Estimation of infection rate in epidemic models with multiple populations

Dissertação para obtenção do Grau de Mestre em Matemática e Aplicações Especialização em Actuariado, Estatística e Investigação Operacional

Linear amplification with multiple nonlinear devices

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e Computadores

Conception and first results of a cross-sectional national study on the demography, disease characteristics and socioeconomics status of the Portuguese patients with multiple sclerosis : the PORT-MS study

RESUMO: Introdução e objetivos: Não existia um estudo multicêntrico que descrevesse as características dos doentes com EM, da doença em si, ou do seu tratamento, em Portugal.Métodos: Doentes McDonald 2010 positivos foram sequencialmente recrutados em 7 centros entre Maio e Novembro 2014. Aplicou-se um Caderno de Recolha de Dados incidindo na demografia, doença, educação e emprego (estudo PORT-MS). Resultados: 561 doentes incluídos. Primeiros sintomas aos 30,2±10,5 anos (RRMS 29,2±10, PPMS 39,4±11,7, p<0,001); diagnóstico 3,2±5,3 anos depois (RRMS 3,0±5,1, PPMS 4,9±2,5, p=0,002); tempo de doença após diagnóstico 9,4±7,2 anos (semelhante RRMS no diagnóstico e PPMS); idade atual 42,9±12,4 anos (grupo RRMS no diagnóstico 42,0±12,1, PPMS 52,5±11,3, p<0,001); EDSS atual 2,5 (RRMS 2.0, PPMS 6.0); proporção feminino:masculino é 2,5:1 (RRMS semelhante, PPMS 1,1:1, p<0,05); no diagnóstico RRMS 90,6%, SPMS 0,9%, PPMS 8,6%; 9,5% dos RRMS encontravam-se em SP na inclusão (nomeadamente os com mais idade no diagnóstico e/ou atualidade ou tempo de doença mais prolongado). PPMS mais frequente em doentes diagnosticados mais tardiamente (p<0,001), onde aumenta também ligeiramente a proporção de mulheres na PPMS. Nas últimas décadas: novos casos mostram estabilidade na proporção de géneros e tipos de doença; idade nos primeiros sintomas e no diagnóstico aumentou ligeiramente, tempo entre eles diminuiu ligeiramente. Proporção sob DMT (Maio 2014): global 84,5%; atualmente RRMS 90,4%; SPMS 70,8%; PPMS 36,8%; progressivas agregadas 48%. Tipo de DMT, amostra global: interferões 56,5%, GA 18,4%, Natalizumab 11,6%, Fingolimod 9,7%. Global: economicamente ativos 61,5%, desemprego 13,5%, 74,1% dos não activos estão reformados por doença. Gravidezes após diagnóstico em 15% mulheres. Casos com história familiar positiva 7,8%. Discussão e conclusões: Incluída cerca de 10% da população portuguesa. Resultados congruentes com dados internacionais. Elevada proporção sob DMT, mesmo EDSS alto e formas progressivas. Terapêuticas de segunda linha sub representadas. Doentes jovens e com doença ligeira com vida económica ativa; restantes essencialmente reformados por doença.---------------- ABSTRACT : Background/aims: In Portugal, there wasn’t a multicentric study on the general characteristics (demography, disease milestones, DMT, socioeconomic status) of Multiple Sclerosis patients. Methods: Patients fulfilling McDonald 2010 criteria were sequentially recruited from May to November 2014 in 7 centers and data was systematically collected. Results: 561 patients included. First symptoms occurred at 30,2±10,5 years-old (RRMS 29,2±10, PPMS 39,4±11,7, p<0,001); diagnosis 3,2±5,3 years later (RRMS 3,0±5,1, PPMS 4,9±2,5, p=0,002); 9,4±7,2 years elapsed since diagnosis (similar for those is RRMS at diagnosis and PPMS); current age 42,9±12,4 years-old (group RRMS at diagnosis 42,0±12,1, PPMS 52,5±11,3, p<0,001); current EDSS 2,5 (RRMS 2.0, PPMS 6.0); females to males 2,5:1 (RRMS similar, PPMS 1,1:1, p<0,05); at diagnosis RRMS 90,6%, SPMS 0,9%, PPMS 8,6%; 9,5% of RRMS reached SP at inclusion (those older at diagnosis, in actuality, or with longer follow-up). PPMS more frequente in patients diagnosed at older ages (p<0,001), also slight increase in females. Along the last decades: new cases have showed stable proportions of gender and disease types; age at first symptoms and diagnosis slightly increased, time between them slightly decreased. Proportion on DMT (May 2014): 84,5% of all; 90,4% of currently in RRMS; 70,8% of SPMS; 36,8% of PPMS; 48% of progressive forms together. Type of DMT, all patients: interferons 56,5%, Glatiramer Acetate 18,4%, Natalizumab 11,6%, Fingolimod 9,7%. Economically active 61,5% of all, unemployment 13,5%, 74,1% of non-active are retired due to disease. Females pregnant after diagnosis 15%. Positive family cases in 7,8%. Discussion/Conclusions: 10% of the national MS population collected. Data generally consistente with international reports. Proportion under DMT relatively high in all disease types, but second line therapies underrepresented. Young patients with mild disease have an active economic life. Those not active are essentially retired due to disease.

What Fidelidade group should do to convey its human centric brand identity values to millennial insurance consumers

Companies are concerned in attracting and retaining Millennial consumers, especially if their relation with this target audience is weak. This happens in the insurance industry in Portugal and in Fidelidade group specifically. The aim of this study is to recommend a strategy for the insurance group to improve its relationship with these consumers, by conveying its human centric values. In order to address this goal, we developed a qualitative research. The main insight is that Millennials may perceive those values in the industry but do not associate them with insurance brands.

Fatigue life assessment of endodontic instruments subjected to multi planar curvature

This present study aimed to investigate the fatigue life of unused (new) endodontic instruments made of NiTi with control memory by Coltene™ and subjected to the multi curvature of a mandibular first molar root canal. Additionally, the instrument‟s structural behaviour was analysed through non-linear finite element analysis (FEA). The fatigue life of twelve Hyflex™ CM files was assessed while were forced to adopt a stance with multiple radius of curvature, similar to the ones usually found in a mandibular first molar root canal; nine of them were subjected to Pecking motion, a relative movement of axial type. To achieve this, it was designed an experimental setup with the aim of timing the instruments until fracture while worked inside a stainless steel mandibular first molar model with relative axial motion to simulate the pecking motion. Additionally, the model‟s root canal multi-curvature was confirmed by radiography. The non-linear finite element analysis was conducted using the computer aided design software package SolidWorks™ Simulation, in order to define the imposed displacement required by the FEA, it was necessary to model an endodontic instrument with simplified geometry using SolidWorks™ and subsequently analyse the geometry of the root canal CAD model. The experimental results shown that the instruments subjected to pecking motion displayed higher fatigue life values and higher lengths of fractured tips than those with only rotational relative movement. The finite element non-linear analyses shown, for identical conditions, maximum values for the first principal stress lower than the yield strength of the material and those were located in similar positions to the instrument‟s fracture location determined by the experimental testing results.

Analysis of outcome and cancer stem cells status in patients with resectable pancreatic adenocarcinoma

RESUMO: Actualmente, a única possibilidade de cura para doentes com adenocarcinoma do pâncreas (PDAC) é a ressecção cirúrgica, no início deste estudo, perguntamo-nos se os predictores clínico-patológicos clássicos de prognostico poderiam ser validados em uma grande cohort de doentes com cancro do pâncreas ressecável e se outros predictores clínicos poderiam ter um papel na decisão de que doentes beneficiariam de ressecção cirúrgica. No capítulo 2, observamos que até 30% dos doentes morrem no primeiro ano após a ressecção cirúrgica, pelo que o nosso objectivo foi determinar factores pré-operatórios que se correlacionam com mortalidade precoce após ressecação cirúrgica com recurso a um instrumento estatisticamente validado, o Charlson-Age Comorbidity Index (CACI), determinamos que um CACI score superior a 4 foi preditivo de internamentos prolongados (p <0,001), complicações pós-operatórias (p = 0,042), e mortalidade em 1 ano pós- ressecção cirúrgica (p <0,001). Um CACI superior a 6 triplicou a mortalidade no primeiro ano pós-cirurgia e estes doentes têm menos de 50% de probabilidade de estarem vivos um ano após a cirurgia. No capítulo 3, o nosso objectivo foi identificar uma proteína de superfície que se correlacionasse estatisticamente com o prognostico de doentes com adenocarcinoma do pâncreas e permitisse a distinção de subgrupos de doentes de acordo com as suas diferenças moleculares, perguntamo-nos ainda se essa proteína poderia ser um marcador de células-estaminais. No nosso trabalho anterior observamos que as células tumorais na circulação sanguínea apresentavam genes com características bifenotípica epitelial e mesenquimal, enriquecimento para genes de células estaminais (ALDH1A1 / ALDH1A2 e KLF4), e uma super-expressão de genes da matriz extracelular (colagénios, SPARC, e DCN) normalmente identificados no estroma de PDAC. Após a avaliação dos tumores primários com RNA-ISH, muitos dos genes identificados, foram encontrados co-localizando em uma sub-população de células na região basal dos ductos pancreáticos malignos. Além disso, observamos que estas células expressam o marcador SV2A neuroendócrino, e o marcador de células estaminais ALDH1A1/2. Em comparação com tumores negativos para SV2, os doentes com tumores SV2 positivos apresentaram níveis mais baixos de CA 19-9 (69% vs. 52%, p = 0,012), tumores maiores (> 4 cm, 23% vs. 10%, p = 0,0430), menor invasão de gânglios linfáticos (69% vs. 86%, p = 0,005) e tumores mais diferenciados (69% vs. 57%, p = 0,047). A presença de SV2A foi associada com uma sobrevida livre de doença mais longa (HR: 0,49 p = 0,009) bem como melhor sobrevida global (HR: 0,54 p = 0,018). Em conjunto, esta informação aponta para dois subtipos diferentes de adenocarcinoma do pâncreas, e estes subtipos co-relacionam estatisticamente com o prognostico de doentes, sendo este subgrupo definido pela presença do clone celular SV2A / ALDH1A1/2 positivo com características neuroendócrinas. No Capítulo 4, a expressão de SV2A no cancro do pâncreas foi validado em linhas celulares primárias. Demonstramos a heterogeneidade do adenocarcinoma do pâncreas de acordo com características clonais neuroendócrinas. Ao comparar as linhas celulares expressando SV2 com linhas celulares negativas, verificamos que as linhas celulares SV2+ eram mais diferenciadas, diferindo de linhas celulares SV2 negativas no que respeita a mutação KRAS, proliferação e a resposta à quimioterapia. No capítulo 5, perguntamo-nos se o clone celular SV2 positivo poderia explicar a resistência a quimioterapia observada em doentes. Observamos um aumento absoluto de clones celulares expressando SV2A, em múltiplas linhas de evidência - doentes, linhas de células primárias e xenotransplantes. Embora, tenhamos sido capazes de demonstrar que o adenocarcinoma do pâncreas é uma doença heterogénea, consideramos que a caracterização genética destes clones celulares expressando SV2A é de elevada importância. Pretendemos colmatar esta limitação com as seguintes estratégias: Após o tratamento com quimioterapia neoadjuvante na nossa coorte, realizamos microdissecação a laser das amostras primarias em parafina, de forma a analisar mutações genéticas observadas no adenocarcinoma pancreático; em segundo lugar, pretendemos determinar consequências de knockdown da expressão de SV2A em nossas linhas celulares seguindo-se o tratamento com gemicitabina para determinação do papel funcional de SV2A; finalmente, uma vez que os nossos esforços anteriores com um promotor - repórter e SmartFlare ™ falharam, o próximo passo será realizar RNA-ISH PrimeFlow™ seguido de FACS e RNA-seq para caracterização deste clone celular. Em conjunto, conseguimos provar com várias linhas de evidência, que o adenocarcinoma pancreático é uma doença heterogénea, definido por um clone de células que expressam SV2A, com características neuroendócrinas. A presença deste clone no tecido de doentes correlaciona-se estatisticamente com o prognostico da doença, incluindo sobrevida livre de doença e sobrevida global. Juntamente com padrões de proliferação e co-expressão de ALDH1A1/2, este clone parece apresentar um comportamento de células estaminais e está associado a resistência a quimioterapia, uma vez que a sua expressão aumenta após agressão química, quer em doentes, quer em linhas de células primárias.----------------------------- ABSTRACT: Currently, the only chance of cure for patients with pancreatic adenocarcinoma is surgical resection, at the beginning of my thesis studies, we asked if the classical clinicopathologic predictors of outcome could be validated in a large cohort of patients with early stage pancreatic cancer and if other clinical predictors could have a role on deciding which patients would benefit from surgery. In chapter 2, we found that up to 30% of patients die within the first year after curative intent surgery for pancreatic adenocarcinoma. We aimed at determining pre-operative factors that would correlate with early mortality following resection for pancreatic cancer using a statistically validated tool, the Charlson-Age Comorbidity Index (CACI). We found that a CACI score greater than 4 was predictive of increased length of stay (p<0.001), post-operative complications (p=0.042), and mortality within 1-year of pancreatic resection (p<0.001). A CACI score of 6 or greater increased 3-fold the odds of death within the first year. Patients with a high CACI score have less than 50% likelihood of being alive 1 year after surgery. In chapter 3 we aimed at identifying a surface protein that correlates with patient’s outcome and distinguishes sub-groups of patients according to their molecular differences and if this protein could be a cancer stem cell marker. The most abundant class of circulating tumor cells identified in our previous work was found to have biphenotypic features of epithelial to mesenchymal transition, enrichment for stem-cell associated genes (ALDH1A1/ALDH1A2 and KLF4), and an overexpression of extracellular matrix genes (Collagens, SPARC, and DCN) normally found in the stromal microenvironment of PDAC primary tumors. Upon evaluation of matched primary tumors with RNA-ISH, many of the genes identified were found to co-localize in a sub-population of cells at the basal region of malignant pancreatic ducts. In addition, these cells expressed the neuroendocrine marker SV2A, and the stem cell marker ALDH1A1/2. Compared to SV2 negative tumors, patients with SV2 positive tumors were more likely to present with lower CA 19-9 (69% vs. 52%, p = 0.012), bigger tumors (size > 4 cm, 23% vs. 10%, p= 0.0430), less nodal involvement (69% vs. 86%, p = 0.005) and lower histologic grade (69% vs. 57%, p = 0.047). The presence of SV2A expressing cells was associated with an improved disease free survival (HR: 0.49 p=0.009) and overall survival (HR: 0.54 p=0.018) and correlated linearly with ALDH1A2. Together, this information points to two different sub-types of pancreatic adenocarcinoma, and these sub-types correlated with patients’ outcome and were defined by the presence of a SV2A/ ALDH1A1/2 expressing clone with neuroendocrine features. In Chapter 4, SV2A expression in cancer was validated in primary cell lines. We were able to demonstrate pancreatic adenocarcinoma heterogeneity according to neuroendocrine clonal features. When comparing SV2 expressing cell lines with SV2 negative cell lines, we found that SV2+ cell lines were more differentiated and differ from SV2 negative cell lines regarding KRAS mutation, proliferation and response to chemotherapy. In Chapter 5 we aimed at determining if this SV2 positive clone could explain chemoresistance observed in patients. We found an absolute increase in SV2A expressing cells, with multiple lines of evidence, in patients, primary cell lines and xenografts. Although, we have been able to show evidence that pancreatic adenocarcinoma is a heterogeneous disease, our findings warrant further investigation. To further characterize SV2A expressing clones after treatment with neoadjuvant chemotherapy in our cohort, we have performed laser capture microdissection of the paraffin embedded tissue in this study and will analyze the tissue for known genetic mutations in pancreatic adenocarcinoma; secondly, we want to know what will happen after knocking down SV2A expression in our cell lines followed by treatment with gemcitabine to determine if SV2A is functionally important; finally, since our previous efforts with a promoter – reporter and SmartFlare™ have failed, we will utilize a novel PrimeFlow™ RNA-ISH assay followed by FACS and RNA sequencing to further characterize this cellular clone. Overall our data proves, with multiple lines of evidence, that pancreatic adenocarcinoma is a heterogeneous disease, defined by a clone of SV2A expressing cells, with neuroendocrine features. The presence of this clone in patients’ tissue correlates with patient’s disease free survival and overall survival. Together with patterns of proliferation and ALDH1A1/2 co-expression, this clone seems to present a stem-cell-like behavior and is associated with chemoresistance, since it increases after chemotherapy, both in patients and primary cell lines.

Contributos para a avaliação económica de medicamentos em Portu

RESUMO: A importância da avaliação económica na área da saúde tem sido crescente ao longo das últimas três décadas. É um campo de análise multi-disciplinar pelo qual se têm vindo a interessar investigadores de todo o mundo e onde, naturalmente, a economia da saúde tem contributos significativos para dar. Contudo, é uma área ainda relativamente inexplorada em termos de investigação metodológica em Portugal. Espera-se com este trabalho colmatar algumas das lacunas existentes e lançar pontes para investigação futura. Neste estudo pretendem-se desenvolver contributos autónomos, numa óptica de resolução de problemas concretos, para a avaliação económica de medicamentos em Portugal. Em primeiro lugar abordou-se a importância da estimação dos custos nos estudos de avaliação económica em saúde. Em segundo lugar determinou-se o custo de um doente com esclerose múltipla, por nível de severidade em Portugal com recurso à metodologia de estudos custo da doença. Em terceiro lugar, aplicando a mesma metodologia, determinou-se o custo de um doente com psoríase, por nível de severidade, em Portugal. Em quarto lugar mediu-se o impacto da psoríase na qualidade de vida relacionada com a saúde dos indivíduos afectados através de dois instrumentos genéricos de medição da qualidade de vida relacionada com a saúde (SF-36 e EQ-5D) e de dois instrumentos específicos (DLQI e PDI). Em quinto lugar, foi feita a adaptação de um modelo sobre a prevenção do tromboembolismo venoso em cirurgia ortopédica ao contexto nacional com recurso a dados recolhidos numa amostra de hospitais portugueses. Foi ainda apresentada uma estimativa do impacto orçamental decorrente da inclusão, ou não, de um novo medicamento na prática nacional. ABSTRACT: The importance of economic evaluation in the health care sector has been growing in the last three decades. It is a multidisciplinary field of research that attracts attention from researchers working all over the world. It is should be noted that health economics can contribute significantly to the development of this area. However, in Portugal is difficult to find methodological contributes in this field of research. With this work one hope to be able to overcome some of the existing shortcomings. This study seeks to develop independent contributions for the economic evaluation of pharmaceuticals in Portugal, aiming to help to solve existing problems. Firstly, one approached the importance of the accurate estimation of costs in economic evaluations of pharmaceuticals. Secondly, the cost of patient with multiple sclerosis was estimated, by severity level, in Portugal, using a cost of illness methodology. In third place, employing the same methodology, the cost of patient with psoriasis by severity level was estimated for Portugal. In fourth place, the impact of psoriasis in the quality of life related to health was ascertained by two generic measures (SF-36 and EQ-5D) and two specific measures (DLQI and PDI). Lastly, a model on the prevention of venous thromboembolism in orthopedic surgery was adapted for Portugal through the inclusion of data collected in a sample of Portuguese hospita

Immune reconstitution after autologous hematopoietic stem cell transplantation

Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the B–T cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkin’s lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relações entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreensão deste complexo sistema biológico. Tal é particularmente verdade no caso das interacções entre os linfócitos B e T, quer durante o desenvolvimento celular, quer ao nível das funções celulares efectoras. A compreensão da interdependência entre linfócitos B e T e a possibilidade de manipular esta relação pode ser directamente aplicável a situações em que a imunidade está deficiente, como é o caso das doenças neoplásicas ou da imunossupressão após radio ou quimioterapia. O trabalho apresentado nesta dissertação iniciou-se com o desenvolvimento de um novo método laboratorial para medir directamente a diversidade do reportório celular (Capítulo III). Reduções da diversidade do reportório dos receptores de células T têm sido relacionadas com um estado de imunodeficiência. O método desenvolvido utiliza “gene chips”, aos quais hibridizam os ácidos nucleicos codificantes das cadeias proteicas dos receptores linfocitários. A diversidade é calculada com base na frequência de hibridização do ácido nucleico da amostra aos oligonucleótidos presentes no “gene chip”. De seguida, e utilizando este novo método e outras técnicas de quantificação celular examinei, num modelo animal, o papel que as células policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitário T no timo, especificamente na aquisição de um reportório diverso de receptores T (Capítulos IV e V). Testei, então, a hipótese de que a presença no timo de péptidos mais diversos, como a imunoglobulna policlonal, induzisse a génese de precursores T mais diversos. Demonstrámos que a diversidade do compartimento T é aumentado pela presença de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molécula, representam as moléculas autólogas mais diversas presentes nos organismos vertebrados. Estes péptidos são apresentados por células apresentadoras de antigénio às células precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a génese da diversidade dos receptores. Também demonstrámos que a presença de um reportório mais diverso de linfócitos T se associa a um incremento da função imunológica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeição mais eficientes de um maior número de agressores internos e externos. Demonstrámos que ratinhos com receptores de células T (RCT) com maior diversidade rejeitam transplantes cutâneos discordantes para antigénios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportório T (Capítulo V). Por outro lado, uma redução da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivência de transplantes cutâneos incompatíveis para o antigénio H-Y (antigénio minor de histocompatibilidade), indicando uma diminuição da função linfocitária T. Além disso, a reconstituição da diversidade dos linfócitos T em ratinhos com uma diversidade de reportório T diminuída, induzida pela administração de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuição significativa da sobrevivência dos enxertos cutâneos (Capítulo V). Estes resultados sugerem que o aumento do reportório de células T contribui para uma melhoria das funções celulares T e poderão ter implicações importantes na terapêutica e reconstitutição imunológica em contexto de SIDA, neoplasias, autoimunidade e após tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hipótese clínica de que doentes com neoplasias hematológicas sujeitos a transplantação de precursores hematopoiéticos e com recuperação imunológica precoce após transplante teriam uma sobrevivência mais longa do que doentes que não recuperassem tão bem a sua imunidade. Analisámos a sobrevivência global e sobrevivência sem doença de 42 doentes com linfoma não Hodgkin de células do manto sujeitos a transplante autólogo de precursores hematopoiéticos (Capítulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfócitos imediatamente após o transplante autólogo, apresentaram uma sobrevivência global e sem progressão mais longa do que doentes que não recuperaram contagens linfocitárias tão precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutição imunológica robusta após transplante de presursores hematopoiéticos, sobre a sobrevivência de doentes com neoplasias hematológicas. Do mesmo modo, estudámos o efeito que a recuperação de níveis séricos normais de imunoglobulina policlonal tem na sobrevivência de doentes com outras neoplasias hematológicas de linfócitos B, como o mieloma múltiplo,após transplante autólogo de precursos hematopoiéticos (Capítulo VII). A sobrevivência livre de doença dos 110 doentes com mieloma múltiplo analizados está associada com a sua capacidade de recuperar níveis séricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importância da imunoglobulina policlonal para a génese de competência imunológica. Também estudámos o impacto de um sistema imunitário eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma não Hodgkin (LNH) (Capítulo VIII). Os resultados mostram que doentes com valores mais elevados de linfócitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doença) ao rituximab, do que doentes com valores mais baixos. Estas observações ilustram a necessidade de um sistema imunitário competente para o benefício clínico da terapêutica com rituximab em doentes com LNH. Em conclusão, o trabalho apresentado nesta dissertação demonstra que as células B e a imunoglobulina policlonal promovem a diversidade das células T no timo e melhoram a função linfocitária T periférica. Concomitantemente, também demonstrámos que, no contexto de reconstituição imune, por exemplo, após transplante autólogo de precursores hematopoiéticos em doentes com linfomas de células do manto, o número absoluto de linfócitos é uma factor independente da sobrevivência. Os resultados demonstram, também, a importância dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princípio se prova pelo facto de que doentes com mieloma múltiplo sujeitos a transplante autólogo de precursores hematopoiéticos que recuperam valores normais séricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparação com doentes que não recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicações na prática clínica dado que a maioria dos tratamentos de doenças neoplásicas implica imunossupressão e, subsequente, recuperação imunológica. Estes estudos podem ser um instrumento fundamental para uma melhor compreensão do sistema imune e guiar uma escolha mais eficiente de opções terapêuticas bem como contribuir para a concepção de futuros estudos clínicos.

A utilização de actos complementares de diagnóstico: uma análise económica

Dissertação submetida para obtenção do grau de Doutor em Saúde Pública Especialidade de Economia da Saúde

Atherogenesis and atherosclerosis in primary antiphospholipid syndrome

ABSTRACT: In the late seventies the term “Haematological Stress Syndrome” defined some haematological abnormalities appearing in the course of acute and chronic disorders, such as raised plasma levels of fibrinogen (FNG) and factor VIII, reduced fibrinolytic activity and hyperviscosity. In the early nineties the “Membrane stress syndrome hypothesis” proposed the unification of the concepts of haematological stress syndrome with those of oxidation, inflammation and immune activation to explain the pathogenesis of the antiphospholipid syndrome (APS) Antiphospholipid antibodies, coagulation, fibrinolysis and thrombosis. This chapter investigated the occurrence of the “Haematological Stress Syndrome” and thrombosis in 144 participants positive for aPL detected by clotting and immune tests. Among the clotting assays for the detection of lupus anticoagulant, dilute Russell's viper venom time better correlated with a history of venous thrombosis than activated partial thromboplastin time (p<0.0002 vs p<0.009) and was the only test correlated with a history of arterial thrombosis (p<0.01). By regression analysis, serum levels of IgG anticardiolipin antibodies (aCL) associated with the number of venous occlusions (p<0.001). With regards to FNG and von Willebrand factor (vWF), the former rose by 36% (95% CI; 21%, 53%) and the latter by 50% (95% CI; 29%, 75%) at the first venous occlusion and remained unchanged after subsequent occlusions. At variance FNG rose by 45% (95% CI; 31%, 60%) per arterial occlusion and vWF by 27% (95% CI; 10%, 47%) per arterial occlusion throughout. The coagulation/fibrinolytic balance was cross-sectionally evaluated on 18 thrombotic PAPS patients, 18 subjects with persistence of idiopathic aPL and in healthy controls. Markers of thrombin generation prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and of fibrin turnover D-Dimer (D-D) were higher in thrombotic (p=0.006)and non-thrombotic subjects (p=0.0001) than in controls as were those of D-D (p<0.0001 and p=0.003 respectively). TAT levels did not differ. Gender analysed data revealed blunted tPA release (hence a negative venous occlusion test) in thrombotic females but neither in thrombotic males (p=0.01) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p<0.0002) and control females (p<0.02). The activity of factor XIII (FXIIIa) was evaluated was evaluated in 29 patients with PAPS, 14 persistent carriers of aPL without thrombosis, 24 thrombotic patients with inherited thrombophilia, 28 healthy controls and 32 patients with mitral and aortic valve prosthesis as controls for FXIII only. FXIIIa was highest in PAPS (p=0.001), particularly in patients with multiple (n=12) than single occlusion (p=0.02) and in correlation with PAI (p=0.003) and FNG (p=0.005). Moreover FXIIIa was strongly associated with IgG aCL and IgG anti-2GPI (p=0.005 for both) in the PAPS group and to a lesser degree in the aPL group (FXIIIa with IgG aCL, p=0.02, with IgG anti-2GPI, p=0.04). Altogether these results indicate: 1) a differential relationship of aPL, vWF and FNG with venous and arterial thrombosis; 2) heightened thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs; 3) enhanced FXIIIa that may contribute to atherothrombosis via increased fibrin/fibrinogen cross-linking. Lipid profile, lipid peroxidation and anti-lipoprotein antibodies in thrombotic primary antiphospholipid syndrome. Given the atherogenic lipid profile of SLE, the same possibility was explored in PAPS by comparing high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol (CHO), apolipoprotein AI (ApoAI), apolipoprotein B (ApoB), triglycerides (TG), anti-lipoprotein antibodies, beta-2-glycoprotein I complexed to oxidized low-density lipoprotein (oxLDL-2GPI) and C-reactive protein (CRP) in 34 thrombotic PAPS patients compared to 36 thrombotic patients with inherited thrombophilia (IT), to 18 subjects persistently positive for antiphospholipid antibodies (aPL) with no underlying autoimmune or non-autoimmune disorders and to 28 healthy controls. Average concentrations of HDL (p<0.0001), LDL (p<0.0001), CHO (p=0.0002), ApoAI (p=0.002) were lower in PAPS whereas average TRY was higher (p=0.01) than other groups. Moreover PAPS showed higher IgG anti-HDL (p=0.01) and IgG anti-ApoAI (p<0.0001) as well as greater average oxLDL-2GPI (p=0.001) and CRP (p=0.003). Within PAPS, IgG anti-HDL correlated negatively to HDL (p=0.004) and was an independent predictor of oxLDL-2GPI (p=0.009). HDL and ApoAI correlated negatively with CRP (p=0.001 and p=0.007, respectively). IgG anti-HDL may hamper the antioxidant and anti-inflammatory effect of HDL favouring low-grade inflammation and enhanced oxidation in thrombotic PAPS. Indeed plasma 8-epi-prostaglandin F2α (a very specific marker of lipid peroxidation) was significantly higher in 10 patients with PAPS than 10 age and sex matched healthy subjects (p=0.0002) and strongly related to the titre of plasma IgG aCL (r=0.89, p=0.0004). Hence oxidative stress, a major player in atherogenesis, also characterises PAPS. Nitric oxide and nitrative stress in thrombotic primary antiphosholipid syndrome. Oxidative stress goes hand in hand with nitrative stress and to address the latter plasma nitrotyrosine (NT, marker of nitrative stress), nitrite (NO2-) and nitrate (NO3-) were measured in 46 thrombotic PAPS patients, 21 asymptomatic but persistent carriers of antiphospholipid antibodies (PCaPL), 38 patients with inherited thrombophilia (IT), 33 patients with systemic lupus erythematosus (SLE) and 29 healthy controls (CTR). Average crude NT was higher in PAPS and SLE (p=0.01) whereas average plasma NO2- was lower in PAPS and average NO3- highest in SLE (p<0.0001). In PAPS, IgG aCL titer and number of vascular occlusions negatively predicted NO2-, (p=0.03 and p=0.001, respectively) whereas arterial occlusions and smoking positively predicted NO3- (p=0.05 and p=0.005). Moreover CRP (an inflammatory marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade marker) positively predicted NT (p=0.004). Nitric oxide metabolites relates to type and number of vascular occlusions and to aPL titers, whereas nitrative stress relates to low grade inflammation and both phenomena may have implications for thrombosis and atherosclerosis in PAPS Inflammation and immune activation in thrombotic primary antiphospholipid syndrome. To investigate inflammation and immune activation in thrombotic PAPS high-sensitivity CRP (hs-CRP), serum amyloid A (SAA), oxLDL-2GPI, CRP bound to oxLDL-2GPI (CRP-oxLDL-2GPI) (as inflammatory markers) neopterin (NPT) and soluble CD14 (sCD14) (as immune activation markers) were measured by ELISA in 41 PAPS patients, in 44 patients with inherited thrombophilia (IT) and 39 controls (CTR). Compared to other groups, PAPS presented with higher plasma concentrations of inflammatory, hs-CRP (p=0.0004), SAA (p<0.01), CRP-oxLDL-2GPI (p=0.0004) and immune activation markers, NPT (p<0.0001) and sCD14 (p=0.007). By regression analysis SAA independently predicted thrombosis number (p=0.003) and NPT independently predicted thrombosis type (arterial, p=0.03) and number (p=0.04). These data confirm that low-grade inflammation and immune activation occur and relate to vascular features of PAPS. Antiphosholipid antibodies, haemostatic variables and atherosclerosis in thrombotic primary antiphospholipid syndrome To evaluate whether IgG aCL titre, haemostatic variables and the lipid profile bore any relationship to the intima media thickness (IMT) of carotid arteries high-resolution sonography was applied to the common carotid (CC), carotid bifurcation (CB) and internal carotid (IC) of 42 aPL subjects, 29 with primary thrombotic antiphospholipid syndrome and 13 with persistence of aPL in the absence of any underlying disorder. The following were measured: plasma FNG, vWF, PAI, homocysteine (HC), CHO, TG, HDL, LDL, platelet numbers and aCL of IgG and IgM isotype. By multiple regression analysis, IgG aCL titre independently predicted IMT at all carotid segments examined (p always <0.005). Plasma FNG and HC independently predicted IMT at the CB (p=0.001 and p<0.0001, respectively) and IC (p=0.03 and p<0.0001, respectively). These data strongly support an atherogenic role for IgG aCL in patients with aPL in addition to traditional risk factors. The atherosclerosis hypothesis was investigated in an age and sex-matched case-double-control study including 49 thrombotic PAPS patients (18 M, 31 F, mean age 37 ± 11), 49 thrombotic patients for IT and 49 healthy subjects. Average IMT was always greater in PAPS than control patients (CC: p=0.004, CB: p=0.013, IC: p=0.001). By dividing participants into age tertiles the IMT was greater in the second (CC: p=0.003, CB: p=0.023, IC: p=0.003) and third tertiles (CC: p=0.03, CB: p=0.004, IC: p=0.007). Conclusion: Coagulation activation, fibrinolysis depression, hightened fibrin turnover, oxidative and nitrative stress in parallel with low grade inflammation and immune activation characterise thrombotic PAPS: all these are early atherogenic processes and contribute to the demonstrated premature atherosclerosis that should be considered a clinical feature of PAPS.

Narrative Bifurcation, Cloud Computing Interfaces and Hitchcok

From a narratological perspective, this paper aims to address the theoretical issues concerning the functioning of the so called «narrative bifurcation» in data presentation and information retrieval. Its use in cyberspace calls for a reassessment as a storytelling device. Films have shown its fundamental role for the creation of suspense. Interactive fiction and games have unveiled the possibility of plots with multiple choices, giving continuity to cinema split-screen experiences. Using practical examples, this paper will show how this storytelling tool returns to its primitive form and ends up by conditioning cloud computing interface design.

Characterization of processed materials by electrical currents: development of equipment and applications

Dissertação para obtenção do Grau de Mestre em Engenharia Mecânica

Gold nanoparticles for plasmid delivery

Gene therapy presents an ideal strategy for the treatment of genetic as well as acquired diseases, such as cancer and typically involves the insertion of a functioning gene into cells to correct a cellular dysfunction or to provide a new cellular function. Gene delivery vectors are based in two models: viral and non-viral. Viral vectors have high transfection efficiency but their major barrier is immunogenicity. Since the non-viral vectors have no immunogenicity, these have been widely studied. Gold nanoparticles have been proposed as optimal delivery systems of genetic material, due their small size, high surface-to-volume ratio and the ability to be functionalized with multiple molecules. In the present work, an AuNP-based formulation was developed to deliver a plasmid in a colorectal cancer cell line, containing as reporter gene the gene encoding to EGFP. The delivery system resulted from the functionalization of 14 nm AuNP with a PEG layer (4300114 PEG chains/AuNP), which increases stability and biocompatibility of AuNPs; quaternary ammonium groups which provide positive charges that allow electrostatic binding of plasmid, which is considered the therapeutic agent to be transported into cells. The system developed was characterized by UV-vis spectroscopy, DLS, TEM and by electrophoretic mobility, yielding a formulation with 113.5 nm.Transfection efficiency of the formulation developed was evaluated through PCR and through EGFP expression by fluorescence microscopy and fluorescence spectroscopy. The internalization was observed 3h post transfection; however a low level of EGFP expression was achieved. After 24h of incubation, EGFP expression increases just 3 times compared to non-transfected cells. The commercial system (Lipofectamine) expressed EGFP 5 times more than the system developed AuNP@PEG@R4N+@pEGFP. This difference could be related to lower translocation to the nucleus.

Determinants of mother-baby relationship evaluated during pregnancy

ABSTRACT Background Mental health promotion is supported by a strong body of knowledge and is a matter of public health with the potential of a large impact on society. Mental health promotion programs should be implemented as soon as possible in life, preferably starting during pregnancy. Programs should focus on malleable determinants, introducing strategies to reduce risk factors or their impact on mother and child, and also on strengthening protective factors to increase resilience. The ambition of early detecting risk situations requires the development and use of tools to assess risk, and the creation of a responsive network of services based in primary health care, especially maternal consultation during pregnancy and the first months of the born child. The number of risk factors and the way they interact and are buffered by protective factors are relevant for the final impact. Maternal-fetal attachment (MFA) is not yet a totally understood and well operationalized concept. Methodological problems limit the comparison of data as many studies used small size samples, had an exploratory character or used different selection criteria and different measures. There is still a lack of studies in high risk populations evaluating the consequences of a weak MFA. Instead, the available studies are not very conclusive, but suggest that social support, anxiety and depression, self-esteem and self-control and sense of coherence are correlated with MFA. MFA is also correlated with health practices during pregnancy, that influence pregnancy and baby outcomes. MFA seems a relevant concept for the future mother baby interaction, but more studies are needed to clarify the concept and its operationalization. Attachment is a strong scientific concept with multiple implications for future child development, personality and relationship with others. Secure attachment is considered an essential basis of good mental health, and promoting mother-baby interaction offers an excellent opportunity to intervention programmes targeted at enhancing mental health and well-being. Understanding the process of attachment and intervening to improve attachment requires a comprehension of more proximal factors, but also a broader approach that assesses the impact of more distal social conditions on attachment and how this social impact is mediated by family functioning and mother-baby interaction. Finally, it is essential to understand how this knowledge could be translated in effective mental health promoting interventions and measures that could reach large populations of pregnant mothers and families. Strengthening emotional availability (EA) seems to be a relevant approach to improve the mother-baby relationship. In this review we have offered evidence suggesting a range of determinants of mother-infant relationship, including age, marital relationship, social disadvantages, migration, parental psychiatric disorders and the situations of abuse or neglect. Based on this theoretical background we constructed a theoretical model that included proximal and distal factors, risk and protective factors, including variables related to the mother, the father, their social support and mother baby interaction from early pregnancy until six months after birth. We selected the Antenatal Psychosocial Health Assessment (ALPHA) for use as an instrument to detect psychosocial risk during pregnancy. Method Ninety two pregnant women were recruited from the Maternal Health Consultation in Primary Health Care (PHC) at Amadora. They had three moments of assessment: at T1 (until 12 weeks of pregnancy) they filed out a questionnaire that included socio-demographic data, ALPHA, Edinburgh post-natal Depression Scale (EDPS), General Health Questionnaire (GHQ) and Sense of Coherence (SOC); at T2 (after the 20th weeks of pregnancy) they answered EDPS, SOC and MFA Scale (MFAS), and finally at T3 (6 months after birth), they repeated EDPS and SOC, and their interaction with their babies was videotaped and later evaluated using EA Scales. A statistical analysis has been done using descriptive statistics, correlation analysis, univariate logistic regression and multiple linear regression. Results The study has increased our knowledge on this particular population living in a multicultural, suburb community. It allow us to identify specific groups with a higher level of psychosocial risk, such as single or divorced women, young couples, mothers with a low level of education and those who are depressed or have a low SOC. The hypothesis that psychosocial risk is directly correlated with MFAS and that MFA is directly correlated with EA was not confirmed, neither the correlation between prenatal psychosocial risk and mother-baby EA. The study identified depression as a relevant risk factor in pregnancy and its higher prevalence in single or divorced women, immigrants and in those who have a higher global psychosocial risk. Depressed women have a poor MFA, and a lower structuring capacity and a higher hostility to their babies. In average, depression seems to reduce among pregnant women in the second part of their pregnancy. The children of immigrant mothers show a lower level of responsiveness to their mothers what could be transmitted through depression, as immigrant mothers have a higher risk of depression in the beginning of pregnancy and six months after birth. Young mothers have a low MFA and are more intrusive. Women who have a higher level of education are more sensitive and their babies showed to be more responsive. Women who are or have been submitted to abuse were found to have a higher level of MFA but their babies are less responsive to them. The study highlights the relevance of SOC as a potential protective factor while it is strongly and negatively related with a wide range of risk factors and mental health outcomes especially depression before, during and after pregnancy. Conclusions ALPHA proved to be a valid, feasible and reliable instrument to Primary Health Care (PHC) that can be used as a total sum score. We could not prove the association between psychosocial risk factors and MFA, neither between MFA and EA, or between psychosocial risk and EA. Depression and SOC seems to have a clear and opposite relevance on this process. Pregnancy can be considered as a maturational process and an opportunity to change, where adaptation processes occur, buffering risk, decreasing depression and increasing SOC. Further research is necessary to better understand interactions between variables and also to clarify a better operationalization of MFA. We recommend the use of ALPHA, SOC and EDPS in early pregnancy as a way of identifying more vulnerable women that will require additional interventions and support in order to decrease risk. At political level we recommend the reinforcement of Immigrant integration and the increment of education in women. We recommend more focus in health care and public health in mental health condition and psychosocial risk of specific groups at high risk. In PHC special attention should be paid to pregnant women who are single or divorced, very young, low educated and to immigrant mothers. This study provides the basis for an intervention programme for this population, that aims to reduce broad spectrum risk factors and to promote Mental Health in women who become pregnant. Health and mental health policies should facilitate the implementation of the suggested measures.

Optimal choice between even- and uneven-aged forestry: the case of non-industrial private forest owners

An infinite-horizon discrete time model with multiple size-class structures using a transition matrix is built to assess optimal harvesting schedules in the context of Non-Industrial Private Forest (NIPF) owners. Three model specifications accounting for forest income, financial return on an asset and amenity valuations are considered. Numerical simulations suggest uneven-aged forest management where a rational forest owner adapts her or his forest policy by influencing the regeneration of trees or adjusting consumption dynamics depending on subjective time preference and market return rate dynamics on the financial asset. Moreover she or he does not value significantly non-market benefits captured by amenity valuations relatively to forest income.