Sequencing CYP2D6 for the detection of poor-metabolizers in post-mortem blood samples with tramadol

Tramadol concentrations and analgesic effect are dependent on the CYP2D6 enzymatic activity. It is well known that some genetic polymorphisms are responsible for the variability in the expression of this enzyme and in the individual drug response. The detection of allelic variants described as non-functional can be useful to explain some circumstances of death in the study of post-mortem cases with tramadol. A Sanger sequencing methodology was developed for the detection of genetic variants that cause absent or reduced CYP2D6 activity, such as *3, *4, *6, *8, *10 and *12 alleles. This methodology, as well as the GC/MS method for the detection and quantification of tramadol and its main metabolites in blood samples was fully validated in accordance with international guidelines. Both methodologies were successfully applied to 100 post-mortem blood samples and the relation between toxicological and genetic results evaluated. Tramadol metabolism, expressed as its metabolites concentration ratio (N-desmethyltramadol/O-desmethyltramadol), has been shown to be correlated with the poor-metabolizer phenotype based on genetic characterization. It was also demonstrated the importance of enzyme inhibitors identification in toxicological analysis. According to our knowledge, this is the first study where a CYP2D6 sequencing methodology is validated and applied to post-mortem samples, in Portugal. The developed methodology allows the data collection of post-mortem cases, which is of primordial importance to enhance the application of these genetic tools to forensic toxicology and pathology.info:eu-repo/semantics/publishedVersio

A Tale of two Smart Cities : Investigating the Echoes of New Public Management and Governance Discourses in Smart City Projects in Brazil

This study investigates the influence of discourses of New Public Management and e-Governance on the manner in which Information Technology (IT) has been conceived in recent smart city initiatives in Brazil. A critical discourse analysis is conducted as the methodological approach to investigate the role of IT in smart city discourses of two cities. The main result has shown that the role of technology within the two cases strongly reflects the discourses of New Public Management and e-governance, in which there is clearly a latent tension between top-down and bottom-up approaches to smart cities

Characterization of GlobalFiler loci in Angolan and Guinean populations inhabiting Southern Portugal

We analyzed the GlobalFiler short tandem repeat (STR) loci for 152 and 70 unrelated individuals from Angolan and Guinean immigrant populations inhabiting Southern Portugal, respectively. After Bonferroni correction, no significant deviations from the Hardy-Weinberg equilibrium and linkage disequilibrium were detected for either population. For the Angolan population, SE33 was the most informative marker. In contrast, D5S818 and D13S317 were the least informative loci. The combined power of discrimination was 99.9999999999999999999999961907%. For the Guinean population, SE33 and D21S1 were the most informative loci, while D13S317 was the least. The combined power of discrimination was 99.99999999999999999999997915%. No significant differences were observed between Angolan, Guinean, and Afro-American populations for any of the analyzed STRs. The South African population presented significant differences at D22S1045 and D10S1248 when compared to Angola, and at D22S1045 when compared to Guinea-Bissau. The MDS plot and neighbor-joining tree analysis revealed that Angolan and Guinean populations are genetically close to African-American and South African populations, and genetically different from Korean, Mexican, European (including American-Caucasian), and Middle Eastern populations.eu-repo/semantics/submittedVersio

Digital-by-Default: Exclusion through Digital Public Service Channels

The use of digital channels has been promoted by governments as a way of improving services and reducing costs. This article aims to discuss how new forms of exclusion can be created by policies based on a digital-by-default orientation. To this end, more than 1.2 million service records are analyzed, as well as being considered socioeconomic indicators and spatial factors of the city of São Paulo. Results show that there is a statistically significant correlation between income and digital channels usage, as well as between digital channels usage and the requested service attendance time. It is also verified that the districts of the central region of the city tend to have high income, high rates of human development and high digital channels usage, while the periphery’s districts tend to have low income, lower rates of human development and high traditional channels usage. It is concluded that, by prioritizing digital service channels, new forms of social exclusion are being created and that a portion of the population may be forgotten by the government

Chromosome Instability in Asthma an Antioxidant Treatment as a Possible Pharmacological Strategy?

This article belongs to the Proceedings of The 1st International e-Conference on Antioxidants in Health and Diseas, publicado em: Med Sci Forum. 2021;2(1):13. doi:10.3390/CAHD2020-08577Asthma is a disease with multiple phenotypes and different degrees of severity. Severe inflammation appears when oxidative stress (OS) overwhelms the antioxidant defense. Knowing the role of OS in the increase of chromosome instability (CI) and, consequently, in cancer predisposition, it is important to carry out studies to establish limits for OS harmful effects. The aim of this study was to evaluate OS-related CI in asthma patients and the possible protective effect of antioxidants. For that purpose, spontaneous and OS-induced lymphocyte cultures from patients with mild and severe asthma, and from normal controls, were performed. Antioxidant-enriched cultures from severe patients were posteriorly performed. A hundred metaphases per culture were scored in order to estimate the frequency of CI. Our results showed that lymphocytes from severe patients had increased CI, both in spontaneous and OS-induced cultures. However, in lymphocytes from mild patients there were no differences compared to controls. This suggests that in patients subjected to extreme OS conditions, a genotoxic effect may occur at the cellular level. When lymphocytes from these patients were treated with antioxidants, a decrease in CI was observed. Understanding how CI correlates to asthma patients’ clinical situation may be pivotal to the design of future preventive measures.info:eu-repo/semantics/publishedVersio

Analysis of 17 STR data on 5362 southern Portuguese individuals—an update on reference database

The main objective of this work consisted of the updating of allele frequencies and other relevant forensic parameters for the 17 autosomal STR loci provided by the combination of the two types of kits used routinely in our laboratory casework: AmpF/STR Identifiler® and the Powerplex® 16 Systems. This aim was of significant importance, given that the last study on these kits within the southern Portuguese population dates back to 2006, and, as a consequence, it was necessary to correct the deviation caused by population evolution over the last ten years so that they might be better applied to our forensic casework. For this reason genetic data from 5362 unrelated Caucasian Portuguese individuals from the south of Portugal who were involved in paternity testing casework from 2005 to 2014 was used. Of all the markers, TPOX proved to be the least polymorphic, and Penta E the most. Secondly, this up-to-date southern Portuguese population was compared not only with the northern and central Portuguese populations, but also with that of southern Portugal in 2006, along with populations from Spain, Italy, Greece, Romania, Morocco, Angola and Korea in order to infer information about the relatedness of these respective populations, and the variation of the southern Portuguese population over time.info:eu-repo/semantics/publishedVersio

Y-Filer Plus® genetic characterization of caucasian individuals from South Portugal

Trabalho apresentado sob a forma de poster na reunião cientifica - "Haploid Markers 2016 Update on DNA variation" Maio 20-21, Berlin, GermanyDue to their paternal inheritance, Y-STRs offers particular perspectives for identification and kinship analysis and are also a precious tool in sexual assault cases with relatively high amount of female DNA and also in mixtures from multiple male donors. Nonetheless their value, there are some limitations to their use in forensic investigations since their ability to discriminate between individuals is considerably lower than that of the autosomal STRs set, mainly in cases with close or distant patrilineal relatives.One of the most recently developed Y-STR kit, Y-Filer Plus® (Life Technologies, Foster city, USA), allows forensic geneticists to study 27 Y-chromosomal loci. All the 16 markers included in the Y-Filer® kit (Life Technologies, Foster city, USA), plus 9 additional markers: DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYF387S1 and DYS533, six of which (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) are characterized as “rapidly mutating”, and can differentiate between unrelated individuals and possibly between male relatives.Allelic frequencies were estimated with Arlequin v. 3.5. Gene and Haplotype diversities were estimated according to Nei formula. The discrimination capacity was also calculated by dividing the number of different haplotypes by the total number of individuals in the sample. The fraction of unique haplotypes was determined as the percent proportion of unique haplotypes. In conclusion, the recently introduced Y-Filer Plus® system provides innovative discriminatory power for forensic applicationN/

Potential of Portuguese vine shoot wastes as natural resources of bioactive compounds

Since annually a high amount of wastes is produced in vine pruning, the aim of this study was to evaluate the potential of vine shoots from two Portuguese grape varieties (Touriga Nacional - TN and Tinta Roriz - TR) to be used as a natural source of phenolic compounds. To reach this goal, three techniques were explored, namely microwave-assisted extraction (MAE), subcritical water extraction (SWE) and conventional extraction (CE). The phenolic composition of the extracts, antioxidant and biological activities were evaluated by spectrophotometry and chromatography. MAE and SWE produced the highest concentrated extracts. TR vine shoot variety had the highest antioxidant activity and total phenolic (32.1±0.9mggallicacidequivalents/g dry sample), as well as flavonoid content (18.7±1.2mgepicatechinequivalents/g dry sample). For the first time, the biological activity of the vine shoot extracts was tested. Results demonstrated that all of them had antimicrobial potential against different bacteria and yeasts, and the ability of inhibiting α-amylase and acetylcholinesterase enzymes, with MAE TR extracts being the most efficient. HPLC analysis enabled the identification of different phenolic compounds, with gallic acid, catechin, myricetin and kaempferol-3-O-rutinoside being the main contributors to the phenolic composition. Portuguese vine shoot wastes could serve as easily accessible source of natural antioxidants for the food or pharmaceutical industries.Manuela M. Moreira (SFRH/BPD/97049/2013) and M. F. Barroso (SFRH/BPD/78845/2011) are grateful for the postdoctoral fellowships financed by POPH-QREN – Tipologia 4.1 – Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. The authors are also grateful for the financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 through the project UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265; the project 6818 - Transnational Cooperation, Agreement between Portugal (FCT) and Serbia (MSTD) is also acknowledged. This work was also supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). The supply of the vine shoots is acknowledged to Sogrape Vinhos, S.A.info:eu-repo/semantics/publishedVersio

The role of DNA concentrations in forensic casework results : regression models application

Póster apresentado no 28th Congress of International Society For Forensic Genetics (ISFG 2019), Praga, República Checa, 9-13 de Setembro de 2019In forensic DNA typing, short tandem repeats (STRs) are the most frequently genotyped markers in order to distinguish between individuals and to relate them to a crime or to exonerate the innocent. In recent years, new controversies have arisen with the advent of more sensitive techniques, allowing profiles to be recovered from minimum amounts of DNA, hence, bringing challenges to weight of evidence evaluation for forensic DNA profiles obtained from low template DNA samples. Introduction of interpretation models, or even new weight of evidence software should be accompanied by a measure of uncertainty that is part of any biological analysis. Specially, due to stochastic effects, the reliability of the obtained profiles might differ between machinery, workflow and also PCR settings in use in different laboratories. In this work we try to understand the relation between Peak Area, DNA concentration and also size marker, using adequate regression models. Buccal swabs from 180 individuals, with unknown identity, were selected for this study. DNA was extracted with prep-n-go™ buffer and quantified using Quantifiler® Trio DNA Quantification kit in a 7500 Real-Time PCR System (Applied Biosystems). STR amplification was performed with Powerplex Fusion 6C amplification kit (Promega). Amplified PCR products were separated and detected in an Applied Biosystems® 3500 Genetic Analyzer using manufacturer’s conditions. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Statistical analysis was performed with R Studio. Our results allow having an important overview about the relation between DNA concentrations, peak area, and size of the studied genetic markers.N/

Forensic genetic analysis of South Portuguese population with the six dye Powerplex® Fusion 6C

Poster apresentado no 28th Congress of International Society For Forensic Genetics (ISFG 2019), Praga, República Checa, 9-13 de Setembro de 2019As an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlex® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y –STRs (DYS391, DYS576 and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymised. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlex® Fusion 6C System, according to manufacturer’s guidelines. Fragment analysis was carried out in an Applied Biosystems® 3500 Genetic Analyser. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters were calculated with adequate software. In conclusion, our population information was updated in order to apply most recent data in our casework weight of evidence.N/