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Detecting Nitrogen Responsive Genes for Improvement of Nitrogen Use EfficiencyYingyu, Chen 23 December 2011 (has links)
A principal concern in crop agriculture is yield, and a key factor for crop growth is the availability of nitrogen. The large amount of nitrogen fertilizer required by plants is a major cost to farmers. Moreover, environmental issues such as groundwater pollution arise from the utilization of nitrogen fertilizers. Therefore, improvement in the nitrogen use efficiency (NUE) of plants is of urgent importance for sustainable and efficient agriculture. Although hybrid varieties have increased crop yields in low N conditions, the molecular mechanism of plant adaptation to N stress is not completely understood. Herein, the study of responses to N limitations in the natural signalling pathways of model plants facilitates the understanding of complex responses in plants to N stress, and this information can be used to further improve NUE. In this research, the transcriptomes of three model plants Arabidopsis, maize, and rice were compared under diverse N growth conditions. An evaluation of the response of the three plants to varying N levels was also conducted. From a statistical point of view, three distinct methods of detecting differential expression were utilized to reduce the likelihood of false positives due to the tens of thousands of genes simultaneously studied. Furthermore, the performance of three statistical approaches was compared during detection of the N-responsive genes. Finally, a clustering analysis (agglomerative hierarchical clustering) was performed on the genes that significantly responded to N levels as identified by a more biologically intuitive method called Rank Products (RP).
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Identification of Virulence Factors in Edwardsiella IctaluriLu, Jingjun 11 May 2013 (has links)
Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is one of the most important diseases impacting the US catfish industry. Though this disease has been very common, progress has been slow to find an economical and practical treatment method. Our long-term goal is to determine the mechanisms of E. ictaluri virulence in ESC. The overall objective of this study was to identify E. ictaluri genes required for host encounter and serum resistance and to determine their roles in pathogenesis. The central hypothesis is that E. ictaluri must differentially regulate its genes to invade fish and evade host defenses, thus, mutation of these differentially expressed genes (DEG) should cause attenuation of E. ictaluri virulence. To test this hypothesis, we first determined the global gene expression patterns of the wild type (wt) E. ictaluri 93-146 and EiAKMut02 mutant during catfish encounter and serum exposure using microarray analysis. Results indicated that in E. ictaluri wt, 377 and 16 DEGs were identified during host encounter and serum exposure, respectively. In EiAKMut02, 82 and 296 DEGs were identified during host encounter and serum experiment. Through functional analysis using Blast2GO, PSORTb, Host Pathogen Interaction Database (HPIDB), and Microbe Virulence Database (MVirDB), 38 DEGs in 9 KEGG pathways have been identified as potential virulence factors. The KEGG pathways represented were 1) bacterial secretion system including T3SS and T6SS, 2) ABC transporters including cystine transport system, iron complex transport system, d-methionine transport system, arginine transport system, thiamine transport system, and molybdate transport system, 3) protein export, 4) flagellar assembly, 5) two-component system, 6) bacterial chemotaxis, 7) ascorbate and aldarate metabolism, 8) phosphotransferase system, and 9) metabolic pathways. In order to understand their role in the E. ictaluri virulence, selected DEGs were inrame deleted by allelic exchange, and their virulence and efficacy were characterized in channel catfish fingerlings. Our results showed that the virulence of E. ictaluri ssaV and yscR mutants was completely attenuated while their efficacies were moderate in catfish fingerlings. These results support that the T3SS and T6SS, ABC transporters, protein export, and flagella seem to be important in E. ictaluri virulence.
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Desenvolvimento Diferencial Casta-Específico das Pernas Posteriores de Apis mellifera. / Differential Hind Leg Development in Apis mellifera Castes.Bomtorin, Ana Durvalina 11 March 2009 (has links)
A diferenciação morfofisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval, que estimula o aumento da produção de Hormônio Juvenil naslarvas que originarão rainhas. Dentre as diversas diferenças morfológicas entre operárias e rainhas encontramse estruturas especializadas para a coleta de pólen e própolis, localizadas na região da tíbia e do basitarso das pernas posteriores de operárias. A diferenciação das pernas tem início entre o quarto e o quinto estágio do desenvolvimento larval. Utilizandose Microscopia Eletrônica de Varredura o presente trabalho relata a presença das cerdas formando as estruturas castaespecíficas na fase de pupa de olho marrom. A partir de estudos de hibridação de microarrays de cDNA com amostras de RNA de A. mellifera de diversas fases do desenvolvimento larval, foram encontrados 91 genes com ortólogos conhecidos em Drosophila, diferencialmente expressos entre rainhas e operárias no período crítico da diferenciação de castas. Destes, cinco estão relacionados com o desenvolvimento de apêndices: ataxin2 (atx2), cryptocephal (crc), dachshund (dac), grunge (gug) e Retinoic and fat acid Binding Protein (RfaBP). O perfil destes genes, e ainda, ultrabithorax (ubx), distalless(dll) e abdominalA (abdA) (estes porsuassuasfunções durante a diferenciação das pernas de insetos) foram analisados por RTPCR em Tempo Real em pernas posteriores de operárias e rainhas desde o quarto estágio larval até o estágio de pupa de olho branco. Apenas ubx e abdA foram encontrados mais expressos em operárias ao final do desenvolvimento larval e início do desenvolvimento pupal. Estudossimilares dos genes abdA, dac, dll e ubx nossegmentos das pernas de pupas de olho branco indicam a tíbia como domínio de expressão de dac. Imunolocalizações utilizando um anticorpo contra um epitopo conservado entre Ubx e AbdA, FP6.87, em pernas posteriores de prépupas de operárias e rainhasrevelam a presença destas proteínas na tíbia apenas de operárias e diferencialmente localizadas no basitarso de operárias e rainhas. Os dados acima apresentados apontam Ubx, um gene Hox, como pontochave na regulação da formação das estruturas castaespecíficas. / Diphenism in the honey bee, Apis mellifera,resultsfromdifferential feeding of female larvae. Among the morphological differences, the hind legs of workers have structures that is used for carrying pollen and propolis, e.g. the corbicula, while the queens hind legslack thisstructures. The corbicula is an expanded region of the tibia deprived of bristles, which has a single bristle in the middle that seems to have a sensorial function. Using scanning electronic microscopy, we found that the leg structures and bristles of the corbicula are already formed in browneyed pupa. Microarray analysis has demonstrated that five of 240 differentiallyexpressed genesin developing castes are potentially related to the caste differences in leg development (ataxin2, cryptocephal, dachshund, grunge and Retinoic and fat acid Binding Protein). Using qPCR, we analyzed the expression of abdominalA, ataxin2, cryptocephal, grunge, Retinoic and fat acid Binding Protein and ultrabithorax genes during hind leg development. cryptocephal, ataxin2, grunge and Retinoic and fat acid Binding Protein genes, which are involved in imaginal disc elongation and bristle formation and are inhibited by juvenile hormone, were not found to be differentially expressed. However, ultrabithorax and abdominalA are over expressed in workersin the early pupalstage. By using immunohistochemistry, Ubx was localized in the tibia and basitarsus of prepupae of workers and in the basitarsus of pre pupae of queens. The pattern of Ubx expression suggests that this Hox gene is a key player in leg structuresformation and caste differentiation in A.mellifera.
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Transcription Regulation and Candidate Diagnostic Markers of Esophageal Cancer.Essack, Magbubah. January 2009 (has links)
<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>
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The effect of normalization methods on the identification of differentially expressed genes in microarray dataKristinsson, Vilhelm Yngvi January 2007 (has links)
<p>In this thesis the effect of normalization methods on the identification of differentially expressed genes is investigated. A zebrafish microarray dataset called Swirl was used in this thesis work. First the Swirl dataset was extracted and visualized to view if the robust spline and print tip loess normalization methods are appropriate to normalize this dataset. The dataset was then normalized with the two normalization methods and the differentially expressed genes were identified with the LimmaGUI program. The results were then evaluated by investigating which genes overlap after applying different normalization methods and which ones are identified uniquely after applying the different methods. The results showed that after the normalization methods were applied the differentially expressed genes that were identified by the LimmaGUI program did differ to some extent but the difference was not considered to be major. Thus the main conclusion is that the choice of normalization method does not have a major effect on the resulting list of differentially expressed genes.</p>
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Transcription Regulation and Candidate Diagnostic Markers of Esophageal Cancer.Essack, Magbubah. January 2009 (has links)
<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>
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Transcription regulation and candidate diagnostic markers of esophageal cancerEssack, Magbubah January 2009 (has links)
Philosophiae Doctor - PhD / Esophageal cancer (EC) ranks among the ten most frequent cancers worldwide. Mortality rates associated with EC are very similar to the incidence rates due to the relatively late stage of diagnosis and the poor efficacy of treatment. The aim of this study was to enhance our insights of putative transcriptional circuitry of EC genes, thereby potentially positively impacting our knowledge of therapeutic targets, providing indications as to more appropriate lines of treatment, and additionally allowing for the determination of putative candidate diagnostic markers for the early stage detection of EC.
This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer. DDEC contains known and novel information for 529 differentially expressed EC genes compiled using scientific publications from PubMed and is freely accessible for academic and non-profit users at http://apps.sanbi.ac.za/ddec/. The novel information provided to users of the DDEC is the lists of putative transcription factors that potentially control the 529 manually curated genes. The value of the information accessible through the database was further refined by providing precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with
pharmacological effects, disease concepts and human anatomy. This feature has the capacity to display potential associations that are rarely reported and thus difficult to identify, and it enables the inspection of potentially new ‘association hypotheses’ generated based on the precompiled reports.
This study further illustrates how the biocurated esophageal squamous cell
carcinoma (ESCC) genes in the database may represent a reliable starting point for exploring beyond current knowledge of the transcriptional circuitry of estrogen related hormone therapy. The genes were used to develop a method that identified 44 combinations of transcription factors (TFs) that characterize the promoter sequence of estrogen responsive genes implicated in ESCC. These significantly over-represented combinations of TFs were then used to increase confidence in the 47 novel putative estrogen response genes that may be related to ESCC too. Coincidently, two of the novel putative estrogen response genes were verified by current (2009), experimental publications. / South Africa
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Estudo da expressão das proteínas cromodomínio-helicase e SET/TAF-Iβ durante o processo de estrobilização de Mesocestoides cortiCosta, Caroline Borges January 2013 (has links)
A cromodomínio-helicase (CHD) e a SET/TAF-Iβ (chaperona de histonas) são proteínas conhecidas por estarem envolvidas em processos de remodelagem de cromatina e controle da expressão gênica. Em organismos como Caenorhabditis elegans, Drosophila melanogaster, camundongo e o homem, estas proteínas estão associadas ao controle de vários processos de desenvolvimento. Em Mesocestoides corti, um modelo de parasito cestódeo, sequências relacionadas à CHD e à SET/TAF-Iβ foram identificadas em uma seleção de genes diferencialmente expressos em larvas e vermes estrobilizados. Visando à identificação de marcadores moleculares de processos e estágios de desenvolvimento da Classe Cestoda, os padrões de expressão de ortólogos das proteínas CHD e SET/TAF-Iβ (McCHD e McSET/TAF) em M. corti foram investigados. Inicialmente as sequências codificadoras da McCHD e McSET/TAF foram amplificadas por RT-PCR, clonadas em vetor de expressão modificado (pGEX-TEV) e expressas em Escherichia coli para produção de proteínas recombinantes. Análises por imunoblot e imuno-histoquímica foram realizadas utilizando anticorpos gerados contra versões recombinantes das proteínas-alvo do estudo. Foram analisados quatro estágios de desenvolvimento de M. corti: larvas (tetratirídeos, TT), TT após 24h de indução ao processo de estrobilização (o qual confere o desenvolvimento da larva em adulto) (24h-Ind), TT após 72h de indução (72h-PI) e vermes estrobilizados (VE). Em imunoblots, a McCHD apresentou altos níveis de expressão em três estágios de desenvolvimento de M. corti: TT, 72h-PI e VE, sendo detectado um menor nível de expressão no estágio 24h-Ind quando comparado aos demais. Já a McSET/TAF apresentou um aumento gradual no seu nível de expressão após a indução (nos estágios de 24h-Ind, 72h-PI e VE), não sendo detectada em TT. Em secções longitudinais foram observados um maior nível de expressão da McCHD em estágios iniciais de desenvolvimento (TT e 24- Ind) enquanto que McSET/TAF foi observado um maior nível de expressão nos estágios intermediários e no final do desenvolvimento de M. corti. Para ambas as proteínas a localização foi citoplasmática e não houve diferenças de distribuição nos tecidos analisados. Para compreender melhor as diferenças encontradas nos níveis de expressão das proteínas, análises por PCR em tempo real (RT-qPCR) foram realizadas para quantificação dos níveis de transcritos dos genes McCHD e McSET/TAF. Foram encontradas diferenças significativas nos níveis de expressão gênica de McCHD e McSET/TAF somente em dois estágios: o inicial (TT) e o adulto (VE), conforme análises pelo teste de Duncan. Níveis maiores de expressão gênica de ambos os genes foram encontrados no último estágio de desenvolvimento (VE) de M. corti. A caracterização de genes e proteínas envolvidas no processo de estrobilização de platelmintos da classe Cestoda contribuirá para o melhor entendimento de rotas do desenvolvimento de cestódeos, podendo auxiliar também na determinação de novos alvos terapêuticos para o tratamento de cestodíases. / Chromodomain-helicase (CHD) and the SET/TAF-Iβ histone chaperone are proteins known to be implicated in processes of chromatin remodeling and regulation of gene expression associated with the control of developmental processes in different organisms, such as Caenorhabditis elegans, Drosophila melanogaster, mouse and human. In Mesocestoides corti, a model cestode parasite, CHD and SET/TAF-Iβ related sequences were isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Aiming at identifying molecular markers of processes and stages of development of Class Cestoda, the expression patterns of M. corti CHD and SET/TAF-Iβ orthologous proteins (McCHD and McSET/TAF) were investigated. Initially, the coding sequences of McCHD and McSET/TAF were amplified by RT-PCR, cloned into a modified expression vector (pGEX-TEV) and expressed in Escherichia coli for recombinant proteins production. Analysis by immunoblotting and immunohistochemistry was performed using antibodies raised against recombinant versions of the proteins target the study. We analyzed four developmental stages of M. corti: bona fide tetrathyridia (TT), tetrathyridia 24 h after strobilation induction (24h-Ind), strobilating worms 72 h after induction (72h-PI), and fully strobilated worms (VE). Immunoblots showed high levels of expression McCHD in three developmental stages of M. corti: TT, 72h-PI e VE, and detected a lower level of expression in stage 24-Ind in comparison to others. The McSET/TAF showed a gradual increase in their level of expression after induction (stages of 24h-Ind, 72h- PI e VE) was not detected in TT. In longitudinal sections were observed an increased level of expression of McCHD in early stages of development (TT and 24-Ind) while McSET/TAF there was a higher level of expression in the intermediate and late stages of development of M. corti. Both McCHD and McSET/TAF showed a cytoplasmic location and a uniform pattern of immunostaining, no differences in distribution between the tissues. For a better understanding the differences in levels of protein expression, analysis by real-time PCR (RT-qPCR) was performed to quantify the transcript levels of genes McCHD and McSET/TAF. There were significant differences in levels of gene expression McCHD and McSET/TAF only in two stages: the initial (TT) and adult (VE), as analysis by Duncan test. Higher levels of gene expression of both genes were found in the last stage of development (VE) of M. corti. The characterization of genes and proteins involved in the process of strobilation flatworms of the class Cestoda contribute to a better understanding of the development of cestodes routes and may also assist in the determination of new therapeutic targets for the treatment of cestodiasis.
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Identificação de genes candidatos relacionados a traços de desempenho em transcriptomas do camarão marinho Litopenaeus vannamei (Penaeidae, Decapoda)Santos, Camilla Alves 28 November 2016 (has links)
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Previous issue date: 2016-11-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The present work had as general objective to perform the genomic annotation of Expressed Sequences
(ESTs) of Litopenaeus vannamei shrimp, available in the database of Project ShEST and to evaluate the
polymorphism of mined SSR and SNP tags. These markers were located in the main chain of protein genes
with function related to performance traits and were validated in SPF (Specific Pathogen Free) shrimp
families submitted to selection for rapid growth and survival. In addition to the EST-SSR and EST-SNP loci,
obtained by Sanger sequencing, Next Generation Sequencing (NGS) analyzes were included in the initial
proposal of work with the objective of expanding the set of SNPs available and verifying the differential gene
expression. The new assembly of ESTs was performed and produced a set of 2.984 unigenes with protein
products for 41% of them, with 1.983 SSRs and 3.472 SNPs being identified. Among the loci with gene
product identified, 231 were enzymes with 127 unique EC numbers inserted in 94 KEGG metabolic pathways.
Loci validation showed that the loci of the 60S ribosomal (SSR-EST) and crustacyanin (SNP-EST) proteins were
polymorphic in the animals sampled from Genearch. Statistical analyzes were conducted to verify the
existence of a possible association between the genotypes and the analyzed weight phenotypes, although
no association was observed. In addition, cross-species amplification tests were performed on seven species
of marine and two freshwater prawns, demonstrating successful transferability for these species. The RNAseq
approach was included in the present work with the purpose of increasing the number of SNPs detected
in candidate genes with performance-related function and identifying differentially expressed (DE) genes in
animals under experimental conditions. A second transcriptome was assembled from the muscle and
hepatopancreas tissues of L. vannamei individuals (i) evaluated for rapid growth and survival and (ii) exposed
to the White Spot Syndrome Virus (WSSV). A total of 63.105 transcripts were generated, with an average
size of 2.511 bp and N50 of 3.464 bp. More than 15.500 SNPs were identified (frequency > 50%). Functional
annotation was also performed on the bases of SwissProt, Gene Ontology (GO) and KEGG. Differential gene
expression analyzes were performed on the animal samples evaluated for growth and response to WSSV
infection. The data generated showed differences in the expression profile between the genes of (i) high and
low growth animals, (ii) the hepatopancreas and muscle and (iii) the uninfected (healthy) and infected (ill)
animals by WSSV, considering the effect of the tissue. Two-hundred and seven DE genes were identified for
growth, 5.816 for hepatopancreas and muscle and 1.017 for ill and healthy animals. / O presente trabalho teve como objetivo geral realizar a anotação genômica de sequências expressas (ESTs)
de Litopenaeus vannamei, disponíveis no banco de dados do Projeto ShEST e avaliar o polimorfismo de
marcas SSR e SNP mineradas. Esses marcadores estavam localizados na cadeia principal de genes de
proteínas com função relacionada a traços de desempenho e foram validados em famílias de camarões SPF
(Specific Pathogen Free) submetidas à seleção para rápido crescimento e sobrevivência. Adicionalmente aos
locos SSR-EST e SNP-EST, obtidos por sequenciamento Sanger, análises de Sequenciamento de Próxima
Geração (NGS) foram incluídas na proposta inicial de trabalho com o objetivo de ampliar o conjunto de SNPs
disponíveis e verificar a expressão gênica diferencial. A montagem de novo das ESTs foi realizada e produziu
um conjunto de 2.984 unigenes com produtos proteicos para 41% destes, sendo identificados 1.983 SSRs e
3.472 SNPs. Dentre os locos com produto gênico identificado, 231 eram enzimas com 127 EC numbers únicos
inseridos em 94 vias metabólicas do KEGG. A validação dos locos SSR-EST e SNP-EST mostrou que os locos
das proteínas 60S ribossomal (SSR-EST) e crustacianina (SNP-EST) apresentaram-se polimórficos nos animais
amostrados da Genearch. Análises estatísticas foram conduzidas para verificação da existência de uma
possível associação entre os genótipos e os fenótipos de peso analisados, embora não tenha sido observada
associação. Além disso, testes de amplificação heteróloga foram realizados em sete espécies de camarões
marinhos e duas de água doce, demonstrando sucesso na transferabilidade para estas espécies. A
abordagem de RNA-seq foi incluída no presente trabalho com o propósito de ampliar o número de SNPs
detectados em genes candidatos com função relacionada a traços de desempenho e identificar genes
diferentemente expressos (DE) em animais sob condições experimentais. Foi realizada a montagem de novo
de um segundo transcriptoma, dos tecidos músculo e hepatopâncreas de indivíduos de L. vannamei (i)
avaliados para rápido crescimento e sobrevivência e (ii) expostos ao vírus da Síndrome da Mancha Branca ou
White Spot Syndrome Virus (WSSV). Foram gerados 63.105 transcritos, com tamanho médio de 2.511 pb e
N50 de 3.464 pb. Foram identificados mais de 15.500 SNPs (frequência > 50%). Também foi realizada a
anotação funcional nas bases do SwissProt, Gene Ontology (GO) e KEGG. Análises de expressão diferencial
gênica foram realizadas nas amostras dos animais avaliados para crescimento e resposta a infecção pelo
WSSV. Os dados gerados demonstraram diferenças no perfil de expressão entre os genes (i) de animais de
alto e baixo crescimento, (ii) do hepatopâncreas e músculo e (iii) dos animais não-infectados (saudáveis) e
infectados (doentes) pelo WSSV, considerando-se o efeito do tecido. Foram identificados 207 genes DE para
crescimento, 5.816 para a comparação entre os tecidos e 1.017 para os animais doentes e saudáveis. / FAPESP: 2012/13069- 6 / FAPESP: 2012/17322-8
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Estudo da expressão das proteínas cromodomínio-helicase e SET/TAF-Iβ durante o processo de estrobilização de Mesocestoides cortiCosta, Caroline Borges January 2013 (has links)
A cromodomínio-helicase (CHD) e a SET/TAF-Iβ (chaperona de histonas) são proteínas conhecidas por estarem envolvidas em processos de remodelagem de cromatina e controle da expressão gênica. Em organismos como Caenorhabditis elegans, Drosophila melanogaster, camundongo e o homem, estas proteínas estão associadas ao controle de vários processos de desenvolvimento. Em Mesocestoides corti, um modelo de parasito cestódeo, sequências relacionadas à CHD e à SET/TAF-Iβ foram identificadas em uma seleção de genes diferencialmente expressos em larvas e vermes estrobilizados. Visando à identificação de marcadores moleculares de processos e estágios de desenvolvimento da Classe Cestoda, os padrões de expressão de ortólogos das proteínas CHD e SET/TAF-Iβ (McCHD e McSET/TAF) em M. corti foram investigados. Inicialmente as sequências codificadoras da McCHD e McSET/TAF foram amplificadas por RT-PCR, clonadas em vetor de expressão modificado (pGEX-TEV) e expressas em Escherichia coli para produção de proteínas recombinantes. Análises por imunoblot e imuno-histoquímica foram realizadas utilizando anticorpos gerados contra versões recombinantes das proteínas-alvo do estudo. Foram analisados quatro estágios de desenvolvimento de M. corti: larvas (tetratirídeos, TT), TT após 24h de indução ao processo de estrobilização (o qual confere o desenvolvimento da larva em adulto) (24h-Ind), TT após 72h de indução (72h-PI) e vermes estrobilizados (VE). Em imunoblots, a McCHD apresentou altos níveis de expressão em três estágios de desenvolvimento de M. corti: TT, 72h-PI e VE, sendo detectado um menor nível de expressão no estágio 24h-Ind quando comparado aos demais. Já a McSET/TAF apresentou um aumento gradual no seu nível de expressão após a indução (nos estágios de 24h-Ind, 72h-PI e VE), não sendo detectada em TT. Em secções longitudinais foram observados um maior nível de expressão da McCHD em estágios iniciais de desenvolvimento (TT e 24- Ind) enquanto que McSET/TAF foi observado um maior nível de expressão nos estágios intermediários e no final do desenvolvimento de M. corti. Para ambas as proteínas a localização foi citoplasmática e não houve diferenças de distribuição nos tecidos analisados. Para compreender melhor as diferenças encontradas nos níveis de expressão das proteínas, análises por PCR em tempo real (RT-qPCR) foram realizadas para quantificação dos níveis de transcritos dos genes McCHD e McSET/TAF. Foram encontradas diferenças significativas nos níveis de expressão gênica de McCHD e McSET/TAF somente em dois estágios: o inicial (TT) e o adulto (VE), conforme análises pelo teste de Duncan. Níveis maiores de expressão gênica de ambos os genes foram encontrados no último estágio de desenvolvimento (VE) de M. corti. A caracterização de genes e proteínas envolvidas no processo de estrobilização de platelmintos da classe Cestoda contribuirá para o melhor entendimento de rotas do desenvolvimento de cestódeos, podendo auxiliar também na determinação de novos alvos terapêuticos para o tratamento de cestodíases. / Chromodomain-helicase (CHD) and the SET/TAF-Iβ histone chaperone are proteins known to be implicated in processes of chromatin remodeling and regulation of gene expression associated with the control of developmental processes in different organisms, such as Caenorhabditis elegans, Drosophila melanogaster, mouse and human. In Mesocestoides corti, a model cestode parasite, CHD and SET/TAF-Iβ related sequences were isolated in a screening for genes differentially expressed in larvae (tetrathyridia) and adult worms. Aiming at identifying molecular markers of processes and stages of development of Class Cestoda, the expression patterns of M. corti CHD and SET/TAF-Iβ orthologous proteins (McCHD and McSET/TAF) were investigated. Initially, the coding sequences of McCHD and McSET/TAF were amplified by RT-PCR, cloned into a modified expression vector (pGEX-TEV) and expressed in Escherichia coli for recombinant proteins production. Analysis by immunoblotting and immunohistochemistry was performed using antibodies raised against recombinant versions of the proteins target the study. We analyzed four developmental stages of M. corti: bona fide tetrathyridia (TT), tetrathyridia 24 h after strobilation induction (24h-Ind), strobilating worms 72 h after induction (72h-PI), and fully strobilated worms (VE). Immunoblots showed high levels of expression McCHD in three developmental stages of M. corti: TT, 72h-PI e VE, and detected a lower level of expression in stage 24-Ind in comparison to others. The McSET/TAF showed a gradual increase in their level of expression after induction (stages of 24h-Ind, 72h- PI e VE) was not detected in TT. In longitudinal sections were observed an increased level of expression of McCHD in early stages of development (TT and 24-Ind) while McSET/TAF there was a higher level of expression in the intermediate and late stages of development of M. corti. Both McCHD and McSET/TAF showed a cytoplasmic location and a uniform pattern of immunostaining, no differences in distribution between the tissues. For a better understanding the differences in levels of protein expression, analysis by real-time PCR (RT-qPCR) was performed to quantify the transcript levels of genes McCHD and McSET/TAF. There were significant differences in levels of gene expression McCHD and McSET/TAF only in two stages: the initial (TT) and adult (VE), as analysis by Duncan test. Higher levels of gene expression of both genes were found in the last stage of development (VE) of M. corti. The characterization of genes and proteins involved in the process of strobilation flatworms of the class Cestoda contribute to a better understanding of the development of cestodes routes and may also assist in the determination of new therapeutic targets for the treatment of cestodiasis.
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