Molecular determinants of nonaqueous biocatalysis

Dissertation presented to obtain the Ph.D degree in BiochemistryOver the last thirty years, the tremendous biotechnological potential of nonaqueous biocatalysis has boosted research efforts in this area. Numerous studies have tried to elucidate how enzymes work in these nonconventional media and many properties are now well understood. However, when this thesis was initiated, some aspects of this field were poorly characterized at the molecular level. In particular, the molecular determinants of protein-ion interactions, enzyme stability, and molecular memory, are important issues which were lacking a thorough molecular analysis. These three subjects are herein investigated using molecular simulation methodologies.(...

Perfil de emergência em prótese fixa

A procura por um resultado que alie estética e função tem-se tornado uma exigência cada vez maior na área da reabilitação oral. Para ser considerada um sucesso, uma prótese parcial fixa deve atingir um balanço harmónico entre aspetos funcionais, estéticos e biológicos. Obter um padrão estético próximo do natural requer um delicado manuseamento dos tecidos moles, tanto durante os procedimentos cirúrgicos, como no momento da confecção restauração definitiva. O tecido gengival deve fazer parte do planeamento protético e como tal, existem condutas clínicas que possibilitam obter resultados estéticos satisfatórios. Nesta monografia realça-se a finalidade e a importância do condicionamento gengival, bem como técnicas existentes que permitem a transferência do perfil de emergência do pôntico provisório para o definitivo.The search for a result that combines aesthetics and function became an increasing requirement in oral rehabilitation. To be considered a success, a partial fixed prosthesis must achieve a harmonic balance between functional, aesthetic and biological aspects. To get an aesthetic pattern around the natural, requires a delicate handling of soft tissue during surgical procedures, till the moment of making definitive restoration. The gingival tissue should be part of the prosthetic planning and there are pipes that allow dentists to obtain satisfactory aesthetic results. This monograph highlights the purpose and importance of gingival conditioning as well as existing techniques that enable the transfer of the gingival emergence profile from the provisional to the final restoration

Margem muro

Anais do V Encontro de Iniciação Científica e I Encontro Anual de Iniciação ao Desenvolvimento Tecnológico e Inovação – EICTI 2016 - 05 e 07 de outubro de 2016 – Sessão Linguística, Letras e ArtesDurante a sabida trajetória do coletivo de nossa espécie pela extensão da esfera terrestre, poucas manifestações de nossa característica industria se mostraram tão resilientes (e em tamanha cumplicidade) quanto o objeto “muro”, tão pluralmente encarnado, ante as distintas carências humanas — desde seu advento. Suas propriedades plásticas — para além de suas funções basilares — constituem caracteres emblemáticos que sobremaneira transcendem sua palpável existência em significações outras, igualmente relevantes. Pensá-lo enquanto constante — em relação ao sabido desvelar da experiência humana — nos empodera a investigar sua intrínseca relação com os campos da expressão e da arte, tanto nos primórdios de nossa experiência quanto no presente estado de nossa marcha. O projeto presente, portanto, se caracteriza pela investigação aprofundada do fenômeno que, em um momento anterior (nos 6 meses que antecederam a feitura da presente empresa), verificamos em pesquisa de campo: a ocupação do muro por meios artístico-expressivos na região das três fronteiras (Brasil, Argentina e Paraguai). Partindo, por conseguinte, dos questionamentos originados desta primeira experiência, propomos assim uma análise deste objeto com o fito de melhor situar os processos com os quais nos deparamos em campo, nos entornos da fronteira

Translating Breast Cancer Immune Features into Biomarkers and Therapies

Breast cancer remains one of the main causes of cancer-related deaths in women worldwide. In the past years, advances in breast cancer treatment have been made, namely with the introduction of preoperative neoadjuvant chemotherapy (NACT) in selected cases of inflammatory/inoperable or advanced tumors (size larger than 2 cm and/or disease extension to the axillary lymph node). This treatment is effective in reducing the size of the primary tumor, allowing breast conservation. However, less than half of the patients achieve a pathological complete response and residual disease after NACT is a strong predictor of relapse. Hence, it is essential to find a suitable marker of response to this treatment, to promptly direct NACT non-responder patients to alternative therapies. Tumor infiltrating lymphocytes (TILs), specifically CD8+ cytotoxic T cells (CTLs) have been appointed as biomarkers of response. However, the clinical usefulness of this biomarker is still controversial and their evaluation is not yet implemented. This controversy could be explained by the fact that tumor cells can escape the immune system by releasing cytokines or expressing immune checkpoint inhibitors, dampening CTLs activity. HLA-DR, a T cell activation marker, could be a more reliable biomarker of response to NACT, than the presence of CTLs per se, since it may reflect the overall immune status of the tumor microenvironment and their functionality. To get more insights into the immune component of the tumor microenvironment, fresh biopsies, surgical specimens and blood were collected from breast cancer patients. In order to assess the differences between breast cancer aggressiveness, the patients were divided in two groups: the ones that have metastasis in the axillary lymph node and the ones that don’t have. Although the immunophenotype was similar in both groups of patients, a significant difference was observed for the expression level of HLA-DR in CTLs and regulatory T cells (Tregs). Indeed, patients without axillary lymph node metastasis had higher level of HLA-DR in CTLs and lower in Tregs when compared to patients with axillary lymph node metastasis. Given this result, we wondered if this immune trait could be used to predict response to NACT. In biopsies of two independent cohorts of breast cancer patients selected for NACT, we observed that high HLA-DR expression level in CTLs was strongly correlated with good response to NACT, with a high sensitivity (94.12% and 80% in cohort 1 and 2, respectively) and specificity (100% and 85.71% in cohort 1 and 2, respectively). Therefore we propose that HLADR expression level in CTLs above a threshold value, calculated by a ROC curve, would identify patients that would be responders to NACT. Additionally, by multivariable analysis, we noted that HLA-DR expression in CTLs was an independent predictor of response to NACT. Interestingly, HLA-DR expression in CTLs also have the likelihood of being a prognostic marker of breast cancer patients’ outcome, since a progression-free survival analysis revealed that patients with low levels of HLA-DR in CTLs tend to relapse sooner.Moreover, we observed that this immune feature was systemically reflected. Indeed, with the assessment of HLA-DR expression level in circulating CTLs we could differentiate breast cancer aggressiveness and even response to NACT, although in a less striking manner when compared to the analysis of HLA-DR expression level in intratumor CTLs. To characterize HLA-DR+ CTLs, we performed a gene expression as well as a broad surface markers’ expression analysis. When comparing to HLA-DR negative CTLs, HLA-DR+ CTLs had higher expression of cytotoxicity-related molecules Granzyme B, IFN-, Perforin, TNF-, Eomes and lower expression of Tbet. Additionally, they had a high proliferative capacity (high Ki67 levels) and an intermediate level of exhaustion markers, namely PD-1, Tim3 and CD127. These results suggest that HLA-DR+ CTLs had a phenotype closer to effector memory T cells (TEM), which have the capacity to home to tissues and to recirculate, rapidly release effector molecules and differentiate in effector CTLs. Then, we developed a 3D culture platform to shed some light on the functionality of these HLA-DR+ CTLs and their contribution for NACT success. Namely, we used two different breast cancer cell lines (MCF-7 and MDA-MB-231) and allowed them to spontaneously form spheroids. Peripheral blood mononuclear cells (PBMCs) isolated from NACT-responders and non-responders were added to the culture, infiltrating the 3D tumor-like structure. Interestingly, the PBMCs from NACT-responders were able by themselves to reduce the viability of the breast cancer cell line; whereas the PBMCs from NACT non-responders showed no effect. This emphasizes that the immune cells from NACT-responders are activated and have cytotoxic capacities; on the opposite, the immune cells from NACT non-responders are immunosuppressed and cannot exert their cytotoxic function. Furthermore, we added doxorubicin (a NACT agent) to the 3D co-culture and observed that NACT-responders’ PBMCs had a synergistic effect with this drug in decreasing the tumor cells’ viability. On the contrary, the addition of doxorubicin and NACT non-responders’ PBMCs did not alter the viability of the tumor cells. With these in vitro assays we validated the clinical observations. To confirm that the anti-tumor activity of NACT-responders’ PBMCs were indeed due to HLADR+ CTLs, which are more abundant in the blood of these patients than in the blood of NACT non-responders, we took advantage of the 3D system implemented. In fact, sorted HLA-DR+ CTLs, but not HLA-DR negative CTLs, reduce the viability of MCF-7, attesting the cytotoxic capacity and anti-tumor properties of HLA-DR+ CTLs. To investigate if it would be possible to revert the immunosuppressed phenotype of NACT non-responders’ CTLs we stimulated their PBMCs with PMA/ionomycin or by T cell receptor (TCR) engagement. Notably, both stimuli raised the HLA-DR levels in CTLs in NACT nonresponders’ PBMCs allowing these cells to reduce the viability of the breast cancer cells, similarly to NACT-responders’ PBMCs. The observed effect was even more striking with the addition of doxorubicin. This result opens the possibility to develop new treatments for breast cancer patients, whose tumors are not susceptible to NACT regimens alone

Insights into Campylobacter jejuni Desulforubrerythrin catalytic mechanism

Dissertation presented to obtain the Master Degree in Molecular Genetics and BiomedicineThe following work aims to contribute to a better understanding of systems involved in resistance to oxidative stress species, namely hydrogen peroxide. The work is focused in one protein from the pathogen Campylobacter jejuni: desulforubrerythrin. Desulforubrerythrin is a non-heme iron protein in which the catalytic centre harbours a diiron cluster. Besides, the protein has a desulforedoxin domain at the N-terminal and a rubredoxin domain at the C-terminal. With the objective of understanding the protein catalytic mechanism three site-directed mutants, as well the wild type protein, were over expressed in Escherichia coli, purified and studied through biochemical and spectroscopic techniques. The amino acid residues selected for mutations are two tyrosines near the catalytic centre (residues 59 and 127). These residues are strictly conserved in rubrerythrins; moreover in diiron centres containing proteins tyrosines play a role in dissipating oxidizing species of iron (IV) by forming a tyrosil radical. The selected residues were replaced by a phenalanine residue which gave rise to three mutants: Y59F, Y127F and Y59F Y127F. These were characterized having as reference the wild type protein. All proteins have a molecular mass of 24 kDa and are tetramers in solution. The EPR and UV-visible techniques confirmed the presence of the three metallic domains in the wild type and Y59F mutant. The Y127F mutant was successfully used to test a protocol for diiron centre reconstitution in desulforubrerythrin. Finally, crystals of the wild type and, for the first time, of the Y59F and double mutants were obtained. The X-ray data for the mutants were collected with a resolution of 1.9 Å and its structure will be determined

Filtration efficiency of meltblown webs

Dissertação de mestrado em Polymer EngineeringNonwoven fabrics are made from fibers and used across a wide range of applications and products. They are high-tech, innovative and versatile products, indispensable for a lot of application nowadays. Filtration is one of the fastest growing segments in the nonwovens industry, and with the necessity of replacing fiberglass for polymer materials keeping the high filtration efficiency of filter media. The aim of this thesis is to study the effect of different machine settings on filtration efficiency, from polypropylene meltblown webs. The webs were produced by meltblowing in two different processing (A and B), with two different dies configuration, for each one it was varied air flow, air temperature and distance-to-collector (DCD). After collecting samples, they were characterized by basis measures (weight and thickness) and SEM pictures. These information was used for 3D modelling software to obtain the results. Due to problems in processing B, the web samples were obtained with plenty of defects, and it was not possible take clear conclusions about the use of different dies. Conclusions of this project shows highest filtration efficiency results were achieved with higher air flow, higher air temperature and smaller DCD

Sensores em fibra ótica baseados numa fibra de núcleo oco quadrado

In the current work, the development of optical fiber sensors based on an antiresonant hollow core fiber was aimed. The sensing structures were developed in a transmission configuration enhancing two antiresonance propagations, along with the multimode interference and Mach-Zehnder interference. With the objective of using these interferometric components as sensing elements, the sensor was employed in both liquid and air media. Initially, a numerical analysis was addressed to both internal and external antiresonances. A simulation on the effective refractive index of the fundamental core mode was also carried out, achieving results that are in good agreement with both numerical and theoretical models. Furthemore, a comparison between the expected and experimental transmission spectra was established observing, in an overall view, similar modulations. In a posterior stage, a characterization of the hollow square core fiber sensor was performed in glucose aqueous solutions. The sensor response was studied to variations in the refractive index, and calibrated to the wavelength range of the optical source utilized. Moreover, the influence of the sensor length was studied, observing the existance of an optimum length where the maximum sensitivity is enhanced. A temperature independent refractive index detection was also established. Still in the framework of this theme, and with the objective of monitoring the evaporation profile of volatile organic compounds, the sensor was embedded in ethanol aqueous solutions. The monitoring of the sensor response towards the solutions was carried out, allowing to successfully attain a real time variation of the ethanol concentration. Finally, the sensor was characterized to different physical parameters in an air environment, where two broadbands were used. In the first band, 1530nm - 1610nm, a characterization to the temperature, curvature, and strain was performed for the Mach-Zehnder interference, external resonance, and multimode interference. In the second broadband, 900nm - 1300nm, the external and internal resonances were characterize to both temperature and curvature. In both cases, the simultaneous measurement of the parameters under study was also proposed. The studied inline sensor revealed to be highly promising in the several experiments that were conducted, where, in many cases, stood out from other sensors that were already reported in the literature. Its reduced dimensions, high robustness, and capability to simultaneously measure different parameters, and making a temperature discrimination, without needing a complex design configuration, makes it extremely viable in the implementation on several applications.No presente trabalho pretendeu-se explorar o uso de uma fibra de núcleo oco antirressonante, como elementos sensores. Estes dispositivos foram desenvolvidos com uma configuração em transmissão, sendo a propagação efetuada através de duas antirressonâncias. Esta permitiu também o aparecimento de interferência multimodal e de Mach-Zehnder. Com o intuito de usar estas componentes interferométricas como elementos sensores, o dispositivo foi empregue tanto em meios líquidos, como no ar. Posteriormente, foi elaborada uma caracterização ao sensor de fibra de núcleo oco quadrado em soluções aquosas de glucose. A resposta deste foi estudada a variações do índice de refração, e calibrada à região espetral da fonte ótica utilizada. Além disso, foi estudada a influência do comprimento do sensor, tendo-se observado que existe um comprimento para o qual a sensibilidade atingida é otimizada. Adicionalmente, foi elaborada uma caracterização ao índice de refração com compensação da temperatura. Ainda no contexto da análise em meios líquidos, o sensor foi imergido em soluções aquosas de etanol, com o intuito de monitorizar o perfil de evaporação de compostos orgânicos voláteis. Efetuou-se uma monitorização da resposta espetral do sensor relativamente às soluções, o que permitiu obter, de forma satisfatória, uma variação em tempo real da concentração de etanol. Finalmente, realizou-se uma caracterização do sensor a diferentes parâmetros físicos, tendo-se usado duas bandas espetrais distintas. Na primeira banda, 1530nm − 1610nm, caracterizou-se a interferência de Mach-zehnder, a ressonância externa e a interferência multimodal a variações da temperatura, curvatura e tensão. Na segunda banda, 900nm − 1300nm, caracterizou-se as ressonâncias externa e interna a variações da temperatura e curvatura. Em ambos os casos, a medição simultânea dos parâmetros em estudo foi também proposta. O sensor desenvolvido revelou-se altamente promissor nas várias experiências realizadas, onde, em muitas situações, se destacou de outros sensores já reportados. Por ser um dispositivo que apresenta reduzidas dimensões, elevadas robustez, e capaz de medir simultaneamente a vários parâmetros, sem recorrer a configurações complexas, a sua implementação poderá vir ter impacto em diversas aplicações.Mestrado em Engenharia Físic

Assessing Geriatric Attitudes among Medical Students

Introduction: Geriatrics, defined as the medical specialty that studies and treats the elderly population has been assuming an increasing importance. Bearing in mind that the geriatric population occupies a high rate of consumption of health care and knowing that health professionals are the ones who most need to provide the best possible care, there is an urgent need to evaluate their attitudes toward the elderly and infer how these same attitudes interfere with the provision of care. The main objective of this study is to adapt the “UCLA Geriatric Attitudes Scale” in Portuguese to medical students so that it can later be applied to other health professionals in the most varied contexts. Our second objective was the evaluation, through the application of “UCLA Geriatrics Attitude Scale” of the changes, or absence thereof, of the attitudes of students from the 3rd to the 5th years of the Integrated Master’s in Medicine towards the geriatric population. Methods: The eligible population to be studied was composed of 172 students of the 3rd year, whose response rate was 39.53% (68 responses) and 166 students of the 5th year with a response rate of 36.14% (60 responses). Therefore, a total of 128 responses were obtained, for a total of 338 students. After informed consent, the sample responded to a scale that had been previously translated by certified professionals after authorization by the authors. After the application phase of the research questionnaires, the data collected were processed using the software Statistical Package for the Social Science (SPSS), version 26 with a significance level of 0.05. Results: Bearing in mind that higher scores correspond to better attitudes, the following results were obtained: the UCLA-GA scale total median score did not differ between academic years (p=.903), sexes (p=.124) or between students with and without close contact with elderly (p=.070) but was higher in single students comparing with married ones (p=.002). Only the students’ current average grade was a significant predictor, with the UCLA-GA score increasing an average of 0.8 points per each value in the student average grade (ß=0,847; 95% CI 0,201-1,492; p=.011). Conclusion: Considering the growing panorama of an aging population, measures should be implemented to study and to improve attitudes towards older people. Globally, the population sample studied shows very favorable attitudes from the students towards the geriatric population

Optimatization of hybrid renewable energy systems on isolated microgrids : a smart grid approach

Tese de doutoramento, Sistemas Sustentáveis de Energia, Universidade de Lisboa, Faculdade de Ciências, 2016The energy systems of small isolated communities face great challenges related to their autonomy and resilience, when looking for a sustainable energy future. Hybrid renewable energy systems, composed from different technologies, partially or totally renewable, potentiates a growing security of supply for these isolated micro-communities. Moreover, with a smart grid approach, the possibility to reschedule part of the electricity load is seen as a promising opportunity to delay further investments on the grid’s power capacity, enabling a better grid management, through peak load control, but also to promote a more efficient use of endogenous resources, maximizing renewable penetration. To identify the micro-communities main energy challenges, a literature review was taken, reporting the design and implementation of isolated hybrid renewable energy systems. Since electricity and heat energy vectors can be, in part, assured by endogenous resources, a methodology to optimize demand response on isolated hybrid renewable energy systems was developed, using the electric backup of solar thermal systems for domestic hot water supply as flexible loads. This approach is intended to increase energy efficiency of the energy system, reducing grid operation costs and associated CO2 emissions. A model of the electric impact of the implementation of solar thermal systems and heat pumps for domestic hot water supply was developed and tested for the Corvo Island case study, a small and isolated microgrid, located in the mid-Atlantic with around 400 inhabitants and a diesel power plant. An impact of 60% on peak load and 7% on annual electricity demand was found. In order to tackle this significant impact in the grid, a model for optimizing the economic dispatch of the island was developed, testing multiple demand response approaches to the backup loads, from heuristics to genetic algorithms, having this last one performed best to control the peak load and minimize the operation costs. Nonetheless, there was the need to compare and validate the demand response optimization strategies of this developed model with other available modeling tools, which in the end presented similar results. As the pillar of this thesis is the optimization of hybrid renewable energy systems, the influence of the uncertainties associated to renewables forecast had to be studied, in particular its impact on the demand response scheduling. Wind uncertainties demonstrated to have a greater impact on the grid than the solar ones. Finally, the methodology developed incrementally along the thesis and validated in Corvo Island, was tested on different scales and types of isolated systems. It demonstrated to be especially suitable for small systems with less than 20 MW power installed and over 25% renewable generation, with mostly residential load profiles