Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-08-22T19:39:56Z No. of bitstreams: 1 2017 - Let?cia Gabriela Poblete Vidal.pdf: 2525390 bytes, checksum: 162c0cfed2f6b4d8e94cb85c1c0254da (MD5) / Made available in DSpace on 2017-08-22T19:39:56Z (GMT). No. of bitstreams: 1 2017 - Let?cia Gabriela Poblete Vidal.pdf: 2525390 bytes, checksum: 162c0cfed2f6b4d8e94cb85c1c0254da (MD5) Previous issue date: 2017-03-08 / CNPq / The hope of this study was increased the knowledge of biodiversity of myxozoan fish parasites, unknown to some groups. In chapter 1, the goal was provide a synopsis of Myxozoa Grass?, 1970 species in the Americas based on records of the valid species of myxozoans (Myxozoa: Myxosporea) described in the Americas is provided based on a comprehensive survey of the literature since 1893, when the first myxozoan species was reported, until December 2015. The synopsis include the habitat of the host, site of the infection of the parasite, locality, size (?m or mm) and form of the plasmodia, spore measurements, provide specimens to parasitological collections, molecular data and explicit linkage of host. This synopsis was based on original descriptions. In chapter 2, the present work complements the original description of H. friderici Casal Matos and Azevedo, 2003 with new morphological and molecular data with Gill filaments on Leporinus friderici (Bloch, 1794) from the Mogi Gua?? River, state of S?o Paulo. Finaly, in chapter 3, specie of Henneguya was found in the kidneys of Astyanax altiparanae (Characiformes: Characidae) and were analyzed by morphological and molecular studies with analysis of the rDNA of the small subunid of the ribosome (18S). These data identify a new species of Myxozoa. / O presente trabalho teve como objetivo ampliar os conhecimentos sobre a biodiversidade de mixospor?deos parasitos de peixes, visto o escasso conhecimento para esse grupo. No cap?tulo 1 o objetivo foi fornecer uma sinopse de esp?cies de Myxozoa Grass?, 1970 nas Am?ricas com base em um levantamento bibliogr?fico desde 1893, quando a primeira esp?cie de myxospor?deo foi descrita, at? dezembro de 2015. A sinopse inclui uma lista parasito-hospedeiro com dados sobre o habitat do hospedeiro, s?tio de infec??o, localidade, tamanho e formato do cisto, medidas dos esporos e esp?cimes em cole??es e uma lista de parasitos-hospedeiros. Nessa sinopse s?o relatados somente as descri??es originais encontradas nas Am?ricas. No cap?tulo 2, ? inclu?da uma descri??o de H. friderici Casal, Matos e Azevedo, 2003 com novos dados morfol?gicos e moleculares com material proveniente de amostras de filamentos branquiais de Leporinus friderici (Bloch, 1794) do rio Mogi Gua??, estado de S?o Paulo. Finalmente, no cap?tulo 3, uma esp?cie de Henneguya encontrada nos rins de Astyanax altiparanae (Characiformes: Characidae) foi descrita e ilustrada com base na sua morfologia e na an?lise do rDNA da subunidade maior do ribossomo (28S). Estes dados identificam uma poss?vel esp?cie nova de Myxozoa.

Made available in DSpace on 2017-07-10T14:38:32Z (GMT). No. of bitstreams: 1 Bueno_Texto.pdf: 3065183 bytes, checksum: 4147005c285a3d2e9e61bbb9b0e03d77 (MD5) Previous issue date: 2012-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Resumo do Capítulo 1.: As relações filogenéticas e identificação de espécies do gênero Hypostomus ainda não são claras. Considerando isto, a citogenética tem-se mostrado uma ferramenta útil no entendimento da sistemática do gênero. Revisões em Hypostomus indicam que o número diplóide varia de 54 a 84 cromossomos e o aumento do número diplóide foi associado a porcentagens maiores de cromossomos subtelocêntricos e acrocêntricos. Embora exista um número grande de espécies no gênero, existem relativamente poucos artigos relacionados à citogenética de Hypostomus, e a maior parte dos dados é publicada em comunicações de simpósios. Com o objetivo de entender a evolução cromossômica do gênero (correlação entre número diplóide x tipos cromossômicos), H. ancistroides e H. topavae do rio Piquiri, bacia do Alto Paraná, foram citogeneticamente analisados, sendo observados os números diplóides de 68 e 80 cromossomos respectivamente. Dados adicionais sobre o número cromossômico e fórmula cariotípica foram compilados de 27 análises publicadas em artigos e 77 de resumos. Nossa análise não mostrou correlação entre números cromossômicos e porcentagens de cromossomos subtelocêntricos e acrocêntricos na maior parte das espécies, já que existe variação considerável entre estas porcentagens mesmo entre espécies com o mesmo número diplóide, indicando que a proporção entre tipos cromossômicos não está sempre associada ao número diplóide.

A Molecular Approach to Assessing Meiofauna Diversity in Marine Sediments Heather C. Hamilton Abstract The purpose of this study was to determine if a molecular approach could be applied to calculating the diversity of meiofauna in marine sediments from two sites in Tampa Bay, FL, similar to the approach of McCaig et al, 1999 in calculating the diversity of microbes in pastureland soils. The approach includes extracting total DNA directly from the sediment and amplifying the 18S rRNA gene by PCR. Clone libraries from the 18S gene would be created for each site and 300 sequences from each clone library would be obtained. These sequences would then be phylogenetically analyzed and assigned to an OTU, from which diversity indices can be calculated. The phylogenetic analysis of the sequences from the two sites revealed that of the 102 OTUs assigned from the sequences, only 7 OTUs included sequences from both sites, while 93 OTUs contained sequences from one site or from the other. Thus the sites were phylogenetically different from each other. Shannon diversity indices calculated for each site showed a difference between the two sites and paralleled diversity indices for macrofauna data for each site collected by the Hillsborough County Environmental Protection Commission. Sequences from 30 OTUs were completely sequenced and identified by phylogenetic comparison with a metazoan reference alignment. A discrepancy between the sequence data and data collected from preserved samples taken at each site was evident upon analysis: roughly 60% of each preserved sample consisted of nematodes and 10% consisted of copepods, while roughly 30% of the identified OTUs consisted of copepods and 10% consisted of nematodes. This discrepancy could be explained if the OTUs that were not identified consisted of nematode sequences or if a primer bias were present in the PCR amplification such that the regions flanking the primer site in the nematode sequences inhibited primer annealing.

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.

The phylum Chaetognatha is a mysterious group of organisms that has eluded scientists for more than a century because of their unique morphology and developmental characteristics, i.e. protostome (mouth develops from blastopore; e.g. mollusks, annelids, arthropods) versus deuterostome (anus develops from blastopore; e.g. echinoderms and chordates) offer few clues to their evolutionary origins. Some early morphological studies argued that chaetognaths were derived mollusks or nematodes according to gross ultrastructural data, while other studies focused on the coelomic cavity. 33 Although 18S rRNA is widely used in molecular phylogeny studies, it has limits such as long- branch chain attractions and a slow rate of evolutionary change. Long-branch chain attractions are a phenomenon in phylogenetic analyses when rapidly evolving lineages are inferred to be closely related, regardless of their true evolutionary relationships. Hence other genes are used in this study to complement the 18S rRNA such as the cytochrome oxidase genes. The cytochrome oxidase genes are highly conserved throughout all eukaryotic organisms and they are less ambiguous to align as compared to the ribosomal genes, making them better phylogenetic markers as compared to the 18S rRNA gene. This study focuses on using a molecular approach (ARDRA, PCR, phylogenetic tree reconstruction) to determine the phylogeny of pelagic chaetognaths found on Pelican Island, Galveston, Texas. 18S rRNA, Cytochrome Oxidase I and Cytochrome Oxidase II genes were used to help decipher the phylogeny of this group. All analyzed genes in this study (18S rRNA, COI, and COII) grouped the Pelican Island chaetognaths with the protostomes. The maximum parsimony bootstrap tree for the 18S rRNA gene, grouped the samples closest to the arthropods (protostome). For the COI and COII genes, the minimum evolution bootstrap tree grouped the 8 collected samples more closely to two other protostome phyla: the mollusks and annelids (COI) while bootstrapping with the COII grouped the samples with the nematodes (with >66 percent bootstrap). My findings are significant because they reveal phylogenetic results of a protostome lineage for the Chaetognatha using 3 genes, one of which (COII) has not been greatly studied for the Chaetognatha.

Τα ισόποδα αποτελούν τη μοναδική τάξη οργανισμών ανάμεσα σε όλα τα Καρκινοειδή που πέτυχε να εποικήσει όλους τους τύπους ενδιαιτημάτων, από τα βάθη των ωκεανών μέχρι τα βουνά, τις έρημους και τις τροπικές περιοχές. Παρόλα αυτά, οι φυλογενετικές σχέσεις εντός της τάξης των ισοπόδων παραμένουν σε πολλά σημεία ασαφείς. Η οικογένεια Tylidae, που περιλαμβάνει 27 αμφίβια είδη, ανήκει σύμφωνα με τη σημερινή συστηματική κατάταξη στην υπόταξη Oniscidea, τη μοναδική που περιλαμβάνει αντιπροσώπους με χερσαίο ή ημι-χερσαίο τύπο διαβίωσης. Αν και υπάρχουν αμφιβολίες ως προς τη μονοφυλετική προέλευση αρκετών από τις 9 υποτάξεις που περιλαμβάνουν θαλάσσιους αντιπροσώπους, η προέλευση των Oniscidea θεωρείται αδιαμφισβήτητα μονοφυλετική. Εντούτοις, δεν υπάρχει ακόμη συμφωνία ως προς την ακριβή τοποθέτηση του κλάδου των Tylidae στο φυλογενετικό δέντρο των Oniscidea. Ο βασικός στόχος της παρούσας εργασίας ήταν ο προσδιορισμός των φυλογενετικών σχέσεων των αμφιβίων ισοπόδων της οικογένειας Tylidae με τα υπόλοιπα ισόποδα, με έμφαση στις σχέσεις με τα υπόλοιπα Oniscidea. Για το σκοπό αυτό χρησιμοποιήθηκαν μοριακοί δείκτες από 14 γένη ισοπόδων, τα οποία αντιπροσωπεύουν όλους τους τύπους διαβίωσης, από τον αποκλειστικά θαλάσσιο έως τον αποκλειστικά χερσαίο. Η πειραματική προσέγγιση περιλάμβανε τον πολλαπλασιασμό αλληλουχιών του πυρηνικού γονίδιου 18s rDNA και των μιτοχονδριακών 16s rDNA και COI με τη μέθοδο της αλυσιδωτής αντίδρασης πολυμέρασης (PCR), τον προσδιορισμό της αλληλουχίας τους και τη φυλογενετική ανάλυσή τους με μεθόδους μέγιστης φειδωλότητας, μέγιστης πιθανοφάνειας και μπεϊεσιανής συμπερασματολογίας. Οι μιτοχονδριακές αλληλουχίες εμφανίζουν πολύ υψηλά ποσοστά νουκλεοτιδικών υποκαταστάσεων, και σε συνδυασμό με τη χαμηλή αξιοπιστία των δέντρων που παράγονται φαίνεται πως έχουν απωλέσει εντελώς το φυλογενετικό τους σήμα. Η αλληλουχία του 18s rDNA έχει μήκος 2400-3400 bp και αποτελείται από 4 συντηρημένες περιοχές και 3 υπερ-μεταβλητές. Για τις φυλογενετικές αναλύσεις χρησιμοποιήθηκαν μόνο οι συντηρημένες περιοχές, που συνιστούν ένα σύνολο 1597 διακριτών χαρακτήρων. Στα αποτελέσματα από όλες τις υπολογιστικές μεθόδους η οικογένεια Tylidae τοποθετείται στο τελικό δέντρο σε αδελφό κλάδο του τάξου Crinochaeta της υπόταξης Oniscidea. Εντούτοις, παρατηρήθηκε πως η οικογένεια Ligiidae τοποθετείται σε κλάδο μη-συγγενικό προς τα υπόλοιπα χερσαία ισόποδα, υπονοώντας πως η υπόταξη Oniscidea δεν είναι μονοφυλετική. Για να ελέγξουμε αυτήν την υπόθεση, χωρίσαμε τα δεδομένα μας σε αλληλουχίες από θαλάσσιους και σε αλληλουχίες από χερσαίους αντιπροσώπους, πραγματοποιώντας ένα σύνολο από πρόσθετες αναλύσεις. Από τα αποτελέσματα φαίνεται πως η υπόταξη Oniscidea είναι μονοφυλετική και η αντίθετη αρχική υπόθεση οφείλεται σε ‘θόρυβο’ στο φυλογενετικό σήμα των συντηρητικών περιοχών του 18s rDNA. Τέλος, από τις πρόσθετες αναλύσεις προκύπτουν, με ισχυρή στατιστική στήριξη, φυλογενετικά δέντρα που υποστηρίζουν τη συστηματική κατάταξη που είχε προτείνει ο Erhard από τις μορφολογικές του μελέτες, τοποθετώντας τα Tylidae εντός του τάξου ‘Holoverticata’. / Isopods comprise a unique order among the Crustaceans that has settled effectively all possible habitats on the planet. The phylogenetic relationships, though, between the 10 suborders remain unresolved, as many of them might prove to be non-monophyletic taxa. The family Tylidae consists of 27 amphibian species and is traditionally classified in the suborder Oniscidea, which includes all the terrestrial and semi-terrestrial isopods and that is thought to be unambiguously monophyletic. However, the previous phylogenetic studies have proposed many hypotheses concerning the relations between the Tylidae and the other Oniscidea, lacking any plausible consensus. In order to resolve those phylogenetic relations, we used two mitochondrial sequences (16s, COI) and one nuclear (18s) from 14 genera of isopods. Our experimental approach included PCR amplification, sequencing and computational phylogenetic analyses by means of maximum parsimony, maximum likelihood and Bayesian inference. The mitochondrial sequences present extreme values of nucleotide substitutions and evident saturation, a fact that prohibits their use in further analyses. The 18s sequences vary significantly in size (2400-3400 bp) and consist of 4 conserved and 3 hyper-variable regions. Only the conserved regions were used for analysis, resulting to a dataset of 1597 discrete nucleotide characters. Regardless of the method used, the family Tylidae appeared as a sister-clade of the taxon Crinochaeta (suborder Oniscidea) in the cladograms. We noticed, though, that the taxon Diplochaeta (Oniscidea: Ligiidae) appeared (with low bootstrap values) in a distant clade of all the other Oniscidea, a result that does not support the monophyletic origin of the Oniscidea. In order to test the validity of this result, we splitted our dataset and conducted additional, separate analyses for the sequences of the marine isopods and those of the terrestrial isopods. Our results indicate that the suborder Oniscidea is monophyletic, so the initial opposite hypothesis was due to weak phylogenetic signal. Finally, our cladograms support, with significant confidence, the systematic classification that Erhard (1998) had proposed through his studies on morphological characters of the Oniscidea, placing together the family Tylidae and the taxon Crinochaeta under the name ‘Holoverticata’.

Το ριβοσωματικό RNA (rRNA) παίζει σημαντικό ρόλο στη διαδικασία της ακριβούς αποκωδικοποίησης της γενετικής πληροφορίας. Σύμφωνα με πρόσφατες κρυσταλλικές δομές υψηλής ευκρίνειας της μικρής υπομονάδας του προκαρυωτικού ριβοσώματος, η περιοχή αποκωδικοποίησης αποτελείται κυρίως από rRNA, περιλαμβάνοντας κυρίως τις έλικες 18 και 34, καθώς και τις αδενίνες Α1492 και Α1493 της έλικας 44 του 16S rRNA. Επιπροσθέτως, ο ρόλος του rRNA στην ακριβή αποκωδικοποίηση της γενετικής πληροφορίας έχει αποκαλυφθεί εκτενώς μέσω κατασταλτικών ή αντικατασταλτικών μεταλλάξεων, οι οποίες αυξάνουν ή μειώνουν τη λανθασμένη ανάγνωση του mRNA, αντίστοιχα. Πολλές από τις μεταλλάξεις αυτές αφορούν στο 16S rRNA της Escherichia coli. Μεταξύ αυτών είναι η μετάλλαξη C310G στην έλικα 12, η G1206C στην έλικα 34 και η G517A στην έλικα 18. Στο πρώτο μέρος της παρούσης μελέτης εξετάσθηκε το αντίκτυπο των αντίστοιχων μεταλλάξεων com1 (C310G), com6 (G1206C) και rdn2 (G517A) στο 18S rRNA του Saccharomyces cerevisiae. Τα κύτταρα yeast μετασχηματίστηκαν με rDNA πλασμίδια αγρίου-τύπου (wt) ή μεταλλαγμένα, τα οποία έφεραν τις προαναφερθείσες μεταλλάξεις. Τα κύτταρα που ελέγχθηκαν ήταν το αγρίου τύπου (rdnwt), τα μεταλλάγματα rdn2, com1, καθώς και τα διπλά μεταλλάγματα com1rdn2 και com6rdn2. Το αντίκτυπο της μετάλλαξης com6 εξήγχθη έμμεσα από το διπλό μετάλλαγμα com6rdn2, αφού το μετάλλαγμα com6 δεν ήταν διαθέσιμο στο Εργαστήριο (Τα στελέχη αυτά χορηγήθηκαν από το Εργαστήριο της Dr Susan Liebman, University of Illinois at Chicago, USA). Όσον αφορά στη μελέτη ανάπτυξης των στελεχών αυτών, τα στελέχη com1 παρουσίασαν εξαιρετικά αργή ανάπτυξη παρουσιάζοντας 3 φορές αύξηση του χρόνου διπλασιασμού τους σε σχέση με τα κύτταρα rdnwt. Αντίθετα, το μετάλλαγμα rdn2 παρουσίασε παρόμοιο φαινότυπο ανάπτυξης με τα rdnwt. Είναι αξιοσημείωτο ότι τα διπλά μεταλλάγματα com1rdn2 και com6rdn2 παρουσίασαν μικρότερο χρόνο διπλασιασμού από τα κύτταρα rdnwt. Όσον αφορά στη μεταφραστική ακρίβεια, το μετάλλαγμα com1 παρουσίασε 1,5 φορά αύξηση στο ρυθμό λανθασμένης ενσωμάτωσης του αμινοξέος λευκίνη σε μία αυξανόμενη αλυσίδα πολυφαινυλαλανίνης κατά την in vitro μετάφραση ενός poly(U) εκμαγείου, σε σχέση με τα κύτταρα rdnwt. Αντίθετα, η μετάλλαξη rdn2 οδήγησε σε μεταφραστική υπερακρίβεια, αφού δεν βρέθηκε καμία ενσωμάτωση λανθασμένου αμινοξέος ανά 10.000 κωδικόνια mRNA. Το διπλό μετάλλαγμα com1rdn2 εμφάνισε παρόμοια επίπεδα μεταφραστικής ακρίβειας με κύτταρα rdnwt. Έτσι, οι μεταλλάξεις com1 και rdn2 επηρρεάζουν τη μεταφραστική ακρίβεια σε ίσο βαθμό, αλλά προς αντίθετες κατευθύνσεις. Το άλλο διπλό μετάλλαγμα της παρούσης μελέτης com6rdn2 παρουσίασε επίσης παρόμοια επίπεδα μεταφραστικής ακρίβειας με το rdnwt, αποκαλύπτοντας έτσι πως η μετάλλαξη com6 οδηγεί επίσης σε μείωση της μεταφραστικής πιστότητας. Τα αποτελέσματα αυτά υποδηλώνουν ότι το αντίκτυπο των μεταλλάξεων com1, com6 και rdn2 στην ακρίβεια της μετάφρασης είναι ανεξάρτητο και προσθετικό και όχι συνεργιστικό. Επιπρόσθετη επιβεβαίωση προήλθε με την in vitro μετάφραση εκμαγείων poly(U) παρουσία παρομομυκίνης (PM), ενός αμινογλυκοζιτικού αντιβιοτικού γνωστού για την αύξηση της ενσωμάτωσης λανθασμένων αμινοξέων στην αυξανόμενη πολυπεπτιδική αλυσίδα τόσο στα προκαρυωτικά, όσο και στα ευκαρυωτικά κύτταρα. Το στέλεχος com1 παρουσίασε 3 φορές αύξηση στη συχνότητα λάθους κατά τη μετάφραση σε σχέση με το rdnwt, επιβεβαιώνοντας το χαρακτηρισμό του ώς επιρρεπές σε λάθη μετάλλαγμα. Αντίθετα, το στέλεχος rdn2 εμφάνισε μισή περίπου συχνότητα λαθών κατά τη μετάφραση σε σχέση με τα κύτταρα rdnwt, σε συμφωνία με το προηγούμενο χαρακτηρισμό του ως υπερακριβές μετάλλαγμα. Τα αποτελέσματα αυτά επιβεβαιώθηκαν περαιτέρω με in vivo μελέτες ανθεκτικότητας έναντι της PM τόσο σε υγρές όσο και σε στερεές καλλιέργειες (τρυβλία petri). Το μετάλλαγμα com1 βρέθηκε το πιο ευαίσθητο όλων, αφού το 50% αναστολής της ανάπτυξής του παρατηρήθηκε σε μόλις 5 μΜ PM σε σχέση με τα 75 μΜ για το στέλεχος rdnwt. Αντίθετα, η τιμή IC50 της PM για το rdn2μετάλλαγμα βρέθηκε ίσο προς 500 μΜ. Το διπλό μετάλλαγμα com1rdn2 παρουσίασε παρόμοια επίπεδα ευαισθησίας στην PM με rdnwt, ενώ το άλλο διπλό μετάλλαγμα com6rdn2 αποδείχθηκε ως πιο ανθεκτικό (IC50 = 125 μΜ), υποδηλώνοντας ότι η com6 πρέπει να είναι λιγότερο επιρρεπής σε λάθη σε σχέση με την com1 μετάλλαξη. Πειράματα in vivo ανθεκτικότητας στην PM (σε τρυβλία petri) επιβεβαίωσαν τα επίπεδα ευαισθησίας που αποκαλύφθηκαν με τις υγρές καλλιέργειες. Η μελέτη των μεταλλάξεων αυτών ολοκληρώθηκε με πειράματα πρόσδεσης στις θέσεις A και P του ριβοσώματος. Οι μεταλλάξεις που μειώνουν την μεταφραστική πιστότητα οδηγούν και σε αύξηση της ικανότητας πρόσδεσης μορίων αμινο-ακυλο tRNA (aa-tRNA) στη θέση Α του ριβοσώματος, ενώ αντίθετα οι μεταλλάξεις που αυξάνουν την ακρίβεια της μετάφρασης συνοδεύονται και από μείωση της ικανότητας πρόσδεσης aa-tRNAs στη θέση Α του ριβοσώματος. Με τέτοιες μελέτες βρέθηκε ότι η μετάλλαξη com1 οδήγησε σε αύξηση της ικανότητας πρόσδεσης στη θέση Α, αντίθετα με την rdn2, όπου διαπιστώθηκε ακόμη μικρότερη ικανότητα πρόσδεσης και σε σχέση με τα ριβοσώματα αγρίου τύπου. Τα διπλά μεταλλάγματα έδειξαν λίγο μεγαλύτερη ικανότητα πρόσδεσης σε σχέση με αυτά του αγρίου τύπου. Τα αποτελέσματα αυτά επιβεβαίωσαν το χαρακτηρισμό των com1 και com6 ως μεταλλάξεις μείωσης της μεταφραστικής ακρίβειας σε αντίθεση με τη μετάλλαξη rdn2, η οποία οδηγεί σε υπερακρίβεια. Όσον αφορά στην ικανότητα πρόσδεσης aa-tRNAs στη θέση P του ριβοσώματος σε σχέση με τα ριβοσώματα αγρίου τύπου (rdnwt), αυτή βρέθηκε μεγαλύτερη στην περίπτωση της com1 μετάλλαξης και μικρότερη στην περίπτωση της rdn2 μετάλλαξης, υποδηλώνοντας ότι η com1 οδηγεί σε μεγαλύτερο ρυθμό πρωτεϊνοσύνθεσης σε σχέση με την υπερακριβή rdn2 μετάλλαξη. Στο δεύτερο μέρος της εργασίας διερευνήσαμε σε συνεργασία με το Εργαστήριο Βιοχημείας του κ. Χρήστου Γεωργίου (Τμήμα Βιολογίας, Παν. Πατρών), για το άν υπάρχει κάποια συσχέτιση μεταξύ της μεταφραστικής πιστότητας και του οξειδωτικού στρες στα ευκαρυωτικά κύτταρα. Για το σκοπό αυτό χρησιμοποιήσαμε δύο καλά μελετημένα μεταλλαγμένα στελέχη S. cerevisiae στο Εργαστήριό μας με αντίθετες επιπτώσεις στη μεταφραστική ακρίβεια. Αυτά ήταν το sup45 μετάλλαγμα (επιρρεπές σε λάθη με συχνότητα λάνθασμένης ενσωμάτωσης αμινοξεών ίση προς 0,0166) καθώς και το τριπλό μετάλλαγμα sup45rdn4rdn6 (συχνότητα λάθους: 0,0057), στο οποίο γίνεται η επαναφορά της μεταφραστικής ακρίβειας στα επίπεδα των μορίων αγρίου τύπου (0,0036). Οι μεταλλάξεις rdn4 και rdn6 αφορούν στην έλικα 27 του 18S rRNA, ενώ η μετάλλαξη sup45 πρόκειται για μία κατασταλτική μετάλλαξη στο γονίδιο που κωδικοποιεί για τον πράγοντα τερματισμού της πρωτεϊνοσύνθεσης eRF-1. Οι οξειδωτικοί δείκτες που μετρήθηκαν ήταν η μαλονική διαλδεϋδη (MDA: κύριο προϊόν υπεροξείδωσης λιπιδίων), η οξειδωμένη γλουταθειόνη (GSSG), οξειδωμένα μη πρωτεϊνικά μόρια (NPSSR), καθώς και οξειδωμένα πρωτεϊνικά μόρια (PSSP). Οι αντιοξειδωτικοί δείκτες που μετρήθηκαν ήταν τα ανηγμένα πρωτεϊνικά μόρια που φέρουν ελεύθερες σουλφυδρυλομάδες (PSH), καθώς και το κλάσμα PSH/PSSP μέσα στα κυτταρικά εκχυλίσματα. Εν συντομία, το επιρρεπές σε λάθη κατα τη μετάφραση μετάλλαγμα sup45 μείωσε τα επίπεδα των οξειδωτικών δεικτών σε αντίθεση με το πιο ακριβές τριπλό μετάλλαγμα sup45rdn4rdn6, στο οποίο παρατηρήθηκε αύξηση των δεικτών αυτών. Περαιτέρω επιβεβαίωση των αποτελεσμάτων αυτών προήλθε με την χρήση παρομομυκίνης κατά την ανάπτυξη των στελεχών αυτών (μειώνει τη μεταφραστική πιστότητα) και τη διεξαγωγή των μετρήσεων αυτών των δεικτών οξειδωτικού στρες εκ νέου. Τα αποτελέσματα των πειραμάτων με χρήση PM έδειξαν στις περισσότερες περιπτώσεις σημαντική μείωση των επιπέδων οξειδωτικού στρες. Εν κατακλείδι, τα αποτελέσματα του δεύτερου μέρους της Μεταπτυχιακής αυτής Διατριβής υποδηλώνουν ότι στα ευκαρυωτικά κύτταρα η μείωση της μεταφραστικής πιστότητας οδηγεί σε μείωση του οξειδωτικού στρες σε αυτά και το αντίστροφο. / The ribosomal RNA (rRNA) plays a key role in the process of the accurate decoding of genetic information. According to recently derived high-resolution crystal structures of the prokaryotic small ribosomal subunit, the decoding site is mainly composed of RNA, including helices 18 and 34 as well as adenines A1492 and A1493 of helix 44 of the 16S rRNA. Furthermore, the role of ribosomal RNA in the accurate decoding of genetic information has been largely uncovered by suppressor or antisuppressor mutations that increase or decrease misreading respectively. Many of these mutations are in 16S rRNA of Escherichia coli. Among these are C310G in helix 12, G1206C in helix 34 and G517A in helix 18. In the first part of this study we examined the function of the corresponding mutations com1 (C310G), com6 (G1206C) and rdn2 (G517A) in 18S rRNA of Saccharomyces cerevisiae. Yeast cells were transformed with wild-type or mutant rDNA plasmids carrying the afore-mentioned site-directed mutations. The strains examined were the wild-type (rdnwt), mutants rdn2, com1 and the double mutants com1rdn2 and com6rdn2. The properties of mutation com6 were only deduced from the double mutant com6rdn2, since mutation com6 was not available individually. The mutants were first constructed in Prof. Susan Liebman’s laboratory at the University of Illinois at Chicago, USA. Strains com1 were viable but exhibited an extraordinary slow growth phenotype as they displayed a 3-fold increase of their doubling time over the rdnwt. On the other hand, rdn2 mutants displayed a growth phenotype similar to that of rdnwt cells. Interestingly, the double mutants com1rdn2 and com6rdn2 abolished the slow growth phenotype and grew faster than rdnwt. Regarding translational accuracy, com1 mutant displayed a 1.5-fold increase of the rate of misincorporation of the nearcognate amino acid leucine in a growing polyphenylalanine chain with poly(U) as template over rdnwt. In contrast, mutation rdn2 displayed impressive hyperaccuracy as it was found that none nearcognate aminoacid is added in 10.000 mRNA codons. Moreover, the double mutant com1rdn2 exhibited misreading levels similar to those of the rdnwt. Thus, com1 and rdn2 affect the decoding process to an equal degree but in reverse ways. The other double mutant under study, com6rdn2 also displayed an error frequency similar to that of rdnwt revealing that com6 is an error-prone mutation as well. These results imply that the effects of mutations com1, com6 and rdn2 on translational accuracy are independent and additive rather than synergistic. Additional confirmation came by in vitro translation of poly(U) templates in the presence of paromomycin (PM), which is an aninoglycoside antibiotic known to induce suppression of the genetic code. Strain com1 displayed a 3-fold increase of error frequency over rdnwt, confirming its characterization as an error-prone mutant. In contrast, rdn2 displayed an error frequency about half of that observed for the rdnwt, compatible with its characterization as a hyperaccurate mutant. The previous results were confirmed by in vivo resistance studies toward PM in liquid cultures and on Petri plates. Mutant com1 was the most sensitive of all, as 50% inhibition of its growth was observed at only 5 μΜ PM compared to 75 μΜ for rdnwt. In contrast, the IC50 value of PM for the rdn2 mutant was 500 μΜ. The double mutant com1rdn2 exhibited a resistance to PM similar to rdnwt, and the other double mutant com6rdn2 was more resistant (IC50 = 125 μΜ), implying that com6 should be a less error-prone mutation than com1. In vivo resistance to PM (on petri dishes) confirmed the sensitivity levels found in liquid cultures. The study of these mutations was completed by A- and P-site binding experiments. An increase in A-site binding of aa-tRNA may arise from a higher affinity to accept noncognate tRNAs and is related to error-prone mutations, whereas a decrease is related to error-restrictive mutations. Binding of Phe-tRNA to A-site of com1 mutants was much higher than in rdnwt. In contrast, binding to A-site of rdn2 was much lower than in rdnwt. Finally, the double mutants showed an A-site binding capacity slightly higher than the wild-type. These results confirmed that com1 and com6 are error-prone mutations, whereas rdn2 an error-restrictive mutation. With regards to P-site, com1 displayed a higher binding capacity and rdn2 a lower binding capacity compared to wild-type, indicating that com1 may have a higher protein synthesis activity than rdn2. In the second part of this study, we (in cooperation with Dr Christos Georgiou’s lab; Dept. of Biology, University of Patras) investigated whether any correlation between the translational fidelity and oxidative stress exists in eukaryotic cells. We used mutants already studied in our lab for their effects on translational fidelity and other aspects of protein synthesis. These were the sup45 mutant encoding the eRF-1 release factor and the triple mutant sup45rdn4rdn6 which carries, in addition to sup45, mutations rdn4 and rdn6 in helix 27 of the 18S rRNA. Mutant sup45 is error-prone (E.F. = 0.0166), while rdn4, rdn6 are both error-restrictive. The triple mutant sup45rdn4rdn6 diplays an error frequency of 0.0057, equal to the sum of the error frequencies of the individual mutations. The error frequency of the rdnwt is 0.0036. The oxidative markers measured were malondialdehyde (MDA, the main product of lipid peroxidation), oxidized glutathione (GSSG), oxidized non protein molecules (NPSSR), and oxidized protein molecules (PSSP). On the other hand, the antioxidative markers measured were t reduced protein molecules carrying –SH groups (PSH), as well as the ratio PSH/PSSP in the cell. In brief, the error-prone mutant sup45 was shown to decrease the concentrations of the various oxidative markers, while the comparatively error-restrictive triple mutant sup45rdn4rdn6 was shown to increase them. To further test the hypothesis that the oxidative status of a yeast cell may be related to the level of translational accuracy, we repeated the above experiments using PM. Our results with PM showed in most cases a decrease in the concentrations of the various oxidative markers. This is compatible with the notion that an increase in translational errors causes a decrease in oxidative stress and vice versa.

Made available in DSpace on 2016-06-02T20:21:38Z (GMT). No. of bitstreams: 1 711.pdf: 3692942 bytes, checksum: abc9a31cfa8c7e6dda5eb0e7195f3d22 (MD5) Previous issue date: 2005-08-26 / Universidade Federal de Minas Gerais / Family Characidae consists of approximately 30 highly diversified subfamilies that probably do not comprise a monophyletic group. However, a few particular subfamilies may constitute monophyletic groups sharing specializations. In the present work, nine specimens belonging to six genera were analyzed using classic staining techniques as well as fluorescent in situ hybridization (FISH) and GCspecific fluorochromes, such as Chromomycin A3 (CMA3). A diploid number of 2n=58 chromosomes was observed in Myleus micans, with 26M+18SM+8ST+6A; 2n=50 in Astyanax lacustris with 8M+20SM+16ST+6A; 2n=50 in A. altiparanae with 8M+20SM+12ST+10A; 2n=50 in Astyanax scabripinnis with 12M+24SM+2ST+6A; 2n=50 in Orthospinus franciscensis with 12M+30SM+2ST+6A; 2n=52 in Piabina argentea with 8M+14SM+16ST+14A; 2n=52 and three cytotypes in Serrapinnus heterodon, with 17M+20SM+14ST+1A, 16M+20SM+14ST+2A and 15M+20SM+14ST+3A; 2n= 52 in Serrapinnus piaba with 16M+20SM+14ST+2A; and 2N=50 in Hasemania nana with 8M+42SM. FISH results with the 18S rDNA probe indicated the presence of these genes in the chromosomes where NORs were active, besides additional sites, as seen in M. micans, A. scabripinnis, P. argentea and S.piaba. Similar to the NOR sites, the 5S rDNA regions were evidenced in different numbers and positions throughout the studied species. The data obtained corroborate the chromosome diversity often reported for Characidae, reinforcing its polyphyletic condition. / A família Characidae consiste de aproximadamente 30 subfamílias, altamente diversificadas e, provavelmente, não compreendendo um grupo monofilético. No entanto, algumas subfamílias, em particular, podem constituir grupos monofiléticos, compartilhando especilalizações. No presente trabalho foram analisadas 9 espécies pertencentes a 6 gêneros de Characidae, utilizando técnicas clássicas de coloração, bem como hibridação fluorescente in situ (FISH) e fluorocromos GC específicos, como Cromomicina A3 (CMA3). Foi observado um número diplóide 2n=58 cromossomos em Myleus micans, com 26M+18SM+8ST+6A; 2n=50 em Astyanax lacustris com 8M+20SM+16ST+6A; 2n=50 em A. altiparanae com 8M+20SM+12ST+10A; 2n=50 em Astyanax scabripinnis com 12M+24SM+2ST+6A; 2n=50 em Orthospinus franciscensis com 12M+30SM+2ST+6A; 2n=52 em Piabina argentea com 8M+14SM+16ST+14A; 2n=52 e três citótipos em Serrapinnus heterodon, com 17M+20SM+14ST+1A, 16M+20SM+14ST+2A e 15M+20SM+14ST+3A; 2n= 52 em Serrapinnus piaba com 16M+20SM+14ST+2A; e 2N=50 em Hasemania nana sendo 8M+42SM. Resultados após a FISH com a sonda de rDNA 18S indicaram a presença desses genes nos cromossomos onde foram identificadas as NORs ativas, além de sítios adicionais, como visto em M. micans, A. scabripinnis, P. argentea e S. piaba. Assim como os sítios de NORs, as regiões de rDNA 5S foram evidenciadas em diferentes números e posições entre as espécies estudadas. Os dados obtidos corroboram a diversidade cromossômica comumente relatada para Characidae, reforçando sua condição polifilética.