Prognostic modelling of breast cancer patients: a benchmark of predictive models with external validation

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Electrotécnica e de Computadores – Sistemas Digitais e Percepcionais pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

O regime fiscal cooperativo – análise crítica e desafios

Dissertação de Mestrado apresentada ao Instituto de Contabilidade e Administração do Porto para a obtenção do grau de Mestre em Contabilidade e Finanças, sob orientação de Doutora Deolinda Meira e Doutora Nina Aguiar

A influência da dor no joelho na incapacidade e proprioceção em adultos mais velhos

Introdução: A dor no joelho apresenta uma etiologia multifatorial, sendo a idade um fator de risco importante. A dor no joelho poderá estar relacionada com alterações na propriocetividade do joelho. Objetivo (s): Comparar a influência dor unilateral com bilateral do joelho na incapacidade e proprioceção em adultos mais velhos. Métodos: Estudo transversal com uma amostra de 11 indivíduos com dor no joelho, divididos em grupo com dor unilateral (GDU=6) e grupo com dor bilateral (GDB=5). Utilizou-se, a Knee injury and Osteoarthritis Outcome Score (KOOS) para analisar a dor, rigidez e outros sintomas, atividades de vida diária, desportivas e de lazer e qualidade de vida. Foi medida a sensação de posição articular passiva e ativa, bem como a sensação de discriminação de carga. Foram utilizados os testes de Mann-Whitney e de correlação de Spearman, com um nível de significância de 0,05. Resultados: Nas diferentes dimensões da KOOS apesar de não se ter verificado diferenças significativas entre os grupos, o GDU apresenta scores menores, que traduzem uma maior incapacidade. Na sensação de posição articular e na sensação de discriminação de carga não se verificaram diferenças significativas entre os grupos. Conclusão: A dor no joelho ser unilateral ou bilateral não influencia nem a incapacidade nem a proprioceção nos adultos mais velhos.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM),and lower results with egg antigen, 77.8% (IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r=0.552 (p=0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

Neural mechanisms of stimulus generalization in auditory fear conditioning

Dissertation presented to obtain the Ph.D degree in Neuroscience Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Empreendedorismo feminino em Portugal

Dissertação apresentada ao Instituto Politécnico do Porto para obtenção do Grau de Mestre em Gestão das Organizações, Ramo Gestão de Empresas Orientada por Prof. Doutora Maria Clara Dias Pinto Ribeiro

O controlo da tuberculose em Portugal : uma apreciação crítica epidemiológica gl

RESUMO - Com tanto ruído informativo e peças de informação incompletas e descontextualizadas, relativos ao controlo da tuberculose em Portugal, a chegarem ao cidadão e aos profissionais de saúde, não é fácil que eles disponham do conhecimento necessário ao desempenho dos respectivos papéis nesse controlo. O presente artigo pretende contribuir para um ganho real em conhecimento quanto (1) ao progresso no controlo da tuberculose em Portugal, enquadrado na evolução desse controlo noutras regiões pertinentes, e (2) aos aspectos do conhecimento disponível e da intervenção na população portuguesa que suscitem especial atenção, para melhoria no futuro imediato. Tem como estratégia integrar, e elevar ao nível de conhecimento, a informação disponibilizada nas fontes mais credíveis e pertinentes, valorizada no contexto da validade das mesmas fontes e da coerência dos diversos componentes. Relata o resultado de um exercício independente de apreciação crítica, com uma perspectiva epidemiológica. São observados: a situação actual, sobretudo a relativa aos anos de 2006 e 2007, e o desempenho do Programa Nacional de Controlo da Tuberculose (PNT), ambos relativizados à evolução recente e ao panorama internacional. O exercício de observação e revisão independentes baseia-se numa selecção de informação oficial e segue o mesmo método de abordagem que a O.M.S. faz nos seus relatórios anuais, à semelhança de outros exercícios já antes realizados. O controlo da tuberculose tem prosseguido a sua tímida, mas firme, evolução favorável, aproximando-se do nível já conseguido nos países seus vizinhos da Europa Ocidental. Em 2007, Portugal contribuiu para os 9 milhões de casos novos anuais, estimados no mundo, com 2916 casos notificados. A este número corresponde a taxa de incidência notificada de 25,7 por 100 000 habitantes e uma redução de 14% em relação ao ano anterior. Esta evolução afigura-se animadora, ainda que seja desejável um impacte mais acentuado do PNT, conforme é de esperar considerando o grau de desenvolvimento do País. A taxa de detecção de casos novos estimada é elevada e continua uma das melhores da Europa Ocidental — o que desfavorece artificialmente a imagem notificada do País, relativamente aos países com pior capacidade de detecção. A taxa de sucesso terapêutico melhorou de novo, situando- -se acima da meta de 85% preconizada pela O.M.S, para um bom controlo da tuberculose. Uma das consequências importantes é que se consegue um melhor aproveitamento da detecção habitualmente alcançada. O conhecimento no seu conjunto aponta para que o grau de controlo possa e deva realmente ser melhorado, sendo imperiosa a discriminação positiva das áreas geográficas e dos grupos populacionais em que tende a concentrar-se a emergência de maior número de casos e de resistências aos medicamentos. Deverão assim ser reforçados selectivamente tanto os meios de detecção e de intervenção clínica, como a qualidade da organização local da intervenção, para o cumprimento efectivo da estratégia DOTS. Enquanto programa vertical que atravessa os diversos níveis do sistema de cuidados de saúde, o desempenho do PNT sofre os efeitos das atribulações desses serviços, sobretudo os de cuidados primários, funcionando como uma «situação-marcadora» quanto ao desempenho do sistema de saúde. A evidência é de que é nesta primeira linha de cuidados que se decide o sucesso na detecção e no tratamento dos casos de tuberculose, reflectindo-se também aí o grau de desenvolvimento social e os comportamentos das populações, por sua vez determinantes do risco de doença e do sucesso terapêutico. ------------------- ABSTRACT - It is not easy that both the citizen and health professionals get enabled with the required knowledge, in order do play the corresponding roles in the control of tuberculosis, considering all the information noise and incomplete, out of context information pieces about the subject, that reach them. This paper is envisaging to contribute for a real gain in knowledge, regarding: (1) the progress in tuberculosis control in Portugal, framed by the evolution of such control in other pertinent regions and (2) the available knowledge and intervention aspects in the Portuguese population that require a special attention, for improvement. The article’s strategy is to integrate, and raise to a knowledge level, information provided by the most accredited and pertinent sources, interpreted as a function of the validity context of the same sources and of the coherence of the several components. Two aspects are observed: the current situation, in particular concerning years 2006 and 2007, and the performance of the National Programme for the Tuberculosis Control (PNT), both made relative to the recent evolution and to the international panorama. This independent observation and revision exercise is based on a selection of official information and follows the same approach that the World Health Organization (W.H.O.) uses in its annual reports, like other similar exercises previously undertaken. The control of tuberculosis is evolving in a shy, but firm, fashion, getting closer to the level already attained by the neighbor countries, in Western Europe. Portugal has contributed with 2916 new notified cases, to the 9 million annual cases estimated in the world, in 2007. This number corresponds to an incidence rate, for notified cases, of 25.7 per 100000 population, and to a reduction of 14% in one year. Such evolution seems encouraging, although a greater impact of PNT is desirable, as expected in relation to the degree of the Country development. Estimated new cases detection rate is high and keeps being one of the best in Western Europe — and this artificially disadvantages the notified image of the Country, as compared with other countries having a worst detection capacity. Treatment success rate has improved again and it is above the 85% target proposed by W.H.O., so that a good control of the disease is achieved. One of the important consequences is a better use of the attained detection. Altogether, knowledge suggests that the degree of control can and must be in fact better; and that a positive discrimination of geographic areas and population groups, in which a greater number of new cases and drug resistances tend to concentrate, is mandatory. Therefore, either clinical detection and intervention resources, or the quality of the local intervention organization have to be reinforced, if a total fulfillment of DOTS strategy is to be obtained. As a vertical programme that crosses the several levels of the health care system, PNT performance suffers the effects of services tribulations, mainly primary care, thus acting as a «markersituation » as to this system performance. Evidence shows that it is in this first line of care that success in both detection and treatment of tuberculosis cases is decided; and that this level also reveals the degree of social developmen

American trypanosomiasis and electrocardiographic alterations among industrial workers in São Paulo, Brazil

In this article, we evaluate electrocardiographic (ECG) alterations among urban workers from a mechanical-metallurgical industry in São Paulo, Brazil. In a cross-sectional survey carried out in 1980, we found 2.2% positive serological testing for Trypanosoma cruzi infection among 27,081 workers. A comparison between seropositive workers and a random sample of seronegative workers frequency-matched by age and occupation revealed that seropositive workers had a much lower educational level, and that a higher proportion of seropositive workers had ECG abnormalities (42.7%) when compared to those negative for T. cruzi infection (19.8%). The high frequency of ECG abnormalities suggests the need to provide medical assistance to these workers, without any kind of discrimination and to bring in a form of management that would decrease this dangerous risk to the workers and people around them.

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.

Ankylosing spondylitis: genomic and functional characterization of candidate genes and their repercussion in clinical practice

RESUMO: Introdução: A espondilite anquilosante (EA) é uma doença inflamatória crónica caracterizada pela inflamação das articulações sacroilíacas e da coluna. A anquilose progressiva motiva uma deterioração gradual da função física e da qualidade de vida. O diagnóstico e o tratamento precoces podem contribuir para um melhor prognóstico. Neste contexto, a identificação de biomarcadores, assume-se como sendo muito útil para a prática clínica e representa hoje um grande desafio para a comunidade científica. Objetivos: Este estudo teve como objetivos: 1 - caracterizar a EA em Portugal; 2 - investigar possíveis associações entre genes, MHC e não-MHC, com a suscetibilidade e as características fenotípicas da EA; 3 - identificar genes candidatos associados a EA através da tecnologia de microarray. Material e Métodos: Foram recrutados doentes com EA, de acordo com os critérios modificados de Nova Iorque, nas consultas de Reumatologia dos diferentes hospitais participantes. Colecionaram-se dados demográficos, clínicos e radiológicos e colhidas amostras de sangue periférico. Selecionaram-se de forma aleatória, doentes HLA-B27 positivos, os quais foram tipados em termos de HLA classe I e II por PCR-rSSOP. Os haplótipos HLA estendidos foram estimados pelo algoritmo Expectation Maximization com recurso ao software Arlequin v3.11. As variantes alélicas dos genes IL23R, ERAP1 e ANKH foram estudadas através de ensaios de discriminação alélica TaqMan. A análise de associação foi realizada utilizando testes da Cochrane-Armitage e de regressão linear, tal como implementado pelo PLINK, para variáveis qualitativas e quantitativas, respetivamente. O estudo de expressão génica foi realizado por Illumina HT-12 Whole-Genome Expression BeadChips. Os genes candidatos foram validados usando qPCR-based TaqMan Low Density Arrays (TLDAs). Resultados: Foram incluídos 369 doentes (62,3% do sexo masculino, com idade média de 45,4 ± 13,2 anos, duração média da doença de 11,4 ± 10,5 anos). No momento da avaliação, 49,9% tinham doença axial, 2,4% periférica, 40,9% mista e 7,1% entesopática. A uveíte anterior aguda (33,6%) foi a manifestação extra-articular mais comum. Foram positivos para o HLA-B27, 80,3% dos doentes. Os haplótipo A*02/B*27/Cw*02/DRB1*01/DQB1*05 parece conferir suscetibilidade para a EA, e o A*02/B*27/Cw*01/DRB1*08/DQB1*04 parece conferir proteção em termos de atividade, repercussão funcional e radiológica da doença. Três variantes (2 para IL23R e 1 para ERAP1) mostraram significativa associação com a doença, confirmando a associação destes genes com a EA na população Portuguesa. O mesmo não se verificou com as variantes estudadas do ANKH. Não se verificou associação entre as variantes génicas não-MHC e as manifestações clínicas da EA. Foi identificado um perfil de expressão génica para a EA, tendo sido validados catorze genes - alguns têm um papel bem documentado em termos de inflamação, outros no metabolismo da cartilagem e do osso. Conclusões: Foi estabelecido um perfil demográfico e clínico dos doentes com EA em Portugal. A identificação de variantes génicas e de um perfil de expressão contribuem para uma melhor compreensão da sua fisiopatologia e podem ser úteis para estabelecer modelos com relevância em termos de diagnóstico, prognóstico e orientação terapêutica dos doentes. -----------ABSTRACT: Background: Ankylosing Spondylitis (AS) is a chronic inflammatory disorder characterized by inflammation in the spine and sacroiliac joints leading to progressive joint ankylosis and in progressive deterioration of physical function and quality of life. An early diagnosis and early therapy may contribute to a better prognosis. The identification of biomarkers would be helpful and represents a great challenge for the scientific community. Objectives: The present study had the following aims: 1- to characterize the pattern of AS in Portuguese patients; 2- to investigate MHC and non-MHC gene associations with susceptibility and phenotypic features of AS and; 3- to identify candidate genes associated with AS by means of whole-genome microarray. Material and Methods: AS was defined in accordance to the modified New York criteria and AS cases were recruited from hospital outcares patient clinics. Demographic and clinical data were recorded and blood samples collected. A random group of HLA-B27 positive patients and controls were selected and typed for HLA class I and II by PCR-rSSOP. The extended HLA haplotypes were estimated by Expectation Maximization Algorithm using Arlequin v3.11 software. Genotyping of IL23R, ERAP1 and ANKH allelic variants was carried out with TaqMan allelic discrimination assays. Association analysis was performed using the Cochrane-Armitage and linear regression tests as implemented in PLINK, for dichotomous and quantitative variables, respectively. Gene expression profile was carried out using Illumina HT-12 Whole-Genome Expression BeadChips and candidate genes were validated using qPCR-based TaqMan Low Density Arrays (TLDAs). Results: A total of 369 patients (62.3% male; mean age 45.4±13.2 years; mean disease duration 11.4±10.5 years), were included. Regarding clinical disease pattern, at the time of assessment, 49.9% had axial disease, 2.4% peripheral disease, 40.9% mixed disease and 7.1% isolated enthesopathic disease. Acute anterior uveitis (33.6%) was the most common extra-articular manifestation. 80.3% of AS patients were HLA-B27 positive. The haplotype A*02/B*27/Cw*02/DRB1*01/DQB1*05 seems to confer susceptibility to AS, whereas A*02/B*27/Cw*01/DRB1*08/DQB1*04 seems to provide protection in terms of disease activity, functional and radiological repercussion. Three markers (two for IL23R and one for ERAP1) showed significant single-locus disease associations. Association of these genes with AS in the Portuguese population was confirmed, whereas ANKH markers studied did not show an association with AS. No association was seen between non-MHC genes and clinical manifestations of AS. A gene expression signature for AS was established; among the fourteen validated genes, a number of them have a well-documented inflammatory role or in modulation of cartilage and bone metabolism. Conclusions: A demographic and clinical profile of patients with AS in Portugal was established. Identification of genetic variants of target genes as well as gene expression signatures could provide a better understanding of AS pathophysiology and could be useful to establish models with relevance in terms of susceptibility, prognosis, and potential therapeutic guidance.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

Which “place and non-place” do women occupy in social economy organisations?

Scientific literature has strengthened the perpetuation of inequality factors in the labour market based on gender, despite the on-going endeavour of various political bodies and legal norms against the vertical and horizontal segregation of women. National and European statistical data shows the relevance and timeless features of theories of market segmentation associated with the labour market dating back to the 70’s of the 20th century. Hence, the European Community considers as a priority in the Europe 2020 strategy, the definition of “policies to promote gender equality […] to increase labour force participation thus adding to growth and social cohesion”. If we consider that on the one hand, social economy is fairly recognised to be equated with market actors and the State for its economic and social role in tackling the current crisis, and on the other hand, that the ideals of the sector, systematised in the “Framework Law of Social Economy” (Law no. 30/2013 8th of May), particularly in article 5 proposing “the respect for the values […] of equality and non-discrimination […], justice and equity […]”, we aim to reflect on indicators that uncover a vertical and horizontal segregation in the labour market. Departing from a mixed methodological approach (extensive and intensive), subject to the topic of "Social Entrepreneurship in Portugal" in social economy organisations, we detect very high rates of employment feminisation, with a ratio of 1 man (23%) for every 3 women (77%). Women are mainly earmarked for technical and operational activities, arising from the privileged intervention areas, namely education, training, health, elderly, families, poverty, ultimately being underrepresented in statutory boards and, as such, far removed from deliberations and strategic resolutions. This is particularly visible in the existing hierarchy of functions and management practices of the responsibility of male members. Thus, it seems easily verified that the sector is travelling away from the ideals of justice and social equity, which can crystallise the "non-place" of women in the definition of a strategic direction of social economy and in the most invisible/private “place” of the organisational setting.