DMF - Teses de Doutoramento
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- Análise da função de Delta4 na regulação da angiogénese embrionária e neo-angiogénese tumoralPublication . Trindade, Alexandre José Neto; Duarte, António José de Freitas; Costa, Luís Filipe Lopes daO correcto desenvolvimento dos embriões de metazoários segmentados depende do desenvolvimento de um sistema vascular sanguíneo, um processo regulado a vários níveis. Este passo do desenvolvimento embrionário passa por vários eventos morfogenéticos, que resultam na conversão funcional de células progenitoras, responsivas a factores pró-angiogénicos, em redes complexas de vasos sanguíneos, que cobrem todo o organismo e acompanham o crescimento do embrião. O desenvolvimento do sistema vascular é um processo dinâmico, em que as células endoteliais integram continuamente sinais de expansão e amadurecimento da rede vascular. A presença de sinais conflituosos resulta num sistema altamente dinâmico, que responde rapidamente às necessidades mas que não se expande desnecessariamente. Existem várias vias de sinalização que estão envolvidas na regulação da remodelação do sistema vascular embrionário. Neste trabalho avaliou-se a importância de Dll4, um ligando da via de sinalização Notch, neste papel. Produziram-se linhas de ratinhos transgénicos com sobre-expressão condicional de dll4, que revelaram um papel importante deste gene na determinação da identidade endotelial arterial e na indução de quiescência e maturação do sistema vascular. Neste sentido, Dll4 reduz o perfil migratório das células endoteliais, inibe a formação de novas ramificações vasculares, estimula o recrutamento de células de músculo liso, aumenta a produção de matriz extracelular, aumenta a adesão celular homotípica e reduz a proliferação endotelial. Este papel é conseguido, pelo menos parcialmente, por Dll4, através da via Notch, actuar de forma negativa sobre o funcionamento de vias pró-angiogénicas, como a via VEGF e Wnt. A análise da função de Dll4 no desenvolvimento da vasculatura tumoral confirmou o papel anti-angiogénico deste gene. Os resultados revelaram que o bloqueio do funcionamento deste gene resulta numa vasculatura hiper-ramificada, mas hipo-perfundida, que não suporta o crescimento tumoral. Este facto sustenta a hipótese do controlo do funcionamento de Dll4 apresentar potencial terapêutico na luta contra o cancro.
- Células Mato na retina : caracterização morfológica e funcional de uma nova população de células perivasculares autofluorescentes em situação fisiológica e de retinopatiaPublication . Jorge, Maria Luísa Mendes; Pina, José António Rebocho Esperança; Ruberte París, JesúsA retina contém diferentes populações celulares derivadas dos monócitos: macrófagos perivasculares e células da micróglia. Neste trabalho, descreve-se a presença de um novo subtipo de macrófagos residentes, distintos das células da micróglia, localizados no espaço perivascular, em retinas humanas e de murganho. Estas células emitem uma fluorescência específica e expressam constitutivamente receptores scavenger da classe A. Exibem um movimento alternado constante, ao longo dos vasos sanguíneos, providenciando um revestimento adicional a áreas mais delgadas da parede vascular. Para além disso, acumulam peroxidase de rábano e lipoproteínas de baixa densidade acetiladas em circulação, sem rotura da barreira hemato-retiniana. No conjunto, estes resultados sugerem que estas células, com função scavenger, contribuem para o funcionamento da barreira hemato-retiniana. Acumulam, também, ferro, em lisossomas, provavelmente por um mecanismo de endocitose de ferritina, mediada por receptores TIM-2, sugerindo o seu envolvimento na homeostasia do ferro na retina e em situações de alteração desta. Em fases iniciais de degenerescência dos fotorreceptores, estas células migram para o local das lesões, sugerindo a sua participação nos processos de retinopatia. Todos estes aspectos são semelhantes aos descritos para as células Mato cerebrais. Por conseguinte, este estudo estabelece, pela primeira vez, a presença de células Mato na retina.
- Characterization of immune responses in feline mammary carcinoma towards the development of improved diagnostic tools and molecular therapiesPublication . Nascimento, Ana Catarina Gaspar do; Ferreira, Fernando António da Costa; Ferreira, João António Augusto; Correia, Jorge Manuel de JesusFeline mammary carcinoma (FMC) is one of the most common tumors in female cats, being highly malignant and leading to early metastasis. In most of the cases the diagnosis is late and often leads to mastectomy to prevent the formation of new tumors in the remaining mammary glands. Thus, the development of new diagnostic and prognostic tools and molecular therapies are crucial to improve the efficient detection and treatment of these animals. In this study, the main goals were to characterize distinct immune cell subpopulations in the tumor microenvironment (TME) of FMC and to evaluate the PD-1/PDL1 and the VEGF-A/VEGFRs pathways in serum and tissue samples of cats with mammary carcinoma, in order to suggest new diagnostic and prognostic biomarkers and novel therapies for FMC. Results showed that cats with mammary carcinoma with higher densities of stromal CD8+ tumor infiltrating lymphocytes (TILs) had longer disease-free survival and overall survival, suggesting its usefulness as a favorable and novel prognostic biomarker for FMC. Furthermore, and according to the molecular subtype, triple negative (TN) normal-like tumors showed higher densities of stromal CD3+, CD8+ and CD68+ immune cells, and lower expression of PD-L1 in TILs and cancer cells, compared to HER2-positive tumor subtype. These results suggest that HER2-positive tumors had a highly immunosuppressive TME. Additionally, results demonstrated that cats with HER2-positive and TN normal-like molecular subtypes showed higher serum PD-1, PD-L1, VEGF-A, VEGFR-1, VEGFR-2 levels than healthy controls, suggesting that these molecules might be potential biomarkers for the diagnosis of cats with these two aggressive molecular subtypes. Results also suggest that these animals were under systemic immunosuppression, and may benefit from anti-PD- 1/PD-L1 immunotherapies or anti-angiogenic agents. In sum, these results identified a novel prognostic factor for FMC, and new molecules that may serve as promising non-invasive diagnostic biomarkers for cats with HER2-positive and TN normal-like molecular subtypes. Moreover, this project may lead to the development of new drugs, in order to improve the treatment of cats with mammary carcinoma. Additionally, the data obtained support the idea that spontaneous FMC is a suitable cancer model for the study of human breast cancer.
- Clinical and molecular characterization of feline mammary carcinomas overexpressing HER2 proto-oncogene (FMC-HER2+) : new strategies for effective diagnostic and cancer therapyPublication . Silva, Maria João da Costa Soares da; Ferreira, Fernando António Costa; Correia, Jorge Manuel de JesusConsidering the scarce data available in feline mammary carcinoma (FMC) and despite its importance in veterinary clinical practice, this thesis emerges in order to increase the knowledge of this tumor type, especially the FMC-HER2 positive. In the first two studies, the protocols for detection and quantification of the fHER2 and Ki-67 biomarkers were optimized and validated. These studies demonstrated that, in cats, the incidence of fHER2 overexpression were similar to women (about 30%), although no gene amplification was detected. It was also demonstrated that high levels of Ki-67 index were associated with a worse prognosis. Using a panel of protein biomarkers, the FMC were divided into six different groups that demonstrated prognostic value, similarly to what is described in women. In fact, cats with triple negative basal-like or HER2-positive subtypes were associated with shorter overall survival, contrasting with cats presenting luminal A tumors. Moreover, these studies also indicated that luminal B and triple negative basal-like subtypes are the most common in cats. When the metastatic lesions were evaluated, a marked loss of receptor expression was found, which was associated with an increase of the triple negative basal-like subtype, highlighting the importance of immunophenotyping all lesions (primary and metastatic) in cats. Considering these results, the development of diagnostic methodologies that allows the continuous follow-up of the patients would be very useful. Therefore, the last study presented in this thesis evaluates the fHER2 serum levels in cats with FMC using two different immunoassays (ELISA and dot blot). The serum levels of fHER2 were significantly associated with the fHER2 in tissue samples of FMC (assessed by IHC). This is consistent to what is described for humans and suggests that serum quantification could be an important tool for monitoring cats after the surgery. In sum, the results presented herein provide new diagnostic and prognostic tools for veterinary oncology. Considering the high prevalence and similarities with the human counterpart, cat can also represent a potential animal model for the study of luminal B and triple negative subtypes. Considering fHER2-positive FMC more studies are required in order to determine the aetiology of the protein overexpression.
- Contribution to the study of lipid composition and nutritional value of intramuscular fat in ruminant meatsPublication . Alfaia, Cristina Maria Riscado Pereira Mateus; Prates, José António Mestre; Castro, Matilde Fonseca eContribuição para o estudo da composição lipídica e do valor nutricional da gordura intramuscular na carne de ruminantes - A composição em ácidos gordos e o ácido linoleico conjugado (CLA) são temas actuais de grande interesse, com particular relevância para a qualidade da carne e saúde humana. As carnes de ruminantes têm sido consideradas alimentos pouco saudáveis, principalmente devido à sua fracção lipídica com elevada concentração em ácidos gordos saturados. Contudo, as gorduras edíveis dos ruminantes são a principal fonte natural de isómeros CLA, sendo os isómeros bioactivos (c9,t11 e t10,c12) associados a propriedades potencialmente benéficas para a saúde humana. Em Portugal, a informação científica disponível para apoiar a qualidade e a reputação da carne bovina de raças autóctones criadas segundo as especificações de Denominação de Origem Protegida (DOP) continua restrita. Para além disso, os dados disponíveis não permitem ter uma percepção global de como distintos factores influenciam a composição da carne, desde a sua produção até ao consumo. Neste contexto, a nossa investigação foi desenvolvida estudando o efeito dos regimes alimentares dos animais (pastagem×concentrado), processamento tecnológico da carne após o abate (irradiação) e tratamentos térmicos (fervura, microondas e grelhagem), no perfil lipídico, especialmente nos isómeros do CLA, e no valor nutricional de carne de ruminantes. Foram seleccionadas e caracterizadas 4 carnes tradicionais DOP (Carnalentejana, Mertolenga, Barrosã e Arouquesa). Os resultados sugerem que a gordura intramuscular das carnes de novilho relativamente às de vitela, apresentam menor valor nutricional ao longo do ano, devido ao sistema de produção semi-extensivo utilizado nos novilhos. Por sua vez, a Carnalentejana-DOP apresenta uma qualidade nutricional superior comparativamente à carne de vaca de produção intensiva. No sentido de avaliar em detalhe o efeito de diferentes regimes alimentares no valor nutricional dos lípidos da carne, bem como a sua utilidade como discriminantes químicos da origem da carne, realizou-se um ensaio controlado. Os dados obtidos reforçam os resultados anteriores que sugeriam que a carne de animais de pastoreio apresenta melhor qualidade nutricional (teores mais elevados de n-3 PUFA e CLA) do que a carne de animais alimentados a concentrado. Este facto traduz os efeitos benéficos da pastagem no perfil de ácidos gordos da carne. Relativamente às alterações na composição da gordura intramuscular, resultantes do efeito de processamentos seguintes ao abate, a irradiação, uma tecnologia prospectiva para a conservação das carnes, não promoveu modificações significativas. Os tratamentos térmicos, frequentemente utilizados em culinária, induziram um impacto moderado no perfil dos ácidos gordos, como consequência das perdas de humidade e concentração da gordura. Em resumo, a composição lipídica da gordura intramuscular das carnes de bovino DOP estudadas apresenta um valor nutricional acrescentado para os consumidores, considerando os valores de n-3 PUFA, CLA (total e do isómero c9,t11) e da razão n-6/n-3, contribuindo para a sua diferenciação no mercado.
- Distribuição nuclear, dinâmica e função das topoisomerases I, IIα e IIβ na replicação de genomas : estudo experimental no adenovírus serótipo 2-Publication . Ferreira, Fernando António da Costa; Ferreira, João António Augusto; Fonseca, Maria de Carmo S. V. R.Neste trabalho foi estudado o papel das topoisomerases celulares (I, IIα e IIβ) na replicação, usando os adenovírus como modelo. Os adenovírus apresentam um genoma de ADN de cadeia dupla e são responsáveis por infecções respiratórias, gastro-intestinais, oftalmológicas, neurológicas e genito-urinárias. São organismos ubiquitários, infectam passáros e a maioria dos mamíferos, incluindo o Homem. Com efeito, a importância destes vírus tem vindo a aumentar por surgirem associados a novos quadros nosológicos (imunodeficiências, transplantes de órgãos) e por poderem vir a funcionar como vectores terapêuticos em doenças genéticas e na viroterapia do cancro. No presente trabalho (1) analisámos a distribuição das topoisomerases I, IIα e IIβ no núcleo de células infectadas, com utilização de tecnologias modernas de microscopia, (2) caracterizámos funcionalmente os locais de acumulação destas enzimas, (3) testámos se a replicação e a transcrição virais eram necessárias ao seu recrutamento, (4) estudámos a dinâmica das topoisomerases I e IIα in vivo por FRAP, (5) realizámos a analise mutacional da topoisomerase I e (6) quantificámos as concentrações e actividades catalíticas das topoisomerases antes e depois da infecção. Por fim, (7) abordámos a conexão funcional entre estas proteínas celulares e a replicação do vírus por depleção selectiva de cada topoisomerase (siRNA), evitando os efeitos genotóxicos dos fármacos antitopoisomerase. Os resultados obtidos permitem concluir que ambos os tipos de topoisomerases (I e II) são utilizados pelo adenovírus durante a sua replicação, mas que o seu papel é diferencial, com relevo inesperado para as topoisomerases IIα e IIβ. Estes resultados sugerem que as topoisomerases poderão ser potenciais alvos na terapêutica de patologias infecciosas e potenciais factores preditivos na terapêutica viral do cancro.
- Improving feline mammary carcinoma treatment through HER2-related immunochemotherapy agents and biomarkersPublication . Gameiro, Andreia; Ferreira, Fernando António da Costa; Correia, Jorge Manuel de JesusABSTRACT - Improving feline mammary carcinoma treatment through HER2-related immunochemotherapy agents and biomarkers - The feline mammary carcinoma (FMC) is a common tumour, with the HER2-positive and triple-negative being the most aggressive subtypes, as in women. This tumour is usually diagnosed belatedly, with scarce information about its development, and limited therapeutic options beyond mastectomy, presenting the cat a low overall survival time. Thus, this project was developed in order to improve therapeutic options, as well as, to reveal new tumour diagnosis/prognosis biomarkers, related to HER2 protein and taking advantage for the extensive knowledge in protocols used in humans. In order to disclose different therapeutic options for diseased cats, several drugs (TKi – lapatinib and neratinib; mAbs – trastuzumab and pertuzumab; ADC – T-DM1; and mTOR inhibitor – rapamycin, as adjuvant) and combined protocols were tested using FMC cell lines (CAT-M, FMCp and FMCm). These assays revealed promising antiproliferative effects and conserved molecular responses, inducing lower phosphorylation levels of target proteins, when TKi were used, and apoptosis, when mAbs and ADC drugs were evaluated. In parallel, combined protocols presented excellent synergistic responses. Moreover, feline tumour clinical tissue samples were analysed in order to identify her2 mutations as prognosis markers or therapy resistance indicators. The obtained results correlates a her2 mutation, in exon 18 (c.19573 A>T), to larger tumour sizes, a poor prognosis feature. Furthermore, any of the mutations found are described, in woman, as inducing therapeutic resistance. Additionally, considering the close relationship between obesity with increased leptin levels and the development of HER2-positive breast cancer, in woman, and since in cat obesity is a frequent nutritional disorder, the leptin/leptin receptor (ObR) axis was evaluated as possible tumour biomarker. Interestingly, cats with mammary carcinoma presented a decreased free leptin index, being the higher leptin levels associated to poor prognostic features, and serum ObR levels were correlated to an immunosuppressive status. In conclusion, the results obtained support the use of therapeutic drugs targeting the HER2, in order to improve cats’ prognosis, which could contribute for an advance in the veterinary oncology practice. Furthermore, leptin and ObR were suggested as tumour biomarkers, being proposed its use as putative adjuvant therapeutic targets
- Improving the nutritional value of microalgae for feeding pigs through the use of novel enzymesPublication . Coelho, Diogo; Prates, José António Mestre; Fontes, Carlos Mendes Godinho AndradeThe pig industry will face new challenges due to the increase demand for pork, concerning the negative environmental impact and the low content in the beneficial n-3 polyunsaturated fatty acids (PUFA) of pork. Microalgae exhibit a well-balanced nutritional composition, including in n-3 PUFA, and its production has a low environmental impact. Thus, microalgae could be a suitable alternative to traditional feedstuffs of pig industry. Arthrospira platensis and Chlorella vulgaris are the two most studied microalgae, also with the highest commercial expression. However, these microalgae are endowed by recalcitrant cell walls, which impairs the bioavailability of their compounds for pigs. In line with this, in the first part of this study, we developed a two-Carbohydrate-Active Enzyme (CAZYme) and a four-CAZYme mixtures with the ability to disrupt A. platensis and C. vulgaris cell walls, respectively. This process was performed via a high-throughput (HTP) approach, where the enzyme mixtures were selected from a 178 CAZymes and 22 sulfatases library, according to its ability to disrupt the microalgae cell wall, which was evaluated through the release of cell wall sugars, decrease of fluorescence intensity and the release of several microalgae compounds. We verified that this two CAZYme formulations are able to degrade these cell walls and may constitute a good approach to improve the bioavailability of these microalgae nutrients for pig diets. In the second part of this study, we assessed for the first time the effect of a high dietary incorporation level of the selected microalga (5% of C. vulgaris) supplemented or not with the respetive CAZYme mixture, the four-CAZYme mixture developed in the first part, and the commercially available Rovabio® Excel AP, on productive performance, meat quality and composition, health status and liver composition of finishing pigs. We observed that C. vulgaris diets had no effect on productive performance, meat quality traits and pork oxidative stability, but promoted an increase in pork carotenoids content and in n-3 PUFA composition of pork and liver. The action of the four-CAZYme mixture was preponderant for the decrease of blood lipemia. The C. vulgaris diets also promoted the decrease of plasma immunoglobulins content. It is concluded that the use of C. vulgaris in finishing pig diets, at this high incorporation level, improves the nutritional value of pork fat without compromising pig performance and health status of pigs, except the immunosuppressive effect promoted by the microalga, which deserves further investigation
- Mare endometrium fibrosis : epigenetics and novel fibrosis markersPublication . Alpoim-Moreira, Joana; Dias, Graça Maria Leitão Ferreira; Crisóstomo, Maria Rosa Rebordão Cordeiro SimõesABSTRACT - Endometrosis is a major cause of infertility in mares and involves the excessive deposition of extracellular matrix (ECM) in the mare's endometrium, such as collagen type I (COL1), and type III (COL3). In fibrosis there is an imbalance between metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), resulting in dysregulation between collagen degradation and deposition. Transforming growth factor β1 (TGF-β1) is one of the major pro fibrotic signals for myofibroblast differentiation, identified by the expression of α-smooth muscle actin (α-SMA), promoting collagen production. Alterations in fibroblast phenotype are associated with epigenetic alterations in chromatin structure. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. DNA methylation analysis is one of the most used methods to detect epigenetic changes. The aims of this study were to: (i) evaluate if COL1, COL3 and hydroxyproline could serve as possible blood biomarkers of equine endometrosis diagnosis and fertility assessment; (ii) evaluate the existence of a possible correlation between mRNA levels of DNA methyltransferases (DNMTs) and COL1A2 or COL3A1 in different Kenney and Doig’s endometrial categories and at different estrous cycle phases; (iii) evaluate the transcription and locus specific DNA methylation of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 in different mare endometrial categories; (iv) investigate the in vitro epigenetic regulation in endometrial fibroblasts challenged with TGF-β1 and to assess the use of the epigenetic modifier, 5-aza-2′- deoxycytidine (5-aza-dC or decitabine), on endometrial fibroblasts exposed to profibrotic TGF-β1. Serum COL3 concentrations increased with more advanced Kenney and Doig´s endometrial categories. Serum COL3 concentration was also higher in infertile mares, when compared to fertile mares. Both sensibility and specificity of COL3 was most effective to differentiate mares from category I from all other categories. Only COL3 proved to be a possible biomarker of mare endometrial fibrosis. Concerning epigenetic studies, DNMT3B increased with endometrial category grade, and it was independent of estrous cycle phase. There were lower transcription levels of MMP2 and MMP9, simultaneously with higher DNA methylation percentage, in higher endometrial categories. The demethylating drug 5-aza-dC reduced the collagen expression (mRNA and protein) in TGF-β1 treated endometrial fibroblasts. A different approach via epigenetics may provide an alternative means to address mare endometrial fibrosis and a more in-depth knowledge of the pathophysiology of this condition
- New insights into biological effects of conjugated linoleic acid and saturated fats in body fat composition, obesity and related disorders: experimental studies in normal-weight Wistar and obese Zucker ratsPublication . Martins, Susana Isabel Vargas; Prates, José António Mestre; Castro, Matilde da Luz dos Santos Duque Fonseca eThe daily intake of conjugated linoleic acid (CLA) isomers by humans, through diet and supplementation, and the controversial effects of these compounds in human health, were the main motivation for the elaboration of this thesis. Firstly, the present work intended to estimate the daily CLA ingestion by the Portuguese population. Secondly, the biological effects of CLA were exploited using two distinct animal models, normal-weight (Wistar rat) and genetically fat (obese Zucker rat), in combination with saturated fat based diets. The estimative of total CLA intake for the Portuguese population was 73.70 mg/day. The cis(c)9,trans(t)11 and t7,c9 were the most prevalent CLA isomers, with, respectively, 76.10 and 12.56% of the total CLA intake value. Concerning the animal trials, CLA in conjugation with saturated fats revealed beneficial but also deleterious biological effects. In the normal-weight Wistar rat fed a palm oil based diet, the administration of c9,t11 CLA isomer increased the serum triacylglycerols and the size of adipocytes from epididymal and retroperitoneal fat depots. In addition, a CLA mixture of c9,t11 and t10,c12 isomers increased the glycerol membrane permeability of kidney proximal tubules, which may indicate an improvement of glycerol reabsorption pathway. In the obese Zucker rat, CLA (as a mixture) induced changes in fatty acid profile of liver, muscle and adipose depots. CLA supplemented with a vegetable saturated fat diet seemed to promote a more beneficial adipokine serum profile and an alleviation of hepatic steatosis. In contrast, adverse effects of CLA were observed with hypercholesterolaemia promotion. Regardless CLA, the ovine fat diets worsened the insulin resistance and increased the pro-inflammatory serum cytokines. In the liver, different levels of cell death and apoptotic pathways were modulated by CLA, depending on the type of saturated fat present in the diet. The most striking result of this study was that CLA was not able to promote fat loss in both experimental models. Moreover, new mechanisms of CLA action were disclosed in this work, which reinforce the need to further investigate this compound.