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- Influência da temperatura de fixação na análise de DNA in vitro: revisãoPublication . Rego, Beatriz; Duarte, Cláudia; Canhoto, Inês; Marques-Ramos, AnaIntrodução: Uma vez que os tecidos fixados em formalina e impregnados em parafina (FFIP) fazem parte da rotina laboratorial em histopatologia, existindo em grande número, estes constituem uma fonte importante de material biológico para estudos moleculares. O DNA extraído destes tecidos pode, no entanto, encontrar-se degradado, sendo a fixação uma das etapas em que a degradação poderá ocorrer. Isto irá depender de vários fatores, como o tipo de fixador, o seu pH, tempo e temperatura de fixação. Uma vez que existe uma crescente necessidade de DNA para a realização de técnicas moleculares, úteis para a caraterização de mutações em tumores e escolha de terapêuticas mais eficazes, vários autores têm tes-tado os efeitos da fixação na qualidade do DNA extraído, nomeadamente da temperatura. Nesta revisão, iremos procurar compreender se existe uma temperatura ideal de fixação para tecidos FFIP quando se pretendem realizar técnicas de análise de DNA in vitro. Objetivos do estudo: 1) Sistematizar os estudos existentes sobre a influência da temperatura de fixação no DNA extraído de tecidos fixados em formalina e impregnados em parafina; 2) Verificar a existência de um consenso sobre qual a temperatura de fixação ideal para a obtenção de melhores resultados na análise de DNA in vitro.
- Síntese proteica e patogénesePublication . Marques-Ramos, AnaMecanismo canónico da iniciação da síntese proteica: regulação da iniciação da síntese proteica, tradução mediada por IRESs, tradução mediada por CITEs, outras formas da iniciação da síntese proteica: N6-methyladenosine, 5’TOP, tradução dependente de cap sem scanning, controlo da tradução por uORFs, função das proteínas de ligação ao RNA, elementos reguladores da tradução como alvos terapêuticos.
- Imunoterapia finalPublication . Marques-Ramos, AnaSistema Imunitário. Reconhecimento tumoral. Cancer Imunoediting. Evasão à imunidade inata. Evasão à imunidade adaptativa. Estratégias imunoterapia em cancro. CTLA-4. PD-1. Bloqueio dos checkpoints imunes. Regulação das células T. Anticorpos. Adoptive cell therapy. TIL. T cells expressing novel TCRs. Chimeric antigen receptor (CAR-T cells). Citoquinas. Vacinas. Vacinas com células dendríticas. Vacinas personalizadas com neoantigénios. Vacinas oncolíticas. Monitorização do sistema imunitário. Sangue periférico.
- Novo inibidor de motor de alta especificidade em cancro gástricoPublication . Pereira, Miguel; Mendes, Priscila; Palma, João; Brito, Miguel; Ribeiro, Edna; Cervantes, Renata; Marques-Ramos, AnaO mechanistic target of rapamycin (mTOR) é uma quinase que regula o crescimento, proliferação e sobrevivência celulares. A desregulação da via de mTOR está presente em diversas patologias, sendo a sua hiperativação observada em mais de 70% de cancros humanos, incluindo cancro gástrico (CG). Apesar de existirem atualmente diversos inibidores de mTOR (mTORi), a sua utilização clínica ainda é limitada devido à elevada toxicidade associada à inibição inespecífica de outras proteínas e à inibição parcial da via. Assim, é crucial desenvolver uma nova classe de mTORi que iniba específica e totalmente a via do mTOR. As opções terapêuticas atuais em CG são invasivas e agressivas6 e sabendo que neste tipo tumoral há hiperativação da via e sobreexpressão da proteína mTOR1, uma potencial forma de restringir a progressão CG é através da utilização de um mTORi. Objetivo: Desenvolver um composto que iniba especifica e totalmente o mTOR e avaliar o seu efeito terapêutico num modelo animal de tumor gastrointestinal.
- Influence of histochemical stains on DNA obtained from FFPE samplesPublication . Boghenco, O.; Freitas, B.; Pote, Alexandra; Marques-Ramos, AnaIntroduction: Formalin-fixed, paraffin embedded (FFPE) samples are used for diagnostic and prognostic purposes. Histopathological analysis frequently includes not only histomorphological evaluation but also histochemical and molecular studies. In some cases FFPE samples are scarce and it is necessary to use the same histological section for histochemical analysis and DNA extraction. In molecular pathology labs this is a common practice, allowing the analysis of DNA specifically from altered cells. However, histochemical techniques use reagents that may induce chemical modifications on DNA. To perform a literature review about the influence of histochemical stains on DNA integrity. Materials and methods: PubMed and Research Gate were used to survey original articles published until December 2017. Results: For this review articles about the analysis of DNA extracted from stained FFPE sections were considered. The studies demonstrated that: DNA extracted from sections stained with Azure B, toluidine blue and methyl green (MG) was successfully amplified by Polymerase Chain Reaction (PCR) whereas Mayer’s hematoxylin stain inhibits the reaction. Another study demonstrated that DNA amplification by PCR had better results with eosin Y and MG stains comparatively to Mayer’s hematoxylin and May-Grunwald. Banaschak et al. showed that DNA analysis by PCR and capillary electrophoresis was successful with Hematoxylin-Eosin (HE), Periodic Acid Schiff (PAS), Azan and Perl's stains. Phosphotungstic acid hematoxylin (PTHA) and Gomori stains had negative results. Two different studies concluded that DNA is refractory to HE stain as capillary electrophoresis demonstrated similar degradation to that of unstained samples and it was successfully amplified by PCR. Conclusions: Histochemical analysis allows demonstration of cellular components whose alterations are typical from pathological conditions. These techniques encompass reagents that may alter biomolecules. Nevertheless, from the analyzed studies it is possible to conclude that DNA integrity is maintained in techniques such as Azure B, toluidine B, MG, eosin Y, HE, PAS, Azan and Perls. On the other hand, Mayer’s hematoxylin, May-Grunwald, PTHA and Gomori resulted in inhibition of DNA amplification. Since this analysis was not performed in common routine techniques such as Masson’s Trichrome and PAS-Alcian Blue, it is important to deepen the knowledge, performing new studies for future appliance.
- Detection of Helicobacter pylori in extragastric tissues: association of infection with carcinogenesisPublication . Faria, L. M.; Almeida, M. S.; Amaral, M. R.; Marques-Ramos, AnaIntroduction: Helicobacter pylori (HP) is a gram-negative microaerophilic bacterium that has been described as the main pathogen of several benign and malignant diseases of the digestive tract, such as gastric and duodenal ulcers, chronic gastritis, hepatobiliary diseases, gastric lymphoma, and carcinoma. HP was classified as class I human carcinogen in 1994. The relationship between HP infection and extragastric diseases has been investigated over the years with contradictory conclusions. The objective of this work is to perform a bibliographic review on the association between HP infection and the development of extragastric pathologies, particularly of pancreatic carcinoma. It is also intended to determine the best method for the detection of the bacterium. Materials and methods: work was based on the literature from the following databases: b-on, PubMed, SciELO, Wiley Online Library and Scientific Repository of the Polytechnic Institute of Lisbon, under the terms “Helicobacter spp.”, “Helicobacter pylori”, “pancreatic cancer”, “extragastric diseases”, “risk of pancreatic cancer” and “CagA”. From a total of 32 analyzed articles, 18 were selected, excluding those with dubious methods and results that were not performed in human samples and meta-analysis. Results: In the selected studies the presence of HP DNA was detected by Polymerase Chain Reaction (PCR) in more than 50% of the pancreatic and hepatocellular cancer samples. There was also an association between the presence of this bacterium and colonic cancer, primary sclerosing cholangitis, primary biliary cirrhosis, cholecystitis and biliary tract carcinoma. However, detection by enzyme-linked immunosorbent assay provided ambiguous results, with 2 of the studies showing no association between infection and pancreatic cancer, while others showed positive results below 50%. There are also 3 studies that related the seroconversion of cytotoxin-associated gene A negative to pancreatic carcinoma. Other methods like histochemical and immunocytochemistry (ICQ) techniques were compared to each other and the results demonstrated that ICQ had the greatest consistency. Conclusions: The majority of the studies demonstrated an association between HP infection and the development of extragastric diseases. Therefore, HP is a potential risk factor for the etiology of these pathologies. Comparing previous methods, it was possible to observe that PCR was the most sensitive and specific technique for HP detection.
- Impact of particles on pulmonary endothelial cellsPublication . Almeida-Silva, Marina; Cardoso, Jéssica; Alemão, Catarina; Santos, Sara; Monteiro, Ana; Manteigas, Vítor; Marques-Ramos, AnaAccording to the WHO, air quality affects around 40 million people, contributing to around 21,000 premature deaths per year. Severe respiratory diseases, such as asthma and chronic obstructive pulmonary disorder, can be promoted by air pollution, which has already been documented; this is one of the reasons why air quality is a very relevant factor for human health and well-being. Aerosols are an aggregation of solid or liquid particles dispersed in the air and can be found in the form of dust or fumes. Aerosols can be easily inhaled or absorbed by the skin, leading to adverse health effects according to their sizes ranging from the nanometre to the millimeter scale. Based on the PRISMA methodology and using the Rayyan QCRI platform, it was possible to assess more than four hundred research articles. This systematic review study aimed to understand the impact of particles on pulmonary endothelial cells, namely particulate matter in different sizes, cigarette smoke, diesel exhaust particles, and carbon black. The main conclusions were that particles induce multiple health effects on endothelial cells, namely endothelial dysfunction, which can lead to apoptosis and necrosis, and it may also cause necroptosis in lung structure.
- Determinação da eficiência de obtenção de DNA a partir de secções histológicas submetidas a técnicas histoquímicasPublication . Pote, Alexandra; Freitas, Beatriz; Boghenco, Otília; Marques-Ramos, AnaIntrodução: Devido à deteção precoce de lesões, as amostras para análise molecular são cada vez mais reduzidas e a quantidade DNA pode não ser suficiente para obter resultados, tornando-se necessário extrair DNA diretamente de tecidos corados. Apesar da influência das colorações na obtenção de DNA já ter sido estudada, esta é uma área pouco explorada. Objetivo do estudo: Apresentar a recolha de dados referentes à influência de técnicas histoquímicas na integridade do DNA.
- The influence of fixation temperature in in vitro DNA analysisPublication . Canhoto, I.; Duarte, C.; Rego, B.; Marques-Ramos, AnaIntroduction: Formalin-fixed and paraffin-embedded (FFPE) are important sources for molecular studies, namely to identify cancer related biomarkers. Nevertheless, the quality of FFPE-obtained DNA is lower than that of fresh/frozen tissues, since fixation induces several chemical modifications. The impact of tissue fixation duration, fixative type and pH on the integrity of FFPE-extracted DNA has been the subject of several studies. It’s well established that fixation using formalin for less than 72 hours allows extraction of high quality DNA. Although it is known that DNA stability is highly dependent on temperature, the influence of the fixation temperature on the quality of FFPE-extracted DNA is not understood. Objective: Evaluate the influence of the fixation temperature in the quality of FFPE-extracted DNA through a systematic literature review. Materials and methods: The search was performed in PubMed for studies published in English, up to December 2017. The included studies compared two or more fixation temperatures using formalin, to perform DNA analysis. Results: Seven studies met the defined criteria. All compared room temperature (RT) with 4 °C (5 studies), 0-4 °C (1 study) or 37 °C (1 study). DNA integrity was evaluated by agarose gel electrophoresis electrophoresis, PCR, multiplex ligation-dependent probe amplification (MLPA) and whole gene amplification (WGA). In 5 studies the best results were obtained for fixation at 4 °C (electrophoresis, PCR and WGA); whereas RT allowed the best results in 2 cases (PCR and MLPA), one of which demonstrated that fixation at 37 °C preserved DNA similarly to RT. Conclusions: DNA stability is highly temperature-dependent as DNAses are inhibited at low temperatures. Accordingly, it is not surprising that fixation at 4 °C allows the extraction of less degraded DNA in most studies. Nevertheless, RT seems to be also an acceptable temperature, as it allowed successful MLPA and PCR when using small DNA fragments. From these observations one may conclude that tissue fixation must be performed at 4 °C if the goal is to analyse high-molecular-weight DNA. As RT is the standard fixation temperature in diagnostic lab units, these conclusions may imply the adequate adjustments in order to prevent false conclusions from molecular analysis. Nevertheless, additional studies that analyse not only DNA integrity, but also DNA purity, yield and concentration should be done to determine the optimal fixation temperature to perform molecular analysis.
- Workshop de pipetagemPublication . Marques-Ramos, AnaInstrumentos que permitem a medição e transferência precisa e rigorosa de líquidos.