Neste trabalho utilizamos diferentes abordagens para o estudo de genes associados com desenvolvimento e funcionamento do SNC assim como com doenças neurológicas: 1) uma das abordagens consistiu na identificação do alelo associado a uma forma de retardo mental sindrômico com herança recessiva ligada ao cromossomo X, síndrome de Snyder-Robinson, em uma família Brasileira. Utilizando as estratégias de estudo de ligação genética e análise de genes candidatos, identificamos a segunda mutação patogênica no gene SMS (que codifica a enzima espermina sintase) associada à síndrome de Snyder-Robinson. A identificação dessa mutação contribuiu para: delinear e expandir o espectro clínico da síndrome, evidenciar domínios importantes para o funcionamento da proteína espermina sintase, comprovar a importância dessa proteína nos processos cognitivos, e também possibilitar um aconselhamento genético preciso para membros da família; 2) outra abordagem consistiu em analisar (triar mutação) o gene codificador da proteína colibistina (ARHGEF9), a qual está envolvida em sinaptogênese inibitória, em pacientes Brasileiros portadores de hiperecplexia (6 pacientes) e em pacientes portadores de retardo mental associado com epilepsia (22 pacientes). Não identificamos nenhuma alteração patogênica no gene ARHGEF nos 28 pacientes estudados; contudo, o número de pacientes analisados foi muito pequeno. Julgamos que a análise de um número maior de pacientes com essas doenças neurológicas pode vir a revelar novas mutações deletérias em ARHGEF9; 3) a última abordagem consistiu no estudo funcional da proteína colibistina. Com o objetivo de identificar outras proteínas que interagem com a colibistina humana utilizamos o sistema de duplo-híbrido em leveduras e experimentos de co-imunoprecipitação in vitro e in vivo. Identificamos a proteína eIF3-p40 interagindo com a proteína colibistina e também com a proteína gefirina (a qual, por sua vez, também interage com colibistina e está envolvida com funcionamento de sinapses inibitórias). A proteína eIF3-p40 é uma das subunidades do complexo do fator 3 de iniciação de tradução protéica em eucariotos (eIF3). Essas interações ligam as proteínas colibistina e a gefirina à maquinaria de tradução protéica, revelando uma provável nova função dessas proteínas no controle da tradução em sítios pós-sinápticos inibitórios. / In this work we have used different approaches to the study of genes associated with CNS development and function as well as with neurological diseases: 1) one study involved the identification of the allele associated with an X-linked recessive sindromic form of mental retardation, Snyder-Robinson syndrome, in a Brazilian family. Using genetic linkage analysis and candidate gene strategy, we identified the second pathogenic mutation in the SMS gene (that encodes the spermine synthase enzyme) associated with the Snyder-Robinson syndrome. The identification of this mutation contributed to: the delineation and expansion of the clinical spectrum of the syndrome, highlight important domains for spermine synthase protein functioning, demonstrate the importance of this protein in cognitive processes, and also a precise genetic counseling for family members; 2) a second study involved the mutation screening of ARHGEF9, gene encoding the collybistin protein, which is involved in inhibitory synaptogenesis, in Brazilian patients with hyperekplexia (6 patients) and in patients with mental retardation associated with epilepsy (22 patients). We did not identify any pathogenic alteration in the ARHGEF9, gene in the 28 studied patients, but the number of patients analysed was very small. However, the possibility remains that additional mutations in ARHGEF9, may contribute to other cases of hyperekplexia and mental retardation associated with epilepsy; 3) the last study involved the functional analysis of collybistin protein. In order to identify other proteins that interact with human collybistin, we used the yeast two-hybrid system and in vitro e in vivo co-immunoprecipitation experiments. We identified the eIF3-p40 protein as collybistin and gephyrin (another protein involved in the function of inhibitory synapses that also interacts with collybistin) binding partner. The eIF3-p40 protein is one of the subunits of the eukaryotic initiation factor 3 complex (eIF3). These interactions link the collybistin and gephyrin proteins to the protein translation machinery, revealing a putative new role of these proteins in the translation control at inhibitory postsynaptic sites.

Duchenne Muscular Dystrophy (DMD) is a fatal condition occurring in approximately 1 in 3500 male births and is due to the lack of a protein called dystrophin. Initially DMD was considered a skeletal myopathy, but the pathology and consequences of cardiomyopathy are being increasingly recognised. Fibrosis, resulting from continual cycles of degeneration of the muscle tissues followed by inadequate regeneration of the muscles, is progressive in both cardiac and skeletal dystrophic muscle. In the heart fibrosis interferes with contractility and rhythm whereas it affects contractile function and causes contractures in skeletal muscles. This study utilised the mdx mouse which exhibits a pathological loss of muscle fibres and fibrosis characteristic of DMD, to examine a range of mechanisms that can influence muscle function and fibrosis. Ageing and workload both appear to contribute to the development of dystrophic features in cardiac and skeletal muscle of the mdx mouse. Therefore the effect of eccentric exercise on cardiac and skeletal muscle was examined in older mdx mice. Mice ran in 30 minute sessions for five months, 5 days per week. Downhill treadmill running did not exacerbate the contractile function or fibrosis of the mdx heart or the EDL, SOL or diaphragm muscles suggesting that cytokines influence function and fibrosis to a greater extent than workload alone. The role of the cytokine TGF-beta was examined by treating mdx mice with the TGF-beta antagonist pirfenidone at 0.4, 0.8 or 1.2 per cent in drinking water for six months. Pirfenidone improved cardiac contractility (P<0.01) and coronary flow (P<0.05), to levels comparable to control mice, despite no reduction in cardiac fibrosis. Pirfenidone did not reduce fibrosis or improve function in skeletal muscle. A deficiency of neuronal nitric oxide synthase (nNOS) in DMD and mdx mice causes a lowered production of nitric oxide indicating that the substrate of nNOS, l-arginine, may be beneficial to cardiac and skeletal muscle function in mdx mice. Oral l-arginine (5 mg/g bw) improved cardiac contractility, coronary flow and reduced cardiac fibrosis (P<0.05) without improving skeletal muscle function or fibrosis. In contrast, 10 mg/g bw l-arginine improved cardiac function and coronary flow (P<0.01), despite also elevating cardiac collagen. This increment in collagen was prevented by co-administration of prednisone. The experiments described in this dissertation reveal for the first time that pharmacological treatments in mdx mice can improve cardiac structure and function. Further elucidation of the optimum time and doses of such treatments may result in future pharmacological treatments to improve cardiac function and fibrosis in DMD.

No description available.

Atopic dermatitis (AD), ichthyosis vulgaris (IV), and X-linked recessive ichthyosis (XLRI) are characterized by dry skin and impaired skin barrier. AD and IV are related to loss-of-function mutations in FLG (encoding filaggrin), whereas XLRI is caused by deletions or inactivating mutations in the steroid sulphatase gene (STS). Patients regularly use moisturizing creams, but little is known about the creams’ effects on the skin barrier. The present work combines objective scorings, non-invasive techniques, and molecular analyses of skin biopsies to characterize the skin in 57 patients with AD, IV, or XLRI, and in 14 healthy controls. Patients were classified according to their FLG and STS mutation status: AD with FLG+/+ (n = 14), AD with FLG+/– (n = 14), AD/IV with FLG–/– (n = 15), and XLRI with STS– (n = 14), as well as one man with a novel point mutation. Assessments were conducted at baseline and after four weeks of treatment with three different moisturizers applied to volar forearm skin. At baseline, dryness scoring and non-invasive assessments verified impaired skin barrier function in all patients. In patients with AD/IV, microarray analysis identified 300–3000 up- or downregulated mRNA transcripts involved in signalling pathways important for inflammation and barrier repair. The skin phenotype and number of altered transcripts were correlated with the FLG mutation status, with FLG–/– patients displaying the highest transepidermal water loss (TEWL) and the most altered transcript levels. In contrast, despite an equally dysfunctional skin barrier, only limited changes in mRNA transcripts occurred in XLRI patients. Treatment with moisturizers improved skin dryness similarly in all groups, but TEWL behaved differently: it decreased slightly in the AD/IV group and increased in the XLRI group, especially after urea treatment. Only minute effects on skin pH and mRNA expression were observed. In conclusion, FLG mutations elicit pro-inflammatory mechanisms probably aimed at restoring barrier competence. This does not occur in patients with XLRI, presumably because STS deficiency automatically increases the barrier thickness. Moisturizing treatment improves skin dryness in patients with AD, IV, or XLRI, but does not seem to normalize the altered epidermal gene expression profile in AD/IV patients.

No description available.

The biosynthesis of tetrapyrolles in eukaryotes and the alpha-subclass of purple photosynthetic bacteria is controlled by the pyridoxal 5?-phosphate (PLP)-dependent enzyme, 5-aminolevulinate synthase (ALAS). Aminolevulinate, the universal building block of these macromolecules, is produced together with Coenzyme A (CoA) and carbon dioxide from the condensation of glycine and succinyl-CoA. The three-dimensional structures of Rhodobacter capsulatus ALAS reveal a conserved active site serine that moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed, substrate-bound enzyme conformation, and simultaneously to within 3-4 angstroms of the thioester sulfur atom of bound succinyl-CoA. To elucidate the role(s) this residue play(s) in enzyme activity, the equivalent serine in murineerythroid ALAS was mutated to threonine or alanine. The S254A variant was active, but both the KmSCoA and kcat values were increased, by 25- and 2-fold, respectively, suggesting the increase in turnover is independent of succinyl-CoA-binding. In contrast, substitution of S254 with threonine results in a decreased kcat, however the Km for succinyl-CoA is unaltered. Removal of the side chain hydroxyl group in the S254A variant notably changes the spectroscopic properties of the PLP cofactor and the architecture of the PLP-binding site as inferred from circular dichroism spectra. Experiments examining the rates associated with intrinsic protein fluorescence quenching of the variant enzymes in response to ALA binding show that S254 affects product dissociation. Together, the data led us to suggest that succinyl-CoA binding in concert with the hydrogen bonding state of S254 governs enzyme conformational equilibria. As a member of the alpha-oxoamine synthase family, ALAS shares a high degree of structural similarity and reaction chemistry with the other enzymes in the group. Crystallographic studies of the R. capsulatus ALAS structure show that the alkanoate component of succinyl-CoA is bound by a conserved arginine and a threonine. To examine acyl-CoA-binding and substrate discrimination in murine erythroid ALAS, the corresponding residues (R85 and T430) were mutated and a series of CoA substrate analogs were tested. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that calculated for the wild-type enzyme, suggesting this residue is strategic in substrate binding. Hydrophobic substitutions of the residues that coordinate acyl-CoA-binding produce ligand-induced changes in the CD spectra, indicating that these amino acids affect substrate-mediated changes to the microenvironment of the chromophore. Pre-steady-state kinetic analyses of the R85K variant-catalyzed reaction show that both the rates associated with product-binding and the parameters that define quinonoid intermediate lifetime are dependent on the chemical composition of the acyl-CoA tail. Each of the results in this study emphasizes the importance of the relationship between the bifurcate interaction of the alkanoic acid component of succinyl-CoA and the side chains of R85 and T430. From the X-ray crystal structures of Escherichia coli 8-amino-7-oxonoanoate synthase and R. capsulatus ALAS, it was inferred that a loop covering the active site moved 3-6 Å between the holoenzymic and acyl-CoA-bound conformations. To elucidate the role that the active site lid plays in enzyme function, we shuffled the portion of the murine erythroid ALAS cDNA corresponding to the lid sequence (Y422-R439), and isolated functional variants based on genetic complementation in an ALA-deficient strain. Variants with potentially greater enzymatic activity than the wild-type enzyme were screened for increased porphyrin overproduction. Turnover number and the catalytic efficiency of selected functional variants with both substrates were increased for each of the enzyme variants tested, suggesting that increased activity is linked to alterations of the loop motif. The results of transient kinetics experiments for three isolated variants when compared to wild-type ALAS showed notable differences in the pre-steady-state rates that define the kinetic mechanism, indicating that the rate of ALA release is not rate-limiting for these enzymes. The thermodynamic parameters for a selected variant-catalyzed reaction indicated a reduction in the amount of energy required for catalysis. This finding is consistent with the proposal that, in contrast to the wild-type ALAS reaction, a protein conformational change associated with ALA release no longer limits turnover for this variant enzyme.

Rett Syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). The mosaic brain environment in heterozygous (MECP2+/-) females consists of both MeCP2-wildtype (MeCP2+) and Mecp2-mutant (MeCP2-) neurons. To separate possible cell autonomous and cell non-autonomous effects three-dimensional morphological analysis was performed on individually genotyped layer V pyramidal neurons in the primary motor cortex of heterozygous (Mecp2+/-) and wild-type (Mecp2+/+) mature female mice (>8 months old) from the Mecp2tm1.1Jae line. Mecp2+/+ neurons and Mecp2+ were found to be indistinguishable while Mecp2- neurons have significantly reduced basal dendritic length (p<0.05), predominantly in the region 70-130 μm from the cell body, culminating in a total reduction of 15%. Mecp2- neurons have three (17%) fewer total branch points, lost specifically at the second and third branch orders. Thus the reduced total dendritic length in Mecp2- neurons is a result of fewer higher-order branches. Soma and nuclear areas of 30 Mecp2+/- female mice (5-21 months) with X chromosome inactivation (XCI) ratios ranging from 12% to 56% were analyzed. On average Mecp2- somata and nuclei were 15% and 13% smaller than Mecp2+ neurons respectively. The variation observed in the soma and nuclear sizes of Mecp2- neurons was not due to age, but was found to be correlated with the XCI ratio. Animals with a balanced XCI ratio (approximately 50% Mecp2-) were found to have Mecp2- neurons with a less severe cellular phenotype (11-17% smaller than Mecp2+). Animals with a highly skewed XCI ratio favouring expression of the wild-type allele (less than 30% Mecp2-) were found to have a more severe Mecp2- cellular phenotype (17-22% smaller than Mecp2+). These data support indicate that mutations in Mecp2 exert both cell autonomous and cell non- autonomous effects on neuronal morphology. / Graduate

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Deficiência Intelectual (DI) é uma condição definida como um funcionamento intelectual significativamente prejudicado, expresso juntamente com limitações em pelo menos duas áreas do comportamento adaptativo que se manifestam antes dos 18 anos de idade. A prevalência estimada da DI na população em geral é de 2-3% e um número expressivo de casos permanece sem um diagnóstico definitivo. Há um consenso geral de que a DI é mais comum em indivíduos do sexo masculino em relação aos do sexo feminino. Entre as explicações para este excesso está a concentração de genes específicos para a habilidade cognitiva no cromossomo X. MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificador que modulam a expressão gênica pós-transcricional de RNAs mensageiros alvo. Recentemente, estudos têm demonstrado a importância essencial dos miRNAs para o desenvolvimento e funcionamento cerebrais e sabe-se que o cromossomo X tem uma alta densidade de genes de miRNAs. Neste contexto, os miRNAs são candidatos potenciais como fatores genéticos envolvidos na Deficiência Intelectual Ligada ao X (DILX). Neste estudo, foram analisadas as regiões genômicas de 17 genes de miRNAs expressos no cérebro localizados no cromossomo X, com o objetivo de investigar o possível envolvimento de variantes na sequência destes miRNAs na DILX. Para este fim, selecionamos amostras de DNA genômico (sangue periférico) de 135 indivíduos do sexo masculino portadores de DI sugestiva de DILX de um grupo de mais de 1.100 pacientes com DI encaminhados ao Serviço de Genética Humana da UERJ. O critério de inclusão para este estudo era de que os probandos apresentassem um ou mais parentes do sexo masculino afetados pela DI que fossem interligados por via materna. As amostras de DNA dos pacientes foram amplificadas utilizando a técnica de reação em cadeia da polimerase, seguida por purificação e sequenciamento direto pelo método de Sanger dos fragmentos amplificados. Para avaliar a conservação dos 17 miRNAs foi realizada uma análise filogenética in silico incluindo sequências dos miRNAs selecionados de humanos e de outras 8 espécies de primatas estreitamente relacionadas. Não foram encontradas alterações nas sequências nos genes de 17 miRNAs analisados, mesmo diante do padrão genético altamente heterogêneo da população brasileira. Adicionalmente, a análise filogenética destes miRNAs revelou uma alta conservação entre as espécies comparadas. Considerando o papel dos miRNAs como reguladores da expressão gênica, a ausência de alterações e a alta conservação entre primatas sugerem uma forte pressão seletiva sobre estas moléculas, reforçando a sua importância funcional para o organismo em geral. Apesar de não termos encontrado variantes de sequência nos miRNAs estudados, o envolvimento de miRNAs na DI não pode ser completamente descartado. Alterações fora da molécula de miRNA precursor, nos fatores de processamento, nos sítios alvo e variações no número de cópias de genes de miRNAs podem implicar em alteração na expressão dos miRNAs e, consequentemente, na funcionalidade do miRNA maduro. Sendo assim, uma análise sistemática da expressão de miRNAs em pacientes com DILX é urgentemente necessária, a fim de desvendar novos genes/mecanismos moleculares relacionados a esta condição. / Intellectual Disability (ID) is defined as a significantly impaired intellectual functioning, along with limitations in at least two areas of adaptive behavior appearing before 18 years of age. The estimated prevalence of ID in the general population is 2-3% and a significant number of cases remain without a definite diagnosis. There is a general consensus that ID is more common in males compared to females. Among the explanations for this excess it is the concentration of specific genes for cognitive ability on the chromosome X. MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate post-transcriptional gene expression of target messenger RNAs. Recently, studies have demonstrated the essential importance of miRNAs for brain development and function and chromosome X has a high density of miRNA genes. In this context, miRNAs are potential candidates as genetic factors involved in X-Linked Intellectual Disability (XLID). In this study, the genomic regions of 17 brain-expressed miRNA genes located on the chromosome X were analyzed, aiming to investigate the possible involvement of sequence variants in these miRNAs in XLID. For this purpose, genomic DNA samples (peripheral blood) were obtained from 135 male patients with ID suggestive of XLID that were selected from a group of over 1,100 patients with ID referred to to the Human Genetics Laboratory of the State University of Rio de Janeiro. Inclusion criteria were at least two affected males in different generations related through maternal lineage. DNA samples from patients were amplified using the polymerase chain reaction technique, followed by purification and direct sequencing by Sanger method. To assess the conservation of the 17 miRNAs, in silico phylogenetic analysis was performed, including sequences of the chosen miRNAs from human and from other 8 closely related primate species. No sequence changes were found for the 17 miRNA genes analyzed, even in light of the highly heterogeneous genetic nature of the Brazilian population. Furthermore, phylogenetic analysis of the selected miRNAs revealed a high conservation among the compared species. Considering the role of miRNAs as regulators of gene expression, the absence of variations and the high conservation among primates suggest a strong selective pressure on these molecules, emphasizing their functional importance for the body in general. Although we have found no sequence variants in the miRNAs studied, the involvement of miRNAs in ID cannot be completely rejected. Alterations outside the precursor miRNA molecules, in the processing factors, in the target sites and changes in copy number of miRNA genes can result in abnormal expression of miRNAs and, consequently, in the functionality of the mature miRNA. Thus, a systematic analysis of miRNAs expression in patients with XLID is urgently needed in order to uncover new genes/molecular mechanisms related to this condition.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Deficiência Intelectual (DI) é uma condição definida como um funcionamento intelectual significativamente prejudicado, expresso juntamente com limitações em pelo menos duas áreas do comportamento adaptativo que se manifestam antes dos 18 anos de idade. A prevalência estimada da DI na população em geral é de 2-3% e um número expressivo de casos permanece sem um diagnóstico definitivo. Há um consenso geral de que a DI é mais comum em indivíduos do sexo masculino em relação aos do sexo feminino. Entre as explicações para este excesso está a concentração de genes específicos para a habilidade cognitiva no cromossomo X. MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificador que modulam a expressão gênica pós-transcricional de RNAs mensageiros alvo. Recentemente, estudos têm demonstrado a importância essencial dos miRNAs para o desenvolvimento e funcionamento cerebrais e sabe-se que o cromossomo X tem uma alta densidade de genes de miRNAs. Neste contexto, os miRNAs são candidatos potenciais como fatores genéticos envolvidos na Deficiência Intelectual Ligada ao X (DILX). Neste estudo, foram analisadas as regiões genômicas de 17 genes de miRNAs expressos no cérebro localizados no cromossomo X, com o objetivo de investigar o possível envolvimento de variantes na sequência destes miRNAs na DILX. Para este fim, selecionamos amostras de DNA genômico (sangue periférico) de 135 indivíduos do sexo masculino portadores de DI sugestiva de DILX de um grupo de mais de 1.100 pacientes com DI encaminhados ao Serviço de Genética Humana da UERJ. O critério de inclusão para este estudo era de que os probandos apresentassem um ou mais parentes do sexo masculino afetados pela DI que fossem interligados por via materna. As amostras de DNA dos pacientes foram amplificadas utilizando a técnica de reação em cadeia da polimerase, seguida por purificação e sequenciamento direto pelo método de Sanger dos fragmentos amplificados. Para avaliar a conservação dos 17 miRNAs foi realizada uma análise filogenética in silico incluindo sequências dos miRNAs selecionados de humanos e de outras 8 espécies de primatas estreitamente relacionadas. Não foram encontradas alterações nas sequências nos genes de 17 miRNAs analisados, mesmo diante do padrão genético altamente heterogêneo da população brasileira. Adicionalmente, a análise filogenética destes miRNAs revelou uma alta conservação entre as espécies comparadas. Considerando o papel dos miRNAs como reguladores da expressão gênica, a ausência de alterações e a alta conservação entre primatas sugerem uma forte pressão seletiva sobre estas moléculas, reforçando a sua importância funcional para o organismo em geral. Apesar de não termos encontrado variantes de sequência nos miRNAs estudados, o envolvimento de miRNAs na DI não pode ser completamente descartado. Alterações fora da molécula de miRNA precursor, nos fatores de processamento, nos sítios alvo e variações no número de cópias de genes de miRNAs podem implicar em alteração na expressão dos miRNAs e, consequentemente, na funcionalidade do miRNA maduro. Sendo assim, uma análise sistemática da expressão de miRNAs em pacientes com DILX é urgentemente necessária, a fim de desvendar novos genes/mecanismos moleculares relacionados a esta condição. / Intellectual Disability (ID) is defined as a significantly impaired intellectual functioning, along with limitations in at least two areas of adaptive behavior appearing before 18 years of age. The estimated prevalence of ID in the general population is 2-3% and a significant number of cases remain without a definite diagnosis. There is a general consensus that ID is more common in males compared to females. Among the explanations for this excess it is the concentration of specific genes for cognitive ability on the chromosome X. MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate post-transcriptional gene expression of target messenger RNAs. Recently, studies have demonstrated the essential importance of miRNAs for brain development and function and chromosome X has a high density of miRNA genes. In this context, miRNAs are potential candidates as genetic factors involved in X-Linked Intellectual Disability (XLID). In this study, the genomic regions of 17 brain-expressed miRNA genes located on the chromosome X were analyzed, aiming to investigate the possible involvement of sequence variants in these miRNAs in XLID. For this purpose, genomic DNA samples (peripheral blood) were obtained from 135 male patients with ID suggestive of XLID that were selected from a group of over 1,100 patients with ID referred to to the Human Genetics Laboratory of the State University of Rio de Janeiro. Inclusion criteria were at least two affected males in different generations related through maternal lineage. DNA samples from patients were amplified using the polymerase chain reaction technique, followed by purification and direct sequencing by Sanger method. To assess the conservation of the 17 miRNAs, in silico phylogenetic analysis was performed, including sequences of the chosen miRNAs from human and from other 8 closely related primate species. No sequence changes were found for the 17 miRNA genes analyzed, even in light of the highly heterogeneous genetic nature of the Brazilian population. Furthermore, phylogenetic analysis of the selected miRNAs revealed a high conservation among the compared species. Considering the role of miRNAs as regulators of gene expression, the absence of variations and the high conservation among primates suggest a strong selective pressure on these molecules, emphasizing their functional importance for the body in general. Although we have found no sequence variants in the miRNAs studied, the involvement of miRNAs in ID cannot be completely rejected. Alterations outside the precursor miRNA molecules, in the processing factors, in the target sites and changes in copy number of miRNA genes can result in abnormal expression of miRNAs and, consequently, in the functionality of the mature miRNA. Thus, a systematic analysis of miRNAs expression in patients with XLID is urgently needed in order to uncover new genes/molecular mechanisms related to this condition.

Neste trabalho utilizamos diferentes abordagens para o estudo de genes associados com desenvolvimento e funcionamento do SNC assim como com doenças neurológicas: 1) uma das abordagens consistiu na identificação do alelo associado a uma forma de retardo mental sindrômico com herança recessiva ligada ao cromossomo X, síndrome de Snyder-Robinson, em uma família Brasileira. Utilizando as estratégias de estudo de ligação genética e análise de genes candidatos, identificamos a segunda mutação patogênica no gene SMS (que codifica a enzima espermina sintase) associada à síndrome de Snyder-Robinson. A identificação dessa mutação contribuiu para: delinear e expandir o espectro clínico da síndrome, evidenciar domínios importantes para o funcionamento da proteína espermina sintase, comprovar a importância dessa proteína nos processos cognitivos, e também possibilitar um aconselhamento genético preciso para membros da família; 2) outra abordagem consistiu em analisar (triar mutação) o gene codificador da proteína colibistina (ARHGEF9), a qual está envolvida em sinaptogênese inibitória, em pacientes Brasileiros portadores de hiperecplexia (6 pacientes) e em pacientes portadores de retardo mental associado com epilepsia (22 pacientes). Não identificamos nenhuma alteração patogênica no gene ARHGEF nos 28 pacientes estudados; contudo, o número de pacientes analisados foi muito pequeno. Julgamos que a análise de um número maior de pacientes com essas doenças neurológicas pode vir a revelar novas mutações deletérias em ARHGEF9; 3) a última abordagem consistiu no estudo funcional da proteína colibistina. Com o objetivo de identificar outras proteínas que interagem com a colibistina humana utilizamos o sistema de duplo-híbrido em leveduras e experimentos de co-imunoprecipitação in vitro e in vivo. Identificamos a proteína eIF3-p40 interagindo com a proteína colibistina e também com a proteína gefirina (a qual, por sua vez, também interage com colibistina e está envolvida com funcionamento de sinapses inibitórias). A proteína eIF3-p40 é uma das subunidades do complexo do fator 3 de iniciação de tradução protéica em eucariotos (eIF3). Essas interações ligam as proteínas colibistina e a gefirina à maquinaria de tradução protéica, revelando uma provável nova função dessas proteínas no controle da tradução em sítios pós-sinápticos inibitórios. / In this work we have used different approaches to the study of genes associated with CNS development and function as well as with neurological diseases: 1) one study involved the identification of the allele associated with an X-linked recessive sindromic form of mental retardation, Snyder-Robinson syndrome, in a Brazilian family. Using genetic linkage analysis and candidate gene strategy, we identified the second pathogenic mutation in the SMS gene (that encodes the spermine synthase enzyme) associated with the Snyder-Robinson syndrome. The identification of this mutation contributed to: the delineation and expansion of the clinical spectrum of the syndrome, highlight important domains for spermine synthase protein functioning, demonstrate the importance of this protein in cognitive processes, and also a precise genetic counseling for family members; 2) a second study involved the mutation screening of ARHGEF9, gene encoding the collybistin protein, which is involved in inhibitory synaptogenesis, in Brazilian patients with hyperekplexia (6 patients) and in patients with mental retardation associated with epilepsy (22 patients). We did not identify any pathogenic alteration in the ARHGEF9, gene in the 28 studied patients, but the number of patients analysed was very small. However, the possibility remains that additional mutations in ARHGEF9, may contribute to other cases of hyperekplexia and mental retardation associated with epilepsy; 3) the last study involved the functional analysis of collybistin protein. In order to identify other proteins that interact with human collybistin, we used the yeast two-hybrid system and in vitro e in vivo co-immunoprecipitation experiments. We identified the eIF3-p40 protein as collybistin and gephyrin (another protein involved in the function of inhibitory synapses that also interacts with collybistin) binding partner. The eIF3-p40 protein is one of the subunits of the eukaryotic initiation factor 3 complex (eIF3). These interactions link the collybistin and gephyrin proteins to the protein translation machinery, revealing a putative new role of these proteins in the translation control at inhibitory postsynaptic sites.