O palácio como matriz de inscrição na cidade : a propósito do Parque Natural no Rio Seco.

Dissertação para obtenção do grau de Mestre em Arquitetura, apresentada na Universidade de Lisboa - Faculdade de Arquitetura

Veda e vermifugação como alternativas de manejo para desmama de bezerros Nelore em pastagem nativa do Pantanal

The aim of this study was to investigate the integrated effects of native pastures sealing with strategic control of gastrointestinal nematodes on body development of weaned Nellore calves, studied between March 1992 and January 1993 in the Pantanal region, Brazil. Two homogeneous groups of weanling calves were put on contiguous paddocks of native pastures with the same physiognomic characteristics and sealed for three and a half months. The paddock where the non-treated group was put, was previously used by cows that were rearing a calf for contamination of the pasture. The treated group remained with low levels of eggs per gram of feces (EGF) during the whole experimental period. In the non-treated group, the EGF diminished during the assay, ending with similar levels of the treated group. On body development, it was observed a lower body weight loss of the treated group during the dry season and a compensatory weight gain of the non-treated group on the subsequent rainy season. The mean body weight of the two groups was similar at the end of the trial.Entre março de 1992 e janeiro de 1993 realizou-se este estudo com o objetivo de avaliar o efeito integrado da veda do pasto nativo com o controle estratégico de nematóides gastrintestinais no desempenho corporal de bezerros Nelore pós-desmame no Pantanal da Nhecolândia, MS, Brasil. Dois lotes de animais recém-desmamados aos nove meses foram colocados em invernadas contíguas de pastagens nativas com as mesmas características fisionômicas, que foram vedadas por três meses e meio; a invernada do lote controle foi previamente pastejada por vacas com bezerro ao pé para contaminação. O lote tratado permaneceu com níveis muito baixos de ovos por grama de fezes durante todo o período experimental, e no lote controle foram diminuindo no decorrer do ensaio, terminando semelhantes. No desenvolvimento corporal, observou-se menor perda de peso do lote tratado durante a estação seca e ganho de peso compensatório do lote controle na estação chuvosa subseqüente. Os pesos médios dos dois lotes no final do experimento foram semelhantes

Downregulation of circulating miR 802-5p and miR 194-5p and upregulation of brain MEF2C along breast cancer brain metastasization

Breast cancer brain metastases (BCBM) have been under-investigated despite their high incidence and poor outcome. Micro RNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and Next-Generation Sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different time points (5 h, 3, 7 and 10 days) to follow metastasis development. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, raising from 18% at 3 days to 30% at 10 days following malignant cells' injection. Work was focused on those altered prior to metastasis detection, among which were miR-802-5p and miR-194-5p, whose downregulation was validated by qPCR. Using TargetScan and DIANA Tools the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR-802-5p and miR-194-5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development

Phenolic diterpenes from Rosemary supercritical extract inhibit non-small cell lung cancer lipid metabolism and synergise with therapeutic drugs in the clinic

Lung cancer is one of the most deadly and common cancers in the world. The molecular features of patient’s tumours dictate the different therapeutic decisions, which combines targeted therapy, chemotherapy, and immunotherapy. Altered cellular metabolism is one of the hallmarks of cancer. Tumour cells reprogram their metabolism to adapt to their novel requirements of growth, proliferation, and survival. Together with the Warburg effect, the role of lipid metabolism alterations in cancer development and prognosis has been highlighted. Several lipid related genes have been shown to promote transformation and progression of cancer cells and have been proposed as biomarkers for prognosis. Nevertheless, the exact mechanisms of the regulation of lipid metabolism and the biological consequences in non-small cell lung cancer (NSCLC) have not been elucidated yet. There is an urgent necessity to develop multidisciplinary and complementary strategies to improve NSCLC patients´ well-being and treatment response. Nutrients can directly affect fundamental cellular processes and some diet-derived ingredients, bioactive natural compounds and natural extracts have been shown to inhibit the tumour growth in preclinical and clinical trials. Previously, we described a supercritical extract of rosemary (SFRE) (12 - 16% composition of phenolic diterpenes carnosic acid and carnosol) as a potential antitumoral agent in colon and breast cancer due to its effects on the inhibition of lipid metabolism and DNA synthesis, and in the reduction of resistance to 5-FluoroUracil (5-FU). Herein, we demonstrate SFRE inhibits NSCLC cell bioenergetics identifying several lipid metabolism implicated targets. Moreover, SFRE synergises with standard therapeutic drugs used in the clinic, such as cisplatin, pemetrexed and pembrolizumab to inhibit of cell viability of NSCLC cells. Importantly, the clinical relevance of SFRE as a complement in the treatment of NSCLC patients is suggested based on the results of a pilot clinical trial where SFRE formulated with bioactive lipids (PCT/ES2017/070263) diminishes metabolic and inflammatory targets in peripheral-blood mononuclear cells (PBMC), such as MAPK (p=0.04), NLRP3 (p=0.044), and SREBF1 (p=0.047), which may augment the immune antitumour function. Based on these results, SFRE merits further investigation as a co-adjuvant in the treatment of NSCLC.This research was funded by Regional Government of Community of Madrid (IND2017/BIO-7857; P2018/BAA-4343-ALIBIRD2020-CM), Ministerio de Ciencia e Innovación, Spain (PID2019-110183RB-C21); Ramon Areces Foundation (CIVP19A5937); EU Structural Funds and COST Action (CA17118); Synergistic Projects Community of Madrid (NUTRISION-CM/Y2020/BIO-6350) and REACT EU Program (Comunidad de Madrid and The European Regional Development Fund. ERDF. European Union- FACINGLCOVID-CM project). Adrián Bouzas has a predoctoral grant from the industrial predoctoral program of Community of Madrid (IND2017/BIO-7857).Peer reviewe

A study of viral pathogens in bat species in the Iberian Peninsula: identification of new coronavirus genetic variants

Bats have long been associated with multiple pathogens, including viruses affecting humans such as henipaviruses, filoviruses, bunyaviruses and coronaviruses. The alpha and beta coronaviruses genera can infect most mammalian species. Among them, betacoronavirus SARS-CoV, MERS-CoV and SARS-CoV-2, which have caused the three major pandemics in the last two decades, have been proposed to originate in bats. In this study, 194 oral swabs from 22 bats species sampled in 19 locations of the Iberian Peninsula were analysed and characterized by three different PCR tests (coronavirus generic real-time RT-PCR, multiplex conventional PCR, and SARS-CoV-2 specific real-time RT-PCR) to detect bat coronaviruses. Screening with coronavirus generic PCR showed 102 positives out of 194 oral swabs analysed. Then, metabarcoding with multiplex PCR amplified 15 positive samples. Most of the coronaviruses detected in this study belong to alphacoronavirus (α-CoV) genus, with multiple alphacoronaviruses identified by up to five different genetic variants coexisting in the same bat. One of the positive samples identified in a Miniopterus schreibersii bat positive for the generic coronavirus PCR and the specific SARS-CoV-2 PCR was classified as betacoronavirus (-CoV) through phylogenetic analysis. These results support the rapid evolution of coronaviruses to generate new genomic potentially pathogenic variants likely through co-infection and recombination.Los murciélagos se han asociado durante mucho tiempo con múltiples patógenos, incluidos los virus que afectan a los humanos, como los henipavirus, los filovirus, los bunyavirus y los coronavirus. Los géneros de coronavirus alfa y beta pueden infectar a la mayoría de las especies de mamíferos. Entre ellos, se ha propuesto que los betacoronavirus SARS-CoV, MERS-CoV y SARS-CoV-2, que han causado las tres principales pandemias en las últimas dos décadas, tienen su origen en los murciélagos. En este estudio, se analizaron y caracterizaron 194 hisopos orales de 22 especies de murciélagos muestreados en 19 lugares de la Península Ibérica mediante tres pruebas de PCR diferentes (RT-PCR en tiempo real genérica de coronavirus, PCR convencional múltiplex y prueba real específica de SARS-CoV-2). -time RT-PCR) para detectar coronavirus de murciélago. El cribado con PCR genérico de coronavirus mostró 102 positivos de 194 hisopos orales analizados. Después, metabarcode con multiplex PCR amplificó 15 muestras positivas. La mayoría de los coronavirus detectados en este estudio pertenecen al género alfacoronavirus (α-CoV), con múltiples alfacoronavirus identificados por hasta cinco variantes genéticas diferentes que coexisten en el mismo murciélago. Una de las muestras positivas identificadas en unMiniopterus schreibersii bat positivo para la PCR de coronavirus genérico y la PCR específica de SARS-CoV-2 se clasificó como betacoronavirus (-CoV) mediante análisis filogenético. Estos resultados respaldan la rápida evolución de los coronavirus para generar nuevas variantes genómicas potencialmente patógenas, probablemente a través de la coinfección y la recombinación

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Circulating microRNAs as Biomarkers for Breast Cancer Brain Metastasis

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018With the recent developments in the treatment of breast cancer (BC), that have improved prognosis of BC patients, metastatic disease is presently a major concern and the leading cause of death from BC. Particularly brain metastasis represent a serious oncologic problem, not only due to their high incidence in BC, poor prognosis and low survival but also because the brain microenvironment is considered a sanctuary for metastatic growth. While the blood brain barrier (BBB) restricts the entrance of most chemotherapeutics into the brain, brain cells can also contribute for tumor growth by exchanging factors with malignant cells. Altogether, this makes breast cancer brain metastases (BCBM) an uprising concern. Thus, the finding of reliable biomarkers that allow the early detection of BCBM, as well as new potential therapeutic targets, is in order. Recently, microRNAs (miRNAs or miR-) have arisen as powerful and specific biomarkers for different types of cancer and metastasis, having unique expression profiles depending not only on the type of cancer but also on the stage of the disease. Circulating miRNAs can be of special interest, due to their stability and easy quantification in biological fluids, allowing their usage in liquid biopsies for diagnosis and prognosis of oncologic diseases. Based on this, we hypothesized that miRNAs are aberrantly expressed in plasma prior to the detection of brain metastasis. So, the aim of this work was to identify miRNAs that could work as predictive biomarkers of BCBM in liquid biopsies. To this end, brain, lungs, kidneys, liver and plasma samples were collected from vehicle (control) and 4T1-injected female mice, at different timepoints during metastatic progression (5 hours, 3 days, 7 days and 10 days). Human samples of resected brain metastasis from triple negative BC patients were also studied. Analysis of brain and peripheral organs of the mouse model revealed that metastasis are particularly relevant in the brain, mainly in the hippocampus and that well established metastasis are only detectable from 7 days onwards. An initial miRNA screening by next generation sequencing (NGS) showed that plasma miRNAs are altered during brain metastasis development and that at 3 days, no metastasis were detected but a set of 8 miRNAs was deregulated. By real time PCR (RT-PCR), we could validate the downregulation of plasma miR-802-5p and miR-194-5p at three days and could see a trend for miR-92a-1-5p upregulation, and so, we propose that these miRNAs are potential biomarkers for the early detection of BCBM. Moreover, using bioinformatical tools, we predicted targets for these miRNAs, not only for a better understanding of the underlying mechanisms of these miRNAs but also to identify potential new targets for modulation. Matrix metalloproteinase-9 (MMP9) was a predicted target for miR-92a-1-5p. Immunofluorescence analysis of mouse brain sections revealed that MMP9 protein is expressed not only in malignant cells but also in the brain vasculature and in metastasis-associated vessels, pointing to a proangiogenic role of MMP9 Myocyte enhancer factor 2C (MEF2C) was predicted as a common target for both miR-802-5p and miR-194-5p. It was also observed that MEF2C is highly expressed in BCBM and that its expression increases with metastatic progression. Moreover, a nuclear translocation of MEF2C was observed in later stages of tumor progression, which indicates a higher activation of this transcription factor. Furthermore, MEF2C expression was detected in peritumoral astrocytes, pointing to a potential role of MEF2C in the cross-talk between astrocytes and malignant cells to promote tumor growth. High expression of MEF2C was further confirmed in human brain metastases from triple negative BC. In summary, this work revealed the potential in using circulating miRNAs as biomarkers for BCBM, pointing to miR-802-5p, miR-194-5p and miR-92a-1-5p as candidates to be used in liquid biopsies for the early detection of BCBM. It also revealed MEF2C as a new target for modulation and abrogation of BCBM.Com os desenvolvimentos recentes no tratamento do cancro da mama que têm vindo a melhorar o prognóstico dos doentes, o cancro metastático é uma preocupação e a principal causa de morte nos doentes de cancro da mama. Particularmente as metástases cerebrais, representam um problema na área da oncologia, não só devido à sua alta incidência no cancro da mama, mau prognóstico e baixa taxa de sobrevivência, mas também porque o microambiente do cérebro é bastante favorável ao desenvolvimento de metástases. Enquanto que a barreira hematoencefálica restringe a passagem da maioria dos medicamentos quimioterapêuticos, as células do cérebro também podem contribuir para o crescimento metastático através da troca de factores com as células malignas. Assim, as metástases cerebrais provenientes do cancro da mama são, cada vez mais, consideradas uma preocupação no tratamento do cancro e a descoberta de novos biomarcadores que permitam a sua detecção precoce e de novos alvos terapêuticos, é necessária. Recentemente, os microRNAs (miRNAs ou miR-) têm sido estudados como biomarcadores específicos para diferentes tipos de cancro e de metástases, tendo perfis de expressão únicos, dependendo não só do tipo de cancro, mas também do estadio da doença. Os miRNAs circulantes podem ter especial interesse para este efeito, devido à sua grande estabilidade e fácil quantificação em fluidos biológicos, que permite o seu uso em biópsias líquidas para o diagnóstico e prognóstico de doenças oncológicas. Sendo assim, neste trabalho, foi proposto que os miRNAs circulantes podem funcionar como biomarcadores eficientes para metástases cerebrais de cancro da mama, tendo como objectivo a selecção de miRNAs específicos que pudessem funcionar como biomarcardores precoces para este tipo de metástases. Para isto, inocularam-se ratinhos com células 4T1 ou com solução salina, como controlo e colheram-se os encéfalos, rins, pulmões e fígados e amostras de plasma a diferentes tempos depois da inoculação (3 dias, 7 dias e 10 dias). Também foram usadas amostravas humanas de metástases cerebrais de cancro da mama. A análise dos encéfalos e dos órgãos periféricos de ratinho demonstrou que este modelo metastiza fortemente para o encéfalo, ao contrário dos outros órgãos e que metástases bem estabelecidas só são detectáveis a partir dos 7 dias. Resultados de sequenciamento de nova geração mostraram os níveis expressão dos miRNAs no plasma estão alterados durante a formação e que aos 3 dias após a inoculação, apesar de ainda não serem detectáveis metástases, há um conjunto de 8 miRNAs que estão alterados no plasma. Por PCR em tempo real, foi possível validar a diminuição do miR-802-5p e do miR-194-5p aos 3 dias após inoculação, relativamente ao controlo. Foi também observada uma tendência do miR-92a-1-5p para estar aumentado. Assim, propõe-se estes três miRNAs como potenciais candidatos a biomarcadores precoces para a detecção de metástases cerebrais de cancro da mama. Através do uso de ferramentas bioinformáticas, foi feita uma previsão de possíveis alvos para estes miRNAs, não só para se ter uma melhor compreensão dos mecanismos subjacentes dos miRNAs, mas também como um processo de triagem para selecionar novos alvos para modulação em metástases cerebrais. A metaloproteinase da matriz-9 (MMP9) foi um dos alvos previstos para o miR-92a-1-5p. Análise por imunofluorescência em secções de encéfalo de ratinho, revelou que a MMP9 é expressa, não só em células malignas no encéfalo, mas também na vasculatura cerebral e em vasos associados às metástases, apontando para um papel pro-angiogénico da MMP9. Por sua vez, o factor potenciador de miócitos 2C (MEF2C) foi previsto como alvo comum para o miR-802-5p e para o miR-194-5p. Através de análise por imunofluorescência, também em secções de encéfalo de ratinho, mostrou-se pela primeira vez que o MEF2C tem uma expressão elevada em metástases cerebrais de cancro da mama e que a sua expressão aumenta com a progressão das metástases. Foi também observada uma translocação nuclear do MEF2C em fases mais avançadas do tumor, o que pode indicar uma maior activação deste factor de transcrição. Além disso, a expressão do MEF2C foi também detectada, especificamente em astrócitos peritumorais, apontando para um papel do MEF2C na interacção entre as células malignas e os astrócitos, para potenciar o crescimento tumoral. A expressão elevada do MEF2C foi também confirmada em metástases cerebrais humanas provenientes de cancro da mama triplo negativo. Em suma, este trabalho demonstrou o potencial do uso dos miRNAs circulantes como biomarcadores para metástases cerebrais de cancro da mama, apontando o miR-802-5p, o miR-194-5p e o miR-92a-1-5p como candidatos para serem usados nas biópsias líquidas para a detecção precoce deste tipo de metástases. Revelou também o potencial do uso do MEF2C como um novo alvo para modulação em metástases cerebrais de cancro da mama.The studies presented on this master thesis were performed in the research group “Neuron Glia Biology in Health & Disease”, from Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, under the supervision of Maria Alexandra Brito, Ph.D. and Mafalda Videira, Ph.D. A part of the work was performed in the research group “Physiology and Pathology of the Blood-Brain Barrier” Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary, during three scientific mission of the student to Hungary. This study was supported by Fundação para a Ciência e Tecnologia (FCT – UID/DTP/04138/2013 and PTDC/MED-ONC/29402/2017), Portugal, and by National Research, Development and Innovation/Hungarian Scientific Research Fund (NKFIH/OTKA – K-100807 and K-116158), Hungary