A coleção de fungos do Instituto de Medicina Tropical de São Paulo/USP compreende 1047 isolados de fungos, algas e actinomicetos de interesse médico e veterinário, que até o início deste estudo eram mantidos sob repiques sucessivos a cada três meses. Criada em 1925 pelo Prof. Dr. Floriano Paulo de Almeida e, a partir de 1953, mantida pelo Prof. Dr. Carlos da Silva Lacaz, a coleção necessitava de adequação de seu espaço físico e atualização nos métodos de preservação dos isolados. A reforma física foi realizada de acordo com as normas de biossegurança recomendadas pelo manual de procedimentos para manipulação de patógenos. Diversos estudos têm demonstrado que repiques sucessivos propiciam alterações nas características morfológicas, antigênicas, genéticas e de virulência de isolados fúngicos. Deste modo, foram aplicadas metodologias de preservação de longo prazo, como: liofilização, água destilada esterilizada, congelamento a -80ºC e em nitrogênio líquido (-196ºC). Foram selecionados 386 isolados, entre fungos dimórficos (213), filamentosos (hialinos e melanizados - 106) e leveduras (67), relacionados às infecções fúngicas mais prevalentes em nosso meio. Foram utilizados dois métodos de preservação para cada grupo de fungos: as leveduras foram submetidas à liofilização e congelamento em freezer -80ºC; e os fungos dimórficos e filamentosos preservados em água destilada esterilizada e no vapor de nitrogênio líquido. Esses isolados foram inicialmente avaliados quanto à caracterização morfológica e, após aplicação dos métodos de preservação, avaliou-se a viabilidade dos mesmos dentro de um período de dois anos, bem como a morfologia. As leveduras apresentaram 100% de viabilidade pelos dois métodos de preservação, mantendo suas características fenotípicas. Do mesmo modo, os dois métodos aplicados aos fungos filamentosos resultaram em elevados índices de viabilidade, com manutenção das características morfológicas. Em relação aos fungos dimórficos, a manutenção em nitrogênio líquido resultou em índices variáveis de viabilidade, dependendo do gênero avaliado. Do total de isolados do estudo, 75 foram submetidos também à identificação molecular por sequenciamento automatizado, após amplificação da região ITS do DNA ribossomal por PCR. A técnica molecular demonstrou, para alguns desses isolados, discordância na identificação das espécies quando comparada aos métodos fenotípicos. Os resultados deste estudo demonstraram que os repiques sucessivos não são recomendados para a preservação de fungos em coleções, por alterarem as características fenotípicas, bem como a capacidade de produzir esporos. As novas metodologias de preservação adotadas mantiveram a morfologia típica dos gêneros e espécies, permitindo ainda a viabilidade da maioria dos isolados estudados. A identificação molecular demonstrou ser importante ferramenta para autenticação dos isolados / The fungal culture collection from the Instituto de Medicina Tropical de São Paulo/USP comprises 1047 isolates of fungi, algae and actinomycetes with medical and veterinary relevance, which up to now have been preserved by subculturing every three months. Created in 1925 by Prof. Floriano Paulo de Almeida and maintained by Prof. Carlos da Silva Lacaz and his assistants since 1953, this fungal collection needed to be realocated to another area in 2007, as well as updated and revised as to its preservation methods. This new area was reformed according to safety procedures recommended in guidelines for pathogenic microorganisms manipulation. Several studies demonstrated that storage of fungi by subculturing may induce antigenic changes, phenotypic and genetic alterations, in addition to attenuation of virulence. The present study was aimed at utilizing more adequate methods of long-term preservation, such as lyophilization, sterile distilled water, and cryopreservation (freezing at -80°C and storage in liquid nitrogen [-196°C]). Three hundred eight six isolates consisting of dimorphic (213) and filamentous fungi (hyaline and melanized - 106), and yeasts (67) associated with the most prevalent fungal diseases in our setting were randomly selected. Two methods were applied for storage of each fungal group: yeasts were submitted to lyophilization and freezing at -80ºC; dimorphic and filamentous fungi were preserved in sterile distilled water and liquid nitrogen. All isolates were initially evaluated for their morphological features, submitted to the different methods of storage, and during a period of up to two years, reassessed for viability and morphological features. Yeasts showed 100% viability by the two applied preservation methods, maintaining their phenotypic characteristics. Similarly, the two methods applied to filamentous fungi resulted in high rates of viability, with maintenance of morphology. Regarding the dimorphic fungi, the preservation in liquid nitrogen resulted in variable rates of recovery, depending on the genus assessed. From all studied isolates, 75 were also submitted to molecular identification by sequencing after PCR amplification of the ITS region of the ribossomal DNA. This molecular technique showed, for some isolates, a lack of agreement in species identification when compared with the phenotypic methods. The new long-term storage methods used in the study preserved the typical genus and species morphology of the studied isolates, while resulting in high viability rates. The molecular identification proved to be an important tool for the authentication of isolates

O programa GenSeed, descrito previamente pelo nosso grupo, implementa um método de montagem progressiva dirigida por semente, o qual permite reconstruir sequências de DNA para montagem alvo-específicas partindo-se de sequências semente curtas de DNA ou proteína. Esse programa pode ser aplicado para a reconstrução de fragmentos genômicos, extracromossômicos e cDNAs, mas não é adequado para a reconstrução de sequências utilizando sementes e bases de dados derivadas de amostras heterólogas. O presente trabalho teve como objetivo o desenvolvimento do GenSeed-HMM, uma versão do GenSeed, capaz de utilizar HMMs de perfis como sementes para a reconstrução de sequências específicas, e de processar dados gerados pelas novas plataformas de sequenciamento, incluindo leituras curtas. Este trabalho relata a implementação do programa GenSeed-HMM, e sua validação utilizando dados reais de diferentes plataformas de sequenciamento, originados de procariotos, eucariotos, bem como de amostras metagenômicas. / The program GenSeed, previously described by our group, implements a seed-driven progressive assembly method for target-specific assembly of DNA sequences, starting from short DNA or protein seed sequences. The program can be applied for the reconstruction of genomic fragments, extrachromosomal genomes, and cDNAs, but is not adequate for sequence reconstruction using seed sequences and databases derived from heterologous samples. The present work aimed at developing GenSeed-HMM, a new version of GenSeed program that can use profile HMMs as seeds for the reconstruction of specific sequences, and incorporates the ability to work with data generated by the new sequencing platforms, including short reads. This work reports the implementation of GenSeed-HMM program and its validation using real life data produced by different next-generation sequencing platforms, and originated from prokaryotic, eukaryotic and metagenomic samples.

A coleção de fungos do Instituto de Medicina Tropical de São Paulo/USP compreende 1047 isolados de fungos, algas e actinomicetos de interesse médico e veterinário, que até o início deste estudo eram mantidos sob repiques sucessivos a cada três meses. Criada em 1925 pelo Prof. Dr. Floriano Paulo de Almeida e, a partir de 1953, mantida pelo Prof. Dr. Carlos da Silva Lacaz, a coleção necessitava de adequação de seu espaço físico e atualização nos métodos de preservação dos isolados. A reforma física foi realizada de acordo com as normas de biossegurança recomendadas pelo manual de procedimentos para manipulação de patógenos. Diversos estudos têm demonstrado que repiques sucessivos propiciam alterações nas características morfológicas, antigênicas, genéticas e de virulência de isolados fúngicos. Deste modo, foram aplicadas metodologias de preservação de longo prazo, como: liofilização, água destilada esterilizada, congelamento a -80ºC e em nitrogênio líquido (-196ºC). Foram selecionados 386 isolados, entre fungos dimórficos (213), filamentosos (hialinos e melanizados - 106) e leveduras (67), relacionados às infecções fúngicas mais prevalentes em nosso meio. Foram utilizados dois métodos de preservação para cada grupo de fungos: as leveduras foram submetidas à liofilização e congelamento em freezer -80ºC; e os fungos dimórficos e filamentosos preservados em água destilada esterilizada e no vapor de nitrogênio líquido. Esses isolados foram inicialmente avaliados quanto à caracterização morfológica e, após aplicação dos métodos de preservação, avaliou-se a viabilidade dos mesmos dentro de um período de dois anos, bem como a morfologia. As leveduras apresentaram 100% de viabilidade pelos dois métodos de preservação, mantendo suas características fenotípicas. Do mesmo modo, os dois métodos aplicados aos fungos filamentosos resultaram em elevados índices de viabilidade, com manutenção das características morfológicas. Em relação aos fungos dimórficos, a manutenção em nitrogênio líquido resultou em índices variáveis de viabilidade, dependendo do gênero avaliado. Do total de isolados do estudo, 75 foram submetidos também à identificação molecular por sequenciamento automatizado, após amplificação da região ITS do DNA ribossomal por PCR. A técnica molecular demonstrou, para alguns desses isolados, discordância na identificação das espécies quando comparada aos métodos fenotípicos. Os resultados deste estudo demonstraram que os repiques sucessivos não são recomendados para a preservação de fungos em coleções, por alterarem as características fenotípicas, bem como a capacidade de produzir esporos. As novas metodologias de preservação adotadas mantiveram a morfologia típica dos gêneros e espécies, permitindo ainda a viabilidade da maioria dos isolados estudados. A identificação molecular demonstrou ser importante ferramenta para autenticação dos isolados / The fungal culture collection from the Instituto de Medicina Tropical de São Paulo/USP comprises 1047 isolates of fungi, algae and actinomycetes with medical and veterinary relevance, which up to now have been preserved by subculturing every three months. Created in 1925 by Prof. Floriano Paulo de Almeida and maintained by Prof. Carlos da Silva Lacaz and his assistants since 1953, this fungal collection needed to be realocated to another area in 2007, as well as updated and revised as to its preservation methods. This new area was reformed according to safety procedures recommended in guidelines for pathogenic microorganisms manipulation. Several studies demonstrated that storage of fungi by subculturing may induce antigenic changes, phenotypic and genetic alterations, in addition to attenuation of virulence. The present study was aimed at utilizing more adequate methods of long-term preservation, such as lyophilization, sterile distilled water, and cryopreservation (freezing at -80°C and storage in liquid nitrogen [-196°C]). Three hundred eight six isolates consisting of dimorphic (213) and filamentous fungi (hyaline and melanized - 106), and yeasts (67) associated with the most prevalent fungal diseases in our setting were randomly selected. Two methods were applied for storage of each fungal group: yeasts were submitted to lyophilization and freezing at -80ºC; dimorphic and filamentous fungi were preserved in sterile distilled water and liquid nitrogen. All isolates were initially evaluated for their morphological features, submitted to the different methods of storage, and during a period of up to two years, reassessed for viability and morphological features. Yeasts showed 100% viability by the two applied preservation methods, maintaining their phenotypic characteristics. Similarly, the two methods applied to filamentous fungi resulted in high rates of viability, with maintenance of morphology. Regarding the dimorphic fungi, the preservation in liquid nitrogen resulted in variable rates of recovery, depending on the genus assessed. From all studied isolates, 75 were also submitted to molecular identification by sequencing after PCR amplification of the ITS region of the ribossomal DNA. This molecular technique showed, for some isolates, a lack of agreement in species identification when compared with the phenotypic methods. The new long-term storage methods used in the study preserved the typical genus and species morphology of the studied isolates, while resulting in high viability rates. The molecular identification proved to be an important tool for the authentication of isolates

Modification of DNA sequences and their suitable representation is important part of analysis, comparison and another processing. Goal of this paper is finding of suitable methods for representation of genomic and proteomic sequences. Because there is great number of metods, this paper will introduce only some of them. All selected methods, are described in the first part of this paper and they were programed in Matlab. Selected methods are illustrated on coding sequences of the first exon of the b-globin gene of 11 different species. Results are compared withresults from the original papers. Some methods are capable of another processing like cluster analysis. Output of this paper is comparison of results, gained from different methods, and finding the most suitable one.

This thesis discusses about possibilities of construction colour DNA spectrograms and about patterns which can be detected there. Spectrograms as tools of spectral analysis give us a simultaneous view of the local frequency throughout the nucleotide sequence. They are suitable for gene identification or gene regions identification, determination of global character about whole chromosomes and also give us a chance for the discovery of yet unknown regions of potential significance. For purpose of this kind of DNA analysis is possible to use digital signal processing methods. We can apply them on only after conversion of DNA sequence to numerical representation. Selection of correct numerical representation affects how well will be reflected biological features in numerical record which we need for another use in digital signal analysis.

The specific recognition of the DNA is crucial for the correct functioning of the cell. Although its mechanisms are extensively studied, the actual process is not yet fully understood, partly due to the variance observed in readout mechanisms so far. In this work, a particular type of specific recognition is examined: the shape readout in the DNA minor groove. Based on a sta- tistical analysis of three-dimensional structures of protein-DNA complexes acquired from the Protein Data Bank, I propose a previously unrecorded readout mechanism of widened minor grooves by hydrophobic amino acids. In addition, the effect of DNA sequence on the topography of the contacted locus, the preferred secondary structures and the interaction between the protein and DNA are explored, as well as the relative information amount of examined features concerning the DNA deformation. 1

Aim Morinda citrifolia L., commercially known as noni or the Indian mulberry plant, is morphologically variable and the only widely distributed member of the pantropical genus Morinda sensu stricto (Rubiaceae). This large distribution has been attributed partly to the ability of the seeds of the large-fruited M. citrifolia L. var. citrifolia L. to be transported by oceanic drifting. This form of M. citrifolia var. citrifolia has been predicted to be the progenitor colonizer of the island endemic Morinda species. Using a phylogenetic approach and large sampling of the widespread, large-fruited M. citrifolia var. citrifolia, we assessed the potential area of origin of M. citrifolia and tested the hypothesis that the large-fruited M. citrifolia var. citrifolia is an ancestral colonizer. Location Tropics. Methods We performed Bayesian analyses of 22 species of the tribe Morindeae (including 11 individuals of the three currently recognized varieties of M. citrifolia) based on combined nrETS, nrITS, rps16 and trnT-F sequence data. Geographic origins of the studied taxa were mapped onto the Bayesian majority rule consensus tree. Results Nine sequenced individuals of M. citrifolia from diverse geographic locations formed a highly supported clade, which was sister to the Australo-Micronesian clade that included M. bracteata var. celebica and M. latibracteata. These sister clades are part of the broader Asian, arborescent Morinda clade. We found no support for the current varietal classification of M. citrifolia. Main conclusions Our analyses suggest a Micronesian origin of M. citrifolia. This implies that the large-fruited M. citrifolia var. citrifolia might well have been present in the Pacific before the arrival of the Micronesian and Polynesian ancestors from Southeast Asia. The wide distribution of this form of M. citrifolia var. citrifolia is attributed partly to the trans-oceanic dispersal of its buoyant seeds, self-pollination and its ability to produce flowers and fruits year-round. The hypothesis that the widespread, large-fruited M. citrifolia var. citrifolia is the progenitor colonizer of the island endemic Morinda species is inconsistent with its derived position within the Asian, arborescent Morinda clade and with the fact that the nine sampled individuals of M. citrifolia form a clade.

No description available.

DNA is the substance that encodes the genetic information that cells need to replicate and to produce proteins. The detection of DNA sequences is of great importance in a broad range of areas including genetics, pathology, criminology, pharmacogenetics, public health, food safety, civil defense, and environmental monitoring. However, the established techniques suffer from a number of problems such as the bulky size, high equipment costs, and time-consuming algorithms so that they are limited to research laboratories and cannot be applied for in-vivo situations. In our research, we developed a novel sensing scheme for DNA sequence detection, featuring sequence specificity, cost efficiency, speed, and ease of use. Without the need for labels or indicators, it may be ideal for direct in-cell application. The principle is simple. With capture DNA immobilized onto the probe by layer-by-layer selfassembly, the hybridization of a complementary strand of target DNA increases the optical thickness of the probe. Three kinds of sensors were developed. The optical fiber tip sensor has been demonstrated with good specificity and high sensitivity for target DNA quantities as small as 1.7 ng. To demonstrate the potential of this structure for practical applications, tularemia bacteria were tested. Two other micrometric structures were designed with specific advantages for different applications. The micro-fiber Bragg grating interferometer (Micro-FBGI) has the intrinsic temperature compensation capability. The micro-intrinsic Fabry-Perot interferometer (Micro-IFPI)features simple signal processing due to its simple configuration. Successful DNA immobilization and hybridization have been demonstrated onto the 25μm Micro-IFPI. Both structures have great potential for nanometric protrusion, allowing future in-cell DNA direct detection. In addition, its quick response time leads to the potential for express diagnosis. What's more, the idea of nanoscale probe has a broad impact in scanning near-field optical microscopy (SNOM), intracellular surgery in cell sensing, manipulation, and injection. / Ph. D.

O vírus do mosaico amarelo da abobrinha (Zucchini yellow mosaic virus - ZYMV) é provavelmente um dos mais dinâmicos e prejudiciais vírus emergentes de plantas. Desde sua primeira detecção na Itália e na França em 1973 e 1979, respectivamente, o ZYMV tem sido reportado em mais de 50 países variando de condições climáticas tropicais a temperadas em todos os continentes, exceto na Antártida. No Brasil esse potyvirus foi constatado primeiramente nos Estados de São Paulo e Santa Catarina, no início da década de 90 e posteriormente nos Estados do Ceará, da Bahia, do Rio Grande do Norte, do Pará, do Mato Grosso do Sul e do Maranhão. É provável que atualmente ocorra em todos os estados brasileiros onde se cultivam cucurbitáceas. Embora ocorra no Brasil há mais de 20 anos, pouco se conhece sobre as características biológicas, sorológicas e moleculares dos isolados até então encontrados no país. Esse projeto de pesquisa teve por objetivo cobrir parcialmente essa lacuna para fornecer subsídios para programas de melhoramento genético. Foram utilizados 11 isolados coletados em diversos estados brasileiros. A caracterização biológica consistiu no estudo da reação de diversas espécies vegetais inoculadas mecanicamente. A caracterização sorológico consistiu na marcação da proteína capsidial dos isolados com o antissoro policlonal contra o vírus. A caracterização molecular foi feita por meio da análise das sequências de nucleotídeos e aminoácidos deduzidos do gene da proteína capsidial dos isolados. A reação das espécies variou de acordo com o isolado inoculado. Análises de RFLP mostraram que a enzima de restrição Hpa II permitiu diferenciar o isolado fraco ZYMV-M da maioria dos isolados severos. O peso molecular da proteína capsidial dos isolados analisados foi em torno de 36 KDa, típico da proteína capsidial desse potyvirus. A identidade de nucleotídeos do gene da proteína capsidial entre os isolados brasileiros do ZYMV variou de 93% a 100% e a de aminoácidos deduzidos variou de 97% a 100%. Comparados com as sequências correspondentes de isolados do ZYMV de diferentes origens geográficas a identidade de nucleotídeos variou de 82% a 99%, enquanto a de aminoácidos deduzidos ficou entre 87% e 99%. Na árvore filogenética os isolados brasileiros do ZYMV pertencem ao grupo A, subdivisões I ou II, indicando uma variação entre os isolados. Nenhum evento de recombinação foi detectado nos isolados brasileiros. / Zucchini yellow mosaic virus (ZYMV) is probably one of the most dynamics and economically important emerging plant viruses. Since it was first report in Italy and France in 1973 and 1979, respectively, ZYMV has been found in over than 50 countries ranging from tropical to temperate climate conditions in all continents, except in Antartida. In Brazil this potyvirus was first reported in the beginning of 90´s in São Paulo and Santa Catarina States, and then in Ceará, Bahia, Rio Grande do Norte, Pará, Mato Grosso do Sul and Maranhão states. Probably it occurs in all Brazilian states which grow cucurbit species. Although ZYMV occurs in Brazil for more than 20 years, there is little information about the biological, serological and molecular characteristics for the isolates found in the country. The purpose of this work was to cover partially this gap to provide subsidies for genetic breeding programs. Eleven isolates collected in several Brazilian states were used. The biological characterization consisted in the study of the reaction of several vegetal species mechanically inoculated with the virus. The serological characterization consisted in the identification of the molecular weight of the coat protein through western blot analysis. The molecular characterization was done by the analyses of nucleotides and deduced amino acids sequences for the coat protein gene. The host reaction showed slight variation according to the virus. RFLP analyses showed that restriction enzyme HpaII allowed differentiate the mild ZYMV-M isolate from the majority of severe isolates. The coat protein molecular weight for all studied isolates was about 36 KDa, typical of the coat protein of this potyvirus. The nucleotide identity of the coat protein gene among the ZYMV Brazilian isolates ranged from 93% to 100%, and the deduced amino acids sequences ranged from 97% to 100%. Compared to corresponding sequences of ZYMV isolates from different geographical locations the nucleotide sequences identity ranged from 82% to 99%, while the deduced amino acids sequences ranged from 87% to 99%. Phylogenetic analysis place the Brazilian isolates of ZYMV into the group A, subdivision I or II, showing some diversity between the isolates. No recombination event was detected in the Brazilian isolates.