ESS - APCT - Posters apresentados em eventos científicos
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- CytoPath®Easy: screening of cervical cancerPublication . Fernandes, Sílvia; Vilarinho, Ana Sofia; Silva, ReginaRecently, CytoPath®Easy kit was created by DiaPath S.p.A. and started to be commercially available for the screening of cervical cancer. Using this methodology, epithelial cells are immersed in a preservative liquid, and a thin-layer of cells in the slide is obtained through gravity sedimentation and filtration. The main objectives of this study are to evaluate the efficacy for the processing of cervical samples, for the detection of pre-neoplastic lesions and for the nucleic preservation and extraction by the kit. For this purpose, 215 cervical samples obtained by self-sampling were used: 174 were collected and processed by CytoPath® Easy and, as a control, 41 were collected and processed by the Thinprep® method. The samples were processed, stained by the Papanicolaou method, and independently evaluated microscopically for various morphological parameters; nucleic acids were isolated and evaluated for purity and integrity by spectrophotometry. Results obtained showed that both methods have a good performance, allowing the morphological evaluation of the cervical epithelium. However, the statistical analysis reveals that the methods are different from each other, with overall lower results being obtained in the method under study (p<0.001). In turn, both methods allow the extraction of good quality and quantity of DNA. Although some differences were found regarding morphology of the cells fixed and processed by the CytoPath®Easy method, this new methodology reveals efficient for the preservation of nucleic acids. Thus, its use in cervical cancer screening is recommended.
- Optimisation of a molecular methodology for the detection of virulence factors of enterotoxigenic Escherichia coli for the diagnosis of swine colibacillosisPublication . Campos, Ana; Oliveira, Ricardo; Almeida, Carina; Vieira, Filipa Quintela; Silva, Regina AugustaThe most common bacterial pathogen causing enteric infections in pigs is enterotoxigenic Escherichia coli (ETEC). Since pigs represent the largest livestock category in the European Union, ETEC-associated diseases, better known as swine colibacillosis leading to acute diarrhea and eventual death of the animal, result in significant costs to the pig industry. These diseases are traditionally prevented or treated with antibiotics, and this has had a huge impact on the emergence of resistant bacteria, correlating with the emergence of resistant infections in humans. Recognition of this problem has led the authorities to set ambitious goals for the reduction of this type of drug in animal husbandry, leading to the creation of a national project, APTAcoli, which aims to select aptamers (consisting of small single-stranded oligonucleotides capable of binding to target molecules with great affinity and specificity, due to the specific secondary and/ or tertiary structures they can acquire) as an alternative in the treatment of colibacillosis. The present experimental study, which is on the APTAcoli agenda, focused on the optimization of a molecular methodology - Multiplex PCR - for the detection of the main virulence factors of ETEC to be used in an epidemiological study to characterize fecal samples from pigs in Portuguese farms. After using different optimization techniques, the results were two multiplex PCR amplification sets, one for amplification of the main toxigenic factors of ETEC (STa, STb, LT and STx2e) and another for amplification of the main adhesion factors (F4, F5, F6, F18 and F41).
- CINtec PLUS cytology as a cervical cancer screening test at the IPO of PortoPublication . Alves, Ana; Lopes, Ana Paula; Granja, Sara; Quintela Vieira, Filipa; Silva, Regina AugustaCervical squamous cell carcinoma of the cervix is the fourth most common cancer between women and seventh globally. There are many women undergoing colposcopy without real need, so more effective screening tests are needed to decide who among HPV-positive women should receive additional diagnostic evaluation to avoid unnecessary colposcopies. So, this study is intended to evaluate CINtec PLUS cytology as a screening test for cervical cancer in the IPO-FG of the Port of women with hpv test positive other than 16/18, improving the referral for colposcopy. CINtec PLUS cytology is an immunocytochemistry kit that simultaneously detects p16 and Ki-67 proteins. The presence of these proteins in the same cell indicates a deregulation of the cell cycle, and may be a marker of the persistence of HPV infections, their greater probability of progression and the severity of lesions secondary to the infection. An experimental research was carried out, in which the immunostaining of 73 non-16/18 HPV cervico-vaginal hpv samples was performed from 73 women integrated in cervical cancer screening in the North region carried out at the IPO-FG of Porto. The results obtained in CINtec PLUS were compared with those of cytology. 2 samples present unsatisfactory cytology for evaluation, being excluded. Cintec Plus cytology would reduce by 17% the number of women sent for colposcopy (7 (about 10%) by Cintec Plus vs 19 (about 27%) by cytology). Of the 52 NILM samples, 3 (about 6%) were positive for Cintec Plus, so they should have been referred for colposcopy and were not. Of the 10 ASC-US samples 100% are negative for Cintec Plus, as well as 5 LSIL 4 (80%) are negative, of the 3 HSIL 2 (about 67%) are negative. So, 16 women (about 23%) made colposcopy unnecessarily. In conclusion, Cintec Plus cytology would be an interesting tool as a second screening test in non-16/18 hpv cases in place of cytology, decreasing the number of women sent for colposcopy.
- Uncovering the microglia response during neonatal Group B Streptococcus meningitisPublication . Soares, Joana; Lorga, Inês; Bravo, Joana; Summavielle, Teresa; Nova, Manuel Vila; Bonifácio Andrade, ElvaGroup B Streptococcus (GBS) remains the most common bacterial cause of meningitis in neonates. Microglia, the brain resident immune cells, have a critical role in the development of neural circuits. However, the role of GBS infection on microglia activation and neurological sequelae remains poorly characterised. Here, we aimed to evaluate whether GBS induces changes in microglia profile during the acute phase of infection, using a mouse model that mimics key steps of GBS pathophysiology in humans. Female C57BL/6 mice were intra-vaginally inoculated with GBS during gestation, and CFU analysis was performed on postnatal days (P) 1, 3 and 5. Bacterial colonisation was found at all ages, peaking at P3. When analysing the status of microglia by flow cytometry in the whole brain of male pups at P3, an overall activation was observed in the infected group. Mainly, we found a significant increase in microglia frequency, as well as the mean fluorescence intensities (MFIs) of CD45, CD11b and F4/80. Additionally, we also analysed some microglial receptors that are important neuro-immune regulators with relevant functions during development. We observed increased CX3CR1 expression in microglia, whereas Sirp and CD200r were not altered. Moreover, analysing the cortex and hippocampus, relevant regions for cognition, we found similar numbers in Iba1+ cells, a known microglia marker, in the hippocampus of infected pups. In contrast, a significant decrease was observed in the cortex, suggesting altered migration of these cells. Furthermore, microglia phagocytosis was increased in the cortex of infected pups but not in the hippocampus. Interestingly, quantification of neurons revealed a significant decrease in the hippocampus of infected pups while being increased in the cortex, compared with age-match controls. Altogether, our results show that GBS meningitis alters the neonatal microglia profile. Further studies will be necessary to better understand the microglia inflammatory state after GBS infection.
- Regulation of CD47 expression by interferon-gamma under chronic Methamphetamine exposure TitlePublication . Rodrigues, João; Bravo, Joana; Bonifácio Andrade, Elva; Canedo, Teresa; Azevedo, Maria; Summavielle, TeresaExposure to methamphetamine (Meth), a highly addictive widely used psychostimulant, is classically associated with damage to neuronal terminals, but its neurotoxicity can also be mediated via activation of the neuroinflammatory response. Microglia, the resident immune cells of the brain, become highly activated and increase the release of proinflammatory mediators upon exposure to Meth. However, their role in Meth-associated neurotoxicity is still not sufficiently understood. Data from our lab shows that, in the hippocampus, chronic Meth administration leads to microglia homeostasis dysregulation, synapse dysregulation, and downregulation of cluster-differentiation 47 protein (CD47). The crosstalk between CD47 and its receptor, signal regulatory protein α (SIRPα), is an important “don’t eat me signal” that inhibits phagocytosis. CD47 has been shown to protect synapses from excessive microglia-mediated pruning during development and neurodegeneration. Of note, in cancer cells CD47 expression is modulated by interferon-gamma (IFN-γ). Consistently, after chronic Meth, we observed a significant decrease of meningeal T cells, and a decrease in the production of IFN-y by these cells. Here we aim to clarify if IFNγ is regulating CD47 in the brain after chronic Meth administration, and consequently regulating synaptic pruning, using IFNyKO mice and wild-type mice injected with recombinant IFNy via stereotaxic surgery. Preliminary results indicate that IFNy/CD47 does not modulate microglia morphology and number after chronic Meth in the hippocampus. Currently we are evaluating synapses and phagocytosis, and we further expect to clarify the impact of IFNγ /CD47 in the chronic Meth conditioning and in memory.
- Unveiling common molecular pathways linked to ILDs with progressive fibrosing phenotype: the role of MUC5BPublication . Santos, Rita F.; Gonçalves, Melany; Mota, Patrícia Caetano; Cardoso, Catarina Gouveia; Coelho, Andreia L.; Sokhatska, Oksana; Beltrão, Marília; Guimarães, Susana; Delgado, Luís; Soares, Miguel; Morais, António; Saraiva, Margarida; Bastos, Hélder NovaisProgressive fibrosing ILDs (PF-ILDs) comprise a heterogeneous group of lung disorders associated with high morbidity and mortality, that exhibit a continuous worsening phenotype despite standard treatment. Among PF-ILDs are pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (HP), involving complex interactions between host genetics and different environmental triggers, shaping the immune milieu that ultimately drives the fibrotic cascade in a susceptible patient. The MUC5B promoter variant rs35705950 is the common genetic variant associated with the greatest risk of developing IPF. As IPF and fibrotic HP present phenotypic resemblances, we aim to analyze the role of rs35705950 MUC5B single nucleotide polymorphism (SNP) in common molecular pathways linked to PF-ILDs. Herein, taking advantage of our extensive ILD patients’ cohort, we found that MUC5B rs35705950 GT and TT genotypes frequency was dramatically increased in IPF and fibrotic HP compared to healthy controls.
- Teste fragilidade osmótica por citometria de fluxo, o teste de referência do futuro: Estudo comparativo entre três soluções salinasPublication . Santos, Marlene; Schelfhout, Jasmijn; Santos, Ana Helena; Fernandes, Sílvia; Ventura, Vânia; Oliveira, Lurdes; Gonçalves, Marta; Moreira, Raquel; Fonseca, Sónia; Pereira, Filipa Martins; Santos, Filipa; Lau, Catarina; Teixeira, Maria AnjosA avaliação da fragilidade osmótica por citometria de fluxo tem sido introduzida na prática clínica nos últimos anos, utilizando um método descrito por Won et al, em 2009. Esta metodologia afere, em tempo real, o quociente entre os eventos registados antes e após exposição dos eritrócitos a uma solução indutora de hemólise, num determinado período de tempo. O método clássico de determinação da fragilidade osmótica avalia indiretamente, por leitura da absorvância, a proporção de eritrócitos lisados, quando submetidos a stress osmótico em soluções hipotónicas seriadas de cloreto de sódio (NaCl).
- Rastreio organizado do cancro do colo do útero açores – Perspetiva de um novo paradigmaPublication . Maciel, Débora; Fernandes, Sílvia; Silva, Regina A.O cancro do colo do útero é um dos cancros mais frequentes na mulher a nível global. Em 2010, foi implementado o programa de Rastreio Organizado do Cancro do Colo do Útero dos Açores (ROCCA) usando como teste primário o método de citologia líquida para a identificação precoce de lesões precursoras. Em 2020 foi implementada uma nova metodologia de rastreio, o teste de HPV mRNA - AptimaTM HPV Assay (Hologic), seguido de genotipagem. Este trabalho tem como objetivo avaliar a eficácia da implementação do ROCCA, bem como a adequabilidade do método molecular enquanto ferramenta de rastreio.
- Epidemiologia do cancro do colo uterino e o impacto da vacinação contra o HPV: a realidade portuguesa no contexto europeuPublication . Marques, Ana; Fernandes, Sílvia; Silva, Regina A.A atual disponibilidade de vacinas e testes de rastreio molecular para o HPV aumentaram a possibilidade de eliminar globalmente o cancro cervical e outras patologias relacionadas com o vírus. No entanto, têm surgido vários obstáculos à implementação dos programas de vacinação contra o HPV a nível mundial. Este estudo tem como objetivos compilar e analisar estatisticamente a informação acerca da vacinação contra o HPV e políticas de rastreio de cancro cervical, em Portugal e em outros países europeus; e avaliar o impacto na incidência e mortalidade de cancro cervical, ao longo dos anos.
- Avaliação do método histoquímico GASMoC para demonstração de micobactériasPublication . Cunha, Rute; Silva, Susana; Silva, Regina A.O método histoquímico Ziehl-Neelsen (ZN) é o método eleito em Anatomia Patológica e Microbiologia para a demonstração de micobactérias em tecidos e amostras citológicas. É usada uma solução corante de carbol-fuscina que contém fenol, um composto muito corrosivo e tóxico, que provoca efeitos adversos quando em exposição crónica. Recentemente, foi desenvolvido um método alternativo e isento de fenol (GASMoC), testado apenas em amostras de tecido infetado por duas espécies de Mycobacterium.