Os peixes apresentam uma grande diversidade quanto a sua morfologia, seus habitats e também sua biologia. São encontrados em lagos, córregos, estuários e oceanos, constituindo assim mais de 50% do número total das espécies de vertebrados conhecidas atualmente. Essa fauna tem sido objeto de um número expressivo de estudos citogenéticos e moleculares, tendo-se já conhecimento não só das relações cromossômicas, mas também da sistemática de vários grupos. Essas pesquisas têm investigado não somente o número e fórmula cromossômica, mas também a presença de cromossomos sexuais diferenciados, presença de cromossomos supranumerários, padrões de distribuição da heterocromatina, localização das regiões organizadoras de nucléolo, padrões de bandamento de restrição e replicação, permitindo a localização de diferentes classes de DNAs repetitivos, bem como a identificação de homeologias cromossômicas que auxiliam a compreensão da evolução cariotípica dos grupos. Os estudos moleculares, por sua vez, têm se tornado cada vez mais importantes nesse grupo e têm fornecido dados fundamentais não só no que diz respeito à filogenia dos grupos, como também em relação a regiões repetitivas do DNA e sua importância no genoma. A união dessa área com a Citogenética tem permitido uma maior e melhor compreensão sobre os processos evolutivos associados às alterações de seqüências específicas do genoma visíveis tanto a níveis cromossômicos, quanto moleculares. O gênero Gymnotus (Teleostei: Gymnotiformes) inclui representantes com características biológicas peculiares, o que os torna objeto de estudo de diversas áreas da Biologia. Estudos sobre o gênero incluem sua caracterização cariotípica, estudo das regiões organizadoras de nucléolo (RONs) polimórficas, bem como estudos envolvendo marcadores moleculares, os quais conjuntamente com a Citogenética permitiram a análise de filogenética molecular, com inferência na evolução cromossômica, permitindo uma melhor compreensão das relações dentro do gênero. No presente trabalho foram levados a efeito estudos sobre as regiões heterocromáticas e os DNAs repetitivos desse grupo, para uma melhor compreensão da organização e localização dessas seqüências no genoma e a identificação de possíveis marcadores moleculares. Foram efetuados ainda, estudos envolvendo a evolução cariotípica das espécies G. carapo e G. sylvius, localização de genes ribossômicos e análise molecular do gene ribossômico 5S juntamente com seu espaçador não transcrito, propiciando uma melhor compreensão da evolução dessa família gênica em Gymnotus. / Fishes present a great diversity in relation to their morphology, habitat and biology. They are found in lakes, rivers, estuaries and oceans, comprising more than 50% of the total number of known vertebrates. Cytogenetic and molecular aspects of the fish fauna have been extensively studied, providing information about their chromosomal relationships and also about the systematic status of several groups. These researches have focused on the description of both chromosomal number and formula as well as the presence of differentiated sex chromosomes, occurrence of B-chromosomes, patterns of heterochromatin distribution, localization of nucleolar organizer regions, restriction or replication banding profiles allowing to locate distinct classes of repetitive DNAs and to identify chromosomal homeologies in order to understand the karyotypic evolution in distinct groups. On the other hand, molecular studies have become of utmost importance in this group, providing essential data about phylogeny of many groups and about repetitive DNA regions and their role in the genome. The union between this approach and cytogenetics has favored a better comprehension about the evolutionary processes associated with visible alterations in specific sequences within the genome at both chromosomal and molecular levels. The genus Gymnotus is composed of representatives with peculiar biological features, which turn them suitable for studies in a variety of biology approaches. Genetic studies in this genus comprise karyotype characterization, analysis of polymorphic NORs, besides studies of molecular markers that, coupled with cytogenetics, have fostered molecular phylogenetic analyzes with inferences on their chromosomal evolution, which have led to a better understanding about the interrelationships in the group. In the present work, we carried out studies about the heterochromatic regions and the repetitive DNAs in this group for a better comprehension about the organization and localization of these sequences in the genome and identification of potential molecular markers. Furthermore, studies related to the karyotype evolution in the species G. carapo and G. sylvius, location of ribosomal genes and molecular analysis of both 5S ribosomal gene and its non-transcribed spacer were performed to provide a better comprehension about the evolution of this gene family in Gymnotus.

O estudo sobre a cinética de incorporação de uridina em ácidos nucleicos permitiu estabelecer que o tamanho do \"pool\" de precursores permanece constante na presença de concentrações de uridina exógena acima de aproximadamente1 µM. Concluiu-se ainda que todo o sistema de tomada de uridina, medido pela sua incorporação em ácidos nucleicos, apresenta um Km de 5,1 µM e opera a uma velocidade máxima de 11 pmoles/min/l,3 x 107 células. Um estudo análogo, em presença de rifampicina, possibilitou calcular que a meia vida de RNAs mensa - geiros em B. subtilis é de 2 minutos e que 44% da radiatividade incorporada em RNA num determinado instante se encontra na fração de RNA estável (ribossômico e de transferência). A análise do mecanismo de transcrição dos genes para RNA ribossômico foi abordada por meio do estudo do alongamento de cadeias de rRNA já iniciadas, em presença de rifampicina. As relações iniciais de radiatividade incorporada em rRNA 16S e 23S, quando rifampicina e uridina tritiada são adicionadas concomitantemente, bem corno a cinética de decaimento de marcação presente em ambas as espécies de rRNA sugerem que os genes para RNA ribossômico 16S e 23S são cotranscritos nessa ordem, em B. subtilis. Levando-se em consideração essas e outras evidências, propõe-se o seguinte relacionamento estrutural para a unidade de transcrição: [Obs.: Ver no arquivo em PDF] Os resultados experimentais foram comparados com modelos teóricos descritos no Apêndice. A existência de um mecanismo de cotranscrição para os genes responsáveis pela síntese de rRNA em procariotos e eucariotos parece sugerir que esse mecanismo, de fundamental importância, instalou-se precocemente e foi mantido durante a evolução. / The kinetics of uridine incorporation into nucleic acids has shown that the precursor pool size remains constant in the presence of exogenous uridine concentrations above 1 µM, approximately. It has also been shown that the whole system of uridine uptake, as measured by the incorporation of uridine into nucleic acids, has an apparent Km of 5.1 µM and operates at a maximal rate of 11 pmoles/min/ 1.3 x 107 cells. The incorporation, in the presence of rifampicin, made it possible to calculate the half life of the messenger RNA\'s in B.subtilis as being 2 minutes. In any given instant, 44% of the radioactivity incorporated into RNA belongs to the stable RNA fraction (ribosomal and transfer). The analysis of the ribosomal RNA genes transcription mechanism was undertaken by a study of the rRNA chain elongation in the presence of rifampicin. The initial ratios of radioactivity incorporated in 16S and 23S rRNA\'s, when rifampicin and tritiated uridine are concomitantly added, and the decay kinetics of the radioactivity present in both rRNA species, suggest that the 16S and 23S rRNA genes are cotranscribed, in that order, in B.subtilis. Taking into consideration these and other evidences, the following structural relationships are proposed for the transcriptional unit: The experimental results were compared with theoretical models described in the Appendix. The existence of a cotranscription mechanism for the rRNA genes in prokaryotes and eukaryotes seems to suggest that such mechanism, being of fundamental importance, established itself very early and was maintained through evolution.

Anuros há muito tempo são conhecidos como portadores de tripanossomas em todo o mundo. Esses tripanossomas são transmitidos por sanguessugas e insetos hematófagos. Nenhum levantamento foi feito na América do Sul. A taxonomia tradicional resulta em identificações não confiáveis desses tripanossomas, a diversidade genética foi pouco investigada e o a filogenético é pouco conhecida. Neste estudo mostramos grande prevalência e marcada diversidade molecular e morfológica entre tripanossomas de várias espécies de anuros da Amazônia, Floresta Atlântica e Pantanal. O relacionamento filogenético inferido entre tripanossomas do Brasil, América do Norte, Europa e África revelou 5 clados de isolados de anuros. Um deles contem isolados de anuros e flebotomíneos, sugerindo que esses insetos possam ser vetores de tripanossomas de anuros. Em geral, os dados mostraram certo grau de associação entre clados de isolados e filogeografia de anuros e sugeriram que trocas de hospedeiros e adaptação ecológica desempenharam um papel importante na história evolutiva desses tripanossomas. / Amphibians of the order Anura have long been known to be infected with trypanosomes worldwide, infecting frogs and toads and transmitted by leeches and insects. No surveys were carried out in South American anurans. Traditional taxonomy renders unreliable identification of these trypanosomes, genetic diversity was poorly investigated and phylogenetic relationships are far from be understood. In this study, we showed high prevalence and marked morphological and molecular diversity among trypanosomes from several species from Amazonia, Atlantic Forest and Pantanal. Phylogenetic relationships inferred among trypanosomes from Brazil, North America, Europe and Africa disclosed 5 major clades of anuran isolates. One clade contains anuran and sand fly isolates, suggesting that these insects can be vectors of anuran trypanosomes. In general, data showed a certain degree of association between clades of isolates and anuran phylogeography and suggested that host switching and ecological fitting also play a role in the evolutionary history of these trypanosomes.

A proteína ribossomal L10 (RP L10) é uma forte candidata a ser incluída na classe de proteínas supressoras de tumor. Também denominada QM, a proteína em questão é conhecida por participar da ligação das subunidades ribossomais 60S e 40S e da tradução de mRNAs. Possui massa molecular entre 24 a 26 kDa e ponto isoelétrico (pI) 10,5. A seqüência da proteína QM é bastante conservada em mamíferos, plantas, invertebrados, insetos e leveduras indicando que esta possui funções críticas na célula. Com função supressora de tumor, a proteína RP L10 foi estudada em linhagens de tumor de Wilm (WT-1) e em células tumorais de estômago, nas quais se observou uma diminuição na quantidade de seu mRNA. Mais recentemente a RP L10 foi encontrada em baixas quantidades nos estágios iniciais de adenoma de próstata e com uma mutação em câncer de ovário, indicando uma participação no desenvolvimento destas doenças. Como proteína, já foi descrito que esta interage com as proteínas c-Jun e c-Yes, inibindo a ação ativadora de fatores de crescimento e divisão celular. Este trabalho tem um papel importante no estabelecimento da expressão desta proteína solúvel, para estudos posteriores que tenham como objetivo avaliar a ação de regiões específicas que atuam na ligação das subunidades ribossomais 60S e 40S e tradução, bem como nas regiões que se ligam a proto-oncogenes. O cDNA para proteína QM foi amplificado por PCR e clonado no vetor de expressão periplásmica p3SN8. A proteína QM foi expressa em E.coli BL21 (DE3) no citoplasma e periplasma bacteriano e na melhor condição, a expressão de QM de bactérias transformadas pelo plasmídeo recombinante p1813_QM em 25°C ou 30°C, a proteína foi obtida solúvel e com quantidad es muito pequenas de contaminantes. Os ensaios de estrutura secundária demonstraram que a proteína QM tem predominância de a-hélice, mas quando do seu desenovelmento, essa condição muda e a proteína passa a ter característica de folhas β. / The ribosomal protein L10 (RP L10) is a strong candidate to be included in the class of tumor suppressor proteins. This protein, also denominated as QM, is known to participate in the binding of ribosomal subunits 60S and 40S and the translation of mRNAs. It has a molecular weight that varies between 24 and 26 kDa and an isoelectric point of (pI) 10.5. The sequence of the protein QM is highly conserved in mammals, plants, invertebrates, insects and yeast which indicates its critical functions in a cell. As a tumor suppressor, RP L10 has been studied in strains of Wilm\'s tumor (WT-1) and tumor cells in the stomach, where was observed a decrease in the amount of its mRNA. More recently, the RP L10 was found in low amounts in the early stages of prostate adenoma and showed some mutation in ovarian cancer, what indicates its role as a suppressor protein in the development of these diseases. It has also been described that this protein interacts with c-Jun and c-Yes inhibiting growth factors and consequently, cell division. This work has an important role on the establishment of soluble expression of QM to give base information for further studies on expression that aim to evaluate the specific regions where it acts binding the 60S and 40S ribossomal subunits and translation, as well as its binding to proto-oncogenes. The cDNA for QM protein was amplified by PCR and cloned into periplasmic expression vector p3SN8. The QM protein was expressed in E. coli BL21 (DE3) in the region of cytoplasm and periplasm, the best condition was obtained from the expression of the recombinant plasmid QM p1813_QM at 25°C or 30°C, the soluble protein was obtained with small amounts of contaminants. The assays of secondary structure showed that the QM protein is predominantly alpha-helix, but when it loses the folding, this condition changes and the protein is replaced by β- sheet feature.

Made available in DSpace on 2017-07-21T19:59:46Z (GMT). No. of bitstreams: 1 Thais Dulz.pdf: 2466351 bytes, checksum: 208f40d4e86e959ef9f685c1e5a3cac5 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Characidae is considered the largest family and most complex among the Characiforms. The Astyanax genus is one of the most specious between the characids, comprising 142 valid species distributed in the Neotropical Region. Many of these species are restricted to certain river basins. The fish fauna of the Iguaçu River basin is characterized by having a high endemism, possibly because its isolation of the Paraná River basin, occurred about 22 million years, caused by the emergence of the Iguaçu Falls. Were recently described new species of this genus in the Rio Iguaçu, however its systematic position opposite the other Astyanax is still unknown, reinforcing necessary studies aimed at understanding their diversity and their evolutionary relationships. Therefore, this research aimed was to perform the karyotype characterization of the Astyanax species from the middle Rio Iguaçu: Astyanax altiparanae, A. bifasciatus, A. dissimilis, A. minor, A. ribeirae and A. serratus, using cytogenetics as a tool, in order to contribute to a more consistent scenario about the evolution of this group. The analysis were based on conventional Giemsa staining techniques, detection of nucleolar organizing regions by silver nitrate (Ag-NORs), C-banding and fluorescence in situ hybridization (FISH) to ribosomal genes 18S e 5S probes. All species presented 2n = 50 chromosomes, confirming the conservatism to the genus. The karyotypes comprise all chromosomal types and prevalence of chromosomes with two arms, mainly on the metacentric type except for A. ribeirae with the karyotype formula containing high number of acrocentrics. Despite the diploid number remain constant in these species; there were variations in karyotype formula and consequently the fundamental number, ranging from 66 to 92. The C-banding showed differences in the distribution of heterochromatic blocks between species, occurring polymorphism in A. serratus, which was also observed in other studies involving Astyanax sp. D (A. serratus). The six species showed the presence of NORs located in the telomeric region of the short arm of a chromosome pair of the type subtelocentric, characteristic conserved for the genus. Was identified single NORs in A. altiparanae and A. dissimilis, although the most common being multiple NORs, in almost all cases confirmed by 18S rDNA. All species showed 5S rDNA located in the interstitial segments of the chromosomes. Have been observed simple rDNA 5S in A. altiparanae, A. minor and A. serratus, and multiple in A. bifasciatus, A. dissimilis and A. ribeirae. Highlighted A. ribeirae by presenting a high number of rDNA 18S and 5S sites, which proved to be sintenics and co-located. In addition, this species is considered endemic for Ribeira de Iguape basin; however, was first found in the Iguaçu basin, strengthening fauna sharing hypothesis between rivers of the plateau and coastal regions in southern Brazil. The karyotype diversity evident for all species is mainly due to non-Robertsonian rearrangements. This study presented the first cytogenetic data for the genus Astyanax occurring in the Middle Rio Iguaçu region. / A família Characidae é considerada a maior e mais complexa entre os Characiformes. O gênero Astyanax é um dos mais especiosos entre os caracídeos, compreendendo 142 espécies válidas distribuídas na região Neotropical. Muitas destas espécies encontram-se restritas a determinadas bacias hidrográficas. A ictiofauna da bacia do Rio Iguaçu caracteriza-se por apresentar um elevado grau de endemismo, possivelmente devido o seu isolamento da bacia do Rio Paraná, ocorrido há cerca de 22 milhões de anos, causado pelo surgimento das Cataratas do Iguaçu. Recentemente, foram descritas novas espécies deste gênero no Rio Iguaçu, entretanto seu posicionamento sistemático frente aos demais Astyanax ainda é desconhecido, fazendo-se necessários estudos que visem à compreensão de sua diversidade e de suas relações evolutivas. Sendo assim, objetivou-se realizar a caracterização cariotípica de espécies do gênero Astyanax provenientes do médio Rio Iguaçu: Astyanax altiparanae, A. bifasciatus, A. dissimilis, A. minor, A. ribeirae e A. serratus, utilizando como ferramenta a citogenética, a fim de contribuir com um cenário mais consistente acerca da evolução deste grupo. Utilizou-se técnicas baseadas na coloração convencional por Giemsa, detecção de regiões organizadoras de nucléolo por nitrato de prata (Ag-RONs), bandamento C e hibridação in situ fluorescência (FISH) com sondas de genes ribossomais 18S e 5S. Todas as espécies estudadas apresentaram 2n = 50 cromossomos, confirmando o conservadorismo para o gênero. Observaram-se cariótipos compostos por todos os tipos cromossômicos e predominância de cromossomos com dois braços, principalmente do tipo metacêntrico, exceto para A. ribeirae com fórmula cariotípica composta por elevado número de acrocêntricos. Apesar do número diploide se manter constante nestas espécies, houve variações na fórmula cariotípica e, consequentemente no número fundamental, variando de 66 até 92. O bandamento C evidenciou variações na distribuição dos blocos heterocromáticos entre as espécies, com ocorrência de polimorfismo em A. serratus, o que também foi observado em outros estudos envolvendo Astyanax sp. D (A. serratus). As seis espécies estudadas mostraram a presença de RONs localizadas na região telomérica do braço curto de um par cromossômico do tipo subtelocêntrico, característica conservada para o gênero. Ocorreram RONs simples em A. altiparanae e A. dissimilis, as demais espécies possuem RONs múltiplas, em quase todos os casos confirmadas pela FISH com rDNA 18S. Todas as espécies evidenciaram rDNA 5S localizados nos segmentos intersticiais dos cromossomos. Foram observados rDNA 5S em um único par em A. altiparanae, A. minor e A. serratus, e múltiplos em A. bifasciatus, A. dissimilis e A. ribeirae. Destaca-se A. ribeirae por apresentar um elevado número de sítios rDNA 18S e 5S, os quais mostraram-se sintênicos e co-localizados. Além disso, esta espécie é considerada endêmica para bacia do Ribeira de Iguape; no entanto, foi encontrada pela primeira vez na bacia do Iguaçu, reforçando a hipótese de compartilhamento de fauna entre rios do planalto cristalino e regiões costeiras no sul do Brasil. A diversidade cariotípica evidenciada para todas as espécies devem-se principalmente a rearranjos não Robertsoniano. Neste estudo foram apresentados os primeiros dados citogenéticos para o gênero Astyanax ocorrentes na região do médio Rio Iguaçu.

As dermatofitoses apresentam alta prevalência na população em geral, sendo Trichophyton interdigitale (Trichophyton mentagrophytes) a segunda espécie mais frequentemente relatada como causadora de infecção em humanos. O objetivo do trabalho foi determinar a identidade genética, o perfil enzimático e de assimilação de açúcares e a suscetibilidade a antifúngicos de isolados clínicos do gênero Trichopyton. Os isolados foram avaliados por de ensaios enzimáticos, assimilação de fontes de carbono e da atividade antifúngica in vitro. A caracterização genotípica foi realizada através da amplificação e do sequenciamento da região do gene que codifica a região interna do gene DNA ribossomal dos isolados testados. No estudo, todos os isolados secretaram DNase, mas nenhum deles foi capaz de secretar fosfolipase e proteinase. Por outro lado, urease e lipase foram produzidas pela maioria dos isolados testados. Entre os antifúngicos avaliados, a terbinafina e o itraconazol demonstraram melhores resultados de sensibilidade, enquanto o fluconazol apresentou baixa atividade para as amostras. A anfotericina B, apesar de menos eficaz que a terbinafina e o itraconazol, também demonstrou resultados satisfatórios. A combinação entre os antifúngicos tioconazol e terbinafina demonstrou ter atividade antagônica em todos os isolados. Todos os dermatófitos testados assimilaram inulina, sorbitol, manose, glicose e trealose. Os demais carboidratos tiveram assimilação variável entre os isolados. Genotipicamente, todos os isolados do estudo foram identificados como pertencentes à espécie T. interdigitale. Os ensaios enzimáticos e de assimilação de carboidratos apresentaram resultados variáveis entre os isolados testados, inferindo variação intraespecífica entre as amostras de T. interdigitale. O perfil de sensibilidade aos antimicóticos testados seguiu o padrão de resultados observado em estudos anteriores. A identificação genotípica através da amplificação e do sequenciamento da região ITS permitiu garantir a identidade entre os isolados testados. / The prevalence of dermatophytosis among population is high, and Trichophyton interdigitale (Trichophyton mentagrophytes) is the second most frequently reported species to cause infection in humans. This study objective was to evaluate the genetic identity, the enzymatic profile and the assimilation of carbon sources, as well as the antifungal susceptibility of clinical isolates of Trichophyton sp. The isolates were analyzed phenotypically by enzymatic assays, assimilation of carbon sources and antifungal activity in vitro. The genotypic characterization was performed by amplification and sequencing the gene region encoding the internal region of the DNA ribosomal gene of the isolates tested. In our study, all isolates secreted DNase, while none of them was capable of secreting phospholipase, keratinase and proteinase. On the other hand, lipase and urease were secreted by most of the isolates. Of the antifungal agents tested, the best results in terms of sensitivity were found with terbinafine and itraconazole, while the antifungal activity of fluconazole was found to be weak. Amphotericin B, although less effective than terbinafine and itraconazole, also yielded satisfactory results. Evaluation of the drug combination of tioconazole and terbinafine revealed an antagonistic effect for all tested isolates. All isolates assimilated inulin, sorbitol, glucose, trehalose and mannose. Arabinose, dulcitol, galactose and xylose were assimilated by some of the isolates, whereas the most isolates assimilated cellobiose, dulcitol, erytritol and mannitol. Genotypically, all isolates in the study were identified as belonging to Trichophyton interdigitale species. In general, enzyme assays and assimilation of carbohydrates showed variable results among the isolates tested, inferring intraspecific variation between Trichophyton interdigitale samples. The sensitivity profile of the antifungal agents tested in this study was similar to results obtained in previous studies. The genotypic identification, by amplification and sequencing of the ITS region, has ensured the identity of the isolates tested.

Rickettsia rickettsii é o agente etiológico da Febre Maculosa das Montanhas Rochosas, que no Brasil é transmitida pelos carrapatos Amblyomma cajennense e A. aureolatum. Para elucidar os mecanismos de virulência sobre seus vetores, construímos bibliotecas subtrativas utilizando RNA de A. aureolatum infectados ou não com o patógeno. Com a análise bioinformática, foram obtidas 56 sequências únicas com expressão induzida e 12 com expressão reprimida pela infecção. Após a validação dos dados por RT-qPCR 3 genes foram caracterizados por RNAi: uma hebraeína, uma proteína dissulfeto isomerase (PDI) e uma proteína com domínio Kunitz-type. Um maior número de carrapatos adquiriu R. rickettsii quando a expressão gênica da hebraeína e da PDI foi silenciada, sugerindo que elas participam na defesa do carrapato contra a infecção. Nenhum efeito foi observado sobre a transmissão da bactéria para o hospedeiro ou sobre o fitness de carrapatos nos três genes analisados. O presente estudo apontou genes importantes que possibilitam uma melhor compreensão da relação carrapato-riquétsia. / Rickettsia rickettsii is the etiological agent of Rocky Mountain Spotted Fever and, in Brazil, it is transmitted by Amblyomma cajennense and A. aureolatum. To elucidate mechanisms of virulence to its vectors, we construct cDNA libraries with RNA of ticks A. aureolatum infected or not with this pathogen. After bioinformatic analysis, 56 unique sequences were obtained representing up-regulated genes and 12 down-regulated by infection. After data validation by RT- qPCR, 3 genes were characterizated by RNAi: a hebraein, a protein disulfide isomerase (PDI), and a protein with Kunitz-type domain. A higher number of ticks acquired R. rickettsii when the gene expression of hebraein and PDI was silenced, suggesting that both proteins participate in the defense of the tick against infection. No effect on the transmission of the bacterium to the host or on the fitness of ticks was observed after knockdown of the 3 analyzed genes. Data obtained by the present study pointed out important genes that provide information to better understand of the tick-rickettsia relationship.

Duas linhagens filogenéticas principais (LI e L2) foram definidas em Trypanosoma cruzi, com base em sequências de genes de rDNA e mini-exon e análise por RAPD (Souto et al. 1996). Neste trabalho investigamos a estrutura e atividade dos promotores do cistron ribossômico das duas linhagens de T. cruzi. O promotor da cepa Dm28 (L2) foi clonado e sua seqüência foi comparada com seqüências publicadas da região homóloga de CL (L1) e La Cruz (L2). A identidade entre os dois promotores de L2 foi de 98%, e entre estes e o de L1 foi de 82%. O ponto de início de transcrição foi mapeado, apresentando a mesma localização nas duas linhagens. A atividade dos promotores de L1 e L2 foi determinada em construções plasmidiais contendo o gene reporter da cloranfenicol acetil transferase (CAT). Experimentos de expressão transitória mostraram que o promotor de L1 foi funcional apenas em isolados de L1, enquanto que o promotor de L2 foi funcional em ambas as linhagens. A expressão do promotor de L2 em isolados de L1 foi maior do que a do promotor homólogo. Analisamos ainda a atividade dos dois promotores em um grupo de isolados que apresentam dois tipos de cistrons ribossômicos (grupo 1/2). Neste grupo de cepas observamos que ambos os promotores presentes nas construções são funcionais, embora o promotor de L2 induza maior expressão de CAT. Por outro lado, demonstramos que nestes isolados apenas o cistron ribossômico de tipo 2 é expresso in vivo. Neste trabalho analisamos ainda a eficiência de entrada das construções plasmidiais nas cepas de T. cruzi; caracterizamos a atividade de regiões do promotor de L2 e a eficiência do processo de \"trans-splicing\" para gerar mRNA de CAT contendo a seqüência de mini-exon na extremidade 5\'. / Two major phylogenetic lineages (L1 and L2) have been defined in Trypanosoma cruzi based on rDNA and mini-exon sequences and RAPD analysis (Souto et al., 1996). In the present work we have investigated the structure and activity of ribosomal RNA promoters from the two T. cruzi lineages. The promoter region of Dm28 strain (L2) was cloned and its sequence was compared with the homologous regions from the CL (L1) and La Cruz (L2) strains, whose sequences were previously published. The identity found between promoters of the two L2 strains was 98%, while the identity between L2 and L1 promoters was 82%. The transcription start point mapped in the two Lineages showed the same localization. The activity of L1 and L2 promoters was investigated through the use of plasmid constructs bearing bacterial chloramphenicol acetyl transferase (CAT) as reporter gene. Experiments of transient expression showed that the L1 promoter drove high CAT activity in L1 isolates, but essentially no activity in L2 strains. On the other hand, L2 promoter was functional in both Lineages. The expression driven by L2 promoter in L1 isolates was higher than that driven by the homologous promoter. We have also analysed the activity of both L1 and L2 promoters in a particular group of T. cruzi isolates (group 1/2) which contains two types of rRNA cistrons. It was observed that both promoters were functional in this group of strains, although L2 promoter drove higher CAT activity. This is an interesting result since we have shown that in group 1/2 isolates only the type 2 rRNA cistron is expressed in vivo. In the present study, we have also analysed the transfection efficiency of the plasmid constructs in T. cruzi strains; the activity of segments of the L2 promoter; and the efficiency of the \"trans-splicing\" process involved in the generation of mature CAT mRNA containing the mini-exon sequence at the 5\' end.

Ma Hoi-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations --- p.X / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Interactions that stabilize proteins --- p.1 / Chapter 1.2 --- Some common strategies of protein engineering to improve thermostability --- p.6 / Chapter 1.3 --- Ribosomal protein T. celer L30e as a study model for thermostability --- p.7 / Chapter 1.4 --- Extra proline residue is one of the insights by comparing the two proteins --- p.10 / Chapter Chapter Two --- Materials and Methods --- p.13 / Chapter 2.1 --- General Techniques --- p.13 / Chapter 2.1.1 --- Preparation of Escherichia coli competent cells --- p.13 / Chapter 2.1.2 --- Transformation of Escherichia coli competent cells --- p.14 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.14 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.14 / Chapter 2.1.5 --- DNA extraction from agarose gel electrophoresis using Viogene Gene Clean kit --- p.15 / Chapter 2.1.6 --- Plasmid DNA minipreperation by Wizard® Plus SV Minipreps DNA Purification System from Promega --- p.16 / Chapter 2.1.7 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.1.8 --- Ligation of DNA fragments --- p.18 / Chapter 2.1.9 --- Sonication of pellet resuspension --- p.18 / Chapter 2.1.10 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.19 / Chapter 2.1.11 --- Native polyacrylamide gel electrophoresis --- p.20 / Chapter 2.1.12 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.22 / Chapter 2.1.13 --- Protein Concentration determination --- p.22 / Chapter 2.2 --- Cloning the Mutant Genes --- p.22 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.22 / Chapter 2.2.1.1 --- Generation of full length mutant gene by megaprimer --- p.23 / Chapter 2.2.1.2 --- Generation of mutant gene by QuikChange® Site-Directed Mutagenesis Kit from Stratagene --- p.26 / Chapter 2.2.2 --- Restriction Digestion of DNA --- p.27 / Chapter 2.2.3 --- Ligation of DNA fragments --- p.27 / Chapter 2.2.4 --- Screening for successful inserted plasmid clones from ligation reactions --- p.28 / Chapter 2.2.4.1 --- By PCR --- p.28 / Chapter 2.2.4.2 --- By restriction digestion --- p.28 / Chapter 2.2.5 --- DNA sequencing --- p.29 / Chapter 2.3 --- Expression and Purification of Protein --- p.29 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.29 / Chapter 2.3.2 --- Purification of recombinant wild type TRP and mutants --- p.30 / Chapter 2.3.3 --- Purification of recombinant wild type YRP and mutants --- p.32 / Chapter 2.4 --- Thermodynamic Studies by Circular Dichroism (CD) Spectrometry --- p.34 / Chapter 2.4.1 --- Thermodynamic studies by guanidine-induced denaturations --- p.34 / Chapter 2.4.2 --- Themodynamic studies by thermal denaturations --- p.36 / Chapter 2.4.3 --- ACp measurement of the TRP mutants --- p.37 / Chapter 2.4.3.1 --- By Gibbs-Helmholtz analysis --- p.37 / Chapter 2.4.3.2 --- By van't Hoff analysis --- p.37 / Chapter 2.5 --- Crystal Screen for the Mutant T. celer L30e --- p.38 / Chapter 2.5.1 --- T. celer L30e Pro→Ala and Pro→Gly mutants --- p.38 / Chapter 2.5.2 --- Yeast L30e K65P mutant --- p.38 / Chapter 2.6 --- Sequences of Primers --- p.39 / Chapter 2.6.1 --- Primers for TRP and its mutants --- p.39 / Chapter 2.6.2 --- Primers for YRP and its mutantsReagents and buffers --- p.40 / Chapter 2.7 --- Reagents and Buffers --- p.40 / Chapter 2.7.1 --- Reagents for competent cell preparation --- p.40 / Chapter 2.7.2 --- Nucleic acid eletrophoresis buffers --- p.41 / Chapter 2.7.3 --- Media for bacterial culture --- p.41 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.42 / Chapter 2.7.5 --- Buffers for TRP purification --- p.44 / Chapter 2.7.6 --- Buffers for YRP purification --- p.45 / Chapter 2.7.7 --- Buffer for Circular Dichroism (CD) Spectrometry --- p.46 / Chapter Chapter Three --- Results --- p.48 / Chapter 3.1 --- "Cloning, expression and purification of the mutant proteins" --- p.48 / Chapter 3.1.1 --- "Mutagenesis, cloning and purification of the thermophilic proteins - T. celer L30e protein and its mutants" --- p.48 / Chapter 3.1.2 --- "Mutagenesis, cloning and purification of the mesophilic proteins - yeast L30e protein and its mutants" --- p.52 / Chapter 3.2 --- Stability of Pro→Ala/Gly mutants of T. celer L30e at 298K --- p.55 / Chapter 3.2.1 --- Design of alanine and glycine mutants from thermophilic homologue --- p.55 / Chapter 3.2.2 --- "Among alanine mutants, only P59A was destabilized" --- p.55 / Chapter 3.2.3 --- Ala→Gly mutations destabilized the protein --- p.59 / Chapter 3.3 --- Stability of Xaa→Pro mutants of yeast L30e at 298K --- p.61 / Chapter 3.3.1 --- Design of proline mutants from mesophilic homologue --- p.61 / Chapter 3.3.2 --- "K65P, corresponding to P59 in T. celer L30e, stabilized yeast L30e" --- p.62 / Chapter 3.3.3 --- Yeast L30e mutated with thermophilic consensus sequence did not give a more stable protein --- p.65 / Chapter 3.4 --- Temperature dependency of the stability of the mutants of T. celer L30e --- p.67 / Chapter 3.4.1 --- The trend of ΔGU was consistence through 25 to 75°C --- p.67 / Chapter 3.4.2 --- Melting temperatures of T. celer mutants determined by thermal denaturations --- p.68 / Chapter 3.5 --- pH dependency of melting temperatures --- p.75 / Chapter 3.5.1 --- ΔCP values of the P59A/G mutants determined by van't HofF's analyses increased significantly --- p.77 / Chapter 3.6 --- No structural change was observed in the crystal structure of P59A --- p.80 / Chapter Chapter Four --- Discussion --- p.84 / Chapter 4.1 --- The trend of stability from guanidine-induced denaturation agreed with that from thermal denaturations --- p.86 / Chapter 4.2 --- The magnitude of destabilization of P59A and Ala→Gly mutation was consistent with the expected destabilization due to entropy --- p.87 / Chapter 4.3 --- Entropic effect had little effect for residues in flexible region --- p.93 / Chapter 4.4 --- Stabilization forces that compensate the entropic effect --- p.96 / Chapter 4.5 --- Compensatory stabilization due to the release of amide group --- p.99 / Chapter 4.5.1 --- Intra-molecular H-bond in P88A --- p.99 / Chapter 4.5.2 --- Solvent-protein H-bond in P43A --- p.103 / Chapter 4.6 --- Consensus concept was not applicable in our model --- p.110 / Chapter 4.7 --- "Pro→Ala mutation destabilized the protein increase the protein's ACP value, however enthalpy and entropy change were difficult to be decomposed" --- p.111 / Chapter 4.8 --- Concluding Remarks --- p.112 / References --- p.113

Chu Lai On. / Thesis submitted in: July 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 152-172). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Content --- p.7 / Abbreviations --- p.12 / Naming system for mutant proteins --- p.13 / Abbreviations for amino acid --- p.14 / Chapter Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- Structure-function relationship of trichosanthin --- p.18 / Chapter 1.2 --- Properties of acidic ribosomal P-proteins --- p.21 / Chapter 1.3 --- Interaction among P-proteins and trichosanthin --- p.25 / Chapter 1.4 --- Properties of eukaryotic elongation factor 2 and interaction with P-proteins --- p.26 / Chapter 1.5 --- "Objectives and strategy of studying the interaction among trichosanthin, P-proteins and eukaryotic elongation 2" --- p.30 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- General techniques --- p.33 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.33 / Chapter 2.1.2 --- Minipreparation of plasmid DNA using Wizard Plus SV Minipreps DNA purification kit from Promega --- p.34 / Chapter 2.1.3 --- Agarose gel electrophoresis of DNA --- p.36 / Chapter 2.1.4 --- Purification of DNA from agarose gel using Wizard SV Gel and PCR Clean-Up System from Promega --- p.36 / Chapter 2.1.5 --- Polymerase Chain Reaction (PCR) --- p.37 / Chapter 2.1.5.1 --- Basic Protocol --- p.37 / Chapter 2.1.5.2 --- Generation of P2 truncation mutants --- p.38 / Chapter 2.1.5.3 --- Generation of TCS mutants --- p.39 / Chapter 2.1.6 --- Restriction digestion of DNA --- p.41 / Chapter 2.1.7 --- Ligation of DNA fragments --- p.41 / Chapter 2.1.8 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.1.9 --- Staining of protein in polyacrylamide gel --- p.45 / Chapter 2.2 --- Expression and purification of recombinant proteins --- p.46 / Chapter 2.2.1 --- "Bacterial culture, harvesting and lysis" --- p.46 / Chapter 2.2.2 --- Purification of recombinant TCS and mutants --- p.47 / Chapter 2.2.3 --- Purification of acidic ribosomal protein P2 and mutants --- p.48 / Chapter 2.2.4 --- Purification of MBP-fusion proteins --- p.50 / Chapter 2.3 --- Purification of eEF2 from rat livers --- p.51 / Chapter 2.4 --- In vitro binding assay by NHS-activated Sepharose resin --- p.53 / Chapter 2.4.1 --- Coupling of protein sample to NHS-activated Sepharose resin --- p.53 / Chapter 2.4.2 --- In vitro binding of protein sample to coupled NHS-activated resin --- p.54 / Chapter 2.5 --- Ribosome-inactivated activity assay using rabbit reticulocyte lysate in vitro translation system --- p.55 / Chapter 2.6 --- Circular dichroism (CD)spectrometry --- p.57 / Chapter 2.7 --- Isothermal titration calorimetry (ITC) experiment --- p.57 / Chapter 2.8 --- Surface plasmon resonance (SPR) experiment --- p.58 / Chapter 2.8.1 --- Immobilization of P2 onto aminosilane cuvette --- p.58 / Chapter 2.8.2 --- Interaction between eEF2 and immobilized P2 --- p.60 / Chapter 2.9 --- Preparation of Anti-P antibody --- p.61 / Chapter 2.10 --- Western blotting of protein --- p.62 / Chapter 2.11 --- Reagents and buffer --- p.64 / Chapter 2.11.1 --- Reagents for competent cell preparation --- p.64 / Chapter 2.11.2 --- Nucleic acids electrophoresis buffer --- p.65 / Chapter 2.11.3 --- Media for bacterial culture --- p.66 / Chapter 2.11.4 --- Buffers for TCS purification --- p.67 / Chapter 2.11.5 --- Buffers for eEF2 purification --- p.68 / Chapter 2.11.6 --- Reagents for SDS-PAGE --- p.68 / Chapter 2.11.7 --- Reagents and buffers for Western blot --- p.70 / Chapter 2.11.8 --- Reagents and buffers for coupling sample proteins to NHS-activated Sepharose resin --- p.72 / Chapter 2.11.9 --- Reagents and buffers for in vitro binding assay --- p.72 / Chapter 2.11.10 --- Reagents and Buffers for surface plasmon resonance --- p.72 / Chapter 2.12 --- Sequences of primers --- p.73 / Chapter Chapter 3 --- Interaction between TCS and P2 --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Interaction between TCS and P-proteins in rat liver lysate --- p.83 / Chapter 3.3 --- Construction of TCS mutants --- p.85 / Chapter 3.4 --- Expression and purification of TCS mutants --- p.87 / Chapter 3.5 --- Biological assay of TCS mutants --- p.91 / Chapter 3.6 --- Physical interaction of TCS mutants and P2 by surface plasmon resonance (SPR) --- p.94 / Chapter 3.7 --- Discussion --- p.100 / Chapter Chapter 4 --- Mapping the region of P2 that binds TCS and eEF2 --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Construction of P2 truncation mutants --- p.106 / Chapter 4.3 --- Expression and purification of P2 truncation mutants --- p.107 / Chapter 4.4 --- Mapping the region of P2 that binds TCS --- p.111 / Chapter 4.4.1 --- Interaction between TCS and P2 mutants by in vitro binding assay --- p.111 / Chapter 4.4.2 --- Interaction study of TCS and P2 mutant by isothermal titration calorimetry (ITC) --- p.116 / Chapter 4.5 --- Mapping the region of P2 that binds eEF2 --- p.120 / Chapter 4.5.1 --- Purification of eEF2 from rat liver --- p.120 / Chapter 4.5.2 --- Physical interaction of P2 and eEF2 by surface plasmon resonance (SPR) --- p.126 / Chapter 4.5.3 --- Interaction between eEF2 and P2 mutants by in vitro binding assay --- p.128 / Chapter 4.6 --- Mapping the C-terminal region of P2 by MBP-fusion proteins --- p.130 / Chapter 4.6.1 --- Construction and purification of MBP-fusion proteins --- p.131 / Chapter 4.6.2 --- "Interaction among eEF2, TCS and MBP-fusion proteins by in vitro binding assay" --- p.133 / Chapter 4.7 --- Discussion --- p.137 / Chapter Chapter 5 --- Effect of C-17 peptide on TCS biological activity --- p.143 / Chapter 5.1 --- Introduction --- p.143 / Chapter 5.2 --- Ribosome-inactivating activity of TCS with C-17 peptide --- p.145 / Chapter 5.3 --- Discussion --- p.147 / Chapter Chapter 6 --- Conclusion and suggestions for future study --- p.149 / References --- p.152 / Appendix --- p.173