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- Cdkl5 mutant zebrafish shows skeletal and neuronal alterations mimicking human CDKL5 deficiency disorderPublication . Varela, Tatiana; Varela, Débora; Martins, Gil; Conceição, Natércia; Cancela, M. LeonorCDKL5 deficiency disorder (CDD) is a rare neurodevelopmental condition characterized primarily by seizures and impairment of cognitive and motor skills. Additional phenotypes include microcephaly, dysmorphic facial features, and scoliosis. Mutations in cyclin-dependent kinase-like 5 (CDKL5) gene, encoding a kinase essential for normal brain development and function, are responsible for CDD. Zebrafish is an accepted biomedical model for the study of several genetic diseases and has many advantages over other models. Therefore, this work aimed to characterize the phenotypic, behavioral, and molecular consequences of the Cdkl5 protein disruption in a cdkl5 mutant zebrafish line (sa21938). cdkl5(sa21938) mutants displayed a reduced head size, suggesting microcephaly, a feature frequently observed in CDD individuals. Double staining revealed shorter craniofacial cartilage structures and decrease bone mineralization in cdkl5 homozygous zebrafish indicating an abnormal craniofacial cartilage development and impaired skeletal development. Motor behavior analysis showed that cdkl5(sa21938) embryos had less frequency of double coiling suggesting impaired glutamatergic neurotransmission. Locomotor behavior analysis revealed that homozygous embryos swim shorter distances, indicative of impaired motor activity which is one of the main traits of CCD. Although no apparent spontaneous seizures were observed in these models, upon treatment with pentylenetetrazole, seizure behavior and an increase in the distance travelled were observed. Quantitative PCR showed that neuronal markers, including glutamatergic genes were dysregulated in cdkl5(sa21938) mutant embryos. In conclusion, homozygous cdkl5(sa21938) zebrafish mimic several characteristics of CDD, thus validating them as a suitable animal model to better understand the physiopathology of this disorder.
- Zebrafish: an interesting model to study CDKL5 deficiency disorderPublication . Varela, Tatiana; Varela, Débora; Vitorino, Marta; Conceição, Natércia; Cancela, M. Leonor; Martins, GilCDKL5 deficiency disorder is a rare X-linked condition that results in early onset of impairedmotor and cognitive skills such as motor rigidity, stereotypical hand movements and deficient language acquisition aswell as recurrent seizures. It is caused by mutations in the cyclin-dependent kinase-like 5 (CDKL5) gene, which encodes a serine/threonine kinase involved in important neuronal processes such as cell signaling and neuron morphogenesis.
- Regulation of human ZNF687, a gene associated with Paget's disease of bonePublication . Varela, Débora; Varela, Tatiana; Conceição, Natércia; Cancela, M. LeonorMutations in Zinc finger 687 (ZNF687) were associated with Paget's disease of bone (PDB), a disease charac-terized by increased bone resorption and excessive bone formation. It was suggested that ZNF687 plays a role in bone differentiation and development. However, the mechanisms involved in ZNF687 regulation remain un-known. This study aimed to obtain novel knowledge regarding ZNF687 transcriptional and epigenetic regulation. Through in silico analysis, we hypothesized three ZNF687 promoter regions located upstream exon 1 A, 1B, and 1 C and denominated promoter regions 1, 2, and 3, respectively. Their functionality was confirmed by luciferase activity assays and positive/negative regulatory regions were identified using promoter deletions constructs. In silico analysis revealed a high density of CpG islands in these promoter regions and in vitro methylation sup-pressed promoters' activity. Using bioinformatic approaches, bone-associated transcription factor binding sites containing CpG dinucleotides were identified, including those for NF kappa B, PU.1, DLX5, and SOX9. By co-transfection in HEK293 and hFOB cells, we found that DLX5 specifically activated ZNF687 promoter region 1, and its methylation impaired DLX5-driven promoter stimulation. NF kappa B repressed and activated promoter regions 1 and 2, respectively, and these activities were affected by methylation. PU.1 induced ZNF687 promoter region 1 which was affected by methylation. SOX9 differentially regulated ZNF687 promoters in HEK293 and hFOB cells that were impaired after methylation. In conclusion, this study provides novel insights into ZNF687 regulation by demonstrating that NF kappa B, PU.1, DLX5, and SOX9 are regulators of ZNF687 promoters, and DNA methylation influences their activity. The contribution of the dysregulation of these mechanisms in PDB should be further elucidated.
- Transcriptional regulation of human T-box 5 gene (TBX5) by bone- and cardiac-related transcription factorsPublication . Varela, Débora; Conceição, Natércia; Cancela, M. LeonorT-box 5 (TBX5) protein belongs to the T-box family whose members play a crucial role in cell-type specification, morphogenesis and organogenesis. TBX5 is a transcription factor important for cardiac development and upper limbs formation and its haploinsufficiency causes Holt-Oram syndrome (HOS). An increase in TBX5 dosage also leads to HOS, suggesting that TBX5 is a dose-sensitive transcription factor that needs to be tightly regulated but the molecular mechanisms involved remain unclear. In this work we report the cloning and functional analysis of human TBX5 promoter region 1 (upstream of exon 1) and promoter region 2 (upstream of exon 2), that probably regulate the transcription of the different transcript variants. In silico analysis showed several binding sites for cardiac and skeletal related transcription factors (TFs) and their functionality was assessed using promoter-luciferase constructions and TF-expressing vectors. MEF2A (Myocyte enhancer factor 2 A) was shown to positively regulate both TBX5 promoters, while EGR1 (early growth response 1) repressed both promoters. SOX9 (SRY (sex determining region Y)-box 9) repressed only the activity of promoter region 2. Interestingly, YY1 (Yin and yang 1) repressed promoter region 1 (that regulates the expression of variant 1 and 3), but activated promoter region 2 (that regulates the expression of variant 4). In conclusion, this work provides novel insights toward the better understanding of TBX5 transcriptional regulation by cardiac- and skeletal-related TFs.
- Genetic and functional analysis of specific TBX5 mutations associated with Holt-Oram SyndromePublication . Varela, Débora; Cancela, Leonor; Conceição, NatérciaMarine compounds are poorly-explored, however this area is becoming attractive and interesting because of the vast diversity of unique organisms that inhabit marine environments. Recent research has targeted compounds from marine organisms directed towards the discovery of new pharmaceuticals, nutraceuticals, cosmetics and biosensors. This monograph gives an overview of research into marine compounds for cosmetics, highlighting their effects in: photoprotection against UV radiation and associated antioxidant effects, characteristic of carotenoids, phlorotannins, carrageenans and mycosporins; antiphotoaging effects, possessed by carotenoids, mycosporins and fucoidans; and melanogenesis inhibition a notable effect of carotenoids and phlorotannins. A laboratory based study of a biosensor is reported with the view to assessing its utility in screening bioactive compounds. Extraction and screening of biomolecules remains challenging and for this reason research continues into appropriate methodologies for fast identification and extraction of bioactive compounds of marine origin. Among potential screening approaches, biosensors are new tools that can be used to evaluate the properties of biomolecules of pharmacological interest. Biosensors based on a quartz crystal microbalance (QCM), are a very promising technique for monitoring molecular interactions and provide a potential alternative to animal models. In the laboratory based study reported a QCM is used to study the interaction of thyroid hormones (THs) and transthyretin in order to infer if the same approach could be useful in marine compound evaluation. The results indicated that the QCM-I technique may a good tool to screen biochemical and biological activities of small molecules and so may be of interest in to industries such as dermocosmetics and the pharmaceutical industry.
- Epigenetic regulation of ZNF687 by miR-142a-3p and DNA methylation during osteoblast differentiation and mice bone development and agingPublication . Domingos Varela, Débora Cristina; Varela, Tatiana da Conceição Domingos ; Conceição, Natércia; Cancela, M. LeonorZinc finger protein 687 (ZNF687), a transcription factor implicated in osteoblast/osteoclast differentiation and linked to Paget's disease of bone, has unclear mechanisms in bone metabolism. Epigenetic disruptions can affect bone cell activity and contribute to bone-related diseases. This work aimed to elucidate the regulatory role of epigenetics in modulating Zfp687 expression throughout osteoblast differentiation and bone growth/aging in mice. Differentiation of the mouse-derived osteoblast precursor cell line (MC3T3-E1) showed increased expression of osteogenic markers and decreased Zfp687 expression. In the hindlimb bones of C57BL/6J mice, the expression of most bone-forming genes decreased from youth to adulthood, while Zfp687 and Runx2 expression was maintained, being only significantly reduced in old mice in comparison to young mice. Bisulfite sequencing revealed hypomethylation of the Zfp687 promoter during MC3T3-E1 differentiation and bone growth/aging. Bioinformatics predicted miR-142a-3p, miR-122b-5p, and miR-124-3p binding sites in Zfp687 3 ' UTR, and RT-qPCR analysis showed higher expression of these miRNAs in mature osteoblasts. Transfection of a miR-142-3p mimic reduced luciferase activity in the wildtype Zfp687 3 ' UTR but not the mutant 3 ' UTR and downregulated the Zfp687 gene and protein levels. In conclusion, miR-142a-3p directly targets the Zfp687 3 ' UTR, promoting its downregulation during osteoblastogenesis. Furthermore, DNA methylation does not appear to regulate Zfp687 during osteoblast differentiation or bone development in mice.