A nanopore-based stochastic detection method: Single molecule characterisation of nanoparticles using ()- hemolysin

Dissertation presented to obtain the Ph.D degree in Chemistry.

In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

Contributo da curva de débito-volume na detecção da obstrução da via aérea central

RESUMO: Introdução: A obstrução da via aérea central (OVAC) refere-se a um processo patológico que conduz a limitação do fluxo de ar ao nível do espaço glótico e subglótico, traqueia e brônquios principais. O seu correcto diagnóstico e tratamento constituem um território de interesse e preocupação para os profissionais de saúde, e requerem um profundo conhecimento da sua etiologia, fisiologia, diagnóstico e opções terapêuticas dado o potencial em originar significativa morbilidade e mortalidade. A avaliação da OVAC abrange múltiplas vertentes, entre as quais se salienta o componente clínico (sinais e sintomas), a repercussão fisiopatológica (função respiratória) e o estudo imagiológico (TC do tórax e broncoscopia). A compilação destes dados associada à etiologia, constituem factores importantes para estabelecer o prognóstico, determinar a necessidade de tratamento ou delinear uma futura intervenção terapêutica. A broncoscopia é o Gold Standard de avaliação desta condição, mas desde há cerca de 40 anos a curva de débito-volume constitui uma ferramenta não invasiva de detecção de OVAC. Apesar deste método ser utilizado até os nossos dias, poucos têm sido os estudos com o objectivo de verificar a sensibilidade e especificidade da curva de débito-volume na detecção de OVAC, bem como averiguar a relação entre as alterações morfológicas e quantitativas da mesma com a localização, o tipo e o grau da obstrução. Material e Métodos: Entre 1 de Novembro de 2009 e 30 de Abril de 2010, os doentes com indicação para a realização de broncoscopia diagnóstica ou terapêutica na Unidade de Técnicas Invasivas Pneumológicas (UTIP) do Centro Hospitalar Lisboa Norte – Hospital Pulido Valente (CHLN – HPV) foram seleccionados de forma consecutiva de acordo com os critérios de inclusão e exclusão. As avaliações (broncoscopia, curva de débito-volume e avaliação da dispneia) realizaram-se com um intervalo de tempo máximo de sete dias. A broncoscopia flexível foi realizada segundo as normas da British Thoracic Society e as curvas de débito-volume segundo as normas da ATS/ERS TaskForce 2005. Para a avaliação da dispneia recorreu-se à escala de dispneia MRC (Medical Research Council). Um painel de peritos realizou a avaliação da morfologia da curva de débito-volume (sugestiva ou não de OVAC) e um elemento independente a verificação dos critérios quantitativos e morfológicos (variáveis intra e extratorácica e fixa) da curva. O estudo foi aprovado pela Comissão de Ética para a Saúde do CHLN e todos os doentes assinaram um consentimento informado de participação. Resultados: Estudaram-se 82 doentes, 36 (44%) dos quais com OVAC. A predominância foi do género masculino, em relação ao feminino. A sensibilidade e especificidade dos critérios quantitativos da curva de débito-volume na detecção de OVAC foi de 91.3% e 88.9% respectivamente. Quando se utilizaram os critérios morfológicos da curva de débito-volume os valores foram de 93.5% e 30.6%. A agregação dos critérios morfológicos e quantitativos permitiu alcançar uma sensibilidade de 95.7% e especificidade de 86.1%. Nesta amostra, o critério quantitativo com maior ocorrência foi o FEF50/FIF50≥1 (83% dos doentes com OVAC). Este mostrou relacionar-se com todas as localizações de obstrução excepto o terço médio da traqueia. Mostrou, ainda, ter uma relação forte e positiva com o grau e tipo de obstrução (intra e extraluminal). O segundo foi o FEV1/PEF≥8, presente em 36% dos casos de OVAC. Relacionou-se com as obstruções no terço inferior da traqueia e brônquio principal direito (BPD). Também apresentou relação forte e positiva com o grau de obstrução e com os tipos de obstrução anteriormente descritos. Quanto à sintomatologia foi possível associar o grau de obstrução com o de dispneia e a presença de estridor com o grau e localização da obstrução na traqueia. Conclusões: Os resultados deste estudo demonstram que os critérios quantitativos da curva de débito-volume têm elevada sensibilidade e especificidade na detecção de OVAC. O critério FEV50/FIF50≥1 tem um bom poder discriminativo na detecção dessa condição, tendo sido relacionado com a localização, o grau e o tipo de obstrução. O critério FEV1/PEF≥8, embora com menor poder discriminativo, também se relaciona com o grau, a localização e o tipo de obstrução. A morfologia da curva tem uma boa sensibilidade mas baixa especificidade na detecção de OVAC, mas a agregação entre os critérios morfológicos e quantitativos aumenta a sensibilidade e especificidade. A dispneia e o estridor foram relacionados com o grau de obstrução e o último com a localização ao nível da traqueia.-------------ABSTRACT: Introduction: Central airway obstruction (CAO) refers to a pathological process that leads to restriction of airflow at the level of the glottis and subglottis, trachea and main bronchi. It’s proper diagnosis and treatment is an area of interest and concern to health professionals, and requires a deep knowledge of its etiology, physiology, diagnosis and treatment options, concerning the potential to cause significant morbidity and mortality. The evaluation of CAO covers multiple aspects: the clinical component (signs and symptoms), the pathophysiological effect (lung function) and the imaging study (bronchoscopy and chest CT). The compilation of this data associated with the etiology, are important for establishing prognosis, determine the need for treatment or outline a future therapeutic intervention. Bronchoscopy is the gold standard for evaluating this condition, but for the last 40 years the flow-volume loop has been used as a noninvasive tool for detecting CAO. Although this method is still in use, only few studies were made in order to verify its sensitivity and specificity in detecting CAO, and investigate the relation between morphological and quantitative changes of the curve to location, type and degree of obstruction. Methods: Between 1st November 2009 and 30th April 2010, patients with indication to perform diagnostic or therapeutic bronchoscopy in Interventional Pulmonology Unit - Hospital Pulido Valente (CHLN - HPV), were selected consecutively according to the inclusion and exclusion criteria. All assessments (bronchoscopy, flow-volume loop and dyspnea) were carried out within a period of seven days. The flexible bronchoscopy was performed according to the standards of the British Thoracic Society and the flow-volume loops in accordance with the standards of the ATS / ERS Taskforce 2005. For the evaluation of dyspnea was used to MRC dyspnea scale (Medical Research Council). A panel of experts evaluated the morphology of flow-volume loop (suggestive or non-suggestive of CAO) and an independent element established the quantitative criteria and morphological (intra and extrathoracic variables and fixed) of the curve. This study was approved by the Ethics Committee for Health CHLN and all the patients signed an informed consent to participate. Results: We’ve studied 82 patients, 36 (44%) of those with CAO. The majority of the patients were males, compared to females. The sensitivity and specificity of the quantitative criteria of the flow-volume curve in detecting CAO was 91.3% and 88.9% respectively. When we used the morphological criteria of flow-volume loop these values were 93.5% and 30.6%. The combination of quantitative and morphological criteria produced values of 95.7% sensitivity and 86.1% specificity. FEF50/FIF50≥1 was the most representative quantitative criterion (83% of patients with CAO) and it was correlated with all sites of obstruction except in the middle third of the trachea. It has shown a strong and positive association with the degree and type of obstruction (intra and extraluminal). The second was the FEV1/PEF ≥ 8, present in 36% of cases of CAO. It could be correlated with the obstruction in the lower third of the trachea and right main bronchus. It also showed a strong positive relation with the degree and types of obstruction described above. Regarding symptoms, we found a link between the degree of obstruction and dyspnea. The presence of stridor was correlated with the location and the degree of obstruction in the trachea. Conclusion: The results of this study demonstrate that the quantitative criteria of the flow-volume loop have a high sensitivity and specificity in detecting CAO. The criterion FEV50/FIF50 ≥ 1 has a good discriminative power to detect this condition and was related to the location, degree and type of obstruction. The criterion FEV1/PEF ≥ 8, although with a weaker discriminative power, also relates to the degree, location and type of obstruction. The morphology of the curve has a good sensitivity but low specificity in detecting CAO although the combination between the morphological and quantitative criteria increases sensitivity and specificity. Dyspnea and stridor were related to the degree of obstruction and the last one with its location in the trachea.

Parasitological stool sample exam by spontaneous sedimentation method using conical tubes: effectiveness, practice, and biosafety

INTRODUCTION: Spontaneous sedimentation is an important procedure for stool examination. A modification of this technique using conical tubes was performed and evaluated. METHODS: Fifty fecal samples were processed in sedimentation glass and in polypropylene conical tubes. Another 50 samples were used for quantitative evaluation of protozoan cysts. RESULTS: Although no significant differences occurred in the frequency of protozoa and helminths detected, significant differences in protozoan cyst counts did occur. CONCLUSIONS: The use of tube predicts a shorter path in the sedimentation of the sample, increases concentration of parasites for microscopy analysis, minimizes the risks of contamination, reduces the odor, and optimizes the workspace.

A method to correct the flow distortion of offshore wind data using CFD simulation and experimental wind tunnel tests

The assessment of wind energy resource for the development of deep offshore wind plants requires the use of every possible source of data and, in many cases, includes data gathered at meteorological stations installed at islands, islets or even oil platforms—all structures that interfere with, and change, the flow characteristics. This work aims to contribute to the evaluation of such changes in the flow by developing a correction methodology and applying it to the case of Berlenga island, Portugal. The study is performed using computational fluid dynamic simulations (CFD) validated by wind tunnel tests. In order to simulate the incoming offshore flow with CFD models a wind profile, unknown a priori, was established using observations from two coastal wind stations and a power law wind profile was fitted to the existing data (a=0.165). The results show that the resulting horizontal wind speed at 80 m above sea level is 16% lower than the wind speed at 80 m above the island for the dominant wind direction sector.

Almirante: Evaluating the board game: Board game, startup, discounted cash flow, venture capital method, real options

This thesis evaluates a start-up company (Jogos Almirante Lda) whose single asset is a board game named Almirante. It aims to conclude whether it makes sense to create a company or just earn copyrights. The thesis analyzes the board game’s market, as part of the general toy’s market, from which some data exists: European countries as well as the USA. In this work it is analyzed the several ways to finance a start-up company and then present an overview of the valuation of the Jogos Almirante based on three different methods: Discounted Cash Flow, Venture Capital Method and Real Options.

A rapid and simple method to detect ESBL in Enterobacter cloacae based on MIC of cefepime

INTRODUCTION: The aim of this study was to identify a rapid and simple phenotypic method for extended-spectrum β-lactamase (ESBL) detection in Enterobacter cloacae. METHODS: A total of 79 consecutive, non-repeated samples of E. cloacae were evaluated. Four phenotypic methods were applied for ESBL detection, results were compared to multiplex polymerase chain reaction (PCR) as the gold standard reference method: 1) ceftazidime and cefotaxime disks with and without clavulanate, both with boronic acid added; 2) disk approximation using cefepime and amoxicillin/clavulanate; 3) ESBL screening by minimum inhibitory concentration (MIC) ≥ 16µg/mL and 4) by MIC ≥ 2µg/mL for cefepime. RESULTS: Method 4 showed the best combination of sensitivity (100%) and specificity (94%). CONCLUSIONS: MIC ≥ 2µg/mL for cefepime would be very useful for the phenotypic detection of ESBL in samples of E. cloacae.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

The use of a modular titanium endoprosthesis in skeletal reconstructions after bone tumor resections: method presentation and analysis of 37 cases

We analyzed 37 patients who underwent segmental wide resection of bone tumors and reconstruction with a modular titanium endoprosthesis at the Orthopaedic Oncology Group, between 1992 and 1998. Twelve patients were male and 25 were female, with a mean age of 30 years (9 - 81). The mean follow-up was 14 months (2 - 48). The diagnoses were: osteosarcoma (14 cases), metastatic carcinoma (10), Ewing's sarcoma (4), giant cell tumor (4), malignant fibrous histiocytoma (3), chondrosarcoma (1), and aneurysmal bone cyst (1). Eleven articulated total knee, 8 partial proximal femur with bipolar acetabulum, 8 partial proximal humerus, 3 total femur, 2 partial proximal tibia, 2 diaphyseal femur, 2 diaphyseal humerus, and 1 total proximal femur with cementless acetabulum endoprosthesis implant procedures were done. The complications related to the procedure included: infection (5 cases), dislocation (3), module loosening (1), and ulnar nerve paresthesia (1). We used the following criteria for the clinical evaluation: presence of pain, range of motion, reconstruction stability, surgical and oncologic complications, and patient acceptance. The results were good in 56.8% of the cases, regular in 32.4% and poor in 10.8%.

Lag-user method: Using laggards to leverage user innovation

Laggards are the last users to adopt a product. Prior literature on user-led innovation ignores laggards’ impact on innovation. In this paper, we develop the Lag-User Method, through which laggards can generate new ideas. Through six studies with 62 teams in three countries, we apply the method to different technologies and services and present our findings to executives to get managerial insights. Findings reveal that laggards who generate new ideas (lag-users) have different perceptions of user-friendly products and different unfulfilled needs. They prefer simple products. We propose that by involving lag-users in NPD, firms can improve the effectiveness of NPD.

New method for evaluation of cutaneous sensibility in diabetic feet: preliminary report

Diabetic neuropathy is an important complication of the disease, responsible for ulceration and amputation of the foot. Prevention of these problems is difficult mainly because there is no method to correctly access sensibility on the skin of the foot. The introduction of the Pressure-Specified Sensory Device (PSSD TM) in the last decade made possible the measurement of pressure thresholds sensed by the patient, such as touch, both static and in movement, on a continuous scale. This paper is the first in Brazil to report the use of this device to measure cutaneous sensibility in 3 areas of the foot: the hallux pulp, the calcaneus, and the dorsum, which are territories of the tibial and fibular nerves. METHOD: Non-diabetic patients were measured as controls, and 2 groups of diabetic patients - with and without ulcers - were compared. The PSSD TM was used to test the 3 areas described above. The following were evaluated: 1 PS (1-point static), 1 PD (1-point dynamic), 2 PS (2-points static), 2 PD (2-points dynamic). RESULTS: The diabetic group had poorer sensibility compared to controls and diabetics with ulcers had poorer sensibility when compared to diabetics without ulcers. The differences were statistically significant (P <.001). CONCLUSION: Due to the small number of patients compared, the results should be taken as a preliminary report.

A continuous solvent- and oil-free method to prepare injectable PEGylated fibrinogen cell-laden microparticles

Injectable biomaterials with in situ cross-linking reactions have been suggested to minimize the invasiveness associated with most implantation procedures. However, problems related with the rapid liquid-to-gel transition reaction can arise because it is difficult to predict the reliability of the reaction and its end products, as well as to mitigate cytotoxicity to the surrounding tissues. An alternative minimally invasive approach to deliver solid implants in vivo is based on injectable microparticles, which can be processed in vitro with high fidelity and reliability, while showing low cytotoxicity. Their delivery to the defect can be performed by injection through a small diameter syringe needle. We present a new methodology for the continuous, solvent- and oil-free production of photopolymerizable microparticles containing encapsulated human dermal fibroblasts. A precursor solution of cells in photo-reactive PEG-fibrinogen (PF) polymer was transported through a transparent injector exposed to light-irradiation before being atomized in a jet-in-air nozzle. Shear rheometry data provided the cross-linking kinetics of each PF/cell solution, which was then used to determine the amount of irradiation required to partially polymerize the mixture prior to atomization. The partially polymerized drops fell into a gelation bath for further polymerization. The system was capable of producing cell-laden microparticles with high cellular viability, with an average diameter of between 88.1 µm to 347.1 µm and a dispersity of between 1.1 and 2.4, depending on the parameters chosen.

Innovative method for the continuous monitoring of concrete viscoelastic properties since early ages: concept and pilot experiments

The experimental evaluation of viscoelastic properties of concrete is traditionally made upon creep tests that consist in the application of sustained loads either in compression or in tension. This kind of testing demands for specially devised rigs and requires careful monitoring of the evolution of strains, whereas assuring proper load constancy. The characterization of creep behaviour at early ages offers additional challenges due to the strong variations in viscoelastic behaviour of concrete during such stages, demanding for several testing ages to be assessed. The present research work aims to assist in reducing efforts for continuous assessment of viscoelastic properties of concrete at early ages, by application of a dynamic testing technique inspired in methodologies used in polymer science: Dynamic Mechanical Analyses. This paper briefly explains the principles of the proposed methodology and exhibits the first results obtained in a pilot application. The results are promising enough to encourage further developments.

Annoyance and welfare costs from the presence of renewable energy power plants: an application of the contingent valuation method

Sustainability is frequently defined by its three pillars: economically viable, socially equitable, and environmentally bearable. Consequently the evaluation of the sustainability of any decision, public or private, requires information on these three dimensions. This paper focuses on social sustainability. In the context of renewable energy sources, the examination of social sustainability requires the analysis of not only the efficiency but also the equity of its welfare impacts. The present paper proposes and applies a methodology to generate the information necessary to do a proper welfare analysis of the social sustainability of renewable energy production facilities. This information is key both for an equity and an efficiency analysis. The analysis focuses on the case of investments in renewable energy electricity production facilities, where the impacts on local residents’ welfare are often significantly different than the welfare effects on the general population. We apply the contingent valuation method to selected facilities across the different renewable energy power plants located in Portugal and conclude that local residents acknowledge differently the damage sustained by the type, location and operation of the plants. The results from these case studies attest to the need of acknowledging and quantifying the negative impacts on local communities when assessing the economic viability, social equity and environmental impact of renewable energy projects.

Typology based method for choosing old masonry walls inspection procedures

The diagnosis of historic masonry walls is an intricate and complex field and has been an object of research for many years. This paper aims to propose practical methodologies for the diagnosis of historic masonry walls, specifically based on their typological characteristics. In order to develop such procedures, information relating to historic masonry typologies in Portugal, classified as rural, urban and military was gathered and techniques for the assessment of historic masonry were studied. All information was integrated to develop a pattern typology oriented methodology. Developed methodology was tested and validated in a small diagnosis campaign carried out in the Guimarães Castle. Methodology was proven to be advantageous and although the study is limited and focused on the Portuguese architectural specificities, it still holds global classifications, and therefore can be useful for any diagnosis procedure of a historic masonry wall.