Introdução: A rede lisossômica é um complexo de vias metabólicas que influenciam processos como degradação de organelas danificadas ou senescentes, processamento de antígenos, reutilização de aminoácidos essenciais e, em última instância, um sistema de fundamental importância para a fisiologia celular normal. Nesse contexto, os lisossomos possuem uma relevância muito grande, uma vez que é nessa organela que ocorre a destruição das moléculas envolvidas em todos os processos citados acima. Entre as unidades operacionais nos lisossomos estão as hidrolases lisossômicas, que são mais de 50 enzimas com capacidade, em ambiente ácido, de realizar a quebra de substratos específicos. A GlcNAc-fosfotransferase é um complexo hexamérico (J2K2L2) residente na porção cis do complexo de Golgi que realiza a adição de resíduos de manose-6- fosfato nas cadeias de oligossacarídeos das hidrolases lisossômicas. Com ação subsequente, a enzima descobridora realiza a remoção da manose, expõe os resíduos de fosfato e possibilita que as hidrolases sejam reconhecidas pelos receptores de manose-6- fosfato e direcionadas aos compartimentos lisossomais. As subunidades J e K da GlcNAc-fosfotransferase são codificadas pelo gene GNPTAB, localizado no cromossomo 12, constituído de 21 éxons, e a subunidade L pelo gene GNPTG, localizado no cromossomo 16, constituído de 11 éxons. Alterações patogênicas em GNPTAB podem causar as doenças Mucolipidose II ou III alfa/beta e alterações em GNPTG causam a doença Mucolipidose III gama. O defeito genético leva à atividade residual, ou nula, da enzima que acaba por gerar o extravasamento das hidrolases lisossômicas ao meio extracelular e o acúmulo de substratos nos lisossomos. Objetivos: (1) caracterizar as alterações patogênicas em GNPTAB em um grupo de pacientes brasileiros não relacionados com Mucolipidose II ou III alfa/beta, e (2) definir um protocolo de pesquisa molecular para os pacientes brasileiros. Metodologia: É um estudo transversal, com amostragem por conveniência, e inclui pacientes com diagnóstico clínico e bioquímico de Mucolipidose II ou III. Foi extraído DNA genômico dos pacientes a partir de sangue obtido por punção venosa periférica. O gene GNPTAB foi sequenciado através da técnica de Sanger. As alterações do tipo troca de sentido foram analisadas pelos programas de Bioinformática Polyphen2, Sift e Consurf, e as preditas como patogênicas foram pesquisadas em alelos controles brasileiros e analisadas por estudos funcionais, quando possível. A geração de construtos das alterações p.Ser385Leu e c.3503_3504delTC foi realizada por mutagênese sítio-dirigida e a atividade residual destes foi avaliada 24 horas após expressão em células HEK. Para a análise financeira, valores atuais de equipamentos, reagente de biologia molecular e materiais plásticos foram utilizados para estimar o custo de uma extração de DNA, reação de PCR, purificação com PEG8000 e sequenciamento. Resultados: Foram incluídos 13 pacientes (ML II= 8; ML III= 5) e, adicionalmente, de uma mãe de paciente com diagnóstico clínico e bioquímico de ML II. A análise molecular identificou seis alterações patogênicas novas, as c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly) e p.Try1111*. A análise de bioinformática das alterações do tipo troca de sentido as caracterizaram como prejudiciais para a função da proteína e os resíduos 76 e 385 como estrutural e funcional, respectivamente, além de ambos como altamente conservados entre as espécies. A análise funcional dos mutantes p.Ser385Leu e c.3503_3504delTC identificaram atividades residuais de 1,5% e nula, respectivamente. Também foram identificadas outras seis alterações patogênicas previamente descritas. A alteração c.3503_3504delTC foi a que apresentou a maior frequência (40%, n= 10/25 alelos), seguido pela p.Ile403The (12%, n=3/25 alelos). Quanto às relações genótipo-fenótipo, sete pacientes com ML II possuem genótipos combinados de alterações do tipo mudança de fase de leitura e sem sentido, enquanto que os cincos pacientes com ML III alfa/beta apresentam pelo menos uma alteração do tipo troca de sentido, o que evidencia a relação entre alterações que impactam a funcionalidade da proteína e fenótipos mais graves. A análise retrospectiva definiu o protocolo 1.0 que finalizaria o diagnóstico com um custo médio de R$ 338,45, em uma amostra de 25 pacientes. A análise prospectiva do protocolo 2.0, sobre a mesma amostra de pacientes, indicou que o mesmo finalizaria o diagnóstico com o custo médio de R$ 299,80, uma economia de 25%. Discussão/Conclusão: As novas alterações patogênicas descritas nesse trabalho confirmam a alta heterogeneidade alélica do gene GNPTAB. A análise funcional da alteração p.Ser385Leu confirma sua patogenicidade, que está de acordo com o fenótipo ML II do paciente, e evidencia a necessidade de mais estudos a fim de constatar o motivo desse resíduo ser importante para a proteína. A síntese dos protocolos demonstrou ser uma estratégia interessante e economicamente importante, uma vez que diminui os gastos envolvidos para finalizar o diagnóstico molecular. / Introduction: The lysosomal network is a complex of metabolic ways that influence processes like damaged or senescent organelles degradation, antigen processing, essential amino acid reutilization, and, in the last instance, it is important for normal cell physiology. In this context, lysosomes have a great relevance since it is in this organelle that the destruction of the molecules involved in all the processes mentioned above occurs. The operational units in the lysosomes are the lysosomal hydrolases that are more than 50 enzymes with capacity, in an acid environment, to breakdown specific substrates. GlcNAc-phosphotransferase is a hexameric complex (J2K2L2) located in the cis portion of the Golgi complex that performs the addition of mannose-6-phosphate residues in oligosaccharides chains on lysosomal hydrolases. In a subsequent way, the uncovering enzyme removes the mannose residues, exposes the phosphate residues, and enables the recognition of hydrolases by mannose-6-phosphate receptors. The J and K subunits are codified by GNPTAB gene, which is located in chromosome 12 and consists of 21 exons, and the L subunit, encoded by GNPTG gene, that is located in chromosome 16 and consists of 11 exons. The consequences of pathogenic alterations in GNPTAB are Mucolipidosis II or III alpha/beta diseases and alterations in the GNPTG are Mucolipidosis III gamma disease. The genetic defect leads to residual or absent activity of enzyme which ultimately generates an overflow of lysosomal hydrolases to the extracellular environment and accumulation of substrates in lysosomes. Objectives: (1) to characterize, by sequencing of the GNPTAB gene, the pathogenic alterations in a group of unrelated Brazilian patients with Mucolipidosis II or III alpha/beta, and (2) to define a molecular research protocol for Brazilian patients. Methodology: It is a crosssectional study with convenience sampling, and it includes patients with biochemical and clinical diagnosis of Mucolipidosis II or III. The DNA was amplified by PCR technique and sequencing by Sanger technique. All patients in the present study had all exons amplified. The missense alterations were analyzed by Polyphen2, Sift, and ConSurf softwares, and the alterations predicted as pathogenic were studied through research in Brazilian control alleles. The p.Ser385Leu and c.3503_3504delTC were evaluated by site-direct mutagenesis and the residual activity was evaluated 24 hours after expression in HEK cells, through radioactive assays. For cost-price analysis, current values for equipment, molecular biology reagents, and plastic materials were utilized to estimate the cost of DNA extraction, PCR reaction, PEG8000 purification, and sequencing. Results: Of the 13 patients, 8 were clinically diagnosed with Mucolipidosis II and 5 with Mucolipidosis III alpha/beta and, additionally, a mother of one patient with biochemical and clinical diagnosis of Mucolipidosis II was also analyzed. The DNA analysis identified six novel pathogenic alterations in GNPTAB: c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly), and p.Tyr1111*. The bioinformatics analysis of missense alterations were characterized as damaging for protein function, and residues 76 and 385 as structural and functional, respectively, and both as highly conserved among the species. The functional analysis of mutants p.Ser385Leu and c.3503_3504delTC showed the residual activity of GlcNAcphosphotransferase of 1.5% and 0%, respectively. Six others pathogenic alterations previously described were also identified. The alteration c.3503_3504delTC showed the highest frequency (40%, n=10/25 alleles) followed by p.Ile403The (12%, n=3/25 alleles). The retrospective analysis defined the 1.0 protocol that finalized the molecular diagnosis at the cost of R$ 338,45 per sample, in a group of 25 patients. The prospective analysis of 2.0 protocol, in the same patients, indicated that it would finalize diagnosis at the cost of R$ 299,80 per sample, a saving of 25%. Discussion/Conclusion: The novel pathogenic alterations described confirm the high allelic heterogeneity of GNPTAB gene. In the genotype-phenotype relationship, 7 patients with Mucolipidosis II have combined genotype of frameshift or nonsense alterations, or both, and 5 Mucolipidosis III alpha/beta patients have at least one missense alteration, that shows the correlation between alterations that cause impact on the protein function and severe phenotype. The functional analysis of alteration p.Ser385Leu confirms its pathogenicity and makes evident the need of more studies in order to determine the reason this residue is so important for protein function. Protocol synthesis proves to be an interesting and economically important strategy, once it decreases costs to conclude the molecular diagnosis.

Considerable effort has recently been directed toward the miniaturization of quantitative-polymerase-chain-reaction [QPCR] instrumentation in an effort to reduce both cost and form factor for point-of-care applications. Notable gains have been made in shrinking the required volumes of PCR reagents, but resultant prototypes retain their bench-top form factor either due to heavy heating plates or cumbersome optical sensing instrumentation. In this thesis, we describe the use of complementary-metal-oxide semiconductor (CMOS) integrated circuit (IC) technology to produce a fully integrated qPCR lab-on-chip. Exploiting a 0.35-µm high-voltage CMOS process, the IC contains all of the key components for performing qPCR. Integrated resistive heaters and temperature sensors regulate the surface temperature of the chip to 0.45°C. Electrowetting-on-dielectric microfluidic pixels are actively driven from the chip surface, allowing for droplet generation and transport down to volumes of less than 1.2 nanoliters. Integrated single-photon avalanche diodes [SPAD] are used for fluorescent monitoring of the reaction, allowing for the quantification of target DNA with more than four-orders-of-magnitude of dynamic range with sensitivities down to a single copy per droplet. Using this device, reliable and sensitive real-time proof-of-concept detection of Staphylococcus aureus (S. aureus) is demonstrated.

No description available.

Este estudo descreve a análise molecular de pacientes da doença de Gaucher (DG) e Tay-Sachs (DTS) no Brasil. Foram estudados nove casos de formas clássicas da DTS que mostraram uma prevalência da mutação IVS7+1g>c, já descrita em pacientes Portugueses e dez casos das variantes de início juvenil e tardio da DTS, mostrando heterogeneidade genética. Nos casos da variante B1, percebeu-se uma maior incidência da mutação R178H, também descrita previamente em pacientes Portugueses. A presença das mesmas mutações nos casos Brasileiros e Portugueses se deve provavelmente à ancestralidade comum. Uma família com quatro pacientes da variante de início tardio da DTS mostrou uma extensa variabilidade clínica intrafamilial e identificou relevantes aspectos do diagnóstico e das implicações dos programas de triagem populacional. A análise por RFLP de nove mutações causadoras da DG, em 262 pacientes permitiu detectar 76% das alterações e mostrou uma prevalência das mutações N370S e L444P, similar à descrita em diversas outras populações. Os pacientes com variantes neuronopáticas da doença apresentaram uma alta freqüência da mutação G377S, que também é encontrada em pacientes Portugueses. Os pacientes apresentando a G377S indicaram a existência de um provável mecanismo de efeito de dose alélica para essa mutação. Foi observada uma alta freqüência de alelos resultantes da recombinação do gene GBA com seu pseudogene. Diversas outras relações genótipo-fenótipo puderam ser verificadas, mostrando uma baixa penetrância do genótipo N370S/N370S e corroborando a importância do diagnóstico molecular da DG, devido a seu valor preditivo. A análise de mutações raras no gene GBA usando as técnicas de RFLP, dHPLC e seqüenciamento de DNA possibilitou detectar mutações em 84% dos alelos de 54 pacientes. Foram identificadas 14 novas mutações causadoras da DG. Diversas relações genótipo-fenótipo puderam ser verificadas, conferindo valor preditivo para a detecção dessas mutações. Por fim, a análise da associação da DG e da DP permitiu encontrar uma freqüência significativamente maior de portadores das principais mutações no gene GBA em pacientes parkinsonianos (2/65=3%), com aparecimento precoce da doença, comparados a um grupo controle de 267 indivíduos. Esse trabalho fornece nova evidência de que mutações no gene GBA são um raro, mas consistente fator de risco para a DP. / This study describes the molecular diagnosis of Gaucher (GD) and Tay-Sachs disease (TSD) patients in Brazil. Nine cases of the classic infantile form of TSD were analyzed disclosing a prevalence of the IVS7+1g>c mutation, described previously in Portuguese patients. Ten cases of juvenile and late-onset TSD forms were diagnosed showing genetic heterogeneity. Among the B1 variant cases, there was a predomenance of mutation R178H that was also associated to Portuguese ancestry. The presence of the same mutations in Brazilian and Portuguese cases are probably due to common ancestry. A family with 4 affected patients of late onset TSD showed and extensive intrafamilial clinical variability, highlighting relevant characteristics of diagnosis and implications of heterozygote screening programs. Among 263 GD patients, the detection of nine mutations by RFLP revealed 76% of the mutant alleles and a preponderance of N370S and L444P, similar to other populations. The type 3 neuronopathic patients presented a high frequency of mutation G377S, that is also described among Portuguese cases. The patients with G377S indicated the possibility of an allele dose effect for this mutation. Recombinant alleles, presenting pseudogene mutations were detected at a high frequency. Several genotype-phenotype correlations could be verified, highlighting a low penetrance of genotype N370S/N370S and corroborating the importance of molecular diagnosis in GD cases, due to its predictive value. The search for rare mutations at the GBA gene, using dHPLC and DNA sequencing after RFLP analysis, allowed the detection of 84% of the alleles among 54 patients. Fourteen new GD causing mutations were described. Several genotype-phenotype correlations could be established, confering prective value to the identification of these mutations. Finally, the study of the association of GD and Parkinson disease (PD) lead to the detection of a significant increase in the frequency of GBA mutations carriers, among 65 PD patients (2/65=3%) with earlier disease onset compared to a control group (n=267 individuals). This work confers further evidence for the fact that GBA mutations are a rare but consistent risk factor for PD.

O vírus da hepatite E (HEV) é um patógeno emergente de distribuição global, causador de hepatite aguda e crônica em humanos e infecções assintomáticas em animais. No Brasil a prevalência de infecção por HEV em humanos e animais ainda é pouco compreendida, assim como as características de virulência, patogenicidade e de infecção inter-espécies de isolados do genótipo 3, zoonótico, circulantes no país também são desconhecidas. O estudo foi dividido em duas etapas, com os objetivos de 1) contribuir no diagnóstico laboratorial molecular do HEV a partir do desenvolvimento de um protocolo de RT-PCR em tempo real (RT-qPCR) para pesquisa do HEV em amostras biológicas, e 2) contribuir com a compreensão das características moleculares, sorológicas, clínico-laboratoriais, ultrassonográficas e histopatológicas associadas à infecção experimental em macacos-da-noite (Aotus azarai infulatus) por um isolado do genótipo 3 suíno do HEV previamente detectado na Amazônia oriental brasileira. O protocolo de RT-qPCR foi desenvolvido com a caracterização da curva de detecção e aplicado em concomitância com testes sorológicos para avaliação diagnóstica restrospectiva de 318 (n = 318) amostras de soros humanos suspeitos de hepatite E. O HEV-RNA não foi detectado em nenhuma das amostras humanas testadas, mas foi determinada soroprevalência de 3,4% e 5,9% de anti-HEV IgM e IgG, respectivamente, o que indicou baixa prevalência de infecção por HEV, mesmo entre pacientes com suspeita clínica e/ou laboratorial de hepatite E na Amazônia brasileira. O estudo experimental em macacos-da-noite foi desenvolvido durante 12 semanas e os animais infectados, por via intravenosa (n=3) e oral (n=3) (e dois controles), foram avaliados semanalmente para determinação dos parâmetros clínicos, bioquímicos, hematológicos, sorológicos (pesquisa de anti-HEV IgM e IgG por enzimaimunoensaio) e moleculares (HEV-RNA soro e fezes por RT-qPCR). Adicionalmente, os animais também foram submetidos a avaliação hepática mensal por ultrassonografia, histopatologia e pesquisa hepática de antígenos do HEV por imunohistoquímica. Os seis macacos-da-noite infectados apresentaram o HEV-RNA em amostras de soro e/ou fezes, e alguns apresentaram evidências de soroconversão, detecção hepática do antígeno viral por imunohistoquímica associada a alterações clínicas e laboratoriais de hepatite aguda oligossintomática. Assim, o protocolo RT-qPCR demonstrou ser aplicável na pesquisa molecular do HEV em amostras de humanos e animais, representando uma importante ferramenta de diagnóstico laboratorial. O estudo experimental permitiu a primeira validação de um primata neotropical como modelo experimental para estudos de infecção com o genótipo 3 do HEV. / Hepatitis E virus (HEV) is an emerging pathogen with global distribution that causes acute and chronic hepatitis in humans and asymptomatic infections in animals. In Brazil, the prevalence of HEV infection in humans and animals is still poorly understood, and the characteristics of virulence, pathogenicity and inter-species infection of the genotype 3 isolates circulating in the country are unknown. The study was divided in two stages that aimed to 1) contribute to the molecular diagnosis of HEV infection by the development of a real-time RT-PCR protocol (RT-qPCR) for HEV-RNA research in biological samples, and 2) to contribute to understanding of molecular, serological, clinical-laboratory, ultrasonographic and histopathological features of HEV genotype 3 in owl monkeys (Aotus azari infulatus) experimental infected with isolate of swine HEV genotype 3 previously detected in the Eastern Brazilian Amazon. The RT-qPCR protocol was developed with characterization of a quantification standard curve and later applied concurrently with serological tests in the retrospective evaluation of 318 (n = 318) human serum samples of hepatitis E suspected cases. HEV-RNA was not detected in any of human tested samples, but seroprevalence of 3.4% and 5.9% was determined for anti-HEV IgM and IgG, respectively, that indicated a low prevalence of HEV infection, even among patients with clinical and/or laboratory suspicion of hepatitis E in the Brazilian Amazon. The experimental study in owl monkeys was developed during12 weeks and the animals were infected by intravenous (n = 3) and oral (n = 3) routes (and two negative controls) were evaluated for determination of clinical, biochemical, hematological, serological (anti-HEV IgM and IgG by enzyme immunoassay) and molecular (HEV-RNA serum and stool by RT-qPCR) parameters weekly. Additionally, the animals were also evaluated by hepatic ultrasonography, histopathology and immunohistochemistry research of HEV antigens in liver monthly. The six infected owl monkeys presentend HEV-RNA in serum and/or stool, and some monkeys presented with evidence of seroconversion, liver detection of HEV antigens by immunohistochemistry associated with clinical and/or laboratory findings of oligosymptomatic acute hepatitis. Thus, the RT-qPCR protocol demonstrated to be applicable in the molecular investigation of HEV infection in human and animal samples, and it also represented an important laboratory diagnostic tool. The experimental study allowed the validation of the first neotropical primate model for HEV genotype 3 infection studies.

A audição representa a principal fonte para o aprendizado da fala e linguagem durante a infância e a surdez e a privação de estímulos auditivos pode implicar em dificuldades emocionais e sociais àqueles indivíduos afetados. Aproximadamente 360 milhões de pessoas sofrem de perda auditiva no mundo, o que corresponde a 5,3% da população mundial. A surdez pode se desenvolver em decorrência de causas genéticas (hereditárias), não-genéticas e ambientais. As infecções pré-natais e a exposição a ruídos constituem as causas ambientais mais comuns. Já a surdez hereditária, constitui o transtorno neurossensorial mais comum em humanos, com uma prevalência de 1:1000 nascidos vivos. Mais de 70% dos casos de surdez hereditária constituem casos não-sindrômicos, destes cerca de 70% cursam com surdez congênita ou pré-lingual. Na maioria dos casos, a perda auditiva hereditária é neurossensorial, heterogênea, com diferentes padrões de herança e com uma grande quantidade de genes envolvidos. Estudos têm demonstrado o importante papel dos genes GJB2, GJB6 e SLC26A4 na fisiologia do ouvido interno e alterações nestes genes têm sido relatadas como causa da surdez hereditária. Desta forma, o objetivo deste estudo foi investigar a base genética e o papel do gene SLC26A4 na perda auditiva neurossensorial (PANS) nãosindrômica pré-lingual em pacientes atendidos pelo serviço de Genética Médica do Hospital das Clínicas de Ribeirão Preto. Para isso, uma série de 88 casos foi investigada quanto a características clínicas e moleculares. A amostra abrangeu indivíduos de ambos os sexos, com idade de 2 a 63 anos, provenientes de 88 famílias diferentes, assistidos durante o período de 2003 a 2013. As amostras foram triadas pela técnica de High Resolution Melting (HRM) e em seguida levadas para o seqüenciamento para caracterização das alterações. Na série de casos estudada, 23,9% (21/88) dos pacientes portadores de surdez neurossensorial não-sindrômica pré-lingual evidenciaram alterações nos genes GJB2, GJB6 e SLC26A4 sugeridas como patogênicas. A prevalência de alterações no gene SLC26A4 foi de 28,4% (25/88), não relacionada à Síndrome do Aqueduto Vestibular Alargado (SAVA). Dentre as 11 alterações encontradas neste gene, três constituem mutações não descritas: p.Gly139Arg, p.Ile254Val, p.Asn382Lys. Os genótipos mais freqüentes neste estudo foram a c.35delG/c.35delG no gene GJB2 (5/88), a dupla heterozigose com o gene GJB6 c.35delG/del(GJB6-D13S1854) (3,4%) e chr7:g.107301238C>G/wt no gene SLC26A4 (10,2%). Entretanto, apenas 19,3% dos indivíduos apresentaram genótipos sugeridos como responsáveis pelo fenótipo estudado. Alterações particulares no gene SLC26A4 podem sugerir a explicação para a surdez genética para aproximadamente 9,1% destes casos. Destes, cinco casos de heterozigose preditas como patogênicas (p.Ile300Leu; p.Asn324Tyr e p.Asn382Lys), dois casos de heterozigose composta (chr7:g.107301201T>C/chr7:g.107301238C>G e chr7:g.107301238C>G/p.Gly139Arg) e um caso de dupla heterozigose com GJB2 (chr7:g.107301238C>G/c.35delG). Isto ressalta a importância do gene SLC26A4 para o diagnóstico molecular de surdez hereditária e reforça a sua potencial contribuição para o processo de aconselhamento genético. Entretanto, nossos dados sugerem a necessidade de testes funcionais a fim de elucidar o papel destas alterações para o estabelecimento do fenótipo, como também, a presença de outros genes ou regiões envolvidas naqueles casos em que mutações monoalélicas não foram suficientes para justificar o fenótipo. / The hearing is the main source for learning speech and language during childhood and deafness and deprivation of auditory stimuli can result in emotional and social difficulties to those affected individuals. Approximately 360 million people suffer from hearing loss worldwide which corresponds to 5.3% of the world population. Deafness may develop due to genetic (hereditary), non-genetic and environmental causes. Prenatal infections and exposure to noise are the most common environmental causes. Hereditary deafness is the most common sensorineural disorder in humans, with a prevalence of 1:1000 live births. More than 70% of hereditary deafness cases are nonsyndromic, about 70% of these occur with congenital or prelingual deafness. In most cases, inherited sensorineural hearing loss is heterogeneous, with different patterns of inheritance and with a large number of genes involved. Studies have shown the important role of genes GJB2, GJB6 and SLC26A4 in the physiology of the inner ear and changes in these genes have been reported as cause of hereditary hearing loss. Thus, the aim of this study was to investigate the genetic basis and the role of SLC26A4 gene in non-syndromic prelingual sensorineural hearing loss (SNHL) in patients enrolled in the Medical Genetics Service of Hospital das Clínicas de Ribeirão Preto. For this, a series of 88 cases were submitted to a clinical and molecular investigation. The sample consisted of individuals of both sexes, aged 2-63 years from 88 different families, assisted during the period 2003-2013. The samples were screened by the technique of High Resolution Melting (HRM) and then taken for sequencing to characterize the mutations. In the series of cases studied, 23.9% (21/88) of patients with non-syndromic prelingual sensorineural deafness showed variants in genes GJB2, GJB6 and SLC26A4 suggested as pathogenic. The prevalence of mutations in the SLC26A4 gene was 28.4% (25/88), not related to non-syndromic EVA. Among the 11 mutations found in this gene, three are reported as novel mutations: p.Gly139Arg, p.Ile254Val, p.Asn382Lys. The most frequent genotypes found in this study were the c.35delG/c.35delG in GJB2 gene (5/88), the double heterozygosity with GJB6 gene c.35delG/del(GJB6-D13S1854) (3,4%) and chr7:g.107301238C>G/wt in the SLC26A4 gene (10,2%). However, only 19.3% of subjects presented genotypes suggested as responsible for the studied phenotype. Particular mutations in the SLC26A4 gene may suggest the explanation for the genetic deafness to approximately 9.1% of these cases. Of these, five cases of heterozygosity predicted as pathogenic (p.Ile300Leu; p.Asn324Tyr and p.Asn382Lys), two cases of compound heterozygosity (chr7:g.107301201T>C/chr7:g.107301238C>G and chr7:g.107301238C>G/p.Gly139Arg) and one case of double heterozygosity with GJB2 gene (chr7:g.107301238C>G/c.35delG). Those data highlights the importance of the SLC26A4 gene for molecular diagnosis of hereditary hearing loss and give strength to its potential contribution to the genetic counseling process. However, our data suggest the need for functional tests in order to elucidate the role of these changes to the phenotype, as well as the presence of other genes or regions involved in those cases that monoallelic mutations were not sufficient to justify the phenotype.

This thesis describes biochemical and molecular methods diagnostic for a spectrum of steroid metabolic diseases. Deficiency of any enzyme in the steroid hormone biosynthetic pathways leads to disorders including congenital adrenal hyperplasia, while some cause disorders of sex development (DSD). A gas chromatography mass spectrometry-based analytical technique called urinary steroid profiling (USP) has been shown to be a useful diagnostic test for these diseases and for steroid-secreting adrenocortical tumours. To test the hypothesis that this approach would be effective in our local population, we interpreted 482 USP results using reference intervals set up from 371 local healthy subjects. Characteristic steroid metabolite excretion patterns were found in 39 patients, including 21 patients with 21-hydroxylase deficiency (21OHD) where there were grossly increased 17-hydroxyprogesterone metabolites, 12 patients with 5α-reductase 2 deficiency (5ARD) with extremely low 5α- to 5β-reduced steroid metabolite ratios, and five patients with adrenocortical carcinoma with markedly raised tetrahydro-11-deoxycortisol and 3,16,20-pregnenetriols levels. / The genetic basis of 21OHD in various populations is mainly due to conversion between the CYP21A2 and the CYP21A1P genes but this has not yet been explored in our population. By using DNA sequencing and multiplex ligation-dependent probe amplification, 74 mutations were found in 35 patients with 21OHD. Gross deletion/conversion of the CYP21A2 gene accounted for 27%. c.290-13A/C>G was the most common point mutation (27%), followed by p.Ile172Asn (17.6%). One novel mutation c.1367delA was also detected. Their prevalence in our patients differs from those in other populations. / The most common cause of 46,XY DSD in Western populations is androgen insensitivity syndrome (AIS) but this has not been verified locally. A prospective study was conducted where 64 patients were recruited for comprehensive hormonal profiling and targeted molecular analysis. In this study, a genetic diagnosis was established in 22 patients, with 5ARD being the most common disease, followed by AIS. Traditionally the diagnosis of 5ARD relies on measuring dihydrotestosterone. However, with our experience in diagnosing this condition based on USP and mutational analysis of the SRD5A2 gene, two new diagnostic algorithms for 46,XY DSD were proposed where dihydrotestosterone is not required. / In vitro study is the preferred method for characterising the function of novel genetic variants. However, clinical laboratories rarely have the facilities and resources for it. In silico prediction programmes appear to be practical alternatives but their performance on testing non-synonymous variants in genes related to steroid metabolism has not been verified. Three web-based in silico prediction programmes, namely Sorting Intolerant From Tolerant, PolyPhen-2 and Pathogenic-Or-Not-Pipeline, were tested by analysing 797 published non-synonymous genetic variants in 12 genes related to steroid metabolism. The results of in vitro functional study and/or clinical phenotype were used as gold standards. The performance of these three programmes were: sensitivity (76.6%, 84.1%, 70.0%), specificity (56.6%, 56.3%, 89.4%) and accuracy (70.1%, 75.2%, 76.8%), respectively. / In conclusion, USP is a valuable biochemical phenotyping technique that helps to select patients for subsequent genetic confirmation. Since the mutation spectrum of 21OHD and the aetiological basis of 46,XY DSD in our population differ from the others, laboratory diagnostic algorithms and molecular analytical strategies must be adjusted accordingly. / Chan, On Kei Angel. / Thesis (M.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 250-269). / Appendixes includes Chinese.

Orientador: Helio Jose Montassier / Resumo: As infecções do trato respiratório de aves têm-se constituído em problemas crescentes e com marcantes consequências negativas sobre a produção avícola em várias partes do mundo, notadamente onde a avicultura é mais desenvolvida como no Brasil. Dentre essas enfermidades, destacam-se as micoplasmoses aviárias e a pneumovirose aviária, que, apesar de suas relevâncias em sanidade avícola, não têm sido investigadas de forma sistematizada no Brasil, em especial no que concerne à interação entre esses agentes ou a ocorrência de co-infecção em frangos de corte. O presente estudo investigou a ocorrência de infecção por Mycoplasma gallisepticum (MG) e pelos subtipos A ou B de Metapneumovírus aviário (AMPV) em frangos de corte de plantéis avícolas comerciais mantidos em granjas mais tecnificadas localizadas nas regiões Sudeste e Centro-Oeste do Brasil. Para tanto, as técnicas de PCR e de RT-Nested-PCR foram usadas na detecção e/ou identificação, respectivamente de MG e AMPV em amostras de suabes nasais e traqueais colhidos de 87 lotes de frangos de corte com problemas respiratórios e oriundos de 15 granjas de produção comercial de frangos de corte. Dos lotes amostrados, dois deles em um total de 87 (2,3%) e de uma única granja da região Sudeste, mostraram-se positivos para MG, enquanto que nenhum dos lotes investigados revelou-se positivo para AMPV. A baixa ou nenhuma incidência desses agentes pode ser explicada pela utilização de medidas cada vez mais efetivas para o controle sanitário... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Respiratory infections of poultry may be an increasing and negative problem with poultry production in several parts of the world, especially when poultry farming is more widely used than in Brazil. These diseases include avian mycoplasmosis and avian pneumovirosis, which, despite their relevance in poultry health, have not been systematically investigated in Brazil, especially with regard to the interaction between these agents or the occurrence of co-infection in broilers. The present study investigated the occurrence of infection by Mycoplasma gallisepticum (MG) and A or B subtypes of avian Metapneumovirus (AMPV) in broiler commercial poultry farms located in the Southeast and Center-West regions of Brazil. For this, PCR and RT-Nested-PCR techniques were used in the detection and / or identification, respectively of MG and AMPV in nasal and tracheal swab samples collected from 87 lots of broilers with respiratory problems and from 15 commercial production of broilers. Of the sampled lots, two of them in 87 (2.3%) were positive for MG, whereas none of the lots were positive for AMPV. The low or no incidence of these agents can be explained by the use of increasingly effective measures for the sanitary control of these agents in the commercial farms of sampled broilers. Our findings also suggest, however, that other bacterial and viral infectious agents not investigated in this study may be involved in the etiology of the respiratory problems of these birds, since those that... (Complete abstract click electronic access below) / Mestre

Includes bibliographies. / [xvii], 173, [38] leaves, [18] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis presents the development of an assay for reverse transcription-polymerase chain reaction (RT-PCR) to be applied to the immunomagnetic isolation of carcinoma cells, as a possible means of detecting small numbers of breast cancer cells in a haemopoietic environment. The messenger RNA expression of two different genes, CD44 and the cytokeratin K19, is assessed for suitability as tumour markers for the Immunobead RT-PCR method, and clinical results using K19 are presented. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1977?

published_or_final_version / Pathology / Master / Master of Medical Sciences