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What does composing music mean today? What is the role of the composer in today’s musical world? Can we continue to talk about “composing music” in every situation of musical creation? Or should we consider recourse to other expressions? What is the composition process in the case of museum exhibitions, or installations? Can a sound artist be considered a composer of music? Is the creation of sound art a form of musical composition? Furthermore, can real-time coding or free improvisation be considered forms of instantaneous musical composition? In a world where technological means make musical creation accessible to all, what is the role of the “traditional composer”? Music is today more diverse than ever before. The range of genres, practices, techniques and technologies, forms of dissemination and reception have changed the way in which music is composed. Music is now almost omnipresent in our society spanning concert halls, museums, digital media, as well as public and private spaces. For each of these listening situations, someone conceives and composes the music, creating the sound and organizing the musical discourse through means of a diversity of approaches, knowledge and technologies. All of these means are crucial given their influences over the final outputs., Pires, I., Magalhães, F., Wanke, R. (Eds.). (2022). Composing Music Today; Nova Contemporary Music Journal (Vol. 2). Centro de Estudos de Sociologia e Estética Musical, Lisbon. ISSN 2795-4803.

What does the expression “old is new” mean in music studies? The discovery of novelty in past music? Or the recovery of old practices in new styles? In reading this volume, it is fascinating to find out the extraordinary variety of formulations that this expression can produce. But these answers above already show a subtle connection. What is the use of an old practice in a new piece, if not the acknowledgment of a newness in an old style? Can we say then that “old is new” may open to any historical connection between musical activities? Certainly, but not only this. If with “old” we mean “obsolete” or “abandoned”, we could think of an act of past recovery, but if “old” suggests attributes such “stereotyped” or “conventional”, we are possibly considering a socio-cultural perspective instead of an historical one. The expression “Old is New” is a challenge to all music fans to go beyond their comfort zone and reveal unprecedented connections, surprising relations and inspiring bridges between history, culture, human gestures and beliefs., Pires, I., Magalhães, F., Wanke, R. (Eds.). (2022). Old is New: The Presence of the Past in the Music of the Present; Nova Contemporary Music Journal (Vol. 1). Centro de Estudos de Sociologia e Estética Musical, Lisbon. ISSN 2795-4803.

Importance: Polypoidal choroidal vasculopathy (PCV) is far less common and studied in a Caucasian population than in an Asian population, and the optimal treatment approach remains to be confirmed. Methods: A 52-week, double-masked, sham-controlled, phase 4, investigator-initiated randomized clinical trial (RCT) in naive symptomatic Caucasian patients with PCV treated with aflibercept in a treat-and-extend regimen (T&E) (intravitreal aflibercept injection [IVAI] T&E). Patients were randomized at week 16 to receive IVAI T&E plus either sham photodynamic therapy (PDT) or standard fluence PDT with verteporfin. The main outcome measures were changes in best-corrected visual acuity (BCVA) from baseline to 52 weeks and polyp occlusion at week 52. Data are presented as median (interquartile range [IQR]) for BCVA, number of IVAI, and change in central retinal thickness (CRT). Results: Of the 50 patients included in the study, 48 patients completed the 52 weeks of follow-up. During this period, a significant median (IQR) BCVA gain of 6 [2-12] Early Treatment Diabetic Retinopathy Study letters was observed for all patients (p < 0.001), after 8 (7-9) injections, with a significant reduction of -93.0 [-154.0, -44.0] mu m in central macular thickness (p < 0.001). Using indocyanine green angiography, a complete occlusion of polypoidal lesions was documented in 72% of the cases. Still, no significant difference was detected between the sham PDT and the aflibercept PDT arms, at week 52, for BCVA change (6.5 [2-11] vs. 5 [2-13] letters (p = 0.98)), number of IVAIs (8.5 [7-9] vs. 8 [7-9] (p = 0.21)), change in CRT (-143 [-184; -47] vs. -89 [-123; -41.5] mu m [p = 0.23]), and rates of complete polyp occlusion: 77 versus 68% (p = 0.53) or presence of fluid: 68 versus 57% (p = 0.56). No serious ocular adverse events were registered in the 2 arms. Conclusions and Relevance: To our knowledge, this is the first RCT to compare aflibercept T&E monotherapy with aflibercept T&E plus verteporfin PDT in a Caucasian population with PCV. Aflibercept monotherapy in a T&E showed to be effective and safe with a significant median BCVA improvement of 6 letters and a complete occlusion of polypoidal lesions in near 3 quarters of the eyes, at 1 year. As only 22% of the eyes underwent PDT treatment, the benefit of combined treatment for PCV in Caucasian patients could not be definitively elucidated from this study.

Peripolar cells are granulated cells located in the vascular pole of the renal corpuscle. Even though these cells have already been described, there are still many unknown histological and physiological characteristics. We carried out histochemical and immunohistochemical analyses of peripolar cells in sheep and compared their number in both normal and injured kidneys, discriminating according to the age of the animal. We tested HE, Toluidine Blue, PAS, and Masson's Trichrome stains to select the best stain for identification and quantification. Masson Trichrome yielded the best results and was selected for this purpose. We identified the cells by the presence of cytoplasmatic granules and by their position in the vascular pole. We found no statistically significant association between the number of peripolar cells and the age of the animal or the occurrence of lesions. In the immunohistochemical analysis, we found that the cells were positive to α-smooth muscle actin and less consistently positive to NSE and S100 protein. Chromogranin A, cyclooxygenase-2, AE1/AE3, and Wide Spectrum Cytokeratin and desmin yielded negative results. We conclude that although there was evidence of a contractile function, there was no evidence to support that peripolar cells have either a neuroendocrine or an epithelial nature.

The therapeutic effect of three polyvinyl alcohol (PVA) membranes loaded with electrically conductive materials - carbon nanotubes (PVA-CNTs) and polypyrrole (PVA-PPy) - were tested in vivo for neuro-muscular regeneration after an axonotmesis injury in the rat sciatic nerve. The membranes electrical conductivity measured was 1.5 ± 0.5 × 10

1. Introduction An essential oil (EO), also defined as essence, volatile oil, etheric oil or aetheroleum, is a complex mixture of volatile constituents biosynthesised by aromatic plants. These compounds are mainly monoterpenes, sesquiterpenes and phenylpropanoids. EOs can be synthesized by all plant organs (root, stem, twigs, leaves, flowers, buds, fruits and seeds) and stored in structures classified into 6 groups: (1) secretory cells; (2) osmophores; (3) secretory cavities; (4) secretory ducts; (5) glandular trichomes; (6) epidermal cells. Essential oils are characteristic as being non-polar or weakly polar liquids, soluble in alcohol and in dimethyl sulfoxide (DMSO), and less dense than water. They may have many biological activities, including anticancer, anti-inflammatory, antimicrobial, antiviral, antioxidant and/or insect repellent. The aim of this study was to determine the efficacy of the essential oils from six species of Peniche (Portugal) sand dunes as potential cytotoxic/cytostatic therapeutic agents in breast and colorectal cancer cell line models. 2. Materials and methods 2.1 Plant material Aerial parts of Artemisia campestris, Crithmum maritimum, Eryngium maritimum, Juniperus turbinata, Otanthus maritimus and Seseli tortuosum were collected during flowering stage in July 2013, in the sand dunes of Consolação beach, Peniche (Portugal). Voucher specimens were deposited in the Herbarium of Medicinal Plants, Faculty of Pharmacy of the University of Coimbra. 2.2 Essential oils isolation Essential oils were isolated by hydrodistillation for 3h using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997). 2.3 Chemical characterization of essential oils The essential oils were chemically characterized by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). 2.4 Cell Culture Human cancer cell lines RKO (colorectal) and MCF7 (breast) were maintained in medium (Dulbecco’s Modified Eagle’s), supplemented with 10% fetal bovine serum (FBS) and 1% sodium pyruvate. Cells were grown at 37˚C, under a 5% CO2 atmosphere and at 90% humidity. The subculture was made three times a week. Cells regularly tested negative for Mycoplasma infection. 2.5 In vitro inhibition of RKO and MCF7 cells growth RKO and MCF7 cell lines were seed into 96 well microtiter plates to determine the activity of every essential oil, at a density of 1x104 and 5x103 cells per well, respectively. The serial doubling dilution of the EO was prepared in DMSO. For stock solution were used 25µL EO and 75µL of DMSO (1:4). The final concentration used was 1 µL/mL and the dilution was 1:10. 2.6 MTS Assay for cellular viability MTS (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dyphenyl-2H-tetrazolium bromide) colorimetric assay measures cell respiration and the amount of formazan produced is proportional to the number of living cells present in culture. It is a sensitive, quantitative and reliable colorimetric assay. This method was employed as test for quantification of cell proliferation after 72 hours incubation at 37˚C and 5% CO2. For this purpose, the medium of each well was removed and 100µL of fresh medium was added in addition to 20µL of MTS solution. After 4h incubation, the absorbance was determined using a microplate reader (Bioteck ELx800) at a test wavelength of 490 nm. 3. Results To determine the antitumor activity of EOs against RKO and MCF7 cells, MTS assay was carried out, and cell viability was calculated comparing to the vehicle (DMSO), corresponding to 100% proliferation (fig. 1-6). For all the EOs tested, antitumor activity was shown, as indicated by a decrease in cell viability – except for J. turbinata and C. maritimum which had the least effect on cell viability for MCF7 and RKO cell lines, respectively (table 1). 4. Discussion and Conclusion In this study, different EOs from sand dune plants have been investigated for their antitumor activity using colorectal and breast cancer cell lines. All essential oils tested showed antitumor activity as measured by their effect on cell viability. The most noticeable decrease in viability was shown by S. tortuosum and E. maritimum EOs, as indicated by IC50 average values of 3.29E-03 and 8.59E-04 (for RKO) and 1.85E-03 and 2.72E-03 (for MCF7), respectively. Some of the tested EOs, such as J. turbinata for MCF7 cell line and C. maritimum for RKO cell line, showed lower activity (IC50 average value of 3.85E+1 for MCF7 cells and 8.76E00 for RKO cells, respectively). There are several studies on the antioxidant activity of plant extracts. For example, Aniya and collaborators (2000) investigated the antioxidant and hepatoprotective action in water extracts of A. campestris (from Okinawa Islands), and observed that these extracts provide protection against liver toxicity. The literature available for potential antitumor activity of EOs is more limited, however, there are some studies, about citrus fruits, cherry, mint and herbs that indicate potential of antitumor activity against different types of human cancers, including breast cancer, liver cancer and melanoma. In these studies, this effect is associated with the specific chemical compounds of the EOs, including monoterpenes (Crowell, 1999). It would be interesting to study the role of individual chemical components from the EOs in any potential effects, namely anticancer activity, in their action. For this, it is necessary to analyse in detail the chemical composition of each EO. These would then be evaluated independently and compared to the complex EO mix. Finally, the data here presented indicates that these EOs have an anti-proliferative effect on the cell lines used. Further work needs to be undertaken to investigate the effect of the EOs in known signalling pathways that regulate cell proliferation and viability, such as apoptosis, DNA damage-induced cell cycle arrest or by decreased proliferation signalling.

An essential oil (EO) is defined internationally as the product obtained by hydrodistillation, of a plant or some parts of it. They are aromatic oily liquids, volatile, characterized by a strong odour, rarely coloured, and generally with a lower density than that of water. They can be synthesized by all plant organs (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots) and therefore extracted from these parts, where they are stored in secretory cells, cavities, ducts, epidermal cells, osmophores or glandular trichomes. EOs have a complex composition. The great majority of components identified in EOs include monoterpenes and sesquiterpenes. Nevertheless, phenylpropanoids are also important components of some essential oils. EOs extracted from Thymbra capitata and Helichrysum italicum collected in Algarve (Portugal) were investigated for their antitumor activity, specifically looking at effect on viability of human colorectal cancer (RKO) and breast cancer (MCF7) cells using MTS assay. The serial doubling dilution of the essential oil was prepared in DMSO. Both EOs tested, at final concentration 1 µL/mL and dilution 1:10, lead to a decrease in cell viability for both RKO (colorectal) and MCF7 (breast) cancer cell lines. Specifically, average IC50 values were 2.82E-03 µL/mL and 1.08E-02 µL/mL for T. capitata and 1.03E-01 µL/mL and 1.30E+01 µL/mL for H. italicum (IC50s for RKO and MCF7 cell lines, respectively). It has long been recognised that some EOs have antimicrobial, antibacterial, antioxidant, antiviral, antimycotic, antiparasitic, insecticidal activities. However, studies into their anti-tumoral activities are scarce. This study, indicates that Thymbra capitata and Helichrysum italicum EOs exhibited antitumor activity against RKO and MCF7 cells, deserving further research into both the mechanism of the observed anti-tumour activity and potential cancer therapeutic applications.

Mestrado APNOR A satisfação no trabalho e atitudes de mindfulness têm sido objeto de estudo da área da psicologia social, do trabalho e das organizações. Na última década temos assistido a um crescente número de investigações, as quais destacam o papel do mindfulness no contexto de trabalho, na eficiência de atividades e na promoção da saúde física e psicológica. Estas evidências têm demonstrado que as práticas meditativas melhoram a atenção plena, além de promoverem a saúde psicológica em ambientes de trabalho. Objetivou-se avaliar a satisfação laboral, atitudes de mindfulness e bem-estar dos trabalhadores, definindo dois Objetivos de Estudo (OE): Identificar os níveis de bem-estar e satisfação no trabalho dos trabalhadores em estudo; identificar as atitudes de mindfulness dos trabalhadores em estudo, procedendo-se a um estudo quantitativo, transversal em termos temporais, descritivo e correlacional numa amostra de 272 respondentes, todos eles com atividade profissional. Utilizou-se como instrumento de recolha de dados um questionário online, contendo quatro partes: questões socioprofissionais; a Mindful Attention Awareness Scale; a Satisfaction with Life Scale; e a Generic Job Satisfaction Scale, estas últimas três nas suas versões validadas para a população portuguesa. Os resultados evidenciam que a prática de exercícios de meditação e/ou mindfulness na atividade diária influencia positivamente a satisfação com a vida e o bem-estar, bem como a satisfação no trabalho, havendo diferenças estatisticamente significativas entre quem pratica e quem não. Também o estado civil dos respondentes é determinante para o nível de satisfação com a vida e com o trabalho. Os resultados do presente estudo são corroborados por diversos estudos internacionais de renome, o que nos leva a concluir que programas de introdução da meditação e/ou mindfulness nos currículos académicos, implementados com sucesso nesses vários países, poderão ser facilmente transpostos para a realidade portuguesa, já que os níveis e as diferenças estatisticamente significativas encontrados, mostram semelhanças entre os respondentes dos vários países. Considera-se que, como futura linha de investigação, deveria de ser implementado um programa de MBCT e MBSR, como unidade curricular optativa, nos cursos de enfermagem, enquanto piloto para uma abrangência maior futuramente. Job satisfaction and mindfulness attitudes have been the subject of study in the area of social psychology, work and organizations. In the last decade we have witnessed an increasing number of investigations, which highlight the role of mindfulness in the work context, in the efficiency of activities and in the promotion of physical and psychological health. This evidence has shown that meditative practices improve mindfulness, in addition to promoting psychological health in work environments The objective was to assess job satisfaction, attitudes of mindfulness and well-being of workers, defining two Study Objectives (OE): To identify the levels of well-being and job satisfaction of the workers under study; and, To identify the attitudes of mindfulness of the workers under study, proceeding to a quantitative, transversal study in temporal terms, descriptive and correlational in a sample of 272 respondents, all of them with professional activity. An online questionnaire was used as an instrument for data collection, containing four parts: socio-professional issues; the Mindful Attention Awareness Scale; the Satisfaction with Life Scale; and the Generic Job Satisfaction Scale, the latter three in their versions validated for the Portuguese population. The results show that the practice of meditation and / or mindfulness exercises in daily activity, positively influence satisfaction with life and well-being, as well as job satisfaction, with statistically significant differences between those who practice and those who do not. Respondents' marital status is also determinant for their level of satisfaction with life and work. The results of the present study are corroborated by several renowned international studies, which leads us to conclude that programs of introduction of meditation and / or mindfulness in academic curricula, successfully implemented in these various countries, can be easily transposed to the Portuguese reality, since the levels and the statistically significant differences found, show similarities between the respondents of the various countries. It is considered that as a future line of research, an MBCT and MBSR program should be implemented, as an optional curricular unit, in nursing courses, as a pilot for greater coverage in the future.

A pedunculated cauliflower-like mass was detected on the left posterior limb of a subadult male red deer (Cervus elaphus) after a hunt in Portugal. Histologically the lesion was classified as cutaneous fibropapilloma. The identification of Cervus elaphus papillomavirus (CePV-1 variant) was based on sequencing of the L1 gene. The L2 sequence revealed a nine-nucleotide deletion, as already reported in the Italian and Hungarian CePV-1, further supporting the theory that this is a distinctive genomic characteristic of this viral variant, as this feature has been found in distinct cases from geographically distant countries. In addition, a coinfection with bovine papillomavirus was evidenced by amplification and sequencing of the E5 gene, confirming the ability of Delta papillomaviruses to cross-infect different animal species and providing more evidence that wildlife may act as reservoir for papillomaviruses affecting domestic species. Papillomavirus infection in red deer has been sporadically described in different European countries; in this work, we describe the identification of a CePV-1 variant infection associated with a red deer fibropapilloma in Portugal.

Tese de Doutoramento em Ciências Veterinárias A disciplina de oncologia veterinária tem assistido a enormes progressos nos últimos anos e a doença oncológica constitui hoje a principal causa de morte de animais de estimação seniores. Novos e sofisticados tratamentos estão hoje disponíveis no tratamento da doença oncológica fruto de avanços tecnológicos e da alteração de atitudes dos proprietários de animais domésticos que cada vez mais os encaram como membros da família exigindo assim o melhor tratamento disponível e com fortes preocupações relativas à qualidade de vida dos mesmos. O papel da enzima COX tem vindo a ser demonstrado de uma forma crescente na carcinogénese tanto em pessoas como em animais domésticos sendo um importante fator de prognóstico em vária neoplasias e potencialmente um alvo terapêutico. O objetivo deste estudo consistiu em analisar as expectativas de proprietários de animais de companhia com doença oncológica relativamente a serviços veterinários e avaliar se elas difeririam da população geral de donos de animais de companhia. Nesse sentido foi utilizada a metodologia SERVQUAL, um instrumento popularmente utilizado em marketing de serviços, para avaliar expectativas e perceções de consumidores e, assim, quantificar indiretamente a satisfação desses mesmos consumidores. De facto, esta metodologia é comumente utilizada em serviços de saúde quer como ferramenta de gestão, quer como ferramenta de investigação científica. Através da análise de inquéritos SERVQUAL distribuídos a proprietários de animais de companhia submetidos a tratamentos oncológicos e tratamentos não oncológicos concluiu-se que o primeiro grupo possuía expetativas específicas sobre o que constituiria um serviço veterinário ideal atribuindo maior importância a questões relacionadas com a ´segurança´ as quais não foram correspondidas pela equipa veterinária que por sua vez atribuíram maior importância a questões relacionadas com a tangibilidade dos serviços. Esta melhor compreensão das necessidades dos donos de animais com cancro permite melhor ajustar os serviços veterinários gerando maiores satisfações e melhores “compliances” e resultados dos tratamentos. A COX-2 é uma enzima diretamente implicada em diversas etapas da carcinogénese tais como a iniciação, a promoção, a invasão, a inibição da apoptose e a metastização. Os anti-inflamatórios com as suas propriedades de inibição da COX-2 são potencialmente candidatos óbvios a serem incluídos em terapêuticas de cancro providenciando analgesia e consequente melhoria da qualidade de vida (QoL) e, simultaneamente, atividade anticancerígena. Neste trabalho são revistas as ações da COX-2 nas diversas etapas chave da carcinogénese e é apresentada uma revisão sistemática sobre o valor prognóstico das COX em diversas neoplasias malignas do cão e do gato. Através da análise da literatura constatou-se que a expressão de COX-2 está associada a uma diminuição da sobrevida total no melanoma canino, no osteossarcoma canino e nos tumores mamários da cadela, mas não nos mastocitomas, carcinomas da próstata e carcinomas nasais. Esta revisão permitiu também constatar a escassez de estudos complementares para cada tipo de neoplasia e ausência de estudos para diversos grupos de neoplasias comuns, assim como identificar algumas falhas metodológicas em vários estudos potencialmente conduzindo a riscos de “bias”. De seguida investigámos os mecanismos de ação da COX-2 em 2 grupos de neoplasias importantes em cães: mastocitomas e melanomas- onde demonstrámos a associação da expressão da COX-2 com variáveis clinico-patológicas de agressividade elucidando o papel da COX-2 na carcinogénese destas 2 neoplasias apontando para a contribuição na angiogénese e alteração no microambiente inflamatório peri-tumoral no caso dos melanomas e proliferação e angiogénese nos mastocitomas. Embora a bibliografia publicada não tenha não tenha associado a COX-2 a um prognóstico negativo nos mastocitomas caninos, o nosso trabalho encontrou uma associação entre a intensidade da expressão de COX-2 e a menor sobrevida total em cães com mastocitomas. Finalmente descrevemos um estudo observacional onde a ação de um inibidor COX-2 específico- firocoxib- utilizado sozinho, ou combinado num esquema metronómico com clorambucil, foi avaliada num conjunto de 30 neoplasias em estádio avançado constatando-se atividade clínica com registo de 1 remissão completa e 2 remissões parciais, com um bom perfil de segurança e com melhorias significativas dos scores de QoL. Em conclusão, demonstrámos as especificidades das expectativas dos donos de animais de companhia e a importância dos clínicos se ajustarem às suas necessidades de forma a melhor garantir o cumprimento dos planos de tratamento. Identificámos a COX-2 como um importante fator de prognóstico em várias neoplasias malignas caninas e identificámos a utilidade potencial do uso de inibidores COX-2 no tratamento e melhoria da qualidade de vida de animais com neoplasias em estádio clínico avançado. A expressão de COX-2 poderá de futuro ser usada na clínica como marcador de previsão de agressividade em neoplasias de prognóstico incerto tais como mastocitomas de grau intermédio e o uso de inibidores COX-2 no tratamento de diversas neoplasias caninas deverá ser equacionado e avaliado em ensaios clínicos randomizados avaliando como resultados finais a resposta clínica ou a melhoria da qualidade de vida dos animais. Veterinary oncology field has experienced huge progresses in recent years and oncologic disease constitutes today the leading cause of death among senior pets. New and sophisticated therapies are readily available today for the treatment of oncologic disease resulting from technologic advances and from an attitude shift of pet owners that now regard them as members of the family and so demand the best medical care available and with special concern on Quality of Life issues. COX-2 enzyme role in carcinogenesis has been increasingly demonstrated both in people as well as in animals being an important prognostic factor in several neoplasia and potentially constituting a therapeutic target. The goal of this work consisted in analysing pet owner’s expectations towards veterinary services and evaluate if they differ from general pet owner population. For that we used the SERVQUAL methodology, a popular marketing instrument used to evaluate consumer’s expectations and perceptions allowing to indirectly estimate service satisfaction. In fact this methodology is already routinely used in health care either as a management or research tool. Through SERVQUAL questionnaires distributed to pet owners of animals with oncologic disease and non-oncologic disease we concluded that the former had different expectations regarding an optimum veterinary care service giving extra importance to items related with assurance which were not recognized by the veterinary staff who in turn gave higher importance to questions related to the tangible dimension. This better understanding of the needs of pet owners with cancer will allow for better adjustment of veterinary services offer making way to better client satisfaction, better treatment compliance rates and better outcomes of treatments. COX-2 is an enzyme directly implicated in several hallmarks of carcinogenesis such as initiation, promotion, invasion, apoptosis inhibition and metastization. NSAIDs with its COX-2 inhibitory properties are obvious potential candidates to be included in cancer therapy providing analgesia with consequent improve in QoL and at the same time having anti-cancer activity. In this work we reviewed COX-2 role in several key steps of carcinogenesis and we performed a systematic review on the prognostic value of COX in several malignant neoplasias in the dog and cat. Through review of the literature we observed that COX-2 is associated with an overall decrease in survival in canine melanoma, canine osteosarcoma and canine mammary tumours, but not in canine mast cell tumours, prostatic carcinoma and nasal carcinoma. This systematic review also allowed to highlight the scarcity of complementary prognostic studies for each type of neoplasia and the absence of any study in particular important groups of neoplasias and also permitted to identify methodologic limitations of published studies potentially leading to increased risk of bias. Next we investigated mechanisms of action of COX-2 in 2 important neoplasias in dogs: mast cell tumours and melanomas. Here we showed the association between COX-2 expression and several clinic-pathological variables of aggressiveness enlightening the role of COX-2 in these 2 cancers: acting on angiogenesis and peritumoural inflammatory microenvironment modification in melanomas and proliferation and angiogenesis in mast cell tumours. Although published literature hadn´t found association between COX-2 expression and diminished OS in canine mast cell tumours our work did find such association when taking in account COX-2 intensity of expression. Finally we described an observational study where the action of firocoxib (a COX-2 specific inhibitor) used alone or combined with chlorambucil in a metronomic regimen, was evaluated in 30 end stage various types of canine neoplasia. Clinical response was observed and we recorded 1 complete remission and 2 partial remissions with a good safety profile and with significant improvements in QoL scores of patients. In conclusion we demonstrated the specific needs and expectations of owners of pets with oncologic disease and the importance for the clinicians to make adjustments in order to meet those demands and achieve client satisfaction and compliance of treatment plans. We identified COX-2 as an important prognostic factor in several neoplasias and the potential role of COX-2 inhibitors in the treatment of these malignancies or as palliative care to improve QoL in end stage disease. COX-2 expression could in the future be used to help ascertain the clinical behaviour of neoplasias of uncertain prognostic such as intermediate grade mast cell tumours and the use of COX-2 inhibitors in the treatment of canine malignancies should be contemplated and evaluated throw randomized clinical trials considering as final results clinical activity or improvements in QoL.

Tese de Doutoramento em Ciências Veterinárias A inflamação faz parte dos principais “hallmarks” associados ao cancro e tem um papel relevante na carcinogénese mamária, relacionando-se com a agressividade tumoral e pior prognóstico. Nos tumores mamários dos canídeos (TMC) os estudos que se debruçam sobre o efeito das células imunitárias no prognóstico são escassos. Neste contexto, o presente trabalho foi realizado com o objetivo de fomentar o conhecimento sobre o papel das vias de sinalização compartilhadas pelo infiltrado de linfócitos T/macrófagos e importantes biomarcadores tumorais na progressão, agressividade e prognóstico destes tumores. A imunossupressão associada ao infiltrado de linfócitos T no tumor tem sido explicada pela capacidade inibitória das células T reguladoras (Treg) que impedem as atividades citotóxicas anti-tumorais. Com base nestas evidências, caracterizámos por imunohistoquímica as células Treg intratumorais (FoxP3+), bem como algumas citoquinas relacionadas com elas: TGFβ e IL-35. Os nossos resultados demonstraram que a maior imunorreactividade para o FoxP3 está presente em tumores com fenótipos mais agressivos: elevado grau histológico de malignidade (GHM), presença de embolos intravasculares e metástases nos linfonodos. Adicionalmente, mostrámos que as células Treg FoxP3+ intratumorais estão associadas com uma menor sobrevida total (ST), por análise univariada e multivariada. As células Treg secretam citoquinas inibidoras (TGFβ e IL-35), induzindo a tolerância aos tumores. Os nossos resultados sugerem uma associação entre o TGFβ e parâmetros de agressividade tumoral, refletindo o seu envolvimento na transformação maligna. Demonstraram também uma correlação positiva entre o FoxP3 intratumoral, os níveis de TGFβ, VEGF e CD31. Além disso, tumores com abundante TGFβ e com elevada expressão simultânea de TGFβ/FoxP3, FoxP3/VEGF, TGFβ/VEGF foram associados ao menor tempo de ST e a classe de tumores TGFβ/FoxP3 elevados revelou-se como um fator independente de mau prognóstico na análise multivariada. A IL-35 é uma citoquina secretada pelas células Treg que inibe a proliferação e função das células T. Demonstrámos que a sobre-expressão da IL-35 está associada com o estádio clínico mais avançado e com um prognóstico desfavorável na análise univariada e multivariada. Foi possível constatar, pela 1ª vez nos TMC, o valor prognóstico independente das células FoxP3+ Treg e da classe de tumores TGFβ/FoxP3 elevados. A IL-35 evidenciou-se ainda como um novo biomarcador capaz de prever a evolução clínica dos TMC. No seguimento deste trabalho, realizámos um amplo estudo que incidiu sobre as vias de sinalização compartilhadas pelo infiltrado de linfócitos T/macrófagos e importantes biomarcadores tumorais no microambiente dos TMC. Com esse propósito estudámos biomarcadores celulares importantes na carcinogénese mamária (COX-2, EGFR e c-kit), que estão frequentemente sobre-expressos ou mutados nos TMC. Constatámos uma associação significativa da imunoexpressão elevada da COX-2 com os linfócitos T CD3+ e macrófagos MAC387. Tumores com elevada expressão de COX-2/CD3 e COX-2/MAC estão associados com variáveis de agressividade tumoral e menor ST. Demonstrámos ainda que a expressão simultânea de COX-2+/EGFR+ está associada com maior número de células T CD3+ intratumorais e foi observada uma associação significativa e uma correlação positiva entre os linfócitos T CD3+ e a expressão de c-kit. Tumores com alta expressão de c-kit apresentaram maior número de células T CD3+ estando associados a um GHM elevado, presença de êmbolos intravasculares, metástases nos linfonodos e menor ST. Os resultados demonstram que as vias de sinalização que envolvem a COX-2, o EGFR e o c-kit são importantes, não só para a remodelação do microambiente do tumor mamário, mas também como alvos para a terapia imunológica anti-tumoral. Para complementar o trabalho, foi realizada uma experiência de co-cultura in vitro onde se pretendia demonstrar a potencial participação bidirecional das células tumorais e células imunitárias na regulação da COX-2. Os resultados mostraram que a co-cultura entre a linha celular de carcinoma mamário de cão Sh1b e as células mononucleares de sangue periférico de cão (PBMCs) induziram uma tendência para a sobre-expressão de COX-2 nas células cancerígenas. Por sua vez, a expressão de COX-2 pelos PBMCs, entre eles predominantemente macrófagos CD68+, foi significativamente atenuada pela cocultura com Sh1b. Em conformidade, a co-cultura com CD68+ THP1 diferenciada (dTHP1) provocou um aumento da produção intracelular de COX-2 pelas células Sh1b. A expressão intracelular de COX-2 pela dTHP1 diminuiu quando estas células foram tratadas com meio condicionado das células Sh1b. Estes resultados representam um avanço significativo na compreensão do papel da COX-2 na remodelação do microambiente tumoral nos TMC, nomeadamente através da imunomodulação dos macrófagos associados ao tumor. Globalmente, o presente trabalho demonstrou que, à semelhança do cancro de mama da mulher, também nos TMC as células inflamatórias no microambiente do tumor são capazes de orquestrar programas facilitadores da progressão tumoral. Cancer related inflammation is part of the major cancer hallmarks and has an important role in mammary carcinogenesis being involved in tumor aggressiveness and poor clinical outcome. In dogs only few studies have yet concentrated on the influence of immune cells in clinical outcome of dog mammary tumor patients. In this context, the present work was conducted with the main aim of up-to-date knowledge about the roles of the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in canine mammary tumors (CMT) progression, aggression and prognosis. Immunosuppression associated with tumor infiltrating T-lymphocytes (TILs) has been explained by the compartment of regulatory T-cells (Treg) that inhibit anti-tumor cytotoxic activities. Based on these evidences, first, we assessed, by immunohistochemistry, the characterization of intratumoral Treg cells, as well as some cytokines related by them: TGFβ and IL-35. Our results demonstrated that FoxP3 was present in tumors with more aggressive phenotypes: high histological grade of malignancy (HGM), presence of neoplastic intravascular emboli and presence of lymph node metastasis. Additionally, also showed that intratumoral FoxP3+ Treg cells were associated with shorter overall survival (OS), both in univariate and multivariate analysis. Tregs can secrete inhibitory cytokines (TGFβ and IL-35), inducing tumors tolerance. Present work suggests a link between TGFβ and parameters of tumor aggressiveness, reflecting its involvement in CMT malignant transformation. Our data demonstrated also a positive correlation between intratumoral FoxP3, TGFβ levels, VEGF and CD31. Moreover tumors with abundant TGFβ and with concurrent high expression of TGFβ/FoxP3, FoxP3/VEGF, TGFβ/VEGF were associated with shorter OS time and the TGFβ/FoxP3 tumors class retained the association with worse survival in multivariate analysis, arising as an independent predictor of poor prognosis. IL-35 is a Treg cellsecreted cytokine that inhibits T-cells proliferation and function and to the best of our knowledge, this is the first study that investigates the role of IL-35 in CMT development and clinical outcome. Our results showed that IL-35 overexpression was significantly associated with advancement of tumor stage and unfavorable prognosis by univariate and multivariate survival analysis. Interestingly our findings indicate, for the first time in dog mammary tumors, the independent prognostic value of FoxP3+ Treg cells and TGFβ/FoxP3 tumors class. Additionally the IL-35 seems to be a new biomarker to predict the CMT clinical outcome. For a more comprehensive approach about the role of immune cells in dog mammary carcinogenesis, we perform a wide study focused on the intertwined signaling pathways shared by T-lymphocytic/macrophage infiltrates and important tissue biomarkers in mammary tumor microenvironment. We focused on relevant cell biomarkers in mammary carcinogenesis, the COX-2, EGFR and c-kit which are often overexpressed or mutated in this type of tumor. Present data demonstrated a significant association of high COX-2 immunoexpression with CD3+ T-lymphocytes and MAC387 macrophages. Tumors with concurrent high COX-2/CD3 and high COX-2/MAC expression were associated with variables of tumor aggressiveness and shorter OS. Current results also demonstrated that the concurrent COX-2+/EGFR+ expression was associated with higher numbers of intratumoral CD3+ T-cells. Furthermore a significant association and a positive correlation between CD3+ T-lymphocytes and c-kit expression were observed. Tumors with high c-kit expression showed higher counts of CD3+ T-cells and were associated with high HGM, presence of neoplastic intravascular emboli, presence of lymph node metastasis, angiogenesis and shorter OS. The findings indicate that COX-2, EGFR and c-kit pathways are important not only for the remodeling of mammary tumor microenvironment but also could be a very important targets for tumor immunological therapy. Finally, in an in vitro co-culturing experiment, was our aim to prove the potential bidirectional crosstalk between tumor cells and immune cells on COX-2 regulation. Our data showed that co-culturing of canine mammary carcinoma cell line Sh1b and canine peripheral blood mononuclear cells (PBMCs) induced a trend of COX-2 overexpression in mammary cancer cells. In turn, COX-2 expression by PBMCs, among them predominantly CD68+ macrophages, was significantly attenuated by co-culture with Sh1b. In accordance, co-culture with CD68+ differentiated THP1 (dTHP1) prompted an intracellular production of COX-2 in Sh1b cells. The intracellular COX-2 expression from dTHP1 decreased when they were treated with conditioned medium from cultured Sh1b cells. Present results represents a significant advance on understanding the possible role of COX-2 in inducing a cancer tolerogenic microenvironment in CMT namely through a cancer associated macrophages immunomodulation. Overall present work demonstrated that, similarly to human breast cancer, also in CMT the inflammatory responses in mammary cancer sites are able to orchestrate hallmark-facilitating programs in tumor microenvironment. FCT (“Fundação para a Ciência e a Tecnologia”) and POPH-QREN/FSE (“Programa Operacional Potencial Humano-Quadro de Referência Estratégico Nacional/Fundo Social Europeu”)

Dissertação de Mestrado Integrado em Medicina Veterinária, Ciências Veterinárias Os tumores melanocíticos são neoplasias comuns em equinos, afetando cerca de 80% dos animais de pelagem ruça com mais de 15 anos. Apesar de ser uma lesão de fácil diagnóstico, o seu comportamento biológico é bastante imprevisível. A caderina epitelial é a principal molécula de adesão celular entre melanócitos e queratinócitos, regulando o crescimento e proliferação dos primeiros. Assim, alterações na função ou expressão desta molécula surgem associadas à aquisição de um fenótipo celular invasivo e consequente formação de metástases. O presente trabalho pretendeu avaliar, por imunohistoquímica, a presença da caderina-E em 38 neoplasias melanocíticas de equinos, classificando a sua expressão de acordo com a extensão, localização e intensidade de marcação. Foi efetuada uma o diagnóstico prévio de acordo com os critérios descritos pela OMS, incluindo a verificação da existência de êmbolos vasculares. Todos as neoplasias foram classificadas como melanomas. Foi utilizado um anticorpo monoclonal de murganho anti-caderina E como anticorpo primário. A marcação foi positiva em 36 tumores e 24 destes apresentaram uma extensão difusa, com uma elevada percentagem de células imunorreativas (> 75%). Nas células tumorais, a marcação foi citoplasmática (n=21), membranar (n=7) ou mista (n=8). A maioria dos tumores com imagens de invasão vascular (75%) teve marcação citoplasmática. Em relação à intensidade de marcação, as amostras distribuíram-se de forma idêntica pelos diferentes graus de intensidade. Foi constante a diminuição de intensidade de marcação com a distância à epiderme. Nenhum dos parâmetros da avaliação imunohistoquímica teve uma associação estatisticamente significativa com as características histopatológicas das amostras. Na maioria desta série de tumores melanocíticos de equinos foi evidente a expressão de caderina-E nas células neoplásicas, com uma localização citoplasmática, considerada por vários autores como anómala e um indicador de agressividade. Apesar do reduzido número de amostras em estudo, este é o primeiro estudo da expressão da caderina-E em neoplasias melanocíticas de equinos. São necessários mais trabalhos, com um maior número de casos, acompanhamento clínico destes e estudos de prognóstico, para avaliar o significado destes resultados e investigar uma possível relação da alteração do padrão de marcação de caderina-E com a agressividade tumoral. Melanocytic tumours are a common lesion in horses, affecting about 80% of grey-colored coated animals, over 15 years old of age. Although it is a lesion easily diagnosed, its biologic behavior is very unpredictable. Epithelial cadherin is the major component involved on the cellular adhesion between melanocytes and keratinocytes, with an important role as a regulator of melanocytes growth and proliferation. Altered function or expression of this adhesion molecule has been implicated in tumour cell invasion and metastasis formation. The aim of the present study was to analyze, through immunohistochemistry, E-cadherin expression on thirty-eight equine melanocytic tumours, according to its labeling extension, location and intensity. The samples were previously processed and subjected to histopathological analysis, according to the criteria described by WHO. Vascular invasion and other histopathological parameters were also evaluated. Mouse anti-cadherin E monoclonal antibody was used as the primary antibody. E-cadherin was expressed in thirty-six tumours, and twenty-four of these presented a diffuse labelling, with a high number of reactive cells (>75%). The labelling location on the neoplastic cells was either cytoplasmatic (n=21), membranous (n=7) or heterogeneous (n=8). The majority of the tumours with evidences of intravascular invasion (75%) presented a cytoplasmatic labelling. Regarding the staining intensity, the samples got equally distributed through the three different intensity degrees. Aside from its location or intensity, we realized that the deep components of the tumours showed a decreased degree of E-cadherin expression. We found no significant association between E-cadherin expression and none of the histopathological variables evaluated. In summary, E-cadherin was most frequently expressed in equine melanocytic tumours with a cytoplasmatic labelling, thought to be an abnormal finding and a malignancy factor in the referred lesions. Even though it was based on a low sample number, this study represents the first one focused on E-cadherin expression in equine melanocytic tumours. Further investigations should consider a higher number of animals and include a closer monitoring of its clinical evolution, in order to confirm the suggested association between an altered E-cadherin expression and a malignant behavior of these tumours.

Com o objetivo de contribuir à documentação histórica das práticas das artes musicais no contexto cinematográfico europeu, este trabalho analisará de forma breve as transformações ocorridas no cinema italiano desde a chegada dos talkies2 até a década de 1960. Para isso será utilizado como estudo de caso, o trabalho do compositor romano Ennio Morricone juntamente com o Gruppo di Improvvisazione Nuova Consonanza no filme Un tranquillo posto di campagna (1968), dirigido por Elio Petri. Sempre no âmbito músico-fílmico, será igualmente considerado o contexto histórico e social das práticas em cada período citado, que vai dos anos de 1920 aos de 1960, sendo esta última década a de principal interesse neste estudo. Aiming to contribute to the historical documentation of the musical practices in the art of cinema in Europe, it will be analysed in this work the transformations ocurred in Italian cinema from the arrival of the talkies4 until the 1960 decade. In order to do so, it will be useful as a case study the work of roman composer Ennio Morricone along with the Gruppo di Improvvisazione Nuova Consonanza for the film Un tranquillo posto di campagna (1968), directed by Elio Petri. Always in the music-filmic practices context, some light will be shed over the historical and social scopes for each period mentioned – from the 1920’s to the 1960’s – the latter being of the greatest interest for us here.