Phosphoglucose isomerase variability of Cerastoderma glaucum as a model for testing the influence of environmental conditions and dispersal patterns through quantitative ecology approaches

Extreme conditions of coastal lagoons could directly modify the genetic patterns of species. The aim of this work was to investigate the influence of environmental conditions and small scale dispersal patterns on the phosphoglucose isomerase (PGI*) genetic variability of Cerastoderma glaucum from the Mar Menor coastal lagoon. For this purpose, 284 cockles were collected around the perimeter of the lagoon. Vertical polyacrylamide gel electrophoresis was used to scan for PGI* polymorphisms, giving a total of seven alleles. The spatial genetic distribution of the PGI* variability, which seems to be marked by the main circulation in the lagoon, discriminates four hydrological basins. In the central basin, a gradient of allelic composition reflects the circulation forced by the dominant winds and the main channel communicated to the open sea. This result is well supported by the salinity GAM model that defines this gradient. The other three basins are defined by the distribution of fine sand in a more complex model that tries to explain the isolation of the three sites localized inside these basins. The southern, western and northern basins show the lowest degree of interconnection and are considered the most confined areas of the Mar Menor lagoon. This situation agrees with the confinement theory for benthic assemblages in the lagoon. The greater degree of differentiation seen in the Isla del Ciervo population is probably due to recent human intervention on the nearby Marchamalo channel, which has been drained in recent years thus altering the influence of the Mediterranean Sea on the southern basin.

Identificação de indicadores moleculares de bem-estar durante o cultivo de dourada (Sparus aurata, Linnaeus 1758), utilizando técnicas de proteómica

Dissertação de Mestrado, Biologia Marinha, Especialização em Biotecnologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Estudo de refolding oxidativo de proteínas

O folding oxidativo de proteínas consiste na formação de pontes dissulfureto intramoleculares envolvendo a oxidação de grupos tiol no sentido da criação de uma ligação entre duas cisteínas. Esta modificação postransducional é essencial para a estabilidade das proteínas, principalmente em proteínas secretadas para o meio extracelular. In vivo, o folding oxidativo ocorre no retículo endoplasmático e é assistido por uma série de proteínas que atuam como catalisadores. Estas reações em cadeia necessitam da presença de um aceitador final de eletrões. No presente trabalho foram estudadas duas vias que atuam no reticulo endoplasmático para o refolding oxidativo da proteína modelo Ribonuclease A: Uma via envolve a interação entre duas proteínas, a Endoplasmic Recticulum Oxireductase 1 (Ero1) e a Protein Disulfide Isomerase (PDI); A outra via envolve a interação da PDI com a Peroxiredoxin IV (PRDX4). Foi igualmente estudado o refolding oxidativo com uma enzima homóloga da PRDX4, a PRDX2, no sentido de compreender se existe especificidade na interação entre a PRDX4 e a PDI. O estudo do refolding oxidativo da Ribonuclease foi realizado in vitro e avaliado em géis SDS-PAGE-Tricina com o objetivo de verificar a diferença de mobilidades entre a Ribonuclease reduzida e oxidada no gel. Na via da PRDX4/PDI e PRDX2/PDI é necessária a introdução de Glucose e Glucose Oxidase, responsáveis pela produção de peróxido de hidrogénio que atua como aceitador final de eletrões desta via. Em todas as vias foi observado refolding oxidativo da RNase. Na via da Ero1/PDI este foi substancialmente mais rápido e ocorre, embora em muito menor grau, mesmo na ausência da PDI. Na via da PRDX4/PDI o refolding é mais lento e foi constatado que não existe especificidade da PRDX4 para a PDI visto que, na presença da PRDX2, os resultados foram semelhantes aos resultados obtidos com a PRDX4.

A proteomic study of the effects of nutrient restriction in utero on postnatal metabolism

It is widely recognized that protein restriction in utero may cause metabolic and endocrine adaptations, which may be of benefit to the neonate on a short-term basis but may cause adverse long-term conditions such as obesity, Type 2 diabetes, metabolic syndrome, hypertension and cardiovascular diseases. Adequate foetal and early post natal nutrient and energy supply is therefore essential for adult animal health, performance and life span. In this project it was investigated the progressive adaptations of the hepatic proteome in male mink offspring exposed to either a low protein (FL) or an adequate protein (FA) diet in utero fed either on a low protein (LP) or on an adequate (AP) diet from weaning until sexual maturity. Specifically, the aim was to determine the metabolic adaptations at selected phases of the animal’s first annual cycle and establish the metabolic priorities occurring during those phases. The three different morphological stages studied during the first year of development included, end of bone growth at 4 months of age, maximal fat accretion at 6 months of age and sexual maturity at 12 months of age. A reference proteome of mink liver coming from these different animal groups were generated using 2D electrophoresis coupled to MALDI-TOF analysis and the way in which dietary treatment affect their proteome was established. Approximately 330 proteins were detected in the mink liver proteome. A total of 27 comparisons were carried out between all different animal groups which resulted in 20 differentially expressed proteins. An extensive survey was conducted towards the characterization of these proteins including their subcellular localization, the biological processes in which they are involved and their molecular functions. This characterization allowed the identification of proteins in various processes including the glycolysis and fatty acid metabolism. The detailed analysis of the different dietary treatment animal groups was indicative of differences in metabolism and also to changes associated with development in mink.

Study of transcripts and reactive oxygen species involved in the defence responses of Quercus suber against Phytophthora cinnamomi

The present work has the merit of exploring an insight into the activation of defence genes of Quercus suber during response to infection by Phytophthora cinnamomi. Thus, cDNA-AFLP methodology was used to identify gene fragments differentially present in the mRNA profiles of host cells of micropropagated Q. suber plantlets roots infected with zoospores of P. cinnamomi at different post challenge time points. Six candidate genes were selected based on their interesting cDNA-AFLP expression patterns and homology to genes known to play a role in defence. These six genes encode a cinnamyl alcohol dehydrogenase 2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), thaumatin-like protein (QsTLP), chitinase (QsCHI) and a 1,3-beta glucanase (QsGLU). The current work has been successful in evaluation of the expression of these genes by qRT-PCR. Data analysis revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the early hours of inoculation, while transcript profiles of thaumatin-like protein showed decreasing. No expression was detected for 1,3-beta-glucanase (QsGLU). Furthermore, the choice of suitable reference genes in any new experimental system is absolutely crucial in qRT-PCR; for this reason in this study and for the first time a set of potential reference genes were analyzed and validated for qRT-PCR normalization in the patho-system Phytophthora-Q. suber. Four candidate reference genes polimerase II (QsRPII), eukaryotic translation initiation factor 5A(QsEIF-5A), b-tubulin (QsTUB) and a medium subunit family protein of Clathrin adaptor complexes (QsCACs) were evaluated to determine the most stable internal references in Q. suber. Analysis of stability of genes was carried out using Genex software. Results indicated all these four potential reference genes assumed stable expression. Data analysis revealed that QsRPII and QsCACs were the two most stable genes, while genes QsTUB and QsEIF-5A were the third and the fourth most stable gene, respectively. In this study, a plasmid-based quantitative PCR method was developed to measure P. cinnamomi colonization during infection process of Q. suber. Plasmid-based detection of P. cinnamomi showed a gradual accumulation of the pathogen DNA in cork oak root tips up to 24 h post infection. The higher increase in P. cinnamomi/plasmid DNA ratio occurred between 18 and 24 h. One of the primary objectives of this research was to study the effect of cinnamomins (elicitins secreted by P. cinnamomin) on inducing defence mechanism against the pathogen, as recent histological and ultra-structural studies showed that P. cinnamomi was restricted to the outer cortex root fragments pre-treated with capsicien and cryptogein, suggesting that elicitins can stimulate plant defence reactions against P. cinnamomi. To complement these studies and to have a clear view of the nature of the interaction, the role of cinnamomins in the production of the oxidative burst [ROS and ROS scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD)] and in the defence responses was evaluated. Cork oak seedlings were pretreated with alpha-cinnamomin and then inoculated with P. cinnamomi mycelia. Results showed a significant higher production of reactive oxygen species (ROS) (H2O2 and O2•-) in elicitin and non-elicitin treated roots in interaction with P. cinnamomi in comparison to the corresponding control. The plant group inoculated with the pathogen after cinnamomin treatment showed an earlier increase in H2O2 production but this was lower as compared with that group inoculated with P. cinnamomi alone. Also, in elicitin pre-treated group generally, a lower level of O2•− production during infection was observed as compared with inoculated roots with P. cinnamomi alone without elicitin treatment. Furthermore, in this study, we evaluated activities of antioxidant enzymes upon challenge with P. cinnamomi, with and without pretreatment with alpha cinnamomin. Results indicated that the activities of defense enzymes POD, SOD and CAT increased after P. cinnamomi inoculation when compared with those in the control group. Also, in the group treated with alpha-cinnamomin followed by P. cinnamomi inoculation, a higher level of enzymatic activities was detected as compared with elicitin non-treated group, which suggest the protective effect of alpha-cinnamomin against the pathogen due to higher elevated levels of defense enzymes POD, SOD and CAT during the infection period. Furthermore, a sensitive qPCR method was applied to measure the pathogen biomass in elicited and non-elicited Q. suber roots challenged with P. cinnamomi to elucidate the effect of cinnamomins on the colonization of P. cinnamomi. Plasmid-based quantification of P. cinnamomi showed a significant decrease in accumulation of the pathogen DNA in cork oak roots after treatment with alpha and beta-cinnamomins which attest the role of cinnamomins in promoting defense responses in cork oak against P. cinnamomi invasion.