The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studies

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.

Controlo e análise do processo de produção de aguardente de medronho da Serra do Caldeirão

Dissertação de mest., Tecnologia dos Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2010

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed

Background: Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. Methods: 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. Results: Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 ± 1.6, Rt = 5.7 ± 1.4, Ho = 0.63 ± 0.19 and He = 0.69 ± 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. Conclusions: Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.

Estudo da evolução da microbiota em fermentações naturais de azeitona verde do Algarve

Disssertação de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2011

Yeast diversity in the Mediterranean strawberry tree (Arbutus unedo L.) fruits' fermentations

In the Mediterranean region the fruits of the strawberry tree (Arbutus unedo L.) may be fermented and distilled to produce a traditional beverage very much appreciated in Southern Europe. The aim of the present work was to study the diversity of the yeast population and the killer activity of the isolates identified as Saccharomyces cerevisiae, obtained during solid state industrial fermentations of the arbutus berries. The identification of the isolates was performed by the 5.8S rRNA-ITS region restriction analysis and by sequencing the D1/D2 region of the large subunit of the rRNA gene. At the start of the fermentations, various non-Saccharomyces species were detected including Aureobasidium pullulans, Dothichiza pithyophila, Dioszegia zsoltii, Hanseniaspora uvarum and yeasts belonging to the genera Metschnikowia, Cryptococcus and Rhodotorula. However, as the biological processes progressed the number of different species decreased with S. cerevisiae and Pichia membranaefaciens becoming dominant at advanced stages of the must fermentation that is characterized by high concentrations of ethanol. Forty three isolates identified as S. cerevisiae were tested for killer activity against two sensitive reference strains and Zygosaccharomyces bailii. Their killer sensitivity in relation to five killer referenced toxins (K2, K5, K8, K9 and K10) was also studied. Out of the isolates analyzed, 95.3% were sensitive and 4.7% were tolerant against the killer toxins tested. Only three isolates revealed killer activity against one sensitive strain and two of them against the spoiler yeast Z. bailii. The microbiota obtained revealed an interesting potential to be used as starter cultures to overcome unpredictable uncontrolled fermentations of the arbutus fruits as well as in other applications of biotechnological interest. (C) 2012 Elsevier Ltd. All rights reserved.

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