Screening for RAG activity in haematopoietic tumours using a novel reporter strategy

Tese de mestrado, Biologia (Biologia Molecular Humana), 2009, Universidade de Lisboa, Faculdade de CiênciasResumo alargado em português disponível no documentoRecombination-Activating Genes (RAG) 1 and 2, form the site specific recombinase that mediates V(D)J recombination at the antigen receptor loci, a process responsible for lymphocyte diversity. RAG can also interact with degenerated recognition signal sequences (cRSSs) distributed throughout the genome and potentially induce genomic instability. To this date, no available reporter of RAG activity has gathered the versatile features of a molecular tool with a simple method of readout. Our laboratory has recently generated GFPi, a novel episomal/retroviral fluorescence-based molecular tool that allows for fast assessment of RAG activity in various cell types. We have found that the GFPi reporter can not only provide in vitro quantitative measurement transiently, being sensitive to RSS sequence degeneration, but also ex vivo in its integrated form as a stable substrate. We have also optimized the in vitro recombination assay (IVRA) for the detection of a broader window of RAG activity values. This novel assay allowed us to quantify the efficiency of RAG recombination of a selected set of cRSSs, namely a previously undetected cRSS found in the first exon of the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene, which is known to be involved in leukemogenesis. PTEN cRSS was proved to be as functional as other V(D)J loci RSSs, and this activity correlates with a novel mutation which was recently found in a leukaemic patient in this same site. We also made use of the retroviral-based form of GFPi in order to quantify endogenous RAG activity in human haematopoietic tumour cell lines of lymphoid and myeloid origin. Although we have found lineage-specific differences on RAG activity (namely in Reh B-cell line which presented intense RAG activity), we found no signs of promiscuous endogenous RAG activity in myeloid cell lines in a short timespan. Thus, we believe the GFPi reporter gathers all the requisites for an efficient RAG activity reporter, coupled with a fast and direct method of readout. Moreover, we now possess a robust tool that is suitable for a number of applications, namely unravelling the relation between RAG promiscuous activity and leukaemogenesis, which was preliminary addressed in this work

Validation and quality assurance applied to goat milk chemical composition: minerals and trace elements measurements

In the present study, quality assurance programmes were implemented to validate and control the analytical methodologies used for the characterization of minerals and trace elements in goat milk from Portuguese breeds. With the exception of chloride that was determined by potentiometric titration, all the other elements were determined by spectroscopic techniques after different sample decomposition: P was measured by ultraviolet-visible molecular absorption spectrometry, Ca, Fe, K, Mg, Mn, Na and Zn by flame atomic absorption spectrometry and Cd, Co, Cr, Cu, Mo, Ni and Pb by electrothermal atomic absorption spectrometry. The methods performance characteristics, namely specificity, limit of detection, limit of quantification, working range, precision and trueness were evaluated. Measurement uncertainty was expressed in terms of precision and trueness. Precision under intralaboratory reproducibility conditions was estimated from triplicate analysis, and the trueness component was estimated in terms of overall recovery using either skim milk powder certified reference materials or spiked samples. The results obtained are discussed on the basis of the performance criteria required by EC regulations to verify when a method is suitable for food control. The methods used for the characterization of minerals and trace elements in goat milk complied with EC requirements since there was no matrix influence, the Horrat values were < 2.0, recoveries were within the interval 1.00 ± 0.10 for minerals and 1.00 ± 0.20 for trace elements and the combined uncertainty of the results were lower than the maximum standard uncertainty calculated using the uncertainty function approach. In relation to the limits of detection and quantification, the limits obtained for Pb were lower than those specified by EC regulation

Epigenética nas Perturbações de Ansiedade

Introdução: As perturbações de ansiedade são das perturbações psiquiátricas mais comuns, caracterizadas por medo excessivo, ansiedade e evitação de ameaças. São uma das principais causas de incapacidade mundial, com uma prevalência cada vez maior. Está normalmente associada a outras comorbilidades como depressão ou abuso de substâncias. A patogénese das perturbações de ansiedade é complexa, envolvendo interações entre fatores biológicos, influências do ambiente e mecanismos psicológicos. Objetivos: Esta dissertação tem como objetivo reunir a literatura sobre o papel dos fatores epigenéticos no desenvolvimento de perturbações de ansiedade. Métodos: A pesquisa foi realizada na plataforma PubMed entre setembro de 2021 até abril de 2022. Resultados: A análise da literatura sobre as influências da epigenética no desenvolvimento de perturbações de ansiedade implica o envolvimento de genes que regulam o eixo hipotálamohipofise-adrenal, sistemas neurotransmissores ou plasticidade neuronal, e vários estudos sugerem que a disrupção da expressão genética destes genes através de mecanismos de epigenética contribuem para a patogénese de perturbações de ansiedade. Conclusão: A investigação sobre qual a contribuição dos mecanismos epigenéticos para a suscetibilidade ou resistência para o desenvolvimento das perturbações de ansiedade ainda se encontra na sua infância pelo que será necessária mais pesquisa para clarificar detalhes nesta área.Introduction: Anxiety disorders are some of the most common psychiatric disorders. They’re characterized by excessive fear, anxiety, and threat avoidance. They’re one of the leading causes for disability worldwide, with a rising prevalence. It’s usually associated with comorbidities like depression and substance abuse. Anxiety disorder’s pathogenesis is complex, involving interactions between biology factors, environment influences and psychological mechanisms. Objectives: This dissertation aims to report on current literature findings about the role of epigenetic factors on the development of anxiety disorders. Methods: The research was done on the platform PubMed between the dates September 2021 until April 2022. Results: Current literature about epigenetics’ influence on the development of anxiety disorders implicates genes that regulate the axis hypothalamic-pituitary-adrenal, neurotransmitter systems, and neuroplasticity, and several studies suggest the disruption of the expression of these genes through epigenetic mechanisms contribute to the pathogenesis of AD. Conclusion: Research about the epigenetic mechanisms’ contribution towards susceptibility or resilience in developing anxiety disorders is still in its infancy and requires more more work to clarify details in the area

A Novel Quantitative Fluorescent Reporter Assay for RAG Targets and RAG Activity

Recombination-Activating Genes (RAG) 1 and 2 form the site specific recombinase that mediates V(D)J recombination, a process of DNA editing required for lymphocyte development and responsible for their diverse repertoire of antigen receptors. Mistargeted RAG activity associates with genome alteration and is responsible for various lymphoid tumors. Moreover several non-lymphoid tumors express RAG ectopically. A practical and powerful tool to perform quantitative assessment of RAG activity and to score putative RAG-Recognition signal sequences (RSS) is required in the fields of immunology, oncology, gene therapy, and development. Here we report the detailed characterization of a novel fluorescence-based reporter of RAG activity, named GFPi, a tool that allows measuring recombination efficiency (RE) by simple flow cytometry analysis. GFPi can be produced both as a plasmid for transient transfection experiments in cell lines or as a retrovirus for stable integration in the genome, thus supporting ex vivo and in vivo studies. The GFPi assay faithfully quantified endogenous and ectopic RAG activity as tested in genetically modified fibroblasts, tumor derived cell lines, developing pre-B cells, and hematopoietic cells. The GFPi assay also successfully ranked the RE of various RSS pairs, including bona fide RSS associated with V(D)J segments, artificial consensus sequences modified or not at specific nucleotides known to affect their efficiencies, or cryptic RSS involved in RAG-dependent activation of oncogenes. Our work validates the GFPi reporter as a practical quantitative tool for the study of RAG activity and RSS efficiencies. It should turn useful for the study of RAG-mediated V(D)J and aberrant rearrangements, lineage commitment, and vertebrate evolution.Portuguese League Against Cancer, Terry Fox Foundation, FCT fellowships

Referenciação aos cuidados de saúde secundários

Introdução: O processo de referenciação dos Cuidados de Saúde Primários (CSP) para os Cuidados de Saúde Secundários (CSS) é de grande interesse, sobretudo devido a aspectos relacionados com a qualidade dos cuidados prestados e de ordem económica. O Estudo Europeu sobre Referenciação revelou, em Portugal, uma taxa de referenciação de 5,56%. Em Portugal, não existem estudos sobre a qualidade da referenciação. A Unidade Local de Saúde (ULS) de Matosinhos pretende prestar cuidados globais aos utentes colocando em rede os CSP e CSS. Em termos de referenciação, esta ligação necessita de ser aperfeiçoada. Objectivos: Determinar a taxa de referenciação, caracterizar a população referenciada, avaliar as características das referenciações, descrever e analisar a resposta dos CSS. Metodologia: Estudo transversal analítico. Recolha do número de referenciações do CS Senhora da Hora (CSSH) no ano de 2003. Amostra aleatória de 400 referenciações efectuadas para o Hospital Pedro Hispano (HPH). Determinação das estatísticas descritivas da amostra e uso do teste de 2 para comparação de proporções (a = 0,05). Resultados: A taxa de referenciação foi de 10,1%. A idade média da população referenciada foi 45,3 anos; 61,8% do sexo feminino. As especialidades mais referenciadas foram ORL, Ginecologia e Cirurgia. As cartas de referenciação contêm, na sua maioria, o nome do doente (98,9%), objectivo de referenciação (92,8%) e data (94,8%). Contudo, grande parte não referia medicação habitual (71,3%), descrição do exame físico (51,5%) ou identificação do médico (52,3%). Em 50,8% dos casos, a efectivação da consulta verificou-se 1 a 6 meses após a referenciação. Obteve-se informação de retorno em 26,3% das referenciações. Conclusão: Este estudo revelou lacunas na articulação dos cuidados de saúde. O médico deve ter em conta que as cartas de referenciação/retorno são um veículo primordial de comunicação, devendo ser aperfeiçoadas, pois delas depende a qualidade dos cuidados prestados. Sistemas de informação em rede e regras de referenciação protocoladas podem melhorar a articulação dos cuidados

Characterization of mineral and trace elements of milk from indigenous portuguese goat milk: functional potentiality

Mestrado em Engenharia Alimentar - Instituto Superior de AgronomiaIn Portugal, goat milk is mainly used for traditional cheesemaking. However, goat milk does not yet have an “image” in this country, a better scientific knowledge related with its composition and health benefits being needed. The aim of the present study was to determine and compare the concentration of minerals and trace elements from the milk of indigenous Portuguese goat breeds and those of an exotic breed, and relate the results with their production system, nutritional and health benefits. Raw goat milk samples from Portuguese breeds (Serrana - two ecotypes: Transmontana and Ribatejana, Charnequeira, Serpentina , Algarvia) and from Saanen, were collected monthly at their local grazing sites during one lactation period. Major constituents, minerals and trace elements of goat milk were determined for all samples following validation and quality assurance programs. It was concluded that there was no significant differences for goat milk between the indigenous Portuguese breeds, but Serrana milk was distinct from the others, being also ecotype Transmontana significantly different from ecotype Ribatejana. Milk from the Portuguese breeds can be grouped in two main clusters: Serrana-Transmontana, Charnequeira and Serpentina, and Saanen, Algarvia and Serrana-Ribatejana. In general, the Portuguese goat milk is a good source of functional ingredients. - RESUMO - Em Portugal, o leite de cabra é maioritariamente utilizado para o fabrico de queijo e o consumidor não o vê com outra finalidade. Para tal, é necessário mais investigação sobre a composição e vantagens do leite de cabra para a saúde. O objectivo deste trabalho foi determinar e comparar as concentrações de sais e minerais do leite de raças caprinas autóctones e relacioná-las com o sistema produtivo, aspectos nutricionais e possíveis benefícios para a saúde. Durante um período de lactação foram recolhidas mensalmente amostras de leite crú das raças autoctones (Serrana – dois ecotipos: Transmontana e Ribatejana, Charnequeira, Serpentina e Algarvia) e de uma raça exótica (Saanen) nas respectivas áreas geográficas de produção. Determinou-se a composição global, minerais e elementos vestigiais por métodos validados e controlados com programas de garantia da qualidade. Concluiu-se que não há diferenças significativas entre o leite das diferentes raças, mas o leite da Serrana destacou-se das outras, sendo também o leite da Serrana-Transmontana significativamente diferente do leite da Serrana-Ribatejana. A análise de “Clusters” permitiu-nos agrupar as raças em dois grandes grupos: Serrana-Transmontana, Charnequeira e Serpentina num grupo, e Saanen, Algarvia e Serrana - Ribatejana noutro. No geral, o leite de cabra das raças autóctones é uma boa fonte de ingredientes funcionais

Validation et assurance qualité appliquée à la composition chimique du lait de chèvre : mesures des minéraux et éléments traces.

In the present study, quality assurance programmes were implemented to validate and control the analytical methodologies used for the characterization of minerals and trace elements in goat milk from Portuguese breeds. With the exception of chloride that was determined by potentiometric titration, all the other elements were determined by spectroscopic techniques after different sample decomposition: P was measured by ultraviolet-visible molecular absorption spectrometry, Ca, Fe, K, Mg, Mn, Na and Zn by flame atomic absorption spectrometry and Cd, Co, Cr, Cu, Mo, Ni and Pb by electrothermal atomic absorption spectrometry. The methods performance characteristics, namely specificity, limit of detection, limit of quantification, working range, precision and trueness were evaluated. Measurement uncertainty was expressed in terms of precision and trueness. Precision under intralaboratory reproducibility conditions was estimated from triplicate analysis, and the trueness component was estimated in terms of overall recovery using either skim milk powder certified reference materials or spiked samples. The results obtained are discussed on the basis of the performance criteria required by EC regulations to verify when a method is suitable for food control. The methods used for the characterization of minerals and trace elements in goat milk complied with EC requirements since there was no matrix influence, the Horrat values were $<$ 2.0, recoveries were within the interval 1.00 $\pm$ 0.10 for minerals and 1.00 $\pm$ 0.20 for trace elements and the combined uncertainty of the results were lower than the maximum standard uncertainty calculated using the uncertainty function approach. In relation to the limits of detection and quantification, the limits obtained for Pb were lower than those specified by EC regulation.Dans ce travail, des programmes d'assurance de la qualité ont été mis en œuvre afin de valider et de contrôler les méthodes d'analyse utilisées pour caractériser les minéraux et les éléments traces du lait de chèvres de race portugaise. À l'exception du chlorure, qui a été déterminé par titrage potentiométrique, tous les autres éléments ont été déterminés par différentes techniques spectroscopiques après décomposition de l'échantillon : P a été mesuré par spectrométrie d'absorption moléculaire ultraviolet-visible (UV-VIS-MAS), Ca, Fe, K, Mg, Mn, Na, et Zn par spectrométrie d'absorption atomique de flamme (FAAS) et Cd, Co, Cr, Cu, Mo, Ni et Pb par spectrométrie d'absorption atomique électrothermique (ETAAS). Les caractéristiques de performance des méthodes, à savoir la spécificité, limite de détection, limite de quantification, la plage de travail, la précision et la justesse ont été évaluées. Les incertitudes de mesure ont été exprimées en termes de précision et de justesse. La précision sous des conditions de reproductibilité intralaboratoire a été estimée à partir des analyses en triple. La justesse a été estimée en termes de récupération globale, soit en utilisant des poudres de lait matériaux de référence certifiés ou des échantillons dopés. Les résultats obtenus sont discutés sur la base des critères de performance requis par les règlements CE pour vérifier qu'une méthode est adaptée au contrôle des aliments. Les méthodes utilisées pour la caractérisation des minéraux et des éléments traces dans le lait de chèvre se conformaient aux exigences de la CE car il n'y avait pas d'influence de la matrice, les valeurs de Horrat étaient inférieures à 2,0 ; les récupérations étaient dans l'intervalle $1,00 \pm 0,10$ pour les minéraux et dans l'intervalle $1,00 \pm 0,20$ pour les éléments traces et l'incertitude combinée des résultats était inférieure à l'incertitude normalisée maximale, calculée en utilisant la fonction de l'incertitude. En ce qui concerne les limites de détection et de quantification, nos limites calculées pour le plomb étaient inférieures à celles spécifiées par le règlement CE