Browsing by Issue Date, starting with "2010-02"
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- Infecção VIH/SIDA : a situação em Portugal em 31 de Dezembro de 2009Publication . Departamento de Doenças Infecciosas do INSA. Unidade de Referência e Vigilância Laboratorial Epidemiológica. Núcleo de Vigilância Laboratorial de Doenças Infecciosas; Coordenação Nacional Para a Infecção VIH/SIDA
- Genome diversity in the genera Fructobacillus, Leuconostoc and Weissella determined by physical and genetic mappingPublication . Chelo, Ivo M.; Zé-Zé, Líbia; Tenreiro, RogérioPulsed-field gel electrophoresis analysis of chromosomal single and double restriction profiles of 17 strains belonging to three genera of ‘Leuconostocaceae’ was done, resulting in physical and genetic maps for three Fructobacillus, six Leuconostoc and four Weissella strains. AscI, I-CeuI, NotI and SfiI restriction enzymes were used together with Southern hybridization of selected probes to provide an assessment of genomic organization in different species. Estimated genome sizes varied from 1408 kb to 1547 kb in Fructobacillus, from 1644 kb to 2133 kb in Leuconostoc and from 1371 kb to 2197 kb in Weissella. Other genomic characteristics of interest were analysed, such as oriC and terC localization and rrn operon organization. The latter seems markedly different in Weissella, in both number and disposition in the chromosome. Comparisons of intra- and intergeneric features are discussed in the light of chromosome rearrangements and genomic evolution.
- Rab10 regulates phagosome maturation and its overexpression rescues Mycobacterium-containing phagosomes maturationPublication . Cardoso, Carla M. P.; Jordão, Luísa; Vieira, Otilia V.Phagosome maturation follows a defined biochemical program and, in the vast majority of cases, the microbe inside the phagosome is killed and digested. Although, an important number of pathogens, including Mycobacterium tuberculosis, which kills around two million people every year, have acquired the ability to survive, and even replicate by arresting phagosomal maturation. To identify more of the machinery involved in phagocytosis and phagosomal maturation, we investigated the function of Rab10 in engulfment and maturation of inert particles and Mycobacterium bovis bacille Calmette-Guérin (BCG). We showed that Rab10 association with phagosomes is transient and confocal microscopy revealed detectible levels of Rab10 on phagosomal membranes at very early time-points, occurring even before Rab5 acquisition. Rab10 recruitment had strong functional consequence, as the knockdown of endogenous Rab10 by RNA interference or overexpression of Rab10 dominant-negative mutant delayed maturation of phagosomes of IgG-opsonized latex beads or heat killed-mycobacteria. These results can be explained, at least in part, by the involvement of Rab10 in recycling of some phagosomal components. More importantly, overexpression of the constitutively active mutant of Rab10 partially rescued live-Mycobacterium-containing phagosomes maturation. Indeed, we found that the membrane harbouring Mycobacterium acquired early endosome antigen 1 (EEA-1), a marker excluded from phagosomes in control cells. Altogether these results indicate that Rab10, acting upstream of Rab5, plays a prominent role in phagolysosome formation and can modulate Mycobacterium-containing phagosomes maturation.
- Mixed lineage kinase 3 gene mutations in mismatch repair deficient gastrointestinal tumours.Publication . Velho, Sérgia; Oliveira, Carla; Paredes, Joana; Sousa, Sónia; Leite, Marina; Matos, Paulo; Milanezi, Fernanda; Ribeiro, Ana Sofia; Mendes, Nuno; Licastro, Danilo; Karhu, Auli; Oliveira, Maria José; Ligtenberg, Marjolijn; Hamelin, Richard; Carneiro, Fátima; Lindblom, Annika; Peltomaki, Paivi; Castedo, Sérgio; Schwartz, Simó Jr; Jordan, Peter; Aaltonen, Lauri A.; Hofstra, Robert M.W.; Suriano, Gianpaolo; Stupka, Elia; Fialho, Arsenio M; Seruca, RaquelMixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.
- Four years of expanded newborn screening in Portugal with tandem mass spectrometryPublication . Vilarinho, Laura; Rocha, Hugo; Sousa, Carmen; Marcão, Ana; Fonseca, Helena; Bogas, Mário; Osório, Rui VazINTRODUCTION: The Portuguese Neonatal Screening Programme (PNSP) was started in 1979 for phenylketonuria (2,590,700 newborns screened; prevalence 1:11,031) and, shortly after, for congenital hypothyroidism (2,558,455 newborns screened; prevalence 1:3,174). In 2004, expanded neonatal screening was implemented in the National Laboratory. The programme is not mandatory and has 99.8% coverage of the country (including Madeira and the Azores islands). MATERIAL AND METHODS: In the past 4 years, 316,243 neonates were screened with the use of tandem mass spectrometry (MS/MS) to test for selected amino acids and acylcarnitines. RESULTS: During this time, 132 patients were identified with 24 different inherited metabolic diseases (classic forms and variants). To date, the global frequency for all disorders integrated into the PNSP is estimated to be 1:1,380, with 1:2,396 for metabolic disorders. A total of 379 tests (0.12%) were classified as having false positive results, yielding an overall specificity of 99.9%. Despite the low frequency of several disorders, the positive predictive value of the overall MS/MS screening was found to be 26%, reflecting high diagnostic specificity of the method. Diagnostic sensitivity of extended screening for the different groups of disorders was 100%. Eight cases of maternal disorders [three glutaric aciduria type I, one carnitine transporter defect, and four 3-methylcrotonyl coenzyme A (CoA) carboxylase deficiency] were also detected through newborn screening. CONCLUSIONS: Our data support the advantage of a centralised laboratory for screening an elevated number of samples and making decisions if relying on a clinical network able to provide fast treatment and a good outcome in the screened cases.