Estrogen receptor dependent genetic and epigenetic factors of tamoxifen resistance

Resumo: A decisão da terapêutica hormonal no tratamento do cancro da mama baseiase na determinação do receptor de estrogénio alfa por imunohistoquímica (IHC). Contudo, a presença deste receptor não prediz a resposta em todas as situações, em parte devido a limitações do método IHC. Investigámos se a expressão dos genes ESR1 e ESR2, bem como a metilação dos respectivos promotores, pode estar relacionada com a evolução desfavorável de uma proporção de doentes tratados com tamoxifeno assim como com a perda dos receptores de estrogénio alfa (ERα) e beta (ERß). Amostras de 211 doentes com cancro da mama diagnosticado entre 1988 e 2004, fixadas em formalina e preservadas em parafina, foram utilizadas para a determinação por IHC da presença dos receptores ERα e ERß. O mRNA total do gene ESR1 e os níveis específicos do transcrito derivado do promotor C (ESR1_C), bem como dos transcritos ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 foram avaliados por Real-time PCR. Os promotores A e C do gene ESR1 e os promotores 0K e 0N do gene ESR2 foram investigados por análise de metilação dos dinucleotidos CpG usando bisulfite-PCR para análise com enzimas de restrição, ou para methylation specific PCR. Atendendo aos resultados promissores relacionados com a metilação do promotor do gene ESR1, complementamos o estudo com um método quantitativo por matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) suportado pelo software Epityper para a medição da metilação nos promotores A e C. Fez-se a avaliação da estabilidade do mRNA nas linhas celulares de cancro da mama MCF-7 e MDA-MB-231 tratadas com actinomicina D. Baixos níveis do transcrito ESR1_C associaram-se a uma melhor sobrevivência global (p = 0.017). Níveis elevados do transcrito ESR1_C associaram-se a uma resposta inferior ao tamoxifeno (HR = 2.48; CI 95% 1.24-4.99), um efeito mais pronunciado em doentes com tumores de fenótipo ERα/PgR duplamente positivo (HR = 3.41; CI 95% 1.45-8.04). A isoforma ESR1_C mostrou ter uma semi-vida prolongada, bem como uma estrutura secundária da região 5’UTR muito mais relaxada em comparação com a isoforma ESR1_A. A análise por Western-blot mostrou que ao nível da 21 proteína, a selectividade de promotores é indistinguivel. Não se detectou qualquer correlação entre os níveis das isoformas do gene ESR2 ou entre a metilação dos promotores do gene ESR2, e a detecção da proteína ERß. A metilação do promotor C do gene ESR1, e não do promotor A, foi responsável pela perda do receptor ERα. Estes resultados sugerem que os níveis do transcrito ESR1_C sejam usados como um novo potencial marcador para o prognóstico e predição de resposta ao tratamento com tamoxifeno em doentes com cancro da mama. Abstract: The decision of endocrine breast cancer treatment relies on ERα IHC-based assessment. However, ER positivity does not predict response in all cases in part due to IHC methodological limitations. We investigated whether ESR1 and ESR2 gene expression and respective promoter methylation may be related to non-favorable outcome of a proportion of tamoxifen treated patients as well as to ERα and ERß loss. Formalin-fixed paraffin-embedded breast cancer samples from 211 patients diagnosed between 1988 and 2004 were submitted to IHC-based ERα and ERß protein determination. ESR1 whole mRNA and promoter C specific transcript levels, as well as ESR2_ß1, ESR2_ß2/cx, and ESR2_ß5 transcripts were assessed by real-time PCR. ESR1 promoters A and C, and ESR2 promoters 0N and 0K were investigated by CpG methylation analysis using bisulfite-PCR for restriction analysis, or methylation specific PCR. Due to the promising results related to ESR1 promoter methylation, we have used a quantification method by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) together with Epityper software to measure methylation at promoters A and C. mRNA stability was assessed in actinomycin D treated MCF-7 and MDA-MB-231 cells. ERα protein was quantified using transiently transfected breast cancer cells. Low ESR1_C transcript levels were associated with better overall survival (p = 0.017). High levels of ESR1_C transcript were associated with non-favorable response in tamoxifen treated patients (HR = 2.48; CI 95% 1.24-4.99), an effect that was more pronounced in patients with ERα/PgR double-positive tumors (HR = 3.41; CI 95% 1.45-8.04). The ESR1_C isoform had a prolonged mRNA half-life and a more relaxed 5’UTR structure compared to ESR1_A isoform. Western-blot analysis showed that at protein level, the promoter selectivity is undistinguishable. There was no correlation between levels of ESR2 isoforms or ESR2 promoter methylation and ERß protein staining. ESR1 promoter C CpG methylation and not promoter A was responsible for ERα loss. We propose ESR1_C levels as a putative novel marker for breast cancer prognosis and prediction of tamoxifen response.

Genetic diversity of arginine catabolic mobile element in Staphylococcus epidermidis

PLos One, 4(11): ARTe7722

Constraint-based modeling of minimum set covering: application to species differentation

Work presented in the context of the European Master in Computational Logics, as partial requisit for the graduation as Master in Computational Logics

New mechanisms that regulate the expression of genes implicated in the process of ketogenesis

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Genetic characterization of Portuguese Fasciola hepatica isolates

Dissertation presented to obtain the Master Degree in Molecular, Genetics and Biomedicine

Marketing plan for Snack Books

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Validation of alternative additives for paste production

Dissertation presented to confer Master Degree in Chemical and Biochemical Engineering

Atomic force microscopy assisted with electron and ion beam microscopy for the direct measurement of biostructures and DNA samples

Dissertation to obtain a Master degree in Biotechnology

Epidemiovigilância das infeções por Leishmania spp. no efetivo cinotécnico da Guarda Nacional Republicana e em mesocarnívoros silvestres

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Planning and verification of multipath routing protocols

Conventionally the problem of the best path in a network refers to the shortest path problem. However, for the vast majority of networks present nowadays this solution has some limitations which directly affect their proper functioning, as well as an inefficient use of their potentialities. Problems at the level of large networks where graphs of high complexity are commonly present as well as the appearing of new services and their respective requirements, are intrinsically related to the inability of this solution. In order to overcome the needs present in these networks, a new approach to the problem of the best path must be explored. One solution that has aroused more interest in the scientific community considers the use of multiple paths between two network nodes, where they can all now be considered as the best path between those nodes. Therefore, the routing will be discontinued only by minimizing one metric, where only one path between nodes is chosen, and shall be made by the selection of one of many paths, thereby allowing the use of a greater diversity of the present paths (obviously, if the network consents). The establishment of multi-path routing in a given network has several advantages for its operation. Its use may well improve the distribution of network traffic, improve recovery time to failure, or it can still offer a greater control of the network by its administrator. These factors still have greater relevance when networks have large dimensions, as well as when their constitution is of high complexity, such as the Internet, where multiple networks managed by different entities are interconnected. A large part of the growing need to use multipath protocols is associated to the routing made based on policies. Therefore, paths with different characteristics can be considered with equal level of preference, and thus be part of the solution for the best way problem. To perform multi-path routing using protocols based only on the destination address has some limitations but it is possible. Concepts of graph theory of algebraic structures can be used to describe how the routes are calculated and classified, enabling to model the routing problem. This thesis studies and analyzes multi-path routing protocols from the known literature and derives a new algebraic condition which allows the correct operation of these protocols without any network restriction. It also develops a range of software tools that allows the planning and the respective verification/validation of new protocols models according to the study made.

Analyzing Geospatial patterns of syrian refugee flows in southeastern Turkey by use of remote sensing and complementary data

Crisis-affected communities and global organizations for international aid are becoming increasingly digital as consequence geotechnology popularity. Humanitarian sector changed in profound ways by adopting new technical approach to obtain information from area with difficult geographical or political access. Since 2011, turkey is hosting a growing number of Syrian refugees along southeastern region. Turkish policy of hosting them in camps and the difficulty created by governors to international aid group expeditions to get information, made such international organizations to investigate and adopt other approach in order to obtain information needed. They intensified its remote sensing approach. However, the majority of studies used very high-resolution satellite imagery (VHRSI). The study area is extensive and the temporal resolution of VHRSI is low, besides it is infeasible only using these sensors as unique approach for the whole area. The focus of this research, aims to investigate the potentialities of mid-resolution imagery (here only Landsat) to obtain information from region in crisis (here, southeastern Turkey) through a new web-based platform called Google Earth Engine (GEE). Hereby it is also intended to verify GEE currently reliability once the Application Programming Interface (API) is still in beta version. The finds here shows that the basic functions are trustworthy. Results pointed out that Landsat can recognize change in the spectral resolution clearly only for the first settlement. The ongoing modifications vary for each case. Overall, Landsat demonstrated high limitations, but need more investigations and may be used, with restriction, as a support of VHRSI.

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.

Is the Basel III Leverage Ratio countercyclical? A study for Portuguese banks

This work analyses how the leverage ratio behaves through the cycle, vis-à-vis other capital ratios. For a sample of the largest Portuguese banks, the Basel III leverage ratio is indeed countercyclical. This result is relevant from a regulatory perspective, since the introduction of a limit on the leverage ratio will function as a restriction in the banks’ balance sheet size, reducing the economic costs associated with the excessive growth of leverage in periods of economic expansion followed by aggressive deleveraging in the downturn. However, one cannot exclude that restrictions on banks’ leverage incentivize its transference to less regulated intermediaries.

Aspects of Passives in Capeverdean (Santiago’s variety)

The present dissertation intends to study passives in Capeverdean. I argue that Capeverdean have eventive passives with specific morphology, postverbal morphemes, -du and -da, which affix to the verb to form passives and interact with the TMA morphemes available in the language in the same way as in the active voice. I also show that Capeverdean only allows short passives. However, this study demonstrates that the by-phrase, although not expressed phonologically, is implicit and can be tracked through instrumentals and Agent-oriented adverbs. In order to account for this specific property of Capeverdean passives, I assume the existence of a Voice head which introduces the external argument in all finite sentences in Capeverdean, except in unaccusatives, following proposals from Marantz (1984), Kratzer (1996), Sailor & Ahn (2010), Pratas (2014). I also assume that this Voice head is subject to a Doubly Filled Comp Filter, similar to what is proposed in Koopman (1997), which determines that either heads or specifiers can be overt, never both. In the case of passives, I propose that external argument is in Spec,Voice and the passive morphology is lexicalized in Voice0 and that while Spec,Voice is silent, Voice0 is not. This configuration can be explained if it is assumed, following Costa & Martins (2004), that in Capeverdean passives Voice0 is a strong functional head, thus requiring visibility at PF. This restriction, combined with the Doubly Filled Comp filter, imposes that Spec,Voice is silent.

A caracterização do trabalho análogo ao escravo na jurisprudência dos tribunais brasileiros

Based on bibliographical research and the analysis of court rulings, this study investigates the characterization of slave-like labor by Brazilian courts. After the alteration of article 149 of the Brazilian Penal Code, introduced by Law nº 10.803/2003, which typifies the practice of contemporary slavery in Brazil, divergent characterizations of this practice remain. The courts currently employ the broadest concept of contemporary slave labor, in which the crime is characterized by the engagement in one of the following conducts established as a criminal offense: labor with the restriction of freedom, submission to exhaustive working conditions, degrading working conditions, and debt bondage. The engagement in one of the above is therefore enough to constitute a crime. Contemporary slave labor in Brazil is not characterized only by the restriction of the worker’s freedom, as in the case of forced labor or debt bondage, but also through the submission of the workers to situations that offend their human dignity. Individual freedom and the dignity of the human person, fundamental tenets of the Brazilian Federal Constitution, are juridical resources safeguarded by law. Contemporary slavery is not limited to the mere infringement of labor laws, but represents a severe violation of the human rights of the workers involved.