Browsing by Author "Pinto, Patricia IS"
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- 2.P3. Cloning and gene expression of two novel stanniocalcin genes in the puffer fish Tetraodon nigroviridisPublication . Schein, V; Schein, Vanessa; Pinto, Patricia IS; Canario, Adelino V. M.Stanniocalcin 1 (STC1) is a hypocalcemic hormone first described in fishes. Recently a second gene (STC2) was isolated from mammals and fishes. However, in silico analysis of Tetraodon nigroviridis and Takifugu rubripes genomes revealed the presence of a third and a fourth gene. In this study STC1 was cloned from de Corpuscles of Stannius (CS), STC2 from the muscle, and the two novel genes identified, STC1B and STC2B from the brain of Tetraodon. The STC-1B gene encodes an ORF of 287aa and contains 11 conserved cyteine residues and the Nlinked glycosylation site, present in all identified STC1.
- Characterization of estrogen receptor ßb in sea bream (Sparus auratus): phylogeny, ligand-binding, and comparative analysis of expressionPublication . Pinto, Patricia IS; Passos, A. L.; Martins, Rute S. T.; Power, Deborah; Canario, Adelino V. M.Estrogens control many physiological processes in both female and male vertebrates, mostly mediated by speciWc nuclear estrogen receptors (ER). Two ER subtypes (ER and ER ) are present in most vertebrates, including the sea bream (Sparus auratus) a hermaphrodite teleost Wsh. In the present study several variant cDNAs encoding a second sea bream ER (sbER b) is reported. Phylogenetic and Southern blot analysis indicate that sbER b and the previously cloned sbER a (formerly sbER ) are encoded by diVerent genes, which may have arisen by duplication of an ancestral ER gene. Competitive binding assays show that sbER b has high aYnity for 17 -estradiol (KdD1 nM) and speciWcally binds estrogen agonists (diethylstilbestrol and ethynylestradiol) and antagonists (ICI 182,780). In Northern blot sbER , sbER a, sbER b produce several diVerent transcripts in a variety of tissues. RT-PCR showed a partially overlapping but diVerential tissue distribution in both male and female sea bream.
- Core microbiome profiles and their modification by environmental, biological, and rearing factors in aquaculture hatcheriesPublication . Najafpour, Babak; Pinto, Patricia IS; Sanz, Eric Climent; Martinez-Blanch, Juan F.; Canario, Adelino; Moutou, Katerina A.; Power, Deborah16S rRNA gene sequencing and bacteria-and genus-specific quantitative PCR was used to profile microbial communities and their associated functions in water, live feed (microalgae, Artemia, and rotifer), and European sea bass and gilthead sea bream larvae from hatcheries in Greece and Italy. The transfer to larvae of genus containing potential pathogens of fish was more likely with Artemia and rotifer than with microalgae or water, irrespective of geographic location. The presence of potentially pathogenic bacteria (Vibrio and Pseudoalter-omonas) in the core microbiota of water, live feed, and fish larvae, the enrichment of different bacterial resistance pathways and biofilm formation, and the overall low beneficial bacteria load during larval ontogeny emphasizes the risk for disease outbreaks. The present data characterizing microbiota in commercial aquaculture hatcheries provides a baseline for the design of strategies to manage disease and to model or remediate potential adverse environmental impacts.
- Developmental immunotoxicology: What underlies the critical windows of exposure?Publication . Moreira, Catarina; Paiola, Matthieu; Duflot, Aurelie; del Pozo, Raquel; Carla Piazzon, M.; Fouz, Belen; Varo, Inma; Sitja-Bobadilla, Ariadna; Knigge, Thomas; Pinto, Patricia IS; Monsinjon, TiphaineEndocrine disruptors in general and oestrogenic compounds in particular have been widely investigated in view of their effects on several physiological processes and, therefore, their ecotoxicologic relevance. The modulation of the immune system by oestrogens has increasingly sparked interest in the research community, that previously had been mainly centered on the reproductive effects of these hormones. In fact, since the industrialization an increasing variety of endocrine disruptors, such as oestrogenic endocrine disruptors, are retrieved in the environment. These oestrogenic endocrine disrupting chemicals (EEDCs) have also been suggested to increase the prevalence of autoimmune diseases and cancer. With regard to the high degree of similarities in the immune system of jawed vertebrates as well as the conserved immunomodulatory roles of oestrogen, environmental EEDCs possibly have the capacity to alter immune system functions of teleost fish, which may impair their capacity to fight infectious diseases and eventually may contribute, together with overfishing, to wild stock losses. Importantly, the most deleterious effects of EEDCs, both in mammals and teleosts, appear to arise when the exposure occurs during specific periods of the immune system ontogenesis, commonly referred as critical windows of exposure. However, in mammals and especially in teleost fish, these stages of the immune system development as well as the EEDC-action remain to be fully identified and characterised. The concept and the importance of developmental immunotoxicity is presented by addressing the mechanisms of oestrogenic regulation and the mode of action of EEDCs from an immunological perspective. Emphasis is given to the critical windows of development of the immune system during which EEDCs may alter the immune system development and function with long-term consequences on immunocompetence. Results from different classes of vertebrates are compiled, highlighting studies on teleost fish and their relevance for the human immune system. Additionally, new results on the effects of environmentally relevant concentration of exogenous estradiol exposure during European sea bass (Dicentrarchus labrax) development will be presented with regard to oestrogen's ability to trigger effects on immunocompetence, contributing to fill the gaps on vertebrate immunotoxicology.
- Differential involvement of the three nuclear estrogen receptors during oogenesis in European sea bass (Dicentrarchus labrax)Publication . Zapater, Cinta; Molés, Gregorio; Muñoz, Iciar; Pinto, Patricia IS; Canario, Adelino; Gómez, AnaEstrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.
- 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.
- Distribution and cellular localization of sea bream estrogen receptors in calcified tissuesPublication . Pinto, Patricia IS; Estêvão, Dulce; Redruello, Begoña; Power, Deborah; Canario, AdelinoIn teleost fish, estradiol has been shown to induce calcium mobilization from internal stores, in particular the scales. In the present study, we have localized the estrogen receptor proteins (α, βa and βb) in the scales of juvenile and adult sea bream, using immunohistochemistry with ER isoform-specific antibodies. The ERs co-localized in cells of the scale posterior region that also expressed TRACP, the putative osteoclasts. These results suggest that the calcium mobilising action of estradiol on fish scales is via its direct action on osteoclasts.
- Divergent responsiveness of the dentary and vertebral bone to a selective estrogen-receptor modulator (SERM) in the teleost Sparus auratusPublication . Vieira, Florbela A.; Pinto, Patricia IS; Guerreiro, P. M.; Power, DeborahIn teleosts the regulation of skeletal homeostasis and turnover by estrogen is poorly understood. For this reason raloxifene, a selective estrogen-receptor modulator (SERM), was administered to sea bream (Sparus auratus) and its effect on plasma calcium balance and transcript expression in dentary (dermal bone) and vertebra (perichondral bone) was studied. The concentration of total calcium or phosphorus in plasma was unchanged by raloxifene treatment for 6 days. The activity of alkaline phosphatase (ALP) in dentary bone of raloxifene treated fish was significantly (p < 0.05) higher than control fish but it was not changed in vertebral bone. Transcripts for estrogen receptor (ER) a were in very low abundance in the sea bream dentary and vertebra and were unchanged by raloxifene treatment. In contrast, raloxifene caused significant (p < 0.05) up-regulation of the duplicate ERb transcripts in the dentary but did not affect specific transcripts for osteoclast (TRAP), osteoblast (ALP, Runx2, osteonectin) or cartilage (IGF1, CILP2, FN1a). In the vertebra ERbb was not changed by raloxifene but ERba was significantly (p < 0.05) down-regulated as was the skeletal specific transcripts, TRAP, ALP, CILP2, FN1a. In summary, ERbs regulate estrogen sensitivity of the skeleton in sea bream, which responds in a non uniform manner. In common with mammals raloxifene appears to have an anti-resorptive role (in sea bream vertebra), but also an osteoblast stimulatory role, inducing ALP activity in the dentary of sea bream. Overall, the results indicate bone specific responsiveness to raloxifene in the sea bream. Further work will be required to understand the basis of bone responsiveness and the role of E2 and ERs in teleost bone homeostasis.
- 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).
- Duplicated membrane estrogen receptors in the European sea bass (Dicentrarchus labrax): Phylogeny, expression and regulation throughout the reproductive cyclePublication . Pinto, Patricia IS; Andrade, André; Estêvão, M. Dulce; Alvarado, M. Victoria; Felip, Alicia; Power, DeborahThe numerous estrogen functions reported across vertebrates have been classically explained by their binding to specific transcription factors, the nuclear estrogen receptors (ERs). Rapid non-genomic estrogenic responses have also been recently identified in vertebrates including fish, which can be mediated by membrane receptors such as the G protein-coupled estrogen receptor (Gper). In this study, two genes for Gper, namely gpera and gperb, were identified in the genome of a teleost fish, the European sea bass. Phylogenetic analysis indicated they were most likely retained after the 3R teleost-specific whole genome duplication and raises questions about their function in male and female sea bass. Gpera expression was mainly restricted to brain and pituitary in both sexes while gperb had a widespread tissue distribution with higher expression levels in gill filaments, kidney and head kidney. Both receptors were detected in the hypothalamus and pituitary of both sexes and significant changes in gpers expression were observed throughout the annual reproductive season. In female pituitaries, gpera showed an overall increase in expression throughout the reproductive season while gperb levels remained constant. In the hypothalamus, gpera had a higher expression during vitellogenesis and decreased in fish entering the ovary maturation and ovulation stage, while gperb expression increased at the final atresia stage. In males, gpers expression was constant in the hypothalamus and pituitary throughout the reproductive cycle apart from the mid- to late testicular development stage transition when a significant up-regulation of gpera occurred in the pituitary. The differential sex, seasonal and subtype-specific expression patterns detected for the two novel gper genes in sea bass suggests they may have acquired different and/or complementary roles in mediating estrogens actions in fish, namely on the neuroendocrine control of reproduction.