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- Estudo do papel de genes interagindo funcionalmente/fisicamente com Cited2 na biologia das células estaminais embrionáriasPublication . M., Trindade; Bragança, José Eduardo MarquesAs células estaminais embrionárias são caracterizadas por possuírem a capacidade de se auto-renovarem indefinidamente em cultura e por possuírem a capacidade de se diferenciarem em qualquer tecido ou célula de um organismo adulto (pluripotência). As funções das células estaminais embrionárias são controladas por factores extrínsecos e intrínsecos, sendo os factores de transcrição Nanog, Oct4 e Sox2 proteínas chaves para manter a pluripotência. No entanto, existem factores de transcrição auxiliares que também participam na regulação destes e de outros genes importantes para a pluripotência, tais como Zic3, Smad2/3, Tbx3, Klf4 por exemplo. Cited2 é um factor de transcrição importante para múltiplos processos do desenvolvimento embrionário de ratinho, cuja sobrexpressão é capaz de manter as células estaminais de ratinho com características de pluripotência na ausência do factor inibitório da leucemia (LIF) necessário para impedir a diferenciação das células estaminais embrionárias de ratinho em cultura. Neste trabalho experimental os principais objectivos foram verificar o efeito do factor Cited2 no controlo da actividade do promotor de Nanog em conjunto com outros factores de transcrição e determinar a influência da sobrexpressão de Cited2 no estado de metilação da lisina 4 da histona H3 (H3K4) nos promotores dos genes Nanog, Oct4 e Tbx3. Os nossos estudos revelaram que o Cited2 possui interacções com vários domínios do promotor de Nanog. Observou-se também que o Cited2 não altera significativamente o estado de metilação H3K4 dos promotores de Nanog e Oct4, contudo parece estar a influenciar negativamente o estado de metilação de Tbx3. Verificou-se ainda que a sobrexpressão de Cited2 provoca um aumento da actividade do promotor de Nanog, em aproximadamente duas vezes. Nas nossas condições, também observamos um efeito cooperativo modesto mas significativo entre Smad2, Zic3 e Cited2 para activar o promotor de Nanog, enquanto a sobrexpressão de Nanog e p53 em presença ou ausência de Cited2 reduzem a actividade deste promotor.
- Unravelling the evolution of the Allatostatin-Type A, KISS and Galanin Peptide-Receptor gene families in Bilaterians: insights from Anopheles MosquitoesPublication . Felix, Rute C.; Trindade, Marlene; Pires, Isa R. P.; Vera G Fonseca; Martins, Rute S.; Silveira, Henrique; Power, Deborah M.; Cardoso, João CRAllatostatin type A receptors (AST-ARs) are a group of G-protein coupled receptors activated by members of the FGL-amide (AST-A) peptide family that inhibit food intake and development in arthropods. Despite their physiological importance the evolution of the AST-A system is poorly described and relatively few receptors have been isolated and functionally characterised in insects. The present study provides a comprehensive analysis of the origin and comparative evolution of the AST-A system. To determine how evolution and feeding modified the function of AST-AR the duplicate receptors in Anopheles mosquitoes, were characterised. Phylogeny and gene synteny suggested that invertebrate AST-A receptors and peptide genes shared a common evolutionary origin with KISS/GAL receptors and ligands. AST-ARs and KISSR emerged from a common gene ancestor after the divergence of GALRs in the bilaterian genome. In arthropods, the AST-A system evolved through lineage-specific events and the maintenance of two receptors in the flies and mosquitoes (Diptera) was the result of a gene duplication event. Speciation of Anophelesmosquitoes affected receptor gene organisation and characterisation of AST-AR duplicates (GPRALS1 and 2) revealed that in common with other insects, the mosquito receptors were activated by insect AST-A peptides and the iCa(2+)-signalling pathway was stimulated. GPRALS1 and 2 were expressed mainly in mosquito midgut and ovaries and transcript abundance of both receptors was modified by feeding. A blood meal strongly up-regulated expression of both GPRALS in the midgut (p < 0.05) compared to glucose fed females. Based on the results we hypothesise that the AST-A system in insects shared a common origin with the vertebrate KISS system and may also share a common function as an integrator of metabolism and reproduction. Highlights: AST-A and KISS/GAL receptors and ligands shared common ancestry prior to the protostome-deuterostome divergence. Phylogeny and gene synteny revealed that AST-AR and KISSR emerged after GALR gene divergence. AST-AR genes were present in the hemichordates but were lost from the chordates. In protostomes, AST-ARs persisted and evolved through lineage-specific events and duplicated in the arthropod radiation. Diptera acquired and maintained functionally divergent duplicate AST-AR genes.
- PACAP system evolution and its role in melanophore function in teleost fish skinPublication . CR Cardoso, Joao; C. Félix, Rute; Martins, Rute; M., Trindade; G Fonseca, Vera; Fuentes, Xoan; Power, DeborahPituitary adenylate cyclase-activating polypeptide (PACAP) administered to tilapia melanophores ex-vivo causes significant pigment aggregation and this is a newly identified function for this peptide in fish. The G-protein coupled receptors (GPCRs), adcyap1r1a (encoding Pac1a) and vipr2a (encoding Vpac2a), are the only receptors in melanophores with appreciable levels of expression and are significantly (p < 0.05) down-regulated in the absence of light. Vpac2a is activated exclusively by peptide histidine isoleucine (PHI), which suggests that Pac1a mediates the melanin aggregating effect of PACAP on melanophores. Paradoxically activation of Pac1a with PACAP caused a rise in cAMP, which in fish melanophores is associated with melanin dispersion. We hypothesise that the duplicate adcyap1ra and vipr2a genes in teleosts have acquired a specific role in skin and that the melanin aggregating effect of PACAP results from the interaction of Pac1a with Ramp that attenuates cAMP-dependent PKA activity and favours the Ca(2+)/Calmodulin dependent pathway.
- The xenobiotic sensor PXR in a marine flatfish species (Solea senegalensis): Gene expression patterns and its regulation under different physiological conditionsPublication . Marques, Carlos; Roberto, Vania Palma; Granadeiro, Luis; Trindade, Marlene; Gavaia, Paulo; Laizé, Vincent; Leonor Cancela, M.; Fernandez, IgnacioThe pregnane X receptor (PXR) is a nuclear receptor belonging to the NR1I sub-family and a known master regulator of xenobiotic metabolism. New roles have been recently proposed in mammals through its activation by vitamin K (VK) such as regulation of glucose metabolism, bone homeostasis, reproduction, neuronal development and cognitive capacities. In marine fish species little is known about PXR and its potential roles. Here, expression patterns of pxr transcripts and conservation of protein domains were determined in the Senegalese sole (Solea senegalensis), a marine flatfish model species in aquatic ecotoxicology. In addition to a full coding sequence transcript (sspxrl), two variants lacking DNA and/or ligand binding domains (sspxr2 and sspxr3) were also identified. The expression of sspxrl during early development and in adult tissues was ubiquitous, but highest levels were observed in liver, intestine and skin. Expression was also detected by in situ hybridization in chondrocytes and cells from the granular and inner nuclear layers in three month old fish. Finally, sspxrl expression was shown to be differentially regulated under physiological conditions related with fasting, VK and warfarin metabolism. The present work provides new and basic knowledge regarding pxr sequence and expression patterns in a marine flatfish species to unveil the potential impact of xenobiotics on marine fish physiology, and will allow a better and more ecosystemic environmental risk assessment of different pollutants over the marine environments with the development of reporter assays using PXR sequences from evolutionary distantly marine species (such as vertebrate and invertebrate marine species). (C) 2017 Elsevier Ltd. All rights reserved.
- Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralizationPublication . Vijayakumar, Parameswaran; Laizé, Vincent; Cardeira Da Silva, João; Trindade, Marlene; Cancela, M. LeonorMechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28 degrees C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.
- Acute Loss of Cited2 Impairs Nanog Expression and Decreases Self-Renewal of Mouse Embryonic Stem CellsPublication . Kranc, Kamil R.; Oliveira, Daniel; Armesilla-Diaz, Alejandro; Pacheco-Leyva, Ivette; Matias, Ana Catarina; Escapa, Ana Luísa; Subramani, Chithra; Wheadon, Helen; Trindade, Marlene; Nichols, Jennifer; Kaji, Keisuke; Enver, Tariq; Bragança, JoséIdentifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.