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- Disclosing proteins in the leaves of cork oak plants associated with the immune response to Phytophthora cinnamomi inoculation in the roots: a long-term proteomics approachPublication . Coelho, Ana Cristina; Pires, Rosa; Schütz, Gabriela; Santa, Cátia; Manadas, Bruno; Pinto, Patricia ISThe pathological interaction between oak trees and Phytophthora cinnamomi has implications in the cork oak decline observed over the last decades in the Iberian Peninsula. During host colonization, the phytopathogen secretes effector molecules like elicitins to increase disease effectiveness. The objective of this study was to unravel the proteome changes associated with the cork oak immune response triggered by P. cinnamomi inoculation in a long-term assay, through SWATH-MS quantitative proteomics performed in the oak leaves. Using the Arabidopis proteome database as a reference, 424 proteins were confidently quantified in cork oak leaves, of which 80 proteins showed a p-value below 0.05 or a fold-change greater than 2 or less than 0.5 in their levels between inoculated and control samples being considered as altered. The inoculation of cork oak roots with P. cinnamomi increased the levels of proteins associated with protein-DNA complex assembly, lipid oxidation, response to endoplasmic reticulum stress, and pyridine-containing compound metabolic process in the leaves. In opposition, several proteins associated with cellular metabolic compound salvage and monosaccharide catabolic process had significantly decreased abundances. The most significant abundance variations were observed for the Ribulose 1,5-Bisphosphate Carboxylase small subunit (RBCS1A), Heat Shock protein 90-1 (Hsp90-1), Lipoxygenase 2 (LOX2) and Histone superfamily protein H3.3 (A8MRLO/At4G40030) revealing a pertinent role for these proteins in the host-pathogen interaction mechanism. This work represents the first SWATH-MS analysis performed in cork oak plants inoculated with P. cinnamomi and highlights host proteins that have a relevant action in the homeostatic states that emerge from the interaction between the oomycete and the host in the long term and in a distal organ.
- Diversity, expression and mechanism of action of estrogen receptors in sea bream, Sparus auratusPublication . Pinto, Patricia IS; Canario, Adelino V. M.; Sweeney, G. E.Two estrogen receptor subtypes (ERα and ERβ) mediate most estrogen actions in vertebrates, including fishes. Furthermore in teleost fishes two ERβ genes are differentially expressed. In this thesis, an ERβ (sbERβb) was cloned from the hermaphrodite teleost fish sea bream (Sparus auratus) which added to the previously cloned sbERα and sbERβa. sbERβb specifically binds estrogen agonists and antagonists with high affinity. The three sbER genes (α, βa, βb) have a partially overlapping but differential distribution in male and female sea bream tissues and produce multiple transcripts. Two exon2 deleted sbERα variants were also identified with different tissue distribution and hormonal regulation. Estradiol-17β up-regulated the expression of sbERα and down-regulated both sbERβs in liver, suggesting a major role for ERα in vitellogenesis. Agonistic effects were identified for the “pure antiestrogen” ICI 182,780 in several estrogenic responses, probably mediated by sbERα up regulation in liver. The immunolocalization of sbER proteins in sea bream scales suggested that the calcium mobilising actions of E2 in scales are via a direct action on osteoclasts. Subtractive hybridization followed by RT-PCR demonstrated for the first time in fish testis the E2 up regulation of some typical liver E2-induced genes (e.g. vitellogenins and choriogenins).
- The influence of 17β-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labraxPublication . Moreira, Catarina; Paiola, Matthieu; Duflot, Aurélie; Varó, Inma; Sitjà-Bobadilla, Ariadna; Knigge, Thomas; Pinto, P; Monsinjon, TiphaineThe female sex steroid 17β-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Never theless, little is known about the function of oestrogen in immune system development. To further the under standing of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2⋅L− 1 , at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng⋅L− 1 was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αβ T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is estrogensensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.
- Tissue responsiveness to estradiol and genistein in the sea bass liver and scalePublication . Estêvão, M. Dulce; Andrade, André; Santos, Soraia; Power, Deborah; Pinto, Patricia ISAs in mammals, estrogens in fish are essential for reproduction but also important regulators of mineral homeostasis. Fish scales are a non-conventional target tissue responsive to estradiol and constitute a good model to study mineralized tissues effects and mechanisms of action of estrogenic compounds, including phytoestrogens. The responsiveness to estradiol and the phytoestrogen genistein, was compared between the scales and the liver, a classical estrogenic target, in sea bass (Dicentrarchus labrax). Injection with estradiol and genistein significantly increased circulating vitellogenin (for both compounds) and mineral levels (estradiol only) and genistein also significantly increased scale enzymatic activities suggesting it increased mineral turnover. The repertoire, abundance and estrogenic regulation of nuclear estrogen receptors (ESR1, 2a and 2b) and membrane G-protein receptors (GPER and GPER-like) were different between liver and scales, which presumably explains the tissue-specific changes detected in estrogen-responsive gene expression. In scales changes in gene expression mainly consisted of small rapid increases, while in liver strong, sustained increases/decreases in gene expression occurred. Similar but not overlapping gene expression changes were observed in response to both estradiol and genistein. This study demonstrates for the first time the expression of membrane estrogen receptors in scales and that estrogens and phytoestrogens, to which fish may be exposed in the wild or in aquaculture, both affect liver and mineralized tissues in a tissue-specific manner. (C) 2015 Elsevier Ltd. All rights reserved.
- Oestrogen receptor distribution related to functional thymus anatomy of the European sea bass, Dicentrarchus labraxPublication . Paiola, Matthieu; Knigge, Thomas; Picchietti, Simona; Duflot, Aurelie; Guerra, Laura; Pinto, Patricia IS; Scapigliati, Giuseppe; Monsinjon, TiphaineIn jawed vertebrates, the crosstalk between immune and endocrine system as well as many fundamental mechanisms of T cell development are evolutionary conserved. Oestrogens affect mammalian thymic function and plasticity, but the mechanisms of action and the oestrogen receptors involved remain unclear. To corroborate the oestrogenic regulation of thymic function in teleosts and to identify the implicated oestrogen receptor subtypes, we examined the distribution of nuclear and membrane oestrogen receptors within the thymus of the European Sea bass, Dicentrarchus labrax, in relation to its morpho-functional organisation. Immunohistological analysis specified thymus histology and organisation in teleosts and described, for the first time, Hassall's corpuscle like structures in the medulla of sea bass. All oestrogen receptors were expressed at the transcript and protein level, both in T cells and in stromal cells belonging to specific functional areas. These observations suggest complex regulatory actions of oestrogen on thymic function, notably through the stromal microenvironment, comprising both, genomic and non-genomic pathways that are likely to affect T cell maturation and trafficking processes. Comparison with birds, rodents and humans supports the thymic localization of oestrogen receptors and suggests that oestrogens modulate T cell maturation in all gnathostomes. (C) 2017 Elsevier Ltd. All rights reserved.
- Gene transcripts responsive to drought stress identified in Citrus macrophylla bark tissue transcriptome have a modified response in plants infected by Citrus tristeza virusPublication . da Silva, Melina; Pinto, Patricia IS; Guerra, Rui Manuel Farinha das Neves; Duarte, Amilcar; Power, Deborah; Marques, N T.Citrus macrophylla Wester (CM) has valuable agronomic characteristics such as the ability to grow in saline soils, although with low tolerance to prolonged drought stress (DS). To understand the mechanisms that characterize CM response to water scarcity, this study compared transcriptome profile changes in CM stem tissue when exposed to DS and identified a total of 2745 differentially expressed transcripts (DETs, fold change > 2), of which 631 were up-regulated and 2114 were down-regulated. DETs up-regulated by DS were enriched in pathways such as the redox and osmotic system or soluble carbohydrates and in transcripts for low molecular weight proteins such as late embryogenesis abundant protein (LEA). Down-regulated transcripts were mainly assigned to photosynthesis, transport, phenylpropanoids, calcium dependent kinases, brassinosteroids and other hormones including salicylic acid and abscisic acid. To assess the interplay between DS and Citrus tristeza virus (CTV) infection, twelve genes were profiled by quantitative Real-Time PCR (qPCR) analysis in control and CTV-infected CM plants, with or without DS. The twelve analyzed transcripts were significantly correlated (r = 0.82, p < 0.001) with the RNA-Seq results and gave insight into the responses of CM to drought and/or to infection with CTV. Transcriptome results unveiled highly responsive genes to DS in stem tissue, which may be candidates for genetic selection of high drought tolerant plants of CM.
- Efeitos de fitoestrogénios no metabolismo mineral em escamas de robalo e de tilápia moçambicanaPublication . Estêvão, Dulce; Pinto, Patricia IS; Santos, Soraia; Andrade, André; Power, DeborahO rápido desenvolvimento da aquacultura nas últimas décadas fez aumentar a procura por fontes proteicas adequadas para incluir nas rações dos peixes. A soja tem sido muito utilizada com fonte proteica de origem vegetal mas é particularmente rica em fitoestrogénios, incluindo a genisteína (GEN) e a daidzeína (DAI), que são as principais isoflavonas presentes na soja. Os peixes podem estar expostos aos fitoestrogénios no ambiente ou através das dietas que os contêm, como é o caso da soja. Estes compostos podem ter atividades estrogénicas e efeitos disruptivos na reprodução mas o seu impacto nos tecidos mineralizados continua a ser desconhecido. As escamas de peixe são um tecido mineralizado que, tal como o osso de mamíferos, é mantido por ciclos de formação e reabsorção, mediado por osteoblastos (OSB) e osteoclastos (OSC), respetivamente. As escamas são um tecido responsivo aos estrogénios e expressam os recetores de estrogénio nucleares (ERs). As atividades das enzimas fosfatase alcalina (ALP) e fosfatase ácida resistente ao tartrato (TRAP) são usadas como marcadores das atividades dos OSB e OSC, respetivamente, e são modificadas pelo estradiol (E2) nas escamas de várias espécies de peixe. Usando um ensaio in vitro, investigámos o possível impacto da exposição a GEN e a DAI no metabolismo mineral em escamas. O efeito destes compostos foi avaliado através da determinação das atividades de TRAP e ALP em escamas de robalo (Dicentrarchus labrax), uma espécie marinha, e de tilápia moçambicana (Oreochromis mossambicus), mantida em água salgada (AS) e em água doce (AD).
- Factors driving bacterial microbiota of eggs from commercial hatcheries of European Seabass and Gilthead SeabreamPublication . Najafpour, Babak; Pinto, Patricia IS; Moutou, Katerina A.; Canario, A.V.M.; Power, Deborah MaryA comprehensive understanding of how bacterial community abundance changes in fishes during their lifecycle and the role of the microbiota on health and production is still lacking. From this perspective, the egg bacterial communities of two commercially farmed species, the European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata), from different aquaculture sites were compared, and the potential effect of broodstock water microbiota and disinfectants on the egg microbiota was evaluated. Moreover, 16S ribosomal RNA gene sequencing was used to profile the bacterial communities of the eggs and broodstock water from three commercial hatcheries. Proteobacteria were the most common and dominant phyla across the samples (49.7% on average). Vibrio sp. was the most highly represented genus (7.1%), followed by Glaciecola (4.8%), Pseudoalteromonas (4.4%), and Colwellia (4.2%), in eggs and water across the sites. Routinely used iodine-based disinfectants slightly reduced the eggs’ bacterial load but did not significantly change their composition. Site, species, and type of sample (eggs or water) drove the microbial community structure and influenced microbiome functional profiles. The egg and seawater microbiome composition differed in abundance but shared similar functional profiles. The strong impact of site and species on egg bacterial communities indicates that disease management needs to be site-specific and highlights the need for species- and site-specific optimization of disinfection protocols.
- Identification of androgen receptor variants in testis from humans and other vertebratesPublication . Laurentino, S. S.; Pinto, Patricia IS; Tomas, J.; Cavaco, J. E. B.; Sousa, M.; Barros, A.; Power, Deborah; Canario, Adelino V. M.; Socorro, S.The androgen receptor (AR) is a ligand-activated transcription factor member of the nuclear receptor superfamily. The existence of alternatively spliced variants is well recognised for several members of this superfamily, most of them having functional importance. For example, several testicular oestrogen receptor variants have been suggested to play a role in the regulation of spermatogenesis. However, information on AR variants is mostly related to cancer and androgen insensitivity syndrome (AIS) cases. The objective of this study was to investigate the expression of AR variants in the testis from humans and other vertebrates. Four AR variants [ARD2Stop, ARD223Stop, ARD3 and ARD4(120)] were identified in human testis. ARD2Stop and ARD3, with exon 2 or 3 deleted, respectively, were also expressed in human liver, lung, kidney and heart. In addition, ARD2Stop was expressed in rat and gilthead seabream testis, while an ARD3 was detected in African clawed frog testis. This is the first report revealing the existence of AR variants in the testis of evolutionarily distant vertebrate species and in nonpathological tissues. These data suggest the functional importance of these novel AR forms and demonstrate a complexity in AR signalling that is not exclusive of pathological conditions.
- Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bassPublication . Zapater, Cinta; Moreira, Catarina; Knigge, Thomas; Monsinjon, Tiphaine; Gómez, Ana; Pinto, Patricia I. S.Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.