMT2 na função de metilação de citosinas. A importância de seu papel no âmbito ecológico e evolutivo nesse grupo de espécies se reflete na presença de fenômenos peculiares, como a metilação sexo-específica presente em espécies do subgrupo willistoni de Drosophila, descrito por nosso grupo de pesquisa. No presente trabalho realizou-se a modelagem das enzimas DNMT2 de D. melanogaster, D. willistoni e Mus musculus com diferentes metodologias. A partir desses modelos realizaram-se análises comparativas com as estruturas cristalográficas de DNMT2 depositadas no banco de dados PDB, com o objetivo de estabelecer as relações evolutivas e funcionais entre as diferentes enzimas. Adicionalmente, de posse de modelos de DNMT2 - de validada qualidade - de duas espécies pertencentes a diferentes grupos evolutivos de drosofilídeos, realizaram-se estudos de caracterização evolutiva e estrutural das 22 espécies que tiveram seus genomas sequenciados e depositados no bando de dados Flybase, somando-se a essas a sequência de DNMT2 de Drosophila tropicalis (subgrupo willistoni), sequenciado por nosso grupo de pesquisa. Os resultados das análises evolutivas e estruturais sugerem propriedades diferenciais entre as DNMT2 de espécies do subgrupo willistoni em relação às demais. Estes resultados indicam que mesmo em espécies que possuem relações evolutivas próximas possam ocorrer mecanismos adaptativos que estabeleçam gradações na afinidade das DNMT2 por diferentes substratos, sem que para isso ocorram drásticas mudanças na arquitetura da enzima. / The methylation of genomic DNA is a major mechanism of epigenetic regulation in organisms. Among the different classes of DNA MTase the M5C-MTase are as widely distributed in prokaryotes to eukaryotes. In vertebrates there are three different families: DNMT1, DNMT2 and DNMT3a and 3b. The DNMT1 has activity with the hemimethylated DNA. The DNMT3a and 3b are responsible for de novo methylation. While the DNMT2 subfamily has its catalytic sites highly conserved from prokaryotes to eukaryotes, having properties that allow performing functions of both de novo and maintenance methylation. Furthermore, the DNMT2 family can act methylating cytokines, tRNAs or DNA. In mammals, invertebrates and plants, DNMT2 is classified primarily in vivo as a tRNA MTase. However,DNA-MTase activity by these enzymes has been described, even at low levels. The question is the preferred activities of DNMT2 and mechanisms that modulate its activity, because in organisms that do not have the canonical DNA-MTases (DNMT1 and DNMT3), but have cytokines methylated in its genome, the DNMT2 acts as MTase on both substrates. Drosophila species are known as Dnmt2-only. The importance of DNMT2’s role in ecological and evolutionary context in this species group is reflected by presence of a peculiar phenomenon: the sex-specific methylation described by our research group, presents in willistoni subgroup of Drosophila. In this study, DNMT2 of D. melanogaster, D. willistoni and Mus musculus were modeled by different methodologies. comparative analyzes with the crystallographic DNMT2 structures deposited in the PDB database were performed from these models, in order to establish the evolutionary and functional relationships between the different enzymes. Additionally, evolutionary and structural characterization studies of the 22 species, with the validated DNMT2 models of two species belonging to different evolutionary drosophilids groups, were conducted that have had their genomes sequenced and deposited in FlyBase data pack, adding the DNMT2 sequence of Drosophila tropicalis (willistoni subgroup) sequenced by our research group. The results of evolutionary and structural analyzes suggest differences between the DNMT2 properties of subgroup willistoni species from the other drosophilids. These results indicate that even species that have close evolutionary relationships may have adaptive mechanisms that establish gradations of DNMT2 affinity for different substrates, without the need of drastic changes in enzyme architecture.

Dracula orchids are hypothesized to rely on mushroom mimicry for pollination. These orchids look and smell like mushrooms and are pollinated by mushroom-associated flies in the family Drosophilidae. Dracula includes over 130 species, representing a significant radiation, yet there has never been a systematic study of their pollination biology. Elucidating the processes and mechanisms of pollination in these flowers will broaden our understanding of mimicry within the Orchidaceae, a family well known for its diverse pollination strategies, as well as add to the growing literature on the evolution and maintenance of communication signals. In this study we demonstrate the co-occurrence of the mimics and the putative mushroom models, which is important for evolution by natural selection. We also showed that the resemblance to mushrooms is in fact adaptive, a requisite for floral mimicry. We did this by determining that insect visitors are required for pollination and subsequent fruit set with a hand pollination experiment. We also measured increased visitation rates to the orchids when adjacent to mushrooms. The mechanisms whereby plants attract pollinators can be diverse and often multi-modal, particularly in deceptive systems. Dracula orchids are no exception, with both visual and olfactory signals contributing to the overall success in attracting visitors. We used a series of experiments, first selectively masking the visual and olfactory cues successively, and then using 3D-printed artificial flowers to further disentangle these cues and determine their effect in combination. Upon confirmation that both play a role, we dissected each aspect further. We utilized the artificial flowers to determine the roles of color, contrast, and pattern and employed gas chromatography-mass spectroscopy to identify the volatile signals. The results show that fine-scale contrast is critical to the visual component and that these flowers produce the volatile `mushroom-alcohol' (1-octen-3-ol) in their labella. Finally, we specifically address the hypothesis of brood-site mimicry by using a combination of field observations, insect collections, and rearing studies. The flies gain shelter, a rendezvous location, and food from the flowers. However, no mushroom visiting flies hatched from the flowers, suggesting this may be a brood-site mimicry. This dissertation includes previously unpublished co-authored material. / 2015-09-29

The Hox genes encode a family of developmental regulators that are essential for the normal patterning of the animal body axis. Their correct expression is controlled by a number of mechanisms including RNA processing, a molecular system that allows the formation of alternative mRNAs from a single gene. Previous work in the Alonso Lab has demonstrated that RNA processing of the Drosophila Hox gene Ultrabithorax (Ubx) by means of alternative splicing and alternative polyadenylation plays an important role during Drosophila development, but the mechanisms underlying these regulatory processes are not well understood. In this project we found that the Drosophila neural RNA binding protein ELAV has an important role in the control of both Ubx alternative splicing and polyadenylation during embryonic development. Furthermore, by conducting a series of in vitro experiments we demonstrate that ELAV is able to interact with two specific RNA elements located within Ubx intronic sequences and mutation of such elements abolishes the interaction. We also establish that embryos carrying a loss of function mutation in the elav gene produce lower levels of Ubx mRNA and protein suggesting a role of ELAV on Hox gene expression. Finally we investigated the roles that other Drosophila factors including RNA binding proteins, chromatin regulators and splicing regulatory proteins may have on Ubx RNA processing and found several potential regulators. All in all our work contributes to the understanding of the molecular basis of Hox RNA processing control during Drosophila development.