A banana tem grande importância econômica, é a fruta mais produzida no mundo, e o Brasil é o segundo maior produtor. É uma fruta altamente perecível, de rápida maturação, sensível a choques mecânicos, suscetível a descoloração, ao amolecimento excessivo e a patógenos na fase pós-colheita. As mudanças ocorridas durante o amadurecimento levam a uma vida de prateleira muito reduzida e são dependentes da expressão de diversas proteínas. Portanto, a identificação de proteínas associadas com essas modificações pode contribuir para a melhor compreensão da regulação do amadurecimento e auxiliar no aprimoramento das estratégias de conservação pós-colheita e melhoria da qualidade dessa fruta. Através da análise proteômica diferencial podem ser identificadas proteínas com variação de abundância durante o amadurecimento e que estejam envolvidas nesse processo. O presente trabalho teve por objetivo comparar os mapas protéicos de polpa de banana (Musa acuminata cv. nanicão) nas fases pré-climatérica e climatérica, e a identificar spots de proteínas que diferem em abundância nesses dois estádios, através da eletroforese bidimensional acoplada à espectrometria de massas. Neste trabalho foram utilizadas três amostragens distintas de frutas, de modo a minimizar o efeito da variabilidade biológica, não relacionada com o amadurecimento. Para a obtenção dos perfis protéicos foi utilizada a metodologia 2D-DIGE. Os géis obtidos foram analisados com o software PDQuest e para a análise estatística foi utilizado o teste T. Chegou-se a uma lista de 50 spots que foram recortados dos géis, sendo as proteínas digeridas e seqüenciadas por espectrometria de massas. Destas proteínas, 26 tiveram a provável identidade apontada pela comparação com o banco de dados MSDB, empregando o software Mascot. A maioria das proteínas identificadas apresenta provável função durante o amadurecimento da banana e podem estar relacionadas com processos bioquímicos relacionados com a qualidade da fruta. / Banana has great economic importance, is the most widely produced fruit in the world, and Brazil is the second largest producer. It is a highly perishable fruit, ripening fast, sensitive to mechanical shock, susceptible to discoloration, excessive softening and pathogens in the post-harvest. The changes during ripening leads to a very limited shelf life and are dependent on the expression of several proteins. Therefore, the identification of proteins associated with these modifications may contribute to better understanding the regulation of maturation and help to improve strategies for post-harvest preservation and improvement of this fruit. Through differential proteomics analysis could be identified proteins with a range of abundance during ripening and that could be involved in this process. This study aimed to compare the protein maps of banana pulp (Musa acuminata cv. nanicão) in pre-climacteric and climacteric phases, and identify protein spots that differ in abundance in these two stages by two-dimensional electrophoresis coupled to mass spectrometry. In this study we used three different fruits samples, to minimize the effect of biological variability, non-ripening related. To obtain the protein profiles was used 2D-DIGE methodology. The gels were analyzed with the PDQuest software and Student´s t-Test was used to statistical analysis. A list of 50 spots differential acumulated were detected and then were extracted of gels, protein digested and sequenced by mass spectrometry. Of these proteins, 26 had the probable identity indicated by comparison with the MSDB database, using Mascot software. Most of the identified proteins has probable function during banana ripening and may be related to biochemical processes related to fruit quality.

A banana tem grande importância econômica, é a fruta mais produzida no mundo, e o Brasil é o segundo maior produtor. É uma fruta altamente perecível, de rápida maturação, sensível a choques mecânicos, suscetível a descoloração, ao amolecimento excessivo e a patógenos na fase pós-colheita. As mudanças ocorridas durante o amadurecimento levam a uma vida de prateleira muito reduzida e são dependentes da expressão de diversas proteínas. Portanto, a identificação de proteínas associadas com essas modificações pode contribuir para a melhor compreensão da regulação do amadurecimento e auxiliar no aprimoramento das estratégias de conservação pós-colheita e melhoria da qualidade dessa fruta. Através da análise proteômica diferencial podem ser identificadas proteínas com variação de abundância durante o amadurecimento e que estejam envolvidas nesse processo. O presente trabalho teve por objetivo comparar os mapas protéicos de polpa de banana (Musa acuminata cv. nanicão) nas fases pré-climatérica e climatérica, e a identificar spots de proteínas que diferem em abundância nesses dois estádios, através da eletroforese bidimensional acoplada à espectrometria de massas. Neste trabalho foram utilizadas três amostragens distintas de frutas, de modo a minimizar o efeito da variabilidade biológica, não relacionada com o amadurecimento. Para a obtenção dos perfis protéicos foi utilizada a metodologia 2D-DIGE. Os géis obtidos foram analisados com o software PDQuest e para a análise estatística foi utilizado o teste T. Chegou-se a uma lista de 50 spots que foram recortados dos géis, sendo as proteínas digeridas e seqüenciadas por espectrometria de massas. Destas proteínas, 26 tiveram a provável identidade apontada pela comparação com o banco de dados MSDB, empregando o software Mascot. A maioria das proteínas identificadas apresenta provável função durante o amadurecimento da banana e podem estar relacionadas com processos bioquímicos relacionados com a qualidade da fruta. / Banana has great economic importance, is the most widely produced fruit in the world, and Brazil is the second largest producer. It is a highly perishable fruit, ripening fast, sensitive to mechanical shock, susceptible to discoloration, excessive softening and pathogens in the post-harvest. The changes during ripening leads to a very limited shelf life and are dependent on the expression of several proteins. Therefore, the identification of proteins associated with these modifications may contribute to better understanding the regulation of maturation and help to improve strategies for post-harvest preservation and improvement of this fruit. Through differential proteomics analysis could be identified proteins with a range of abundance during ripening and that could be involved in this process. This study aimed to compare the protein maps of banana pulp (Musa acuminata cv. nanicão) in pre-climacteric and climacteric phases, and identify protein spots that differ in abundance in these two stages by two-dimensional electrophoresis coupled to mass spectrometry. In this study we used three different fruits samples, to minimize the effect of biological variability, non-ripening related. To obtain the protein profiles was used 2D-DIGE methodology. The gels were analyzed with the PDQuest software and Student´s t-Test was used to statistical analysis. A list of 50 spots differential acumulated were detected and then were extracted of gels, protein digested and sequenced by mass spectrometry. Of these proteins, 26 had the probable identity indicated by comparison with the MSDB database, using Mascot software. Most of the identified proteins has probable function during banana ripening and may be related to biochemical processes related to fruit quality.

[Truncated abstract] Clinical therapeutic agents to directly inhibit ischaemic neuronal death are presently unavailable. One approach to developing therapeutics is based upon the identification of proteins up-regulated by 'preconditioning', a natural adaptive response utilised by the neural cells to counter damaging insults, such as ischaemia. Thus, my project aimed to firstly identify proteins differentially expressed following erythropoietin (EPO) mediated neuronal preconditioning and secondly to assess whether any of these proteins possessed neuroprotective activity using in vitro ischaemia like models. To achieve the first aim, it was shown that in vitro neuronal EPO preconditioning could: (i) induce cell signal changes in neuronal cultures, (ii) protect neurons against in vitro ischaemia and (iii) induce differential protein expression. Overall, 40 differentially expressed proteins were identified in cortical neuronal cultures following EPO preconditioning. In order to investigate the neuroprotective or neurodamaging activity of proteins induced by EPO preconditioning I developed an adenoviral expression system for use in neuronal cultures. To this end, I assessed the suitability of four promoters (cytomegalovirus [CMV], rous sarcoma virus [RSV], human synapsin 1 [hSYN1], rat synapsin 1 [rSYN1]) previously used to express proteins in neuronal cultures and demonstrated the superiority of the RSV promoter for this purpose. ... Finally, in order to validate this adenoviral expression system, I over-expressed the anti-apoptotic protein Bcl-XL in neuronal cultures and subsequently confirmed its neuroprotective activity in the in vitro ischaemia and oxidative stress models used in my project. Using this adenoviral vector system and the in vitro oxidative stress model I assessed a number of proteins up-regulated by EPO preconditioning. The results of this preliminary study indicated that cyclophilin A (CyPA), peroxiredoxin 2 (PRDX2) and superoxide dismutase 1 (SOD1) over-expression were neuroprotective. It was subsequently verified that adenoviral mediated over-expression of CyPA and PRDX2, v but not SOD1 in cortical neuronal cultures could protect neurons from in vitro ischaemia. I also confirmed that CyPA mRNA increased in the rat hippocampus in response to 3 minutes of global cerebral ischaemia. Interestingly, an increase in CyPA, PRDX2 or SOD1 protein was not observed in the same experimental paradigm. To investigate CyPA's mode of action I confirmed that cultured neurons, but not astrocytes, express the CyPA receptor, CD147. It was also demonstrated that administration of exogenous CyPA protein to neuronal cultures could protect neurons against oxidative and ischaemic injury. I further demonstrated that exogenous administration of CyPA induces a rapid and transient activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway in neuronal cultures. From this observation, I have proposed that the extracellular mediated neuroprotective activity of CyPA occurs via CD147 receptor signalling and activation of ERK1/2 pro-survival pathways. Based on the findings reported in this thesis, the neuroprotective activities of PRDX2 and CyPA warrant further investigation as targets for the development of new therapies to treat cerebral ischaemia.