Percorrer por autor "Corte-Real, Francisco"
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- Caracterização genética dos imigrantes oriundos de Brasil, Cabo Verde, Angola, Moçambique e Guiné Bissau. Impacto da miscigenação na genética forensePublication . Marcelino, Miguel; Afonso Costa, Heloísa; Correia Dias, Helena; Corte-Real, Francisco; Amorim, AntónioPortugal recebeu um grande influxo de imigrantes oriundos de diversos países. Dentro destes destacam-se aqueles vindos de países pertencentes à Comunidade dos Países de Língua Portuguesa (CPLP). Em 2022 contabilizaram-se cerca de 350 000 imigrantes oriundos destes países, aproximadamente 45% do número total de imigrantes a residir em Portugal. A introdução destas populações imigrantes acrescenta variabilidade genética à população portuguesa pela introdução de variantes genéticas características de populações africanas e sulamericanas. Este facto deve ser avaliado para que na valorização das perícias de genética e biologia forenses a população de referência seja a mais representativa da realidade e se possa alcançar o valor de probabilidade mais correto. A valorização quantitativa da prova biológica é feita, calculando a razão da verossimilhança, Likelihood Ratio (LR). O LR é a razão de duas probabilidades condicionais, que indica o número de vezes que é mais provável a ocorrência dos perfis genéticos determinados admitindo a Hipótese 1 como verdadeira - o indivíduo é o contribuidor -, relativamente à ocorrência desses mesmos perfis admitindo a Hipótese 2 como verdadeira - um indivíduo ao acaso da população de referência é o contribuidor. É então necessário o conhecimento prévio das frequências alélicas, genéticas e haplotípicas da população de referência. A escolha da população de referência pode ser particularmente problemática nas perícias que envolvem imigrantes, já que não é óbvio qual a melhor população de referência a ser utilizada, se a do seu país de origem, se a do local onde está atualmente a residir, ou se a população de referência onde se deu a ocorrência (crime, desaparecimento, procriação). As populações de imigrantes a residir em Lisboa foram sendo estudadas desde 2012 no âmbito do projecto de investigação "A população de Lisboa do início do século XXI: caracterização genética dos novos habitantes oriundos do Brasil, Cabo Verde, Angola e Guiné Bissau". Foram determinadas as frequências alélicas de marcadores genéticos autossómicos, particularmente os da European Standard Set (ESS) e do Combined DNA Index System (CODIS). Foram determinadas as frequências alélicas de marcadores genéticos do cromossoma X, as frequências haplotípicas de marcadores genéticos do cromossoma Y e frequências haplotípicas da região controlo do ADN mitocondrial. Relativamente e mais particularmente ao estudo do ADN mitocondrial verificou-se que estes imigrantes apresentam maioritariamente haplótipos pertencentes a haplogrupos tipicamente africanos e sul-americanos. É importante considerar que a miscigenação contínua entre diferentes grupos étnicos gera uma maior diversidade genética sendo necessária a constante atualização das bases de dados referentes às populações de referência para garantir uma representação precisa e fornecer uma valorização da prova biológica consentânea com a realidade populacional.
- LC–MS-MS-MS3 for the determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samplesPublication . Proença, Paula; Teixeira, Helena; Martinho, Beatriz; Monteiro, Carla; Franco, João; Corte-Real, FranciscoDue to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography–tandem mass spectrometry methodology (LC–MS-MS-MS3) was developed for the determination and quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9- tetrahydrocannabinol (THCOH) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood samples. Chromatographic analysis of THC, THC-OH and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm × 2.1 mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid and methanol at a flow rate of 0.4 mL/min, with a run time of 10 min. For the MS-MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5 and 100 ng/mL (r2 > 0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0% and 117.2% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC–MS-MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC–MS3 technique to determine drugs in biological samples.
- Mitochondrial DNA characterization of Brazilian immigrant Population living in LisboaPublication . Marcelino, Miguel; Amorim, António; Corte-Real, Francisco; Afonso Costa, HeloísaMigration is one of the main factors for genetic variability within populations. Currently, the Portuguese population, and particularly the population from Lisboa, welcomes a considerable number of immigrants. Brazilian immigrants are the main foreign community in Portugal, with about 184 000 individuals in 2020. Mithocondrial DNA (mtDNA), due to its unique characteristics such as being exclusively maternal inheritance and suffering no recombination, which results in its slow evolution, is a useful genetic marker to study the evolution of populations. In this study mtDNA sequencing analyzis of 64 Brazilian immigrants who currently live in Lisbon were carried out in order to assess the impact of this population on the Portuguese gene pool. The mtDNA control region were amplified using two pairs of primers - L15971 / H016 and L16555 / H639. The amplified products were then sequenced using BigDye®Terminator v.3.1 Cycle Sequence (AB) and detected in the SeqStudio™ Genetic Analyzer (AB). The results were analysed with the Sequencing Analysis v7. and SeqScape v4. (AB) softwares, where the obtained sequences were compared with the rCRS in order to obtain haplotypes that, with Phylotree, build 17, can be converted in haplogroups.
- Reverse Body Fluid Identification Workflow: A Direct to DNA ApproachPublication . Porto, Maria-João; Ferreira, Joana; Bento, Ana; Bogas, Vanessa; Lopes, Virgínia; Sampaio, Lisa; Gouveia, Nair; Corte-Real, Francisco; Amorim, António; Amorim, António; Andrade Sampaio, Lisa; Bogas, Vanessa; Ventura Teixeira Bento, Ana Margarida; University of Santiago de Compostela (USC)
- SARS-COV-2 Variants in the region of Lisbon: Comparation and validation study between RT-PCR and NGS methodologiesPublication . Franco, Magda; Cainé, Laura; Rodrigues, Joana; Mofreita, Vânia; Nascimento, Rui; Mukan, Olena; Fadoni, Jennifer; Corte-Real, Francisco; Amorim, AntónioSARS-CoV-2 is a recent coronavirus that appeared in the end of 2019. The World Health Organization named the infection, caused by this new coronavirus, Coronavirus Disease 2019 (COVID-19). This disease was considered a pandemic on March 11th 2020 by the same organization. Coronaviruses rapidly acquire new mutations and, consequently, new variants keep emerging. There are some variants with an associated risk in the increase of the transmissibility and that cause more severe disease and reduction in the neutralization by antibodies and, for this reason, are called variants of concern. The aim of this project was to identify the main VOCs and VOIs circulating in the region of Lisbon, applying the methodology of real time RT-PCR in cadavers that tested positive for SARS-CoV-2. To meet this goal, we used three assays: Allplex™ SARS-CoV-2 Variants I, Allplex™ SARS-CoV-2 Variants II and Allplex™ SARS-CoV-2 Variants V. The first one detects defining mutations of the Alpha, Beta and Gamma variants (N501Y, E454K and HV69/70del), the second assay detects mutations present in the Delta, Beta, Gamma and California variants (L452R, K417T, K417N and W152C) and the third one detects defining mutations of the Delta and Lambda variants (L452R, P681R, L452Q and F690S). In addition, we also wanted to understand if these RT-PCR assays were efficient to correctly identify these variants, comparing the results obtained to the reference methods for the determination of variants – Next Genration Sequencing (NGS). Two different NGS methodologies were used to compare the results – NGS-ONT (Oxford Nanopore Technologies) and NGS-Sanger based. We concluded that 30% of the samples belonged to the Alpha variant and 70% belonged to the Delta variant and that, in general, the three RT-PCR assays applied in this study were efficient in correctly identifying these two variants, based on the comparison to the NGS results.