DMF - Teses de Doutoramento
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- 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
- 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
- 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.
- 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
- New insights on the inhibition of neutrophil extracellular traps enzymes in equine endometriumPublication . Amaral, Ana Sofia Pires; Dias, Graça Maria Leitão Ferreira; Skarzynski, Dariusz JanMares physiologically develop a post-breeding endometritis characterized by a fast arrival of neutrophils into the uterine lumen. These neutrophils besides releasing granules of proteolytic and cytotoxic enzymes, may also deliver to the extracellular environment their DNA, histones and enzymes forming neutrophil extracellular traps (NETs). Besides trapping and fighting pathogens, NETs persistence has been also associated to the development of pathological conditions, such as fibrosis. The enzymes found in NETs, such as elastase (ELA), cathepsin G (CAT) and myeloperoxidase (MPO) act as pro-fibrotic factors in equine endometrial fibrosis, by inducing collagen type I (COL1) accumulation. Matrix metallopeptidases (MMPs) are crucial for this extracellular matrix remodeling. Prostaglandins (PG)s E2 and F2α have been described as possessing anti or pro-fibrotic effects. Equine endometrial explants from follicular phase (FP) or mid-luteal phase (MLP) were treated in vitro with ELA, CAT or MPO and their specific inhibitors for 24 or 48h. This work aimed to evaluate the explants response to: (i) ELA inhibition by sivelestat sodium salt (SIV) on COL1A2 transcription and PGE2 and PGF2α secretion; (ii) ELA and SIV treatment on MMP-2 and MMP-9 activity, and the inhibitory effect of SIV on ELA-induced COL1; (iii) CAT and Cathepsin G inhibitor I (β-keto-phosphonic acid; INH) treatment on MMP-2 and MMP-9 activity, and the effect of INH on CAT-induced COL1 production; (iv) the inhibitory effect of 4-aminobenzoic acid hydrazide (ABAH) on MPO-induced COL1 and the effect of MPO and ABAH on MMP-2 and MMP-9 gelatinolytic activity. In FP, COL1A2 transcription decreased in SIV-treated group, simultaneously with reduced pro-fibrotic PGF2α and increased anti-fibrotic PGE2 production. In ELA- and SIV-treated explants, MMPs expression depended on estrous cycle phase and time of treatment. Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h). The effect of INH was observed on CAT-induced COL1 in both phases at 48h. The MMP-2 might be involved in an earlier response to CAT, while MMP-9 in a later response in FP. The inhibitory effect of ABAH on MPO-induced COL1 was detected in FP at 48h. Matrix metallopeptidase-2 appears to be involved in an acute response to MPO treatment in MLP and MMP-9 in FP in a prolonged MPO treatment. The use of specific inhibitors of ELA, CAT or MPO, might be the grounds for future development of specific drugs to be used as prophylaxis or therapy of endometrosis in the mare.
- Notch and Wnt signaling interplay on regulation of early embryo developmentPublication . Batista, Mariana Raposo; Silva, Maria Elisabete Tomé Sousa; Costa, Luís Filipe Lopes daMammalian early embryo development requires action of a complex network of cell signaling pathways that coordinates cellular proliferation and differentiation events. Notch is a major regulator in embryonic and adult scenarios, also interplaying with other signaling pathways, such as Wnt. The objective of this work was to determine Notch signaling status in early embryo development and its influence on cellular differentiation and pluripotency maintenance, and on embryo competence to implant and develop to term. Additionally, the Notch/Wnt interplay was investigated in this scenario. Firstly, we analyzed individual embryo transcription of Notch components and their relation with transcription of pluripotency and differentiation gene markers (Sox2, Oct4, Klf4, Cdx2). Secondly, a pharmacological approach was used to induce Notch signaling (recombinant JAGGED1 and 2) and to inhibit Notch and/or Wnt signaling (DAPT and/or DKK1, respectively). Finally, embryos treated with DAPT and/or DKK1 were transferred to recipient females and implantation competency (at Day5 of gestation) and development to term (Day18) were evaluated. Results showed that transcription of Notch1-2, Jagged1-2 and Hes1 was highly prevalent and dynamic along stages of development. Transcription of Notch1, Notch2, Jagged2 and Hes1 correlated with each other and with that of Sox2, Oct4, Klf4 and Cdx2. In vitro embryo culture supplementation with JAGGED1 had no effect on embryo developmental kinetics, whereas supplementation with JAGGED2 abolished Jagged1 transcription, downregulated Cdx2 transcription and inhibited blastocyst hatching. Notch and Wnt had opposing effects on developmental kinetics, as Notch blockade retarded development and hatching, while Wnt blockade fastened it. We found evidences of Notch and Wnt interplay in early embryos as double blockade produced more severe phenotypes than expected by cumulative effects of single blockades. Notch and double blockade altered trophectoderm cell numbers and inner cell mass to trophectoderm ratio and all blockades altered transcription of Sox2, Oct4, Klf4 and Cdx2 throughout development. Implantation was unaffected by treatment, but Notch and double blockades affected the rate of Day18 developed fetuses. Notch blockade produced lighter and Wnt blockade heavier fetuses. Overall, results indicate that Notch is active in early embryo development where, together with Wnt, plays a significant role in controlling the pace of differentiation and proliferation of the blastocyst, ultimately affecting development to term.
- Virulence characterization and antimicrobial resistance of major bacterial genera from diabetic foot infectionsPublication . Mottola, Carla; Oliveira, Maria Manuela Castilho Monteiro de; Montez, Patrícia Maria Cavaco Silva de SáDiabetes mellitus is a major chronic disease that continues to increase significantly. One of the most important and costly complications of diabetes is the development of foot ulcers, colonized by pathogenic and antimicrobial resistant bacteria, which may be responsible for impairing its successful treatment. Diabetic foot ulcer (DFU) bacterial communities can be organized in polymicrobial biofilms, which may be responsible for its chronicity. The ability of these communities to produce biofilm was evaluated and was higher when compared to biofilm formation by individual species. Staphylococcus aureus is one of the most prevalent species in diabetic foot infections (DFI). Staphylococci isolated from DFU in patients from the Lisbon area were identified, genotyped and screened for virulence and antimicrobial resistance traits. The isolates showed high genomic diversity, were resistant to important clinically antibiotics and expressed relevant virulence determinants. As biofilm formation is one of the most important virulence traits of S. aureus, the antimicrobial susceptibility patterns of biofilm-producing S. aureus strains were also analysed. The minimum biofilm inhibitory and eradication concentrations were determined for ten antimicrobial compounds. Staphylococci biofilms were resistant to antibiotic concentrations ten to thousand times higher than those effective for planktonic cells. Furthermore, the enterococci frequently isolated from DFI, were also identified and characterized, showing high antimicrobial resistance and important virulence traits. Since DFI are often caused by resistant bacteria, it is necessary to find alternatives to antibiotic therapy, such as phage therapy. The inhibitory potential of five bacteriophages, previously characterized, was evaluated against established biofilms formed by S. aureus, P. aeruginosa and A. baumannii. A significant cell reduction after phage exposure was observed, mainly after multiple treatments. DFI are very complex and studies on this topic are scarce. It is necessary to intensify research in order to develop more adequate therapeutic protocols for this type of infection.
- Utilização de murganhos no estudo de novos alvos da terapia oncológica anti-angiogénicaPublication . Carvalho, Catarina Isabel Cruz de; Duarte, António José de Freitas; Ferreira, Fernando António da CostaA angiogénese é um dos marcos do desenvolvimento tumoral, sendo fundamental para o crescimento do tumor primário e para a metastização. A terapia oncológica antiangiogénica surgiu como uma estratégia promissora. Contudo, o aparecimento de tumores que não são sensíveis, o desenvolvimento de resistência e a controvérsia sobre o impacto na metastização, tem suscitado apreensão na utilização desta abordagem terapêutica. Importa por isso esclarecer qual o impacto da anti-angiogénese na metastização e desenvolver novas abordagens terapêuticas anti-angiogénicas ou melhorar as existentes, identificando a melhor fase da progressão tumoral para intervir. Recentemente a terapia anti-Dll4 foi confirmada como uma ferramenta viável na luta contra o cancro. Todavia, importa ainda esclarecer o seu efeito no aporte tumoral de fármacos citostáticos e no processo metastático, o que permitirá consolidar a validação deste ligando como um alvo terapêutico útil e promissor. Compreender melhor os mecanismos moleculares implicados no desenvolvimento vascular embrionário permitirá a identificação de novos alvos terapêuticos. A Tspan18, uma molécula ainda pouco estudada, parece desempenhar funções pró-angiogénicas, mas o seu papel na angiogénese mamífera ainda não foi elucidado. Utilizando um modelo murino transgénico de adenocarcinoma prostático, o TRAMP, verificámos que a combinação de uma terapia anti-angiogénica, o sDll4Fc, com um citostático, a doxorrubicina, conduziu a uma marcada redução do volume tumoral melhorando simultaneamente a concentração do citostático no estroma tumoral. O modelo de metastização experimental com células de melanoma murino B16-F10 permitiu-nos verificar uma redução do número de focos metastáticos pulmonares nos murganhos heterozigóticos para Dll4 especificamente no endotélio. A produção de uma nova linha de murganhos com perda-defunção condicional e induzível da Tspan18 não foi possível, pelo que não conseguimos investigar o papel desta molécula na angiogénese.
- Portable “lab-on-chip” platform for bovine mastitis diagnosis in raw milkPublication . Duarte, Carla Margarida Pinheiro Cardoso; Bexiga, José Ricardo Dias; Freitas, Paulo Jorge Peixeiro deBovine mastitis is an economic burden for farmers mostly because of decreased milk yield, premature culling and cost of veterinary treatments. The identification of mastitis pathogens is of major importance in order for adequate control measures to be taken, to reduce the risk of appearance of chronic infections, and to target antimicrobial therapy. The aim of this study was to develop and validate a sensitive method for magnetic detection of Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis in raw milk samples. Mastitic milk samples were collected aseptically from 81 cows with subclinical mastitis, from 12 Portuguese dairy farms. Ninety one quarter milk samples were selected based on bacteriological results. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analyzed using a lab-on-a-chip magnetoresistive cytometer, with microfluidics sample handling. This immunological recognition was able to detect bacterial presence above 100 cfu/ ml, depending on antibody and targeted bacteria. Comparison with PCR results showed sensitivities of 73% and 41%, specificity values of 25% and 57%, and PPV values of 35% and 54% for magnetic identification of streptococci species with an anti-S. agalactiae antibody and an anti-GB Streptococcus antibody, respectively. Regarding staphylococci species, the sensitivity values found were of 57.1% and 79.3%, specificities of 75% and 50%, and PPV values of 40% and 95.8% for magnetic identification with an anti-S. aureus antibody and an anti-Staphylococcus spp. antibody, respectively. Both bacterial genus studies translated a fair expectation for a “cow-side” use application, making this integrated platform of potential use after further improvements for fast bacteriological infection screening. Some constraints are described as well as the method´s limitations in bacterial quantification.
- 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.