Browsing by Issue Date, starting with "2004-04"
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- Expression of YAP4 in Saccharomyces cerevisiae under osmotic stressPublication . Nevitt, Tracy; Pereira, Jorge; Azevedo, Dulce; Guerreiro, Paulo; Rodrigues-Pousada, ClaudinaYAP4, a member of the yeast activator protein (YAP) gene family, is induced in response to osmotic shock in the yeast Saccharomyces cerevisiae. The null mutant displays mild and moderate growth sensitivity at 0.4 M and 0.8 M NaCl respectively, a fact that led us to analyse YAP4 mRNA levels in the hog1 (high osmolarity glycerol) mutant. The data obtained show a complete abolition of YAP4 gene expression in this mutant, placing YAP4 under the HOG response pathway. YAP4 overexpression not only suppresses the osmosensitivity phenotype of the yap4 mutant but also relieves that of the hog1 mutant. Induction, under the conditions tested so far, requires the presence of the transcription factor Msn2p, but not of Msn4p, as YAP4 mRNA levels are depleted by at least 75% in the msn2 mutant. This result was further substantiated by the fact that full YAP4 induction requires the two more proximal stress response elements. Furthermore we find that GCY1, encoding a putative glycerol dehydrogenase, GPP2, encoding a NAD-dependent glycerol-3-phosphate phosphatase, and DCS2, a homologue to a decapping enzyme, have decreased mRNA levels in the yap4 -deleted strain. Our data point to a possible, as yet not entirely understood, role of the YAP4 in osmotic stress response.
- A aprendizagem por problemas na formação em tecnologias da saúde: um caso de inovação curricularPublication . Dias, Hermínia Brites; Chagas, IsabelA Aprendizagem por Problemas (APP) constitui actualmente uma metodologia educativa com crescente utilização não só em instituições de Ensino Superior como também nos níveis de escolaridade básico e secundário. Apesar da literatura da especialidade ser rica em obras centradas nesta metodologia, os dados de investigação continuam a ser contraditórios e insuficientes para fundamentar a sua implementação eficiente, o que cria uma lacuna que é pertinente preencher. Pretendeu-se com o presente estudo atingir os seguintes objectivos: 1) criar actividades de APP; 2) descrever o processo de integração dessas actividades no funcionamento de uma disciplina essencialmente transmissiva; 3) analisar os efeitos da integração das actividades de APP nos alunos; 4) caracterizar o ambiente de aprendizagem gerado. A natureza dos objectivos enunciados implicou um desenho de investigação em duas fases, uma de criação dos problemas e das actividades, seguida de outra fase de implementação e estudo das implicações educativas da APP. Seguiu-se uma metodologia essencialmente qualitativa baseada em estudo de caso. Os participantes foram os alunos de uma disciplina integrada no curso de Cardiopneumologia da responsabilidade da Escola Superior de Tecnologia da Saúde de Lisboa. A investigadora foi também a docente que implementou as actividades. Como métodos de recolha de dados seguiu-se a observação participante, a entrevista (semi-estruturada e informal), o questionário e a análise documental. A análise dos resultados permitiu identificar um conjunto de factores condicionantes da implementação da APP, assim como alguns dos seus efeitos no desenvolvimento de diferentes competências e nas atitudes dos alunos. Entre os resultados obtidos evidencia-se que a APP pode funcionar como metodologia alternativa à convencional para a aquisição de conhecimentos e que os alunos reconhecem a importância da APP para o desenvolvimento de aptidões relevantes para a futura prática profissional e para as exigências da sociedade actual.
- Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathwaysPublication . Ferreira, Bruno S.; Calado, Cecília; Keulen, Frederikvan; Fonseca, Luís P. P.; Cabral, Joaquim M.S.; R., Da Fonseca M. M.Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.
- Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strainPublication . Calado, Cecília; Ferreira, Bruno S.; R., Da Fonseca M. M.; Joaquim M. S., Cabral; Fonseca, Luís P. P.By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.