Construction of Ms6 derivative mutants as tools for interaction studies ith mycobacteria

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015Mycobacteriophage Ms6 is a temperate phage isolated in 1989 from a spontaneously induced culture of Mycobacterium smegmatis. Although several studies regarding lysis and integration have been published, the host range of Ms6 is not known. In this work we observed that incubation of this phage with fast- and slow-growing mycobacteria did not result in plaque or halos formation, as usually happens in infection plaque assay. However, an absence of clearing in a bacterial lawn does not mean that Ms6 in unable to infect the tested mycobacteria apart from its natural host, M. smegmatis. Taking this into consideration, our main goal was to construct Ms6 derivative mutants with reporter genes, and use them as tools to follow the mycobacteriophage replication inside other mycobacteria. Using the Bacteriophage Recombineering of Electroporated DNA (BRED) technique, we successfully constructed two Ms6 derivative reporter phages, Ms6lysB::egfp and Ms6pin::gfpm2+, by gene replacement. We successfully showed that their reporter proteins, improved green fluorescent proteins (GFP), are respectively expressed in Ms6 lytic and lysogenic life cycle, on M. smegmatis. Consequently, these new mutant phages are very important and useful tools to be used in future studies since they permit to follow a lytic or lysogenic cycle. With Ms6lysB::egfp and Ms6pin::gfpm2+, we expect to determine with more accuracy the ability of Ms6 to infect/replicate in other mycobacteria.O micobacteriófago Ms6 é um fago temperado, isolado em 1989 a partir de uma cultura de Mycobacterium smegmatis induzida espontaneamente. Apesar de vários estudos sobre o sistema de lise e integração terem já sido publicados, a gama de hospedeiros do Ms6 não é ainda conhecida. Neste trabalho verificou-se que a incubação deste fago com micobactérias quer de crescimento rápido quer de crescimento lento, não resultou na formação de placas fágicas nem de halos, tal como acontece num ensaio clássico de infecção em placa. No entanto, a ausência de placas fágicas não significa que Ms6 não tenha capacidade de infectar outras micobactérias para além de M. smegmatis, o seu hospedeiro natural. Tendo este facto em consideração, o principal objectivo deste trabalho foi construir fagos Ms6 mutantes com genes repórter, de modo a usá-los como ferramentas para testar a capacidade do Ms6 se replicar noutras micobactérias. Através da metodologia Bacteriophage Recombineering of Electroporated DNA (BRED), foi possível proceder a uma troca de genes do fago Ms6 por genes repórter e construir, com sucesso, dois fagos recombinantes designados Ms6lysB::egfp e Ms6pin::gfpm2+, capazes de produzir fluorescência quando infectam um hospedeiro sensível. Foi com sucesso que mostrámos que as proteínas verde fluorescentes modificadas (GFP), são expressas em M. smegmatis no ciclo lítico e lisogénico. Consequentemente, estes novos fagos mutantes constituem importantes ferramentas que poderão ser usados em estudos futuros, tendo em conta que permitem seguir o ciclo lítico e lisogénico. Esperamos assim, através dos fagos construídos Ms6lysB::egfp and Ms6pin::gfpm2+, conseguir determinar de uma forma mais precisa, a capacidade de Ms6 infectar/replicar-se em outras micobactérias

Report of the Second International Symposium on Molecular Epidemiology in Childhood Leukaemia and Embryonal Tumours, Rio de Janeiro, Brazil

The recent International Symposium on Molecular epidemiology in Embryonal Tumours and Paediatric Leukaemia was held on 4–6 March 2008 in Rio de Janeiro, Brazil. It proved a very productive meeting in which studies relating to genetics, therapeutical trials, identification of risk factors in acute leukaemia neuroblastoma and Wilms’ tumours were presented. Over 120 participants gathered for three days of fruitful discussions, including representatives of paediatrics, haematology, laboratory, epidemiology and pathology. Debates were held about strategies of applications of important biomarkers for clinical trials. Highlights of each of the scientific presentations are summarized below

A utilização das fundações na eficiência energetica dos edificios

Mestrado em Engenharia CivilO presente trabalho propõe-se divulgar a importância da Geotermia no mundo, na possibilidade de podermos utilizar um recurso natural que existe e foi descoberto há milhares de anos e até aos dias de hoje tem sido pouco utilizado, apresento numa análise mais específica em Portugal onde, como pode e deveria ser utilizado, usando um caso real de um edifício onde foi instalado e a satisfação do cliente é total tanto em termos monetários como nas condições habitacionais do edifício.This paper aims to promote the importance of Geothermal in the world, the possibility that we can use a natural resource that exists and was discovered thousands of years ago and until today has been little used, present a more specific analysis in Portugal where how can and should be used, using a real case of a building where it was installed and customer satisfaction is total both in monetary terms as the housing conditions of the building

Avaliação de metodologias de optimização energética em veículos eléctricos de proximidade

Tese de mestrado integrado. Engenharia Electrotécnica e de Computadores (Major Automação). Faculdade de Engenharia. Universidade do Porto. 200

Challenges in the use of NG2 antigen as a marker to predict MLL rearrangements in multi-center studies

AbstractRearrangements in MLL (MLL-r) are common within very young children with leukemia and affect the prognosis and treatment. Previous studies have suggested the use of the NG2 molecule as a marker for MLL-r but these studies were performed using a small number of infants. We analyzed 148 patients (all less than 24 months, 86 less than 12 months) from various centers in Brazil to determine the predictive power of NG2 within that cohort. We show that NG2 can be used for MLL-r prediction; however, proper staff training and standardized sampling procedures are essential when receiving samples from multiple centers as the accuracy of the prediction varies greatly on a per center basis

A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report.

BACKGROUND: Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression. CASE PRESENTATION: A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died. CONCLUSIONS: To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome

t(10;11)(p12;q23) KMT2A/NEBL

Review on t(10;11)(p12;q23) KMT2A/NEBL, with data on clinics, and the genes involved

CD44 Expression Profile Varies According to Maturational Subtypes and Molecular Profiles of Pediatric T-Cell Lymphoblastic Leukemia.

CD44 is a glycoprotein expressed in leucocytes and a marker of leukemia-initiating cells, being shown to be important in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). In this study, we have (i) identified the aberrant antigenic pattern of CD44 and its isoform CD44v6 in T-ALL; (ii) tested the association with different T-cell subtypes and genomic alterations; (iii) identified the impact of CD44 status in T-ALL outcome. Samples from 184 patients (123 T-ALL and 61 AML; <19 years) were analyzed throughout multiparametric flow cytometry. Mutations in N/KRAS, NOTCH1, FBXW7 as well as STIL-TAL1 and TLX3 rearrangements were detected using standard molecular techniques. CD44 expression was characterized in all T-ALL and AML cases. Compared with AML samples in which the median fluorescence intensity (MFI) was 79.1 (1-1272), T-ALL was relatively low, with MFI 43.2 (1.9-1239); CD44v6 expression was rarely found, MFI 1 (0.3-3.7). T-ALL immature subtypes (mCD3/CD1aneg) had a lower CD44 expression, MFI 57.5 (2.7-866.3), whereas mCD3/TCRγδpos cases had higher expressions, MFI 99.9 (16.4-866.3). NOTCH1 mut and STIL-TAL1 were associated with low CD44 expression, whereas N/KRAS mut and FBXW7 mut cases had intermediate expression. In relation to clinical features, CD44 expression was associated with tumor infiltrations (p = 0.065). However, no association was found with initial treatment responses and overall survival prediction. Our results indicate that CD44 is aberrantly expressed in T-ALL being influenced by different genomic alterations. Unraveling this intricate mechanism is required to place CD44 as a therapeutic target in T-ALL

Participación en la sesión de formación a distancia “Romanofonia e cinema 5: Plans croisés et intercompréhension

Memoria ID2022-069. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023