Made available in DSpace on 2015-04-20T12:31:12Z (GMT). No. of bitstreams: 1 Alexander Sibajev.pdf: 606940 bytes, checksum: 9c6a8d6a72194ece920cce39baa6aa55 (MD5) Previous issue date: 2005-12-14 / The determination of the prevailing species responsible for American Tegumentary Leishmaniasis (ATL) and the detection at species level of the varieties of the protozoan parasite agent of this endemic disease at the Amazon region are important for determination of the clinical evolution and to better indicate the therapeutic conduct due to the different responses to chemotherapeutic drugs by the Leishmania species. By now we are not aware of an available test to Leishmania (Viannia) guyanensis capable of detecting its intra-specific varieties obtained from human clinical presentations or from vertebrate and invertebrate hosts. We succeed to discriminate Leishmania (V.) guyanensis from Leishmania (V.) braziliensis and also from Leishmania (V.) panamensis through a polimerase chain reaction direct genomic DNA amplification aimed at the transcribed internal spacer (ITS) of the ribosomal RNA gene (rDNA). The positive reaction is visualized at the agarose gel electrophoresis by the presence of an aproximatedly 229 nucleotides size band for L. guyanensis that is absent for the two other species L. braziliensis and L. panamensis. The diagnosis can be done using culture material, skin biopsy or squashing the sand fly vector and having the DNA extracted. In a second step of the investigation, the ITS rDNA was sequenced in L. lainsoni; L. naiffi and four L. guyanensis strains, two of them presenting a muco-cutaneous form of Leishmaniasis. These sequences were compared to others available at the Genbank Database, confirming the previous data from multilocus enzyme electrophoresis and ITS restriction fragment lenght polymorphisms analysis, positioning L. lainsoni and L. naiffi as more divergent species as compared to L. braziliensis, L. panamensis and L. guyanensis species inside de Viannia sub-genus. A correlation was observed in grouping nearer the two L. guyanensis strains, respectively IM4243 and IM4235, responsible for the muco-cutaneous form. This protozoan belonging to sub-genus Viannia show clinical and epidemiological importance in South America north region, particularly the Amazon region and this study can be considered an advance in providing a tool to better understand the parasite species silvatic cycle and in providing tools to characterize the main species responsible for the clinical form presentation of tegumentary leishmaniasis in the human population at this region. / A determinação da espécie responsável pela Leishmaniose Tegumentar Americana e a detecção específica das variedades do protozoário agente dessa doença endêmica na Amazônia, é importante para determinação do comportamento clínico e indicação da conduta terapêutica, devido as diversas respostas aos quimioterápicos pelas espécies de Leishmania. No momento se desconhece um teste disponível para a Leishmania (Viannia) guyanensis, que seja capaz também de incluir suas variedades intra-específicas obtidas de casos humanos e de hospedeiros e vetores silvestres. Neste trabalho se conseguiu discriminar alguns isolados de Leishmania (Viannia) guyanensis, de Leishmania (Viannia) braziliensis e também de Leishmania (Viannia) panamensis através da reação em cadeia da polimerase direcionada para a amplificação do espaçador transcrito interno (ITS) do gene do RNA ribossomal. A reação positiva é dada pela visualização de um fragmento amplificado de cerca de 229 nucleotídeos, para L. (V.) guyanensis e ausente para as outras duas espécies. O diagnóstico pode ser feito com o DNA extraído de cultivo do parasita, diretamente das amastigotas da borda de lesão cutânea ulcerada ou do vetor infectado pela promastigota de Leishmania. Numa etapa seguinte a região ITS do rDNA foi sequenciada para L. lainsoni, L. naiffi e quatro cepas de L. guyanensis em que duas apresentavam forma muco-cutânea e duas outras a forma cutânea da leishmaniose. Essas sequências foram comparadas com as disponíveis em banco de dados como o Genbank, resultando na confirmação dos dados obtidos de variabilidade genética por outros autores, quando utilizada a técnica de isoenzimas e ITS-RFLP. O posicionamento de L. lainsoni e L. naiffi se mostrou mais divergente que L. guyanensis, L. braziliensis e L. panamensis, dentro do subgênero Viannia. Foi também encontrada uma relação de proximidade entre os isolados de L. guyanensis IM4243 e IM4235 apresentando lesão cutâneo-mucosa. As leishmânias do sub-gênero Viannia apresentam importância epidemiológica na América do Sul, sobretudo na região amazônica e esse estudo poder ser considerado importante ao desenvolver meios de diagnóstico específico do parasita, detectando sua presença em vetores e reservatórios silvestres em certas áreas e servindo como marcador para a principal espécie responsável pelas formas clínicas de leishmaniose nessa região.

O I. multifiliis é um protozoário ciliado que acomete diferentes espécies de peixes, causador da ictiofitiriase, responsável por altas taxas de mortalidade em pisciculturas e resultando em perdas econômicas. O presente estudo avaliou isolados do parasito oriundos de peixes capturados em seis estados brasileiros, utilizando como marcadores moleculares ribossomais os genes 18S, 5.8S e 28S e os espaçadores intergênicos ITS1 e ITS2 de I. multifiliis, obtidos através da Reação em Cadeia da Polimerase (PCR) e posteriormente, sequenciados. Primers específicos foram desenhados para este estudo. As sequências dos isolados de I. multifiliis encontrados infectando diferentes hospedeiros em diferentes estados brasileiros apresentaram grande similaridade e homologia, sendo muito conservadas e, por este fato, não permitem diferenciação entre isolados deste parasito. Sequências do DNA de isolados de I. multifiliis de diferentes regiões brasileiras foram depositadas no GenBank e representam as primeiras informações para esta espécie no Brasil. / The I. multifiliis is a ciliate protozoan that affects fishes of different species, which causes Ichthyophthiriasis. This disease is responsible for high mortality in fish farms resulting in economic losses. The present study evaluated parasite isolates from four Brazilian states, using nuclear ribosomal molecular makers for 18S,5.8S and 28S genes and the first and second internal transcribed ITS1 and ITS2 intergenic spacers from I. multifiliis. All the markers were evaluated through Polymerase Chain Reaction (PCR) and rDNA sequenced, using novel specific primers; the sequences of different isolated of I. multifiliis, which were collected from different fish species (host) from different Brazilian regions showed high similarity and homology, which were extremely conserved therefore it was not possible to differentiate among the strains from this parasite. All recovered sequences from this study were deposited in GenBank database. These sequences represent a novel contribution for Brazilian isolate of I. multifiliis.

Os organismos do filo Myxozoa são endoparasitas obrigatórios que infectam principalmente peixes em diversas regiões do mundo, estando entre os mais importantes patógenos de peixes, com mais de 2180 espécies descritas porém, pouco se conhece deste parasita em peixes do Brasil. Neste estudo foram realizadas coletas entre abril de 2011 a agosto de 2012, onde foram coletados oito exemplares de piapara (Leporinus obtusidens) e dezessete exemplares de dourado (Salminus brasiliensis), oriundos do rio Mogi-Guaçu, próximo a Cachoeira de Emas, localizada no município de Pirassununga, estado de São Paulo. As análises morfológicas (microscopia de luz, histologia e a análise ultraestrutural), técnicas de biologia molecular (PCR e sequenciamento) foram utilizadas na identificação de duas novas espécies de Henneguya e análise filogenética do gene 18S rDNA foi realizada para avaliar a relação filogenética desta duas novas espécies com outras espécies de mixosporídeos. Duas novas espécies foram encontradas neste estudo. Uma delas foi encontrada infectando a nadadeira de L. obtusidens e é descrita neste estudo como Henneguya sp. 1 e uma outra espécie encontrada infectando a membrana do arco da branquial e na membrana entre os raios da nadadeira de S. brasiliensis, a qual foi aqui descrita como Henneguya sp. 2. A análise histopatológica revelou que Henneguya sp. 1 ocasionou uma leve compressão no tecido adjacente. A análise ultra-estrutural permitiu a compreensão da interface parasita-hospedeiro e o processo de esporogênese das duas espécies descritas; além disso, foi observado a presença de vesículas esbranquiçadas para Henneguya sp. 2, com função ainda desconhecida. Nos estudos filogenéticos, utilizando os métodos máxima parcimônia e máxima verossimilhança para ambas as espécies, foi avaliado no estudo 1 que Henneguya sp. 1, parasita de peixe characiforme, aparece isolado dando origem a um clado irmão de Henneguya spp., parasitas de siluriformes, characifomes e esociformes; já no estudo 2, a análise filogenética da espécie Henneguya sp. 2, também parasita de peixe characiforme, foi observado a formação de um clado com 3 espécies parasitas de characiformes, sendo duas espécies de Henneguya descritas neste estudo e o Myxobolus oliveirai. / The parasites of Myxozoa are endoparasites that infect mainly fishes in several parts of the world, being this group considered one of the most important pathogens of fishes, with more than 2180 species described. However little is known about this group of parasite in Brazil. During this study, eight piapara (Leporinus obtusidens) and seventeen dourado (Salminus brasiliensis) were caught in Mogi-Guaçu river in a period from April 2011 to August 2012, near waterfall Cachoeira de Emas located at Pirassununga city, state of São Paulo, Brazil. Morphological analyses (light microscopy, histology and ultrastructure) and molecular biology techniques (PCR and sequencing) where done to identify two new species of Henneguya and phylogenetic analyses using the 18S rDNA gene were performed to evaluate the phylogenetic relationship of this two new species among others myxosporeans available on GenBank. Henneguya sp. 1 has been described infecting the fin of L. obtusidens and Henneguya sp. 2 has been described infecting the gill arch membrane and the fin membrane of S. brasiliensis. The histopathological analyses revealed that Henneguya sp. 1 occasioned a small compression of adjacent tissue of the host. Ultraestructural analyses showed the host-parasite\'s interface and the sporogenesis of the two Henneguya spp. furthermore Henneguya sp. 2 showed several whitish vesicles with an unknown function. Phylogenetic studies using the maximum parsimony and maximum likelihood methods, showed in the first study Henneguya sp .1 appearing alone as a basal branch of a sister clade composed by Henneguya parasites of siluriforms, characiforms and esociforms fishes, and the second study showed Henneguya sp. 2 grouped with Hennegua sp. 1 and Myxobolus oliveirai, both parasites of characiforms fishes.

O filo Myxozoa compreende organismos parasitários que infectam vertebrados, principalmente peixes, em diversas regiões do Brasil e do mundo. Com mais de 2180 espécies já descritas os mixosporídeos estão entre os patógenos mais importantes que infectam peixes tanto de ambiente natural como de sistemas de criação. Neste estudo, foram realizadas coletas no ano de 2012, onde foram capturados 13 espécimes de piraputanga (Brycon hilarii) e 14 espécimes de piauçu (Leporinus macrocephalus) oriundos de pisciculturas do estado de São Paulo. Foram encontrados mixosporídeos infectando o rim de B. hilarii e o filamento branquial de L. macrocephalus. Para a identificação das espécies de parasitos, foi utilizada a microscopia de luz para a análise morfológica e, também, histopatológica para a observação das alterações decorrentes do parasitismo. A análise ultraestrutural foi realizada para verificar a interação parasita-hospedeiro do mixosporídeo encontrado na piraputanga. A análise filogenética do gene 18S rDNA avaliou a relação entre as espécies identificadas com outras espécies de mixosporídeos já descritas. Uma nova espécie de Myxobolus foi descrita neste estudo infectando o rim de B. hilarii e a caracterização molecular e filogenética de Henneguya leporinicola, encontrado infectando o filamento branquial de L. macrocephalus, foi realizada. No estudo filogenético, utilizando o método de Máxima Verossimilhança para ambas as espécies, verificou-se que as espécies de mixosporídeos se agruparam primeiro de acordo com a ordem do hospedeiro Characiforme e pelo tropismo tecidual. A análise molecular demonstrou diferenças genéticas significativas em comparação com outras espécies já descritas na América do Sul e em outros países. / The Myxozoa phylum comprises parasitic organisms that infect vertebrates, mainly fish, in many areas from Brazil and from the world. With more than 2180 species already described, myxosporeans are among the most important fish pathogens which infect both natural environment and breeding systems. In this study, captures were made in 2012, where 13 specimens of piraputanga (Brycon hilarii) and 14 specimens of piauçu (Leporinus macrocephalus) proceeding from fish farms in the state of Sao Paulo were capured. Myxosporeans were found infecting the kidney of B. hilarii and the gill filament of L. Macrocephalus. For the identification of parasite species, it was used light microscopy for morphological and histopathological analyzes to observe the damage caused by parasitism. The ultrastructural analysis was performed to verify the host-parasite myxosporean interaction found on piraputanga. The phylogenetic analysis of 18S rDNA evaluated the relation between the species identified with other myxosporean species already described. A new Myxobolus species was described in this study infecting the kidney of B. hilarii and molecular characterization and phylogenetic of Henneguya leporinicola, infecting the gill filament of L. macrocephalus, was performed. In phylogenetic study, using the method of Maximum Likelihood for both species, it was observed that the species of myxosporean grouped according to the order of the host Characiform and the tissue tropism. Molecular analysis showed significant genetic differences with other described species in South America and other countries.

O gênero Crotalaria possui aproximadamente 600 espécies descritas, localizadas principalmente nas regiões tropicais e subtropicais. Estas espécies estão classificadas em oito seções e nove subseções botânicas definidas principalmente com base em caracteres florais. Estas seções botânicas podem ser subdivididas em dois grupos principais: um de espécies com flores especializadas e outro de espécies sem especialização das flores. Estudos citogenéticos anteriores mostraram que as características cromossômicas são conservadas dentro de uma mesma seção ou subseção botânica e foi proposto que as espécies com especialização das flores teriam uma organização cromossômica diferente das espécies sem especialização das flores. Dentro deste contexto, foram descritos os cariótipos e a organização do genoma de três espécies e duas sinonímias, da seção Hedriocarpae, subseção Macrostachyae, com a utilização de coloração convencional dos cromossomos pelo método de Feulgen, de coloração com fluorocromos específicos às regiões cromossômicas ricas em GC ou AT e mapeamento físico por hibridação molecular in situ fluorescente (FISH) dos locos de DNA ribossômicos 45S e 5S. A obtenção de marcadores cromossômicos característicos para esta subseção e a construção de mapas citogenéticos possibilitaram a identificação de uma inversão pericêntrica, de eventos de transposição e da diferenciação na natureza das seqüências de DNA da heterocromatina pericentromérica, previamente descrita em espécies com flores especializadas como ricas em GC. Estes resultados revelaram alguns dos eventos citogenéticos ocorridos durante a diversificação do gênero, sendo que a subseção Macrostachyae é caracterizada por uma inversão no cromossomo 1 que pode ter uma origem antiga no gênero e que espécies sem especialização das flores, de fato possuem uma organização cromossômica distinta das espécies com flores especializadas, principalmente quanto à natureza das seqüências de DNA da heterocromatina pericentromérica. / Approximately 600 species of the Genus Crotalaria have been described, the majority is located in tropical and subtropical regions. These species are classified into eight botanical sections and nine subsections according to floral characteristics. The botanical sections can be divided into two major groups: one with species presenting flower specialization and the other without flower specialization. Previous cytogenetic studies showed that chromosomal features are conserved within the same botanical section or subsection, and it has been suggested that species with flower specialization have a different chromosomal organization from those species without flower specialization. In this context, the karyotypes and genome organization of five species (being three synonymy from the Hedriocarpae section, Macrostachyae subsection) were described by conventional chromosome staining using Feulgen, specific fluorochrome staining to GC or AT chromosomal rich regions and physical mapping by fluorescent in situ molecular hybridization of 45S and 5S ribosomal genes. The mapping of specific chromosome markers and construction of cytogenetic maps of this section have allowed us to identify a pericentric inversion, transposition events and a differentiation in the nature of DNA sequences of the pericentromeric heterochromatin, previously described as GC-rich in species with flower specialization. The results reveal diverse cytogenetic events that occurred during the genus diversification: the Macrostachyae subsection showed a pericentric inversion in the chromosome pair 1 (presumably of ancient origin) while species without flower specialization are characterized by a distinct chromosomal organization, mainly concerning the nature of DNA sequences of pericentromeric heterochromatin.

No description available.

L'ARNr 5S est une partie intégrante du ribosome indispensable pour la synthèse des protéines. Cette fonction essentielle nécessite une régulation fine de l'expression des ARNr 5S en fonction des exigences cellulaires. L'ARNr 5S est codé par des gènes organisés en répétitions en tandem principalement situés dans les régions péricentromériques des chromosomes 3, 4 et 5 dans le génome Col-0 d'Arabidopsis thaliana. L’étude approfondie de l’organisation, la dynamique et la régulation épigénétique des gènes d’ARNr 5S reste cependant difficile du fait de l’absence d’assemblage de ces régions hautement répétées dans la séquence du génome d’Arabidopsis disponible dans TAIR10.Dans cette thèse, nous apportons de nouvelles informations sur les gènes d'ARNr 5S chez Arabidopsis. Nous avons confirmé les signatures d'ADN spécifiques par séquençage haut débit, identifié des polymorphismes spécifiques des régions chromosomiques portant des ADNr 5S et ainsi défini de nouvelles séquences de référence. Nous avons également étudié le statut épigénétique des gènes ARNr 5S, montrant que ces gènes sont globalement enrichis en marques répressives. Les loci situés sur les chromosomes 4 et 5 présentent également des modifications post-traductionnelles d’histones et des variants d’histone particuliers et caractéristiques d’une activité de transcription. Nous avons aussi montré que les loci d'ADNr 5S sont très dynamiques au sein de l'espèce Arabidopsis avec des variations entre différent écotypes du nombre de copies de gènes d’ARNr 5S et de l’importance relative de chaque cluster. En utilisant l'écotype Ler et des mutants spécifiques, nous montrons que le locus du chromosome 5 est une source majeure de réarrangements chromosomiques qui affectent l'organisation de la chromatine dans le noyau. Enfin, nous suggérons que des variations de l’état chromatinien des gènes d'ARNr 5S peuvent conduire à une différence de transcription dans les différents écotypes.Cette étude permet d’établir un rôle de l'organisation de la chromatine dans la régulation transcriptionnelle et l'organisation des gènes d'ARNr 5S. / 5S rRNA is an integral component of the ribosome and is essential for protein synthesis. 5S rRNA expression is therefore tightly regulated to suit the cellular requirements. 5S rRNA is encoded by gene arrays organized in tandem repeats mainly situated in the pericentromeric regions of chromosomes 3, 4 and 5 in the Arabidopsis thaliana Col-0 genome. Full genome assembly remains challenging in these highly repeated regions and impedes further investigation of their organization, dynamics and epigenetic regulation.In this thesis, we provide information on 5S rRNA genes in Arabidopsis thaliana. We confirmed specific DNA signatures by deep sequencing and define chromosome-specific polymorphisms and new reference sequences. We also studied the epigenetic status of the 5S rRNA genes showing that these genes are enriched in repressive marks whereas the loci on chromosome 4 and 5 also display peculiar histone modifications and variants characteristic of transcriptional activity. We report that 5S rDNA loci are highly dynamic within the Arabidopsis species with high level of variation in global copy number and cluster proportion between ecotypes. Using the Ler ecotype, in which reorganization events were recorded, and specific mutant backgrounds, we identified chromosome 5 rDNA locus as a major source of variation and observed altered chromatin organization in nuclear space as a consequence of 5S rDNA locus variation. Finally, we suggest that differences in chromatin states at the two main 5S rDNA loci can lead to differential usage of 5S rRNA genes indifferent ecotypes.The analysis provides evidence for a role of chromatin organization in transcriptional regulation and 5S rDNA organization.

A phylogenetic tree based on ITS1, Adh1 and ndhF grouped the species of the genus Sorghum into one distinct monophyletic group, but including two sister lineages, one with x=5, the other with x=10 as basic chromosome numbers. The goal of this study was to elucidate major patterns in Sorghum genome evolution, particularly n=5 vs. n=10 genomes. A very recent molecular cytogenetic study in our laboratory revealed striking structural karyotypic rearrangements between S. bicolor (x=10) and an x=5 Sorghum species, S. angustum; so an immediate objective here was to determine if identical or similar rearrangements exist in other wild Sorghum species. Our approach was [1] to extend similar methods to additional species, i.e., fluorescent in situ hybridization (FISH) analyses of sorghum genomic bacterial artificial chromosome clones and multi-BAC cocktail probes to mitotic chromosomes of S. angustum, S. versicolor, S. brachypodum and S. intrans; and [2] to augment the BAC-FISH findings by comparing telomeric and ribosomal DNA FISH signal distributions to x=5 and x=10 Sorghum species. Signals from in situ hybridizations of BAC-based probes were insufficiently robust and insufficiently localized to delineate FISH signal patterns akin to those discovered previously in S. angustum. Southern blots of the same BACs to restricted DNA of these species revealed relatively moderate affinity to smeared DNA, suggesting homology to non-tandemized sequences. FISH of the A-type TRS (Arabidopsis-like telomeric repeat sequence) revealed its presence is limited to terminal chromosomal regions of the Sorghum species tested, except S. brachypodum, which displayed intercalary signal on one chromosome and no detachable signal at its termini region. The hybridization of 45S and 5S rDNA revealed that the respective sites of tandemized clusters differ among species in terms of size, number and location, except S. angustum versus S. versicolor. Well localized BAC-FISH signals normally occur when signals from low-copy sequences discernibly exceed background signal, including those from hybridization of dispersed repetitive elements. The low level of signal intensity from BAC low-copy sequences relative to the background signal "noise" seems most likely due to low homology and(or) technical constraints. Extensive dispersal of low-copy sequences that are syntenic in S. bicolor seems unlikely, but possible. In conclusion, the result was a lack of clear experimental success with BAC-FISH and an inability to effectively screen for S. angustum-like rearrangements using BAC-FISH. The telomeric and rDNA FISH indicated that the x=5 genomes vary extensively. One can surmise that although the arrangements seen in S. angustum might extend to S. versicolor, they certainly do not extend to S. versicolor, they certainly do not extend to S. intrans or S. brachypodum. It is clear that S. brachypodum has telomeric repeats that are either very short or rely on some sequence other than the A-type TRS.

Published Article / An artificial neural network (ANN) that is able to distinguish between Cheddar cheese produced with milk from mixed and single breed sources was designed. Samples of each batch (4 pure Ayrshire/4 mixed with no Ayrshire milk) were ripened for 92 days and analysed every 14 days. A novel ANN was designed and applied which, based only on Lactococcus lactis counts, provided an acceptable classification of the cheeses. The ANN consisted of a multi-layered network with supervised training arranged in an ordered hierarchy of layers, in which connections were allowed only between nodes in immediately adjacent layers.

This master's thesis is focused on cytogenetic analysis and karyotype variability of the Nemastomatidae family. This family comprises morphologically uniform harvestmen of small sizes, with low mobility, and with center of distribution in Europe. Karyotype differences could play an important role for detection of cryptic diversity in this family. The karyotype analysis is focused mainly on Alpine and Pyrenean endemic species but also on other taxons located in Central Europe. The goal was not only to identify the differences which occur between the specific genera and species, but also eventually between populations. For detection of the specific chromosomal alterations in evolution of the karyotype in Nemastomatidae the fluorescent in situ hybridization (FISH) was used, localizing the positions and amounts of gene clusters for 18S rRNA. From the results we can assume that the number of chromosomes in the family Nemastomatidae can range between 2n = 12- 30. Further it was found out that in Nemastomatidae the biarmed chromosomes are more prevalent, and that the species which have lower amounts of chromosomes contain chromosomes that noticeably differ in size (probably because of chromosomal fusions). These results indicate that with some morphologically uniform species, the knowledge of specific...