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- Identification of a novel mutation in MEF2C gene in an atypical patient with frontotemporal lobar degenerationPublication . Adrião, Andreia; Santana, Isabel; Ribeiro, Carolina; Cancela, M. Leonor; Conceição, Natércia; Grazina, ManuelaThe MEF2C gene encodes a transcription factor known to play a crucial role in molecular pathways affecting neuronal development. MEF2C mutations were described as a genetic cause of developmental disease (MRD20), and several reports sustain its involvement in dementia-related conditions, such as Alzheimer's disease and amyotrophic lateral sclerosis. These pathologies and frontotemporal degeneration (FTLD) are thought to share common physiopathological pathways. In this exploratory study, we searched for alterations in the DNA sequence of exons and boundaries, including 5 '- and 3 '-untranslated regions (5 ' UTR, 3 ' UTR), of MEF2C gene in 11 patients with clinical phenotypes related with MRD20 or FTLD. We identified a heterozygous deletion of 13 nucleotides in the 5 ' UTR region of a 69 years old FTLD patient. This alteration was absent in 200 healthy controls, suggesting a contribution to this patient's disease phenotype. In silico analysis of the mutated sequence indicated changes in mRNA secondary structure and stability, thus potentially affecting MEF2C protein levels. Furthermore, in vitro functional analysis of this mutation revealed that the presence of this deletion abolished the transcriptional activity of the gene in human embryonic cells and rat brain neurons, probably by modifying MEF2C expression. Altogether, our results provide evidence for the involvement of MEF2C in FTLD manifesting with seizures.
- MEF2C orthologues from zebrafish: evolution, expression and promoter regulationPublication . Adrião, Andreia Lúcia Gonçalves; Conceição, Natércia; Cancela, LeonorMEF2C is a crucial transcription factor for cranial neural crest cells development. An abnormal expression of this protein leads to severe abnormalities in craniofacial features. Recently, a human disease (MRD20) was described as a consequence of MEF2C haploinsufficiency. These patients show severe developmental delay, intellectual disability and dysmorphic features. Zebrafish presents two MEF2C orthologues, mef2ca and mef2cb. In this study we demonstrate a highly conserved pattern of chromosome localization for MEF2C between human and zebrafish, a similar protein sequence and tissue expression profile. We have focused our functional analysis on the zebrafish orthologue mef2cb. We identified three new exons through 5' RACE and described two new transcriptional start sites (TSS). These alternative TSS reflect the occurrence of two alternative promoters differentially regulated by nuclear factors related to craniofacial or neuronal development such as Sox9b, Sox10 and Runx2. We also predict that mef2cb gene may be post transcriptionally regulated by analysing the structure of its 5' UTR region, conserved throughout evolution. Our study provides new insights in MEF2C conservation and provides the first evidence of mef2cb regulation by both transcriptional and post transcriptional mechanisms, thus contributing to validate zebrafish as a good model for future studies concerning MEF2C dependent pathologies. (c) 2015 Elsevier Inc. All rights reserved.
- MEF2C: expression, regulation and interaction with target genes in health and diseasesPublication . Adrião, Andreia Lúcia Gonçalves; Cancela, Leonor; Grazina, Manuela; Conceição, NatérciaMEF2C is the first MEF2 family factor to be expressed during embryonic development. Zebrafish has been proven to be an excellent model for human genetic diseases and has two MEF2C orthologues, mef2ca and mef2cb. The involvement of MEF2C in different developmental processes was studied in zebrafish through expression analysis and usage of mutant lines as loss of function models. Alterations in this gene were related to cardiac defects and its haploinsufficiency was linked to a human disease (MRD20) whose patients show abnormal neuronal and craniofacial development. Recently this gene was reported to be involved in dementia conditions such as Alzheimer disease, Parkinson disease and amyotrophic lateral sclerosis. Our work aims to further validate zebrafish as an animal model to study MEF2C related pathologies focusing on craniofacial and neuronal issues. Our in silico analysis demonstrated a high conservation of mef2c pattern of chromosome localization, protein structure and sites of mRNA expression throughout evolution. We also identified two transcriptional start sites for mef2cb that were conserved through evolution. These are related to the occurrence of alternative promoters that appear to be differentially regulated by Sox9b, Sox10 and Runx2, three nuclear factors associated to craniofacial or neuronal development. Through the use of Mef2ca and Mef2cb loss of function mutant lines, we described the craniofacial phenotype resulting from the absence of both mef2c isoforms in zebrafish. We concluded that both orthologues are involved in cartilage, bone and brain development in zebrafish and we described the molecular profile of specific marker genes resulting from mef2c loss of function. Our results allow us to consider zebrafish as a valuable animal model for analysis concerning MEF2C related pathologies. To complement our work we performed a pilot study in which we describe for the first time an alteration in MEF2C gene in a patient with frontotemporal lobar degeneration, suggesting an association between MEF2C and this disease, a result that extends the involvement of this gene to a previously unsuspected human pathology.
- Determinação dos genótipos de Listeria Monocytogenes por PCR em tempo real e avaliação da sua citopatogenicidadePublication . Adrião, Andreia Lúcia Gonçalves; Faleiro, Maria Leonor
- Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L-monocytogenes but is absent from many field isolatesPublication . Jordan, Suzanne J.; Perni, Stefano; Glenn, Sarah; Fernandes, Isabel; Barbosa, Manuela; Sol, Manuela; Tenreiro, Rogerio P.; Chambel, Lelia; Barata, Belarmino; Zilhao, Isabel; Aldsworth, Timothy G.; Adrião, Andreia Lúcia Gonçalves; Faleiro, L; Shama, Gilbert; Andrew, Peter W.Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.
- Zebrafish as a disease model for studying human Rett SyndromePublication . Vitorino, M.; Conceicao, N.; Adrião, Andreia Lúcia Gonçalves; Cancela, M. LeonorBackground: Rett syndrome (RTT) is a severe neurological disorder that affects almost exclusively females. This disease is characterized by normal growth and intellectual development until around the first year, then progression in development starts to slow down, with loss of purposeful use of the hands, distinctive hand movements, slowed brain and head growth, problems with walking, seizures, and intellectual disability. Mutations in the X-linked genes methyl-CpG-binding protein 2 (MECP2) and cyclin-dependent kinase-like 5 (CDKL5) were described in RTT and recently several bone diseases related with decreased bone mass were also described in those patients, starting early in life. Because zebrafish was largely validated as a model for human diseases, the main objective of this work was to investigate if zebrafish can be a good model to study RTT.
- Zebrafish as a model to study craniofacial phenotypes related to human MEF2CmutationsPublication . Adrião, Andreia Lúcia Gonçalves; Conceição, N.; Gavaia, Paulo J.; Cancela, LeonorBackground: MEF2C is a transcription factor with important roles in differentiation and developmental processes, in particular in endochondral bone development. An association between human MEF2C gene mutations and particular phenotypes affecting craniofacial formation has been shown. Recent studies have shown that the zebrafish palate is under a genetic control similar to that controlling amniotic palatal skeleton, demonstrating the utility of this organism to study craniofacial development. Zebrafish has two mef2c genes and different lines of mutants are available. Therefore we have used zebrafish in an attempt to correlate specific MEF2C mutations with the abnormal human craniofacial phenotypes.
- mef2ca and mef2cb Double mutant Zebrafish show altered craniofacial phenotype and motor behaviourPublication . Adrião, Andreia; Mariano, Sara; Mariano, José; Gavaia, Paulo; Cancela, M. Leonor; Vitorino, Marta; Conceição, NatérciaThe transcription factor MEF2C is crucial in neuronal, cardiac, bone and cartilage molecular processes, as well as for craniofacial development. MEF2C was associated with the human disease MRD20, whose patients show abnormal neuronal and craniofacial development. Zebrafish mef2ca;mef2cb double mutants were analysed for abnormalities in craniofacial and behaviour development through phenotypic analysis. Quantitative PCR was performed to investigate the expression levels of neuronal marker genes in mutant larvae. The motor behaviour was analysed by the swimming activity of 6 dpf larvae. We found that mef2ca;mef2cb double mutants display several abnormal phenotypes during early development, including those already described in zebrafish carrying mutations in each paralog, but also (i) a severe craniofacial phenotype (comprising both cartilaginous and dermal bone structures), (ii) developmental arrest due to the disruption of cardiac oedema and (iii) clear alterations in behaviour. We demonstrate that the defects observed in zebrafish mef2ca;mef2cb double mutants are similar to those previously described in MEF2C-null mice and MRD20 patients, confirming the usefulness of these mutant lines as a model for studies concerning MRD20 disease, the identification of new therapeutic targets and screening for possible rescue strategies.