Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-11-07T16:30:36Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) / Made available in DSpace on 2016-11-07T16:30:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) Previous issue date: 2015-05-19 / Algas verdes da classe Trebouxiphyceae estão entre os organismos presentes no continente Antártico, onde a espécie mais relatada é a macroalga verde Prasiola crispa (Lightfoot) Kützing. Considerada um organismo extremófilo, pois se desenvolve com muito sucesso no habitat extremo da Antártica, ainda são raros na literatura dados moleculares sobre esta espécie, o que impede uma avaliação sobre sua taxonomia e posição filogenética. Com o advento das tecnologias de sequenciamento de nova geração, os genomas de organelas tornaram-se uma grande ferramenta para estudos de filogenia, pois fornecem inúmeros dados filogenéticos, sequências de proteínas e nucleotídeos e também informações sobre conteúdo gênico e arquitetura. Neste trabalho, foi determinada a sequência dos genomas do cloroplasto (cpDNA) e mitocondrial (mtDNA) de P. crispa, com o intuito de inferir as relações evolutivas deste organismos com outras espécies de plantas verdes, bem como uma análise estrutural. Os genomas plastidial e mitocondrial foram sequenciados por Macrogen Service (SolexaIllumina Hi-Seq 2500). A montagem, anotação, alinhamento, construção da filogenia e análise sintênica foram realizados in silico com softwares específicos. O cpDNA e mtDNA P. crispa apresentam 196.502 pb e 89.819 pb, respectivamente. Estes genomas acessórios apresentam 21 genes putativos relacionados com a fotossíntese e 18 genes relacionados com o metabolismo oxidativo. A análise filogenômica baseada no cpDNA demonstrou que P. crispa agrupou com alga trebouxiophyceae Prasiolopsis sp. formando o clado Prasiola juntamente com Stichococcus bacilaris. Nossos resultados para filogenômica embasada no mtDNA revelam que P. crispa agrupa com as outras espécies da classe Trebouxiphyceae. A análise de sintenia do cpDNA e mtDNA de P. crispa com a espécies de plantas verdes relacionadas evolutivamente demonstram que estes organismos apresentam poucos blocos gênicos sintênicos. Este trabalho pioneiro com a alga P. crispa, demonstra que os genomas acessórios suprem uma gama de dados moleculares que podem ser utilizados para estudos filogenômicos. Além disto, as informações geradas a partir do sequenciamento do cpDNA e mtDNA de P. crispa fornecem um aporte para estudos futuros mais aprofundados / Green algae from Trebouxiophyceae class are among the organisms in the Antarctic continent, where the most reported species is the green macroalga Prasiola crispa (Lightfoot) Kützing. This algae is considered an extremophile organism because develops successfully in the harsh Antarctic habitat, however studies reporting molecular data of this species are still lacking in the literature, which prevents an assessment of their correct taxonomy and phylogenetic position. With the advent of next generation sequencing technologies, it because easier to obtain molecular information as for example from organelle genomes making them a great tool for taxonomic studies because they provide a great number of, phylogenetic data, nucleotides, protein sequences, gene content and architecture information. In this study, we determined the sequence of the chloroplast (cpDNA) and mitochondrial (mtDNA) genome of P. crispa in order to infer the evolutionary relationships of the organisms with other species of green plants, as well as a structural analysis. Plastid and mitochondrial genome was sequenced by Macrogen Service (Illumina Solexa Hi-Seq 2500). The genome assembly, annotation, sequences alignment, phylogeny construction, and structural analyses were performed in silico with specific softwares. Plastid and Mitochondrial genomes have a total length of 196,502 bp and 89,819 bp, respectively. These genomes presented 21 putative photosynthesis related genes and 18 oxidative metabolism related genes, respectively. Phylogenetic analysis based on the cpDNA demonstrated that P. crispa grouped with Trebouxiophyceae algae Prasiolopsis sp. forming the Prasiola clade along with Stichococcus bacilaris. Our results for phylogenetic analysis grounded in mtDNA show that P. crispa groups with other species of Trebouxiphyceaen alga. Synteny analysis of P. crispa cpDNA and mtDNA with evolutionarily related species of green plants shows that these organisms have few syntenic gene blocks. This pioneering work with P. crispa provided the accessories genomes which suppled a range of molecular data that can be employed to taxonomic studies. In addition, the information generated from the sequencing of cpDNA and mtDNA of P. crispa provide a contribution for further studies.

In this thesis, phylogenomic and comparative structural analyses of several widespread energy converting enzymes were performed. The focus was on the major subfamilies of the enzymes that process nucleoside triphosphates (ATP and GTP) and on some key enzymes of the electron transfer chains. First, we analyzed the P-loop GTPases, RadA/RecA recombinases, chaperone GroEL, branched-chain α-ketoacid dehydrogenase kinases, chaperone Hsc70, actins, and membrane pyrophosphatases. In the each inspected family we could identify (1) members which were potassium-dependent and/or contained K+ ions in the active site, and (2) potassium-independent enzymes with lysine or arginine residues as catalytic groups that occupy the positions of potassium ions in the homologous, K+-dependent enzymes. Based on the results of our analyses, we suggest that the appearance of the K+-binding sites could precede in evolution the recruitment of positively charged residues (lysine or arginine "fingers") with the latter providing more possibilities to control the enzyme reactions. Second, we have described the distinctive features of a phylogenetically separated subfamily of rotary membrane ATPases which we named N-ATPases. The N-ATPases have a specific operon organization with two additional subunits, absent in other rotary ATPases, and a complete set of Na+-binding ligands in the membrane c-subunits. We made a prediction, which was later confirmed, that these enzymes are capable of Na+ translocation across the membrane and may confer salt tolerance on marine prokaryotes. Third, phylogenomic analysis of the cytochrome bc complexes suggests that these enzyme complexes initially emerged within the bacteria and were then transferred to archaea via lateral gene transfer on several independent occasions. Our analysis indicates that the ancestral form of the cytochrome bc complex was a b6f-type complex; the fusion of the cytochrome b6 and the subunit IV to a "long" cytochrome b of the cytochrome bc1 complexes could have happened in different lineages independently. Fourth, our phylogenomic and comparative structural analyses of the cytochrome bc1 complex and of cytochrome c allowed us to trace how these enzymes became involved in triggering of apoptosis in Metazoa. We could trace the emergence of a specific cardiolipin-binding site within the cytochrome bc complex and the evolution of structural traits that account for the involvement of the cytochrome c as a trigger of apoptosis in vertebrates.