Role of the DHH1 Gene in the Regulation of Monocarboxylic Acids Transporters Expression in Saccharomyces cerevisiae

Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170∶301–306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

Estado da Arte da Computação Evolutiva Aplicada à Eletrónica

A Computação Evolutiva enquadra-se na área da Inteligência Artificial e é um ramo das ciências da computação que tem vindo a ser aplicado na resolução de problemas em diversas áreas da Engenharia. Este trabalho apresenta o estado da arte da Computação Evolutiva, assim como algumas das suas aplicações no ramo da eletrónica, denominada Eletrónica Evolutiva (ou Hardware Evolutivo), enfatizando a síntese de circuitos digitais combinatórios. Em primeiro lugar apresenta-se a Inteligência Artificial, passando à Computação Evolutiva, nas suas principais vertentes: os Algoritmos Evolutivos baseados no processo da evolução das espécies de Charles Darwin e a Inteligência dos Enxames baseada no comportamento coletivo de alguns animais. No que diz respeito aos Algoritmos Evolutivos, descrevem-se as estratégias evolutivas, a programação genética, a programação evolutiva e com maior ênfase, os Algoritmos Genéticos. Em relação à Inteligência dos Enxames, descreve-se a otimização por colônia de formigas e a otimização por enxame de partículas. Em simultâneo realizou-se também um estudo da Eletrónica Evolutiva, explicando sucintamente algumas das áreas de aplicação, entre elas: a robótica, as FPGA, o roteamento de placas de circuito impresso, a síntese de circuitos digitais e analógicos, as telecomunicações e os controladores. A título de concretizar o estudo efetuado, apresenta-se um caso de estudo da aplicação dos algoritmos genéticos na síntese de circuitos digitais combinatórios, com base na análise e comparação de três referências de autores distintos. Com este estudo foi possível comparar, não só os resultados obtidos por cada um dos autores, mas também a forma como os algoritmos genéticos foram implementados, nomeadamente no que diz respeito aos parâmetros, operadores genéticos utilizados, função de avaliação, implementação em hardware e tipo de codificação do circuito.

Candida esophagitis: species distribution and risk factors for infection

Although Candida albicans is the main cause of fungal esophagitis, other species such as C. tropicalis, C. krusei and C. stellatoidea have also been implicated. Several studies have identified risk factors for C. albicans esophagitis. However, data for non-C. albicans species is still sparse. The aim of this study was to determine the etiology of Candida esophagitis in our medical centre over an 18-month period. Additionally, we aimed to investigate predisposing conditions for esophageal candidosis caused by different Candida species. A total of 21,248 upper gastroscopies were performed in Santa Casa Complexo Hospitalar between January 2005 and July 2006. The prevalence of Candida esophagitis was 0.74% (n = 158). C. albicans caused the vast majority of infections (96.2%), followed by C. tropicalis (2.5%), C. lusitaniae (0.6%) and C. glabrata (0.6%). There were 81 women (51.3%) and 77 men (48.7%). No case of mixed infection occurred. Concomitant oral candidosis was documented for 10.8% (n = 17). Most of cases (55.1%) involved outpatients. Around one fifth of patients in our cohort had no identifiable risk factors for esophageal candidosis (20.8%). Since nearly all infections were caused by C. albicans we were not able to determine risk factors for esophagitis caused by other Candida species.

ABCC Subfamily Vacuolar Transporters are Involved in Pb (Lead) Detoxification in Saccharomyces cerevisiae

The present work has as objective to contribute for the elucidation of the mechanism associated with Pb detoxification, using the yeast Saccharomyces cerevisiae as a model organism. The deletion of GTT1 or GTT2 genes, coding for functional glutathione transferases (GST) enzymes in S. cerevisiae, caused an increased susceptibility to high Pb concentrations (500-1000 μmol L(-1)). These results suggest that the formation of glutathione-Pb conjugate (GS-Pb), dependent of GSTs, is important in Pb detoxification. The involvement of ATP-binding cassette (ABC) vacuolar transporters, belonging to class C subfamily (ABCC) in vacuolar compartmentalization of Pb, was evaluated. For this purpose, mutant strains disrupted in YCF1, VMR1, YBT1 or BPT 1 genes were used. All mutants tested, without vacuolar ABCC transporters, presented an increased sensitivity to 500-1000 μmol L(-1) Pb comparative to wild-type strain. Taken together, the obtained results suggest that Pb detoxification, by vacuolar compartmentalization, can occur as a result of the concerted action of GSTs and vacuolar ABCC transporters. Pb is conjugated with glutathione, catalysed by glutathione transferases and followed to the transport of GS-Pb conjugate to the vacuole by ABCC transporters.

Molecular characterization of Cryptosporidium spp. from HIV infected patients from an urban area of Brazil

Cryptosporidium spp. are important cause of enteric disease in humans, but may also infect animals. This study describes the relative frequency of several Cryptosporidium species found in human specimens from HIV infected patients in the São Paulo municipality obtained from January to July 2007. Sequence analysis of the products of nested-PCR based on small subunit rRNA and Cryptosporidium oocyst wall protein coding genes revealed 17 (63.0%) isolates of C. hominis, four (14.8%) C. parvum, five (18.5%) C. felis and one (3.7%) C. canis. These findings suggest that, in urban environments of Brazil, the cat adapted C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis whereas the anthroponotic transmission of cryptosporidiosis caused by C. hominis seems to predominate.

Spatial and temporal analysis of recent drought using vegetation temperature condition index: case of Somali regional state of Ethiopia

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Documento de Consenso Sobre Codificação de Exames de Ressonância Magnética Cardíaca em Portugal

Um dos obstáculos a uma utilização mais frequente e apropriada da ressonância magnética cardíaca (RMC) em Portugal tem sido a ausência de códigos específicos que descrevam adequadamente os exames tal como são efetuados actualmente. Este documento de consenso fornece recomendações para a atualização e uniformização dos códigos empregues na RMC. São igualmente feitas recomendações quanto às técnicas e códigos a utilizar nas indicações clínicas mais frequentes.

A Novel Autosomal Recessive GJA1 Missense Mutation Linked to Craniometaphyseal Dysplasia

Craniometaphyseal dysplasia (CMD) is a rare sclerosing skeletal disorder with progressive hyperostosis of craniofacial bones. CMD can be inherited in an autosomal dominant (AD) trait or occur after de novo mutations in the pyrophosphate transporter ANKH. Although the autosomal recessive (AR)form of CMD had been mapped to 6q21-22 the mutation has been elusive. In this study, we performed whole-exome sequencing for one subject with AR CMD and identified a novel missense mutation (c.716G>A, p.Arg239Gln) in the C-terminus of the gap junction protein alpha-1 (GJA1) coding for connexin 43 (Cx43). We confirmed this mutation in 6 individuals from 3 additional families. The homozygous mutation cosegregated only with affected family members. Connexin 43 is a major component of gap junctions in osteoblasts, osteocytes, osteoclasts and chondrocytes. Gap junctions are responsible for the diffusion of low molecular weight molecules between cells. Mutations in Cx43 cause several dominant and recessive disorders involving developmental abnormalities of bone such as dominant and recessive oculodentodigital dysplasia (ODDD; MIM #164200, 257850) and isolated syndactyly type III (MIM #186100), the characteristic digital anomaly in ODDD. However, characteristic ocular and dental features of ODDD as well as syndactyly are absent in patients with the recessive Arg239Gln Cx43 mutation. Bone remodeling mechanisms disrupted by this novel Cx43 mutation remain to be elucidated.

Expressão em Escherichia coli de antigénios do Cell fusing agent virus (Flaviviridae: Flavivirus) como proteína de fusão

RESUMO: O Cell Fusing Agent Vírus (CFAV), considerado como o primeiro “flavivírus específicos de insectos” (ISF), parece estar exclusivamente adaptado aos seus hospedeiros, não replicando em células de vertebrados. Apesar de ter sido identificado há mais de três décadas (1975), a verdade é que muito pouco se conhece sobre a sua biologia. Dado o seu parentesco filogenético com alguns outros flavivírus encontrados naturalmente em mosquitos de diferentes géneros colhidos em diferentes regiões do globo, este vírus poderá ser usado como modelo para o estudo de ISF. No entanto, necessitam do desenvolvimento de ferramentas básicas, tais como clones moleculares ou baterias de soros contendo anticorpos que reconheçam uma ou mais proteínas codificadas pelo genoma viral, produzidas, por exemplo, a partir de antigénios virais produzidos de forma recombinante. Com este trabalho pretendeu-se a optimização de protocolos que permitiram a expressão e purificação parcial de quatro proteínas [duas proteínas estruturais (C e E) e duas não estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas como proteínas de fusão com “caudas” (tags) de hexahistidina nos seus extremos carboxilo. Para a expansão do CFAV foram utilizadas células Aedes albopictus (C6/36). Após a realização da extracção do RNA viral e a obtenção de cDNA, procedeu-se amplificação, por RT-PCR, das regiões codificantes das proteínas C, E, NS3hel e NS5B, utilizando primers específicos. Os quatro fragmentos de DNA foram independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como hospedeira de clonagem e, posteriormente, inseridos em vectores de expressão pET-28b e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de expressão. Após da indução, expressão e purificação das proteínas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas proteínas produzidas através do método Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in vertebrate cells. Although it has been known for more than three decades (1975), the truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced. In this work, we carried out the optimization of protocols that allowed the expression and partial purification of four proteins [two structural proteins (C and E) and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein for c-terminal hexahistidine tags. For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using specific primers. The four DNA fragments were independently inserted into the vector pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS as expression host. After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the authenticity of these proteins produced.

Novel transcription factors regulating the expression of the rice gene OsDREB1B

Dissertation presented to obtain the Ph.D degree in Biology

Food Allergy and Anaphylaxis in Infants and Preschool-Age Children

Food allergy (FA) prevalence data in infants and preschool-age children are sparse, and proposed risk factors lack confirmation. In this study, 19 children’s day care centers (DCC) from 2 main Portuguese cities were selected after stratification and cluster analysis. An ISAAC’s (International Study of Asthma and Allergies in Childhood) derived health questionnaire was applied to a sample of children attending DCCs. Outcomes were FA parental report and anaphylaxis. Logistic regression was used to explore potential risk factors for reported FA. From the 2228 distributed questionnaires, 1217 were included in the analysis (54.6%). Children’s median age was 3.5 years, and 10.8% were described as ever having had FA. Current FA was reported in 5.7%. Three (0.2%) reports compatible with anaphylaxis were identified. Reported parental history of FA, personal history of atopic dermatitis, and preterm birth increased the odds for reported current FA. A high prevalence of parental-perceived FA in preschool-age children was identified. Risk factor identification may enhance better prevention.

Qualidade em obstetrícia : complicações pós-parto e readmissões

RESUMO - A qualidade no sistema de saúde é alvo de grande preocupação por parte dos gestores, necessitando encontrar estratégias que conduzam a uma melhoria dos resultados das unidades hospitalares, em simultâneo com a contenção dos custos. O objectivo geral deste trabalho consistiu na avaliação da qualidade na área de obstetrícia nos hospitais públicos de Portugal Continental, através da análise das taxas de complicações pósparto e de readmissões verificando se existem diferenças por instituição hospitalar, volume, acreditação e região nos anos 2008 a 2010. Os dados foram cedidos pela Administração Central do Sistema de Saúde à Escola Nacional de Saúde Pública, sendo seleccionados os episódios nos GDH 370 a 375 e 650 a 652. Foram analisados 243 686 episódios, verificando-se que a taxa de incidência de complicações é superior nos partos por cesariana, comparativamente com os partos vaginais (1,66% e 1,03%, respectivamente). Os hospitais com maior volume de procedimentos e não acreditados evidenciam maiores taxas de complicação. A taxa de readmissão a 30 dias foi também superior na cesariana comparativamente ao parto vaginal (0,42% e 0,73%, respectivamente). Os hospitais com menor volume de procedimentos e acreditados, regra geral, são os que evidenciam as maiores taxas de readmissão. De salientar que as reduzidas taxas de complicações pós-parto verificadas poderão ser explicadas por diferenças na codificação dos diagnósticos secundários entre hospitais. Podem ainda existir diferenças aqui não consideradas entre os doentes tratados em cada instituição. Os dados apresentados permitem ter um melhor conhecimento acerca da qualidade no tratamento dos casos de obstetrícia nos hospitais públicos em Portugal Continental, dando indicações para a tomada de decisão em gestão em saúde.

Reducing Discomfort While Measuring Crown-Heel Length in Neonates

To assess the degree of discomfort caused by length measurement in neonates, performed with one or both lower limbs extended, on the first and second day after birth, with either one or both lower limbs extended. METHODS: Healthy full-term neonates were systematically sampled during the months of February and March 2004. Crown-heel length was measured, using a 1-mm precision neonatometer, at approximately 8 h and 32 h after birth, with one and both lower limbs extended. The Neonatal Facial Coding System was used to assess discomfort during measurements. Data were analysed by parametric and non-parametric tests as appropriate. RESULTS: Whatever the measurement technique, discomfort scores are significantly higher during the length measurement than at baseline. Whenever length measurements are performed, discomfort scores are significantly higher when extending both lower limbs rather than one lower limb (p < 0.006). The measured length is greater with one lower limb extended; however, the difference decreases over time, being 0.19 cm (95% CI 0.1-0.3; p < 0.001) at approximately 32 h of age. No significant differences in length were found between measurements at approximately 8 or 32 h, regardless of the technique used. The best correlation between length measurements with one or both lower limbs extended was observed at approximately 32 h after birth (r = 0.98). CONCLUSION: Measuring crown-heel length is a distressful procedure for the neonate. Measurements with one lower limb extended result in less discomfort than when both lower limbs are extended, without decreasing the accuracy.

Bronchial Asthma in Children: Clinical and Epidemiologic Approach in Different Portuguese Speaking Countries

BACKGROUND: Geographical differences in asthma prevalence are currently accepted, but evidence is sparse due to the lack of multicentre studies using the same protocol. OBJECTIVES: To compare the prevalence of asthma and atopy among schoolchildren from Portuguese speaking countries (ISAAC and Portuguese Study) and evaluate some environmental variables, such as house dust mite exposure. MATERIAL AND METHODS: Significant random samples of schoolchildren studied with standard validated methods--questionnaires, skin prick tests, methacholine bronchial challenge tests; dust bed sampling for analysis of mite antigens. RESULTS: In the ISAAC study, in the 13-14 year-old age group, statistical significant differences were found, with higher wheezing prevalence in Brazil than in Portugal (two-fold). In the Portuguese Study, atopy prevalence ranged between 6.0 and 11.9% in Sal and S. Vicente (Cape Verde), up to 48.6 and 54.1% in Macau and Madeira. Active asthma had the higher values in Madeira (14.6%), and the lower in Macau (1.3%). Cape Verde had intermediate asthma prevalence (10.6 and 7.0%). The bronchial challenge test was positive in 25, 66 and 70% of asthmatic children from Sal, S. Vicente and Madeira respectively. Significant HDM antigen concentrations (Der p1) were found in Cape Verde and Madeira. CONCLUSIONS: There are significant variations in asthma and atopy prevalence between these pediatric populations. The reasons remain under discussion, but genetics linked to race, seem to play a central role, modulated by environmental and lifestyle variables.