RESUMO
This study endeavors to scrutinize the perspectives of primary school teachers regarding children's rights. Employing qualitative research methods, particularly a case study approach, the research delves into the insights of 14 teachers working in a primary school within the Turkish Cypriot region during the 2022-2023 academic year. Data collection was facilitated through the utilization of a semi-structured interview form, and subsequent analysis was conducted via content analysis. The findings underscore a discernible lack of adequate knowledge among teachers pertaining to children's rights, particularly in the context of violations occurring on social media platforms. In response, recommendations are posited, advocating for the implementation of in-service training programs to enhance teachers' awareness, the integration of children's rights throughout all stages of primary education, collaborative efforts between the Turkish Education Institution and the Information Technologies Communication Authority to raise awareness among families and educators, and the inclusion of a dedicated course on children's rights in the curriculum of the Turkish Cypriot Region Teacher Academy and university faculties of education.
Assuntos
Professores Escolares , Instituições Acadêmicas , Humanos , Professores Escolares/psicologia , Turquia , Criança , Feminino , Masculino , Adulto , Pesquisa Qualitativa , Docentes , Direitos Humanos , Pessoa de Meia-IdadeRESUMO
Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.
Assuntos
Doenças Cerebelares/metabolismo , Cílios/metabolismo , Anormalidades do Olho/metabolismo , Doenças Renais Císticas/metabolismo , Amaurose Congênita de Leber/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Anormalidades Múltiplas , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Doenças Cerebelares/enzimologia , Doenças Cerebelares/genética , Cerebelo/anormalidades , Cílios/enzimologia , Cílios/genética , Proteínas do Citoesqueleto , Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Humanos , Doenças Renais Císticas/enzimologia , Doenças Renais Císticas/genética , Amaurose Congênita de Leber/enzimologia , Amaurose Congênita de Leber/genética , Quinases Relacionadas a NIMA , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Ratos , Ratos Wistar , Retina/anormalidades , Retina/enzimologia , Retina/metabolismoRESUMO
Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA-binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP-seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.
Assuntos
Cromossomos Humanos Par 7/genética , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Deformidades Congênitas dos Membros/genética , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Pré-Escolar , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 7/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/metabolismo , Humanos , Queratinócitos/metabolismo , Deformidades Congênitas dos Membros/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo , Peixe-ZebraRESUMO
BACKGROUND: Sensenbrenner syndrome is a heterogeneous ciliopathy that is characterised by skeletal and ectodermal anomalies, accompanied by chronic renal failure, heart defects, liver fibrosis and other features. OBJECTIVE: To identify an additional causative gene in Sensenbrenner syndrome. METHODS: Single nucleotide polymorphism array analysis and standard sequencing techniques were applied to identify the causative gene. The effect of the identified mutation on protein translation was determined by western blot analysis. Antibodies against intraflagellar transport (IFT) proteins were used in ciliated fibroblast cell lines to investigate the molecular consequences of the mutation on ciliary transport. RESULTS: Homozygosity mapping and positional candidate gene sequence analysis were performed in two siblings with Sensenbrenner syndrome of a consanguineous Moroccan family. In both siblings, a homozygous mutation in the initiation codon of C14ORF179 was identified. C14ORF179 encodes IFT43, a subunit of the IFT complex A (IFT-A) machinery of primary cilia. Western blots showed that the mutation disturbs translation of IFT43, inducing the initiation of translation of a shorter protein product from a downstream ATG. The IFT-A protein complex is implicated in retrograde ciliary transport along axonemal microtubules. It was shown that in fibroblasts of one of the siblings affected by Sensenbrenner syndrome, disruption of IFT43 disturbs this transport from the ciliary tip to its base. As anterograde transport in the opposite direction apparently remains functional, the IFT complex B proteins accumulate in the ciliary tip. Interestingly, similar results were obtained using fibroblasts from a patient with Sensenbrenner syndrome with mutations in WDR35/IFT121, encoding another IFT-A subunit. CONCLUSIONS: The results indicate that Sensenbrenner syndrome is caused by disrupted IFT-A-mediated retrograde ciliary transport.
Assuntos
Proteínas de Transporte/genética , Cílios/metabolismo , Anormalidades Craniofaciais/genética , Displasia Ectodérmica/genética , Flagelos/metabolismo , Transporte Proteico/genética , Proteínas Recombinantes/genética , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Criança , Cílios/genética , Anormalidades Craniofaciais/etnologia , Displasia Ectodérmica/etnologia , Fibroblastos/fisiologia , Flagelos/genética , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Marrocos/etnologia , Mutação , Países Baixos/epidemiologia , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/metabolismo , Irmãos , Síndrome , TransfecçãoRESUMO
Missense mutations in the 3' end of the p63 gene are associated with either RHS (Rapp-Hodgkin syndrome) or AEC (Ankyloblepharon Ectodermal defects Cleft lip/palate) syndrome. These mutations give rise to mutant p63alpha protein isoforms with dominant effects towards their wild-type counterparts. Here we report four RHS/AEC-like patients with mutations (p.Gln9fsX23, p.Gln11X, p.Gln16X), that introduce premature termination codons in the N-terminal part of the p63 protein. These mutations appear to be incompatible with the current paradigms of dominant-negative/gain-of-function outcomes for other p63 mutations. Moreover it is difficult to envisage how the remaining small N-terminal polypeptide contributes to a dominant disease mechanism. Primary keratinocytes from a patient containing the p.Gln11X mutation revealed a normal and aberrant p63-related protein that was just slightly smaller than the wild-type p63. We show that the smaller p63 protein is produced by translation re-initiation at the next downstream methionine, causing truncation of a non-canonical transactivation domain in the DeltaN-specific isoforms. Interestingly, this new DeltaDeltaNp63 isoform is also present in the wild-type keratinocytes albeit in small amounts compared with the p.Gln11X patient. These data establish that the p.Gln11X-mutation does not represent a null-allele leading to haploinsufficiency, but instead gives rise to a truncated DeltaNp63 protein with dominant effects. Given the nature of other RHS/AEC-like syndrome mutations, we conclude that these mutations affect only the DeltaNp63alpha isoform and that this disruption is fundamental to explaining the clinical characteristics of these particular ectodermal dysplasia syndromes.
Assuntos
Anormalidades Múltiplas/genética , Códon sem Sentido , Displasia Ectodérmica/genética , Proteínas de Membrana/genética , Anormalidades da Boca/genética , Biossíntese de Proteínas , Anormalidades Múltiplas/metabolismo , Adolescente , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Criança , Pré-Escolar , Displasia Ectodérmica/metabolismo , Feminino , Humanos , Queratinócitos/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Anormalidades da Boca/embriologia , Anormalidades da Boca/metabolismo , Alinhamento de Sequência , Transcrição Gênica , Ativação TranscricionalRESUMO
Heterozygous mutations in the p63 gene underlie a group of at least seven allelic syndromes, including ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC) and Rapp Hodgkin syndrome (RHS), which involves varying degrees of ectodermal dysplasia, orofacial clefting and limb malformations. Mutations in the AEC and Rapp Hodgkin syndromes cluster in the 3' end of the p63 gene. Previously reported mutations are mainly missense and frameshift mutations in exons 13 and 14, affecting the p63alpha-specific SAM (sterile alpha motif) and TI (transactivation inhibitory) domains. A patient cohort affected by AEC syndrome was evaluated during International Research Symposium supported by the National Foundation for Ectodermal Dysplasias. Nineteen patients underwent full clinical evaluations and 18 had findings consistent with a diagnosis of AEC syndrome. These 19 patients, along with 5 additional relatives had genomic DNA analysis. Twenty-one of the 24 participants from 12 families were found to have mutations in the p63 gene. Eleven different mutations were identified; 10 were novel mutations. Eight were missense mutations within the coding region of the SAM domain. Three other mutations were located in exon 14 sequences, which encode the TI domain. The effects of the mutations in the SAM and TI domains are poorly understood and functional studies are required to understand the pathological mechanisms. However, AEC and RHS mutations in the 5' and 3' ends of the p63 gene point towards a critical role of the DeltaNp63alpha isoform for the AEC/RHS phenotype.
Assuntos
Anormalidades Múltiplas/genética , Fenda Labial/genética , Fissura Palatina/genética , Análise Mutacional de DNA , Displasia Ectodérmica/genética , Pálpebras/anormalidades , Transativadores/genética , Proteínas Supressoras de Tumor/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/patologia , Sequência de Aminoácidos , Animais , Fenda Labial/diagnóstico , Fenda Labial/patologia , Fissura Palatina/diagnóstico , Fissura Palatina/patologia , Estudos de Coortes , DNA/química , DNA/genética , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/patologia , Éxons/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP , Mutação , Isoformas de Proteínas/genética , Proteína 1 com Domínio SAM e Domínio HD , Alinhamento de Sequência , Síndrome , Transativadores/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/químicaRESUMO
Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.