Land use/ land cover change and its impact on soil erosion process in Begnas Tal Rupa Tal watershed using geospatial tools, Kaski district, Nepal

Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Geospatial Technologies

Cork stoppers industry: defining appropriate mould colonization

Aims: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. Methods and Results: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. Conclusions: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. Significance and Impact of the Study: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Cork stoppers industry: defining appropriate mould colonization

Aims: The main aims of this work were the study of cork slabs moulds colonization and the evaluation of the moulds diversity during cork processing steps, in different cork stoppers factories. Simultaneously, it was envisaged to perform an evaluation of the air quality. Methods and Results: Moulds were isolated and identified from cork slabs and cork samples in four cork stoppers factories. The identification was based on morphological characters and microscopic observation of the reproductive structures. Airborne spore dispersion was assessed using a two stage Andersen sampler. It was observed that Chrysonilia sitophila was always present on cork slabs during the maturing period, but mould diversity appeared to be associated to the different factory configurations and processing steps. Conclusions: Spatial separation of the different steps of the process, including physical separation of the maturation step, is essential to guarantee high air quality and appropriate cork slabs colonization, i.e. C. sitophila dominance. The sorting and cutting of the edges of cork slabs after boiling and before the maturing step is also recommended. Significance and Impact of the Study: This study is very important for the cork stopper industry as it gives clear indications on how to keep high quality manufacturing standards and how to avoid occupational health problems.

Identification of mating type genes in the bipolar basidiomycetous yeast rhodosporidium toruloides: first insight into the MAT Locus structure of the sporidiobolales

Eukaryotic Cell, Vol.7, Nº6

Design and testing of a radiation hardened 13-bit 80 MS/s pipeline ADC implemented in a 90nm standard CMOS process

Second International Workshop on Analog and Mixed Signal Integrated Circuits for Space Applications (AMICSA 2008), Sintra, Portugal, Setembro de 2008

Social network analysis applied to knowledge creation and transfer in the portuguese agricultural R&D field: an exploratory study

Paper presented at the 9th European Conference on Knowledge Management, Southampton Solent University, Southampton, UK, 4-5 Sep. 2008. URL: http://academic-conferences.org/eckm/eckm2008/eckm08-home.htm

Innovation through virtual communities of practice: motivations and constraints in the knowledge-creation process

This paper appears in International Journal of Projectics. Vol 4(1), pp. 39-49

Functional domains of Bacillus subtilis transcription factor AraR and identification of aminoacids important for nucleoprotein complex assembly and effector-binding.

Journal of Bacteriology (Apr 2006) 3024-3036

Numerical Method to Predict Void Formation during The Liquid Composite Molding Process

Void formation during the injection phase of the liquid composite molding process can be explained as a consequence of the non-uniformity of the flow front progression. This is due to the dual porosity within the fiber perform (spacing between the fiber tows is much larger than between the fibers within in a tow) and therefore the best explanation can be provided by a mesolevel analysis, where the characteristic dimension is given by the fiber tow diameter of the order of millimeters. In mesolevel analysis, liquid impregnation along two different scales; inside fiber tows and within the open spaces between the fiber tows must be considered and the coupling between the flow regimes must be addressed. In such cases, it is extremely important to account correctly for the surface tension effects, which can be modeled as capillary pressure applied at the flow front. Numerical implementation of such boundary conditions leads to illposing of the problem, in terms of the weak classical as well as stabilized formulation. As a consequence, there is an error in mass conservation accumulated especially along the free flow front. A numerical procedure was formulated and is implemented in an existing Free Boundary Program to reduce this error significantly.

On the hydrogeology of the Lower Tagus Basin and its Cenozoic geologic evolution

The chemical features of the ground water in the Lower Tagus Cenozoic deposits are strongly influenced by lithology, by the velocity and direction of the water movement as well as by the localization of the recharge and discharge zones. The mineralization varies between 80 and 900 mg/l. It is minimal in the recharge zones and in the Pliocene sand and maximum in the Miocene carbonated and along the alluvial valley. Mineralization always reflects the time of permanence, the temperature and the pressure. The natural process of water mineralization is disturbed in agricultural areas because the saline concentration of the infiltration water exceeds that of the infiltrated rainwater. In the discharge zones, the rise of the more mineralized, some times thermal deep waters related to tectonic accidents give rise to anomalies in the distribution of the aquiferous system mineralization model. The diversity of the hydrochemical facies of the ground water may be related to several factors whose identification is some times difficult.

Saúde mental, qualidade de vida e acesso aos cuidados de saúde na comunidade brasileira de Lisboa

Introdução: A utilização de serviços de saúde tem implicações importantes para o estado de saúde das populações. As políticas de imigração adoptadas nos países de destino têm influência no estado de saúde das comunidades imigrantes. Políticas que limitam o acesso de imigrantes aos cuidados de saúde aumentarão a vulnerabilidade e os riscos na saúde. Apesar da imigração promover uma série de rupturas na vida do sujeito, migrar, por si só, não pode ser considerado como factor de risco no âmbito da saúde e da saúde mental. O peso dos determinantes socioeconómicos tem ganho relevância no estudo das migrações, estado de saúde geral e mental. Isto porque, em geral, os imigrantes estão em situação mais precária do que a população autóctone. O estatuto socioeconómico baixo, as condições precárias de habitação e de trabalho, a falta de suporte social e a irregularidade jurídica são indicadores de risco acrescido para a saúde mental. Neste sentido é um desafio de monta os governos estabelecerem medidas sustentadas e, simultaneamente, integradoras dos imigrantes. Em Portugal, considera-se que há escassez de estudos relacionados com a área das migrações e da saúde.Metodologia: Estudo exploratório, descritivo e transversal. A finalidade foi a de identificar o estado de saúde, saúde mental e qualidade de vida da comunidade brasileira residente em Lisboa e o seu acesso aos serviços de saúde. Este estudo teve como principais objectivos a caracterização sociodemográfica, a identificação de variáveis inerentes ao processo migratório, a identificação da auto-apreciação do estado de saúde, a caracterização do acesso aos cuidados de saúde, a identificação do grupo em provável sofrimento psicológico, a comparação entre os resultados dos imigrantes juridicamente regulares e irregulares e a comparação entre a população imigrante e a população portuguesa. Inicialmente, foi prevista a utilização da técnica de amostragem de propagação geométrica ou snowball, pois a amostra tornar-se-ia maior à medida que os próprios inquiridos identificam outros potenciais respondentes. Ao longo do estudo, a metodologia inicial mostrou-se insuficiente para estabelecer uma amostra mais representativa dos imigrantes juridicamente irregulares. Para este feito, foi utilizada a metodologia de amostragem por conveniência e o local escolhido para a recolha da amostra foi o Consulado do Brasil em Lisboa. O instrumento de recolha de dados empregue baseou-se no questionário utilizado no 4º Inquérito Nacional de Saúde. O MHI-5 (Mental Health Index 5) é um instrumento de saúde mental e é parte integrante do inquérito, sendo recomendado pela Organização Mundial de Saúde. Consta de cinco itens relativos à saúde mental e os resultados são classificados através de um indicador que mede a existência de provável sofrimento psicológico. Foram incluídos no estudo 213 brasileiros. De seguida, procedeu-se ao tratamento estatístico dos dados. Resultados: A população inquirida é jovem, a maior parte tem entre 18 e 44 anos. As mulheres representam mais de metade da amostra. A taxa de actividade é elevada e a taxa de desemprego é similar à nacional. A inserção laboral prioritária é nos segmentos pouco qualificados ou de semi-qualificação. Aproximadamente um terço dos inquiridos afirmou ser beneficiário do Sistema Nacional de Saúde. A autoapreciação do estado de saúde é classificada como bastante positiva, assim como a qualidade de vida. O provável sofrimento psicológico, definido no MHI-5 pelo ponto de corte no score 52, atinge 23,3% dos participantes. Os homens apresentam melhores resultados do que as mulheres. Além disso, para os valores mais baixos no MHI-5 foram encontradas relações com as longas jornadas de trabalho e o diagnóstico de doença crónica.Discussão: O presente estudo apresenta limitações em relação à dimensão da amostra e à provável existência de enviesamento pela ausência de aleatorização. Apesar da legislação portuguesa garantir o acesso aos serviços de saúde e garantir a equidade no caso dos imigrantes que fazem descontos para a Segurança Social, apenas um terço referiu ser beneficiário do Sistema Nacional de Saúde. Este dado pode ser justificado por factores como o cumprimento da lei por alguns serviços e, também, pela falta de conhecimento da legislação e da forma de funcionamento do Serviço Nacional de Saúde por parte dos imigrantes. O facto das mulheres representarem o maior grupo em provável sofrimento psicológico é consistente com a literatura. As hipóteses levantadas para explicar este resultado podem ser agrupadas em: artefactos metodológicos, causalidade biológica e determinação social. Em relação ao instrumento, é possível que o MHI-5 se comporte de forma diferente no que diz respeito ao género.-------------------------------------------Introduction: The utilization of health services has important implications for the health state of the populations. The immigration policies adopted in the destiny countries are going to influence the health state of immigrant communities. Policies that limit the access of immigrants to health care are going to increase the vulnerability and the risk factor in health. Although immigration promotes several disruptive actions in ones life, migrating, on its own, cannot be considered as a risk factor for health and mental health. The preponderance of the socioeconomic factors has gained relevance in the study of migrations and also in the study of general health state and mental health. This happens because, in general, immigrants are in a more unfavorable situation compared with the destiny country population. The low socioeconomic status, the poor working and housing conditions, the lack of social support and the juridical irregularity are indicators of the incremented risk to mental health. Therefore, it is a major challenge for governments to find sustainable, and simultaneously, integrative measures for the immigrants. The studies related with the migrations and health in Portugal were considered to be few.Methods: It is an exploratory, descriptive and transversal study. The purpose is to identify the health state, mental health, quality of life and the access to health care of the Brazilian community resident in Lisbon. In addition, this study has as main goals the sociodemographic characterization, the variables identification inherent to the migrating process, the identification of the self-appreciation of health state, the characterization of the access to health care, the identification of the group in probable psychological suffer, the comparison between the results of regular and irregular immigrants and the comparison between the immigrant population and the Portuguese population. Initially it was predicted the utilization of the geometric propagation or “snowball”, as sampling technique, because the sample becomes larger as one answerer identify other potential answering persons. Along with the study, the methodology has shown insufficient to establish a more representative sample of the irregular immigrants. For this latter case, it was used a convenient sample methodology and the place chosen for the sample gathering was the “Consulate of Brazil in Lisbon”. The instrument was based in the questionnaire used in the “4th National Health Inquiry”. The MHI-5 (Mental Health Index 5) is a mental health instrument which is part of the enquiry and it is recommended by the World Health Organization. There are five items related to mental health and the results are classified through an indicator which measures the existence of a probable psychological suffer. It were included 213 Brazilian in the study. After, the statistical treatment of the data took place.Results: The answering population is young and the majority is between the 18 and 44 years of age. The women represent more than one half of the sample. The activity rate is high and the unemployment rate is similar to the national one. The priority labor insertion is in the few qualified or of semi-qualification segments. Approximately, one third of the answering people has stated to be beneficiary of the National Health System. The self-appreciation of the health state as well as the quality of life are classified as fairly positive ones. The probable psychological suffer, as defined in the MHI-5 through the cut point in the score below or equal to 52, reaches 23,3% of the sample population. Men show the better results than women. Further, for the lower values of MHI-5 it was found a relation with the long work periods and chronic disease diagnostic. Discussion: The present study evidences limitations in relation to the sample dimension and in relation to the existence of biases due to the lack of randomness. Although the Portuguese legislation guarantees the access to health services and the equality in the cases of the immigrants that do their Social Security discounts, only one third has mentioned to be beneficiary of the National Health System. This can be justified by several facts such as the non-fulfillment of law by some national services or the lack of knowledge of the legislation or the functioning process of the National Health System. Women representing the bigger group in probable psychological suffer has been coherent with the literature review. The hypothesis set to explain this result might be grouped in: methodological artifacts, biologic cause and social determination. In relation to the instrument used, it may be that MHI-5 behaves in a different way in respect to gender.

Estudo sobre o insight em pessoas com psicose

Para além das variáveis clínicas e sociodemográficas existem concerteza importantes componentes individuais que desempenham um contributo importante no nível de insight apresentado por cada pessoa doente, por exemplo, o nível de inteligência, personalidade, cultura, experiências passadas, memória, etc. A natureza clínica, emocional e/ou intelectual do termo ajuda-nos a compreender a complexidade da dificuldade que existe na sua tradução e, inclusive, na sua compreensão. Daí que as definições atribuídas ao conceito sejam muito distintas e variem consoante a formação teórica do autor/investigador. Pretende-se, a partir dessa identificação/compreensão, promover a qualidade de vida destas pessoas através do desenvolvimento de novas aprendizagens que possibilitem uma cooperação activa. É igualmente fundamental ir ao encontro das capacidade intactas de maneira a possibilitar a aquisição de novos(s) comportamento(s) que tenham um impacte positivo nas queixas, sinais, sintomas, incapacidade e disfuncionalidade apresentados pelo/a utente. Uma vez que a própria conceptualização do termo traduzirá aquilo que se pretende avaliar,será efectuada uma reflexão detalhada acerca dos instrumentos e definições que têm sido mais utilizadas para explorar o insight nas psicoses.Procurei, no meu trabalho de investigação, realçar e promover a importância que cada sujeito, alvo de intervenção, desempenha ao longo do seu processo de recuperação e na prevenção de recaídas. No seguimento dos objectivos acima descritos, para além da revisão teórica efectuada ao fenómeno em termos de conceptualização e estudos desenvolvidos na área de investigação, foi,neste estudo, realizada a contribuição para a validação do instrumento “Assessment of Insight in Psychosis: a re-standartization of a New Scale” de Marková & Berrios (2003).O fenómeno de insight escolhido pela Insight Scale, relata menos as mudanças vividas em relação à doença mental, e mais a actual consciência e articulação de tais mudanças. Tendo como base uma abordagem psicopedagógica, o fenómeno do insight aqui explorado assentou numa perspectiva reabilitativa, actual e multidimensional, que fosse para além das dimensões clínicas tradicionais. Neste sentido é apresentada uma escala original, intitulada “Escala de Avaliação do Insight e Identificação das Necessidades em Pessoas com Psicose”, bem como um modelo de intervenção psicopedagógico breve, assente nos pressupostos descritos ao longo do trabalho.-----------------------------------------ABSTRACT: The importance of insight in people with mental illnesses was first studied in psychiatry, in the first decades of the 20th century, by people as important as Lewis (1934) and Jaspers (1959). However, this field of investigation was left unexplored for many years. Only in the last decade has this phenomenon become the object of numerous scientific investigations, having been given special attention by its investigators. For this reason a significant number of instruments for evalauting insight in psychotic disorders were developed. Since then many papers have been published, which has allowed for a more in depth knowledge on the subject. Therefore, in recent years, the concept of insight has been developed in an attempt to clarify its compexity. A once dichotomic phenomenon, described in terms of presence or absence, became considered multidimensional, which made the identification of different levels of insight and different dimensions possible. Current concepts categorize insight into five dimensions: the awareness of the patient in relation to his/her mental illness, the awareness of the patient in relation to the social consequences of his/her illness, the awareness of the need for treatment, the awareness of the symptoms and the explanation of those symptoms in relation to the illness. The lack of insight in psychiatry, in general terms, and as this phenomenon has been described, the lack of awareness of having a mental illness, represents one of the most common symptoms of schizophrenia and affects a big part of the population that suffer from this illness. It is estimated that bewteen 50 and 80 per cent of patients with schizophrenia do not believe that they are ill, which, consequently has a big impact in the process of adherence to treatment. It is still not possible, however, to identify all the factors that determine the lack of insight in schizophrenics. There are psychological, social and cultural influences that almost certainly play their role in the lack of insight registered in this pathology.Since the impact of scizophrenia is felt in many aspects of the individual’s life, its effective treatment should be directed at various levels, including the improvement of insight. One of the objectives of this study is to explore the relationship between the level of insight in psychosis and the clinical and sociodemographic variables, the psychopathology and its global functioning. As well as the clinical and sociodemographic variables, there are of course important individual components that contribute to the level of insight seen in each patient, for example, their level of inteligence, personality, culture, past experiences, memory, etc. The clinical, emotional and/or intelectual nature of the term helps us understand the difficulty that lies in its interpretation as well as in its comprehension. Therefore, the definitions attributed to the term are very different and vary according to the theoretical training of the investigator. It is intended, from this identification/understanding, to promote the quality of life of these people through the development of new findings that might enable an active cooperation. It is equally fundamental to observe their unimpaired capacities in order to enable the acquisition of new behaviour(s) that have a positive impact on the complaints, signs, symptoms, incapacity and disfunctioning seen in the patient.As the actual comprehension of the term explains what we intend to evaluate, a detailed reflection is made on the instruments and definitions that have been used the most to explore insight in psychosis.In this investigation I tried to underline and promote the importance that each subject, undergoing medical intervention, plays during his/her process of recovery and prevention of relapses. Considering the above mentioned objectives, as well as a theoretical review of the phenomenon in terms of conceptualization and investigative studies developed, this study contributed to the validation of the instrument.The insight phenomenon chosen by the “Insight Scale”, records less changes experienced in relation to the mental illness and more actual awareness and articulation of these changes. Based on a psychopedagogical approach, the insight phenomenon explored here settled on a rehabilitation, current and multidimensional perspective that would go beyond the traditional clinical dimensions. For this reason an original scale entitled “Insight Evaluation Scale and Need Identification in Psychosis Patients” is presented, as well as a psychopedagogical intervention model soon to be used with admitted patients based on the presuppositions described in this study.

Prevalência do consumo de drogas ilícitas nas Forças Armadas estudo epidemiológico num dos ramos

RESUMO: Desde 1976 que as Forças Armadas desenvolvem acções de prevenção do consumo de drogas e álcool. Na década de 80 foi criada capacidade laboratorial e deu-se início a um programa de rastreios toxicológicos. No quinquénio 2001 a 2005, as proporções de resultados positivos, associando todos os tipos de rastreio, variaram entre 3,7% e 1,5%. De Outubro de 2006 a Julho de 2007 realizou-se um estudo analítico transversal, para estimar a prevalência do consumo de drogas (canabinóides, opiáceos, cocaína e anfetaminas) num dos Ramos das Forças Armadas, com base nos despistes realizados pelo seu laboratório. Foi utilizada uma amostra aleatória simples de 1039 militares, profissionais (QP) e contratados (RC), no activo e de ambos os sexos. Desde a nomeação dos militares a rastrear, passando pela cadeia de custódia das amostras até à obtenção do resultado foi utilizado apoio informático específico. O processo de pesquisa utilizou duas técnicas de triagem por imunoensaio e tecnologia de confirmação por GC/MS, de acordo com as recomendações europeias, permitindo estabelecer uma metodologia standard para organizações e empresas. A prevalência estimada, de consumidores de droga, foi de 3,8/1.000, para um erro de 0,37%. O número de casos registado (4) não permitiu a utilização de testes estatísticos que conduzissem à identificação de características determinantes da positividade, mas não deixou de revelar aspectos inesperados. A observação de séries de casos e a realização regular de estudos epidemiológicos, que ajudem a redefinir grupos alvo e a perceber a dimensão, as determinantes e as consequências do consumo de drogas é sugerida, em conclusão.--------------------------------------- RÉSUMÉ: Depuis 1976, les Forces Armées mettent au point des mesures visant à prévenir la consommation de drogues et d'alcool. En 1980, fut créé capacité laboratoriel et ont ensuite commencé un programme de dépistage toxicologique. Au cours des cinq années allant de 2001 à 2005, les proportions de consommateurs, impliquant tous les types de dépistage, allaient de 3,7% à 1,5 %. D'octobre 2006 à juillet 2007, une étude analytique transversale a été organisée pour évaluer la prévalence de l’usage de drogues (cannabis, opiacés, cocaïne et amphétamines) dans une branche de les Forces Armées, basée sur les dépistages faites par un laboratoire militaire, à l'aide d'un échantillon aléatoire de 1039 militaires, professionnels (QP) et sous contract (RC), à l’actif et des deux sexes. Tout au long du procès, de la nomination des donneurs, en passant par la chaine de garde des échantillons, jusqu’à obtention du résultat, il fut employé un appui informatique sécurisé. Le processus de recherche employa deux techniques de tri par imunoessay et la technologie de confirmation GC/MS, selon les recommandations européennes, permettant d'établir une méthodologie standard pour les organisations et les entreprises. La prévalence estimée fut de 3,8/1.000 pour une marge d’erreur de 0,37%. Le nombre de cas enregistrés (4) n'autorise pas l'utilisation de testes statistiques de menant à l'identification de caractéristiques déterminant de la positivité, mais il permet à révéler des aspects inattendus. L'observation de séries de cas et la tenue régulière d’études épidémiologiques, qui contribuent à redéfinir les groupes cibles et de comprendre l'ampleur, les déterminants et les conséquences de l'usage de drogues, est suggéré, en fin de compte.--------------------------------------- ABSTRACT: Since 1976, the Armed Forces, have been developing measures to prevent the use of drugs and alcohol. In 1980, was created laboratory facility which then started a program of toxicological screenings. In the five years running from 2001 to 2005, the proportions of consumers, involving all types of screening, ranged from 3,7% to 1,5%. From October 2006 to July 2007, a cross-sectional study was held to estimate the prevalence of drug use (cannabinoids, opiates, cocaine and amphetamines) in one branch of the Portuguese Armed Forces, based on laboratory screenings, using a random sample of 1039 military, professional (QP) and enlisted (RC), active-duty and of both sexes. Specific computer support was used all the way, from the appointment, including the chain of custody of samples, to the obtaining of the result. The process of search used two techniques for sorting by immunoassay and confirmation technology GC/MS, according to European recommendations, allowing to establish a standard methodology for organizations and companies. The estimated prevalence of drug users was 3.8/1.000 for a 0.37% error (95% confidence interval). The number of cases registered (4) does not permit use of statistical testing leading to the identification of characteristics weighing in the establishing to extrapolate for the population, but it allows revealing unexpected aspects. The observation of series of cases and the regular holding of epidemiological studies, which help redefine target groups and to understand the extent, the determinants and consequences of drug use, is suggested, in conclusion.

Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

Downstream processing development of enveloped viruses for clinical applications: innovative tools for rational process optimization

Dissertation presented to obtain a Ph.D. degree in Engineering and Technology Sciences, Biotechnology at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa