Browsing by Author "Jorge, Lurdes"
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- Aplicação da bioinformática na deteção de fraudes alimentaresPublication . Jorge, LurdesA partir de sequências nucleotidicas do genoma milocondrial de espécies animais existentes na base de dados da EMBL (hUp:l/www.ebLac.ukl), pretende-se delinear uma estratégia de identificação de espécies presentes em alimentos.
- Bioinformática como suporte às biociênciasPublication . Jorge, LurdesNas últimas décadas os progressos verificados a nível da biologia molecular e das tecnologias do DNA (nomeadamente a utilização da PCR-"Polymerase Chain Reaction" e dos processos de sequenciação automática, de ESTs-"Expressed Sequence Tags" e de SNPs-"Single Nucleotide Polymorphisms") levaram ao crescimento exponencial da informação biológica produzida pela comunidade científica. Para tal contribuiu muito o aparecimento de inúmeros projectos de sequenciação de genomas, sobretudo o ''Human Genom Project'', com início em 1990. No ano de 1996 existiam cerca de 280 000 ESTs nas bases de dados (1). Esta avalanche de informação teve de ser armazenada e organizada. No ano de 1998 as três grandes bases de dados de sequências nucleotídicas existentes (no Japão, Reino Unido e Estados Unidos da América, respectivamente "DNA Database of Japan": DDBJ, "European Molecular Biology Laboratory": EMBL, e GenBank) estabelecem um protocolo de colaboração, criando o "International Nucleotide Sequence Database" (INSD). Em cada uma delas se podem anotar novas sequências nucleotídicas decorrentes de trabalhos de investigação, ou corrigir as pré-existentes. Todas as correcções e novas entradas são partilhadas diariamente entre as três bases de dados, pelo que em todas elas a informação disponível é a mesma, embora com algumas diferenças de formato. O acesso aos dados é livre.
- A bioinformática na identificação de genesPublication . Vaz, Madalena; Jorge, LurdesCom o objectivo de identificar genes, avaliaram-se nove EST (“Expressed Sequence Tag”) de uma biblioteca de cDNA de Trichoderma harzianum. Numa primeira etapa, as ESTs foram sequenciadas, sendo as sequências analisadas com programas bioinformáticos de análise de similaridade em bancos de dados, utilizando ferramentas como Blast P e Fasta X. Identificaram-se duas ESTs que se enquadravam nos objectivos, sendo uma delas a EST-1279, usada para a completa elucidação do gene lip2 de Trichoderma harzianum, cuja sequência pode ser acedida na base de dados com os códigos de acesso AM774154. O gene lip2 de Trichoderma harzianum sequenciado apresenta 1992 pb, sendo a sua ORF (“open reading frame”) constituída por 1215 nucleótidos. Estão sequenciados 550 pb da região do promotor e 229 pb do terminador.
- Caracterização de genes através de recursos bioinformáticosPublication . Jorge, Lurdes; Vaz, MadalenaApós descodificação da fase de leitura aberta de um gene, uma série de ferramentas bioinformáticas podem ser utilizadas para a caracterização da sequência deduzida da proteína. Uma pesquisa no website do Expasy Proteomics Server (http://expasy.org/tools) e uma sequência nucleotídica permitem-nos identificar e caracterizar proteínas, identificar motivos, padrões e perfis, inferir a sua estabilidade, localização celular ou função, fazer as predições das estruturas secundária e terciária, procurar sequências similares depositadas em bases de dados e compará-las, estabelecer relações filogenéticas. Neste caso usámos a ORF ("open reading frame") do gene lip2 de Trichoderma harzianum, cuja sequência pode ser acedida na base de dados da EMBL com o número de acesso AM774154.1. A proteína codificada por lip2 tem 404 aminoácidos, massa molecular calculada de 44604,3 Dalton e ponto isoeléctrico global calculado de 5,0. É considerada estável. Uma pesquisa efectuada na base integrada InterproScan incluiu lip2 na família das lipases com serina no centro activo (PROSITE PS00120, posição 210-219), e na das lipases_classe3 (Pfam PF01746, posição 131-190) com uma probabilidade de 1,4.e-31. http://www.ebi.ac.uk/Tools/pfa/iprscan/ A sua estrutura primária está de acordo com o consenso G-x-S-x-G, descrito como centro activo de lipases, inserido na sequência consenso de lipases com serina no centro activo: [LIV]-{KG}-[LIVFY]-[LIVMST]-G-[HYWV]-S-{YAG}-G-[GSTAC]. Os primeiros 25 aminoácidos constituem uma sequência sinal, que sugere a entrada desta proteína no retículo endoplasmático, e uma localização extracelular. No que respeita a modificações pós-traducionais, entre outras, a proteína codificada por lip2 apresenta três possíveis sítios de N-glicosilação, quatro sítios prováveis de N-miristoilação, um de palmitoilação e 28 sítios potenciais de fosforilação. A palmitoilação aumenta a superficie hidrofóbica e a afinidade para os substratos e desempenha um papel importante no transporte de proteína.
- Caracterização de isolamentos portugueses de Albugo candida (Pers.) KuntzePublication . Jorge, LurdesEste trabalho é uma primeira tentativa de caracterização de isolamentos portugueses de Albugo candida (Pers.) Kuntze, causador da ferrugem branca das crucíferas. Dos treze isolamentos iniciais colhidos em campo (cinco em Brassica oleracea L., sete em B. rapa L. e um em Raphanus sativus L.), obtiveram-se os respectivos isolamentos de pústula única (IPU), e procedeu-se a cruzamentos entre IPU provenientes de hospedeiros homólogos para determinação de sistemas de compatibilidade sexual. À excepção de dois IPU provenientes de B. oleracea, Ac 501 e Ac 502, que parecem enquadrar-se num processo de homotalismo secundário, todos os outros são heterotálicos. Os IPU provenientes de B. rapa estudados pertencem todos a um mesmo grupo de emparelhamento ("mating type"). Nos IPU provenientes de B. oleracea foram observados dois grupos de emparelhamento: i) com Ac 501, Ac 502 e Ac 504; e ii) com Ac 503 e Ac 505. Foram avaliadas classes de interacção fenotípica de quarenta amostras de B. oleracea (pertencentes a diferentes variedades botânicas) e oito dos isolamentos iniciais, a nível cotiledonar. O grau de susceptibilidade das amostras aos três isolamentos homólogos testados foi variável. Algumas revelaram susceptibilidade a infecções cruzadas, nomeadamente a couve lombarda "Brusselse Winter", em que mais de 50% dos indivíduos se revelaram susceptíveis aos quatro isolamentos de B. rapa testados.
- Characterization of ligninolytic enzymes and metabolic profile of Cryphonectria parasitica and the isogenic converted strains by CHV1 hypovirusPublication . Abdellaziz, Omar; Jorge, Lurdes; Moura, Luísa; Coelho, Valentim; Gouveia, Maria EugéniaCryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria Hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By this characteristics the mycovirus CHV1 is used in Europe as a biological control agent of Chestnut Blight.The aim of this project is to better understand the effect of the mycovirus on the fungi pathogenicity by comparing the production of some lignin degrading enzymes and the metabolic profiles of some virulent and hypovirulent (converted and original) strains. For qualitative evaluation, several different compounds have been used as indicators for ligninolytic enzymes production. For quantitative evaluation, among 9 strains 5 were chosen for biological tests and cultivation in minimal liquid media and the amount of enzyme produced were analysed. Virulent strains were found to cause more damage in chestnut branches and to produce more lignin degrading enzymes. In apple fruits, some CHV1 strains produced bigger rot lesions than wild type strains did. In parallel, Biolog FF MicroPlates have been used for the first time with Cryphonectria parasitica to assess their metabolic profiles by the utilization of 95 different carbon sources. Carbohydrates, amino acids, amines/amides, miscellaneous and polymers were found to be more consumed by hypovirulent strains; therefore, this may suggest. an adaptation in this fungal strains ecology and field fitness.
- Characterization of ligninolytic enzymes and metabolic profile of cryphonectria parasitica and the isogenic converted strains by CHV1 hypovirusPublication . Abdellaziz, Omar; Jorge, Lurdes; Moura, Luísa; Coelho, Valentim; Gouveia, Maria EugéniaCryphonectria parasitica, the causal agent of chestnut blight, causes necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees. Cryphonectria Hypovirus 1 (CHV1) infects C. parasitica and reduces the fungus virulence (hypovirulence) and alters the fungus morphology in culture (pigmentation and sporulation capacity). By this characteristics the mycovirus CHV1 is used in Europe as a biological control agent of Chestnut Blight.The aim of this project is to better understand the effect of the mycovirus on the fungi pathogenicity by comparing the production of some lignin degrading enzymes and the metabolic profiles of some virulent and hypovirulent (converted and original) strains. For qualitative evaluation, several different compounds have been used as indicators for ligninolytic enzymes production. For quantitative evaluation, among 9 strains 5 were chosen for biological tests and cultivation in minimal liquid media and the amount of enzyme produced were analysed. Virulent strains were found to cause more damage in chestnut branches and to produce more lignin degrading enzymes. In apple fruits, some CHV1 strains produced bigger rot lesions than wild type strains did. In parallel, Biolog FF MicroPlates have been used for the first time with Cryphonectria parasitica to assess their metabolic profiles by the utilization of 95 different carbon sources. Carbohydrates, amino acids, amines/amides, miscellaneous and polymers were found to be more consumed by hypovirulent strains; therefore, this may suggest. an adaptation in this fungal strains ecology and field fitness.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species. Associated with this oomycete is the ink disease of Castanea sativa Mill. Glucan endo-1,3-β-D-glucosidase catalyzes the hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defence by digesting wall components of the fungal pathogen. In oomycetes, glucanases have been studied at biochemical level for their possible role in hyphal tip growth and branching, where there is thought to be a delicate balance between the cell wall synthesis and hydrolysis. Fungal cell wall degrading enzyme production is influenced by a number of factors including the type of strain, the culture conditions and substrate type. The aim of this work was the analysis of homologous expression, in P. cinnamomi, and heterologous expression, in Pichia pastoris, of the endo-1,3-β-D-glucosidase encoding gene ENDO1 produced by P. cinnamomi. The expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production. Different plasmids were used to clone the gene on each organism and we used RT-PCR analysis to examine its expression. The major expression levels occurred at the medium with glucose as carbon source. These and other results will be presented.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is a soil-borne pseudofungus belonging to the Class Oomycetes or „water moulds' in the Kingdom Chromista (Figure 1). Is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species, as well as of some 900 other woody perennial plant species Associated with this oomycete is the ink disease of Castanea Sativa Mill. Glucan endo-1,3-β-D-glucosidase (EC 3.2.1.39) catalyzes de hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defense by digesting wall components of the fungal pathogen. In yeast, 1,3-- glucanases have been studied for their role in germination, sporulation, mating and cell growth since they are regulated in cell cycle dependent manner, and are differentially expressed during vegetative growth, mating and the late stages of sporulating diploid. In plant, 1,3--glucanases have been characterized for their major role in plant defence, as well as for their involvement in germination, microsporogenesis and embryogenesis. In oomycetes, glucanases have been studied on a biochemical level for their possible role in hyphal tip growth and branching where there is thought to be a delicate balance between cell wall synthesis and hydrolyses. In the present work, we obtained a fragment with 1231bp of the endo-1,3--glucanase gene by standard PCR, using conserved primers and the whole genomic sequence with 2586 bp was obtained by amplifying the previous sequence by asymmetric PCR. The gene expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production.
- Effects of Cryphonectria parasitica infection by the hypovirus CHV1 on cellulase activityPublication . Tayechi, Yosri; Jorge, Lurdes; Coelho, Valentim; Gouveia, Maria EugéniaCryphonectria parasitica is the causal agent of Castanea sativa chestnut blight, an economically important disease. Hypovirulence is due to the presence of a virus, the Cryphonectria hypovirus 1 (CHV1), that attenuates fungus pathogenicity by reducing the sporulation, and the enzymatic activity of pathogenesis-related enzymes. In several regions affected by C. parasitica, the release of C. parasitica hypovirulent strains on chestnut blight affected trees of C. sativa, has been used as a biological control. The objective of this work is to evaluate the effect of hypovirulence by CHV1 on the activity of cellulase, a pathogenesis-related enzyme, in different isolates of C. parasitica. For this, several virulent and hypovirulent (donors: RBB111, SR442, Serra05; and converted: Cast13RBB111, Cast13SR442, Curopos15SR442) isolates were grown in appropriate microbiological media, containing Carboxymethyl cellulose (CMC) and the enzyme activity was qualitatively evaluated. Contrary to expected, the hypovirulent donors (RBB111, SR442, Serra05) and converted strains showed higher cellulolytic activity than the virulent ones, evidencing higher ratio halo/growth (RHG). In the two converted Cast13 isogenic isolates, Cast13 RBB111 and Cast13 SR442 the obtained RHG were significantly different (p< 0.01), the last presented higher cellulolytic activity. In virulent strains, only Cp33PAR presented a small RHG, significantly different from the other virulent strains (p<0.05), without reaction.