Caractérisation des mécanismes d’action du microARN 169 chez Arabidopsis thaliana

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Cinética de inibição do etanol e consumo de açúcar, em reator biológico, usando resíduos industriais de citrinos e de alfarroba. Aumento de escala de produção em unidade-piloto

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2013

Development of chitosan packaging films with carvacrol and grape seed extract as antimicrobial and antioxidant agents

In order to produce packaging films with a broad spectrum of action on microorganisms, the effect of two antimicrobial (AM) to be included in the films, carvacrol and GSE were studied separately on different microorganisms. Carvacrol was more effective against the grampositive bacteria than against the gram-negative bacterium. GSE was not effective against yeast. Subsequently, a search for optimal combinations of carvacrol, GSE and the addition of chitosan (as a third component with film forming properties) was carried out. Response surface analysis showed several synergetic effects and three optimal AM combinations (OAMC) were obtained for each microorganism. The experimental validation confirmed that the optimal solutions found can successfully predict the response for each microorganism. The optimization of mixtures of the three components, but this time, using the same concentration for all microorganisms, was also studied to obtain an OAMC with wide spectrum of activity. The results of the response surface analysis showed several synergistic effects for all microorganisms. Three OAMC, OAMC-1, OAMC-2, OAMC-3, were found to be the optimal mixtures for all microorganisms. The radical scavenging activity (RSA) of the different agents was then compared with a standard antioxidant (AOX) BHT, at different concentrations; as also at the OAMC. The RSA increased in the following order: chitosan

Crescimento e condição nutricional de larvas de sardinha em condições controladas

Com o objectivo de determinar o crescimento de larvas de sardinha foram realizadas experiências em condições controladas. Um cardume de sardinhas adultas foi mantido em cativeiro e a desova induzida por meios naturais (aumento da disponibilidade alimentar, regulação da temperatura para cerca de 15º C e salinidade para ~35‰). Quando ocorreram desovas com mais do que 500 ovos num determinado dia, estes foram colocados a eclodir em condições controladas. As larvas de sardinha apresentaram um desenvolvimento morfológico normal (p.e. pigmentação dos olhos e desenvolvimento da boca entre o 3º e 4º dia). Diferentes tipos e combinações de alimentos foram experimentados (nauplios de Acartia grani, rotíferos e Gymnodinium sp.) e analisou-se a condição nutricional das larvas amostradas. Foram efectuadas experiências de inanição para se poder calibrar os resultados obtidos. As taxas de crescimento variaram entre 0.12 e 0.36 mm.dia-1, sendo a mais elevada correspondente a uma experiência de inanição. Observou-se, através da histologia, que dietas ricas em Acartia grani são mais favoráveis para um melhor desenvolvimento larvar. As análises da razão RNA/DNA sugerem que há uma forte possibilidade de os efeitos parentais terem bastante influência no desenvolvimento e crescimento das larvas de sardinha nas duas primeiras semanas de vida, mas não na sua sobrevivência, que dependeu da quantidade e qualidade de alimento fornecida.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

Modeling the suitable habitat of a structural red gorgonian species in the Mediterranean/east Atlantic regions predicts a wider distribution than previously known

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Univerdade do Algarve, 2015

Azeitona de mesa natural da cultivar Maçanilha com baixo teor de cloreto de sódio

Dissertação de mestrado, Tecnologia dos Alimentos, Instituto Superior de Engenharia, Universidade do Algarve, 2014

Contribuição para o estudo de produção de azeitona de mesa da cultivar Cobrançosa: caraterização microbiológica

Dissertação de Mestrado, Tecnologia dos Alimentos, Instituto Superior de Engenharia, Universidade do Algarve, 2016

Differential expression in “rogue” paramutation in peas (Pisum sativum L.)

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Valorização integrada de resíduos e subprodutos da extração do azeite: extração e caraterização de compostos bioativos do bagaço de azeitona

Dissertação de Mestrado, Tecnologia de Alimentos, Instituto Superior de Engenharia, Universidade do Algarve, 2016