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.

Economias de escala em centros hospitalares

RESUMO - Perante o actual contexto de contenção de gastos no sector da saúde e consequente preocupação com a eficiência do sistema, tem‐se assistido a mudanças várias no modelo de gestão e organizacional do sistema de saúde. Destaca‐se a alteração da estrutura hospitalar, com vista à racionalização dos seus recursos internos, onde as fusões hospitalares têm assumido um papel determinante. Em Portugal, nos últimos 10 anos, assistiu‐se a uma significativa redução do número de hospitais (de sensivelmente 90 para 50 unidades), exclusivamente através das fusões e sem quaisquer alterações no número de estruturas físicas existentes. Não obstante os argumentos justificativos desta reforma, a avaliação dos objectivos implícitos é insuficiente. Neste âmbito, pretendeu‐se com este estudo contribuir para a análise do impacte da criação de centros hospitalares na redução de gastos, isto é, verificar se a consolidação e consequente reengenharia dos processos produtivos teve consequencias ao nível da obtenção de economias de escala. Para esta análise usou‐se uma base de dados em painel, onde se consideraram 75 hospitais durante 7 anos (2003‐2009), número que foi reduzindo ao longo do período em análise devido às inúmeras fusões já referidas. Para avaliar os ganhos relativos às fusões hospitalares, ao nível da eficiência técnica e das economias de escala, recorreu‐se à fronteira estocástica especificada função custo translog. Estimada a fronteira, foi possível analisar três centros hospitalares específicos, onde se comparou o período pré‐fusão (2005‐2006) com o período após a fusão (2008‐2009). Como variáveis explicativas, relativas à produção hospitalar, considerou‐se o número de casos tratados e os dias de internamento (Vita, 1990; Schuffham et al., 1996), o número de consultas e o número de urgências, sendo estas variáveis as mais comuns na literatura (Vita, 1990; Fournier e Mitchell, 1992; Carreira, 1999). Quanto à variável dependente usou‐se o custo variável total, que compreende o total de custos anuais dos hospitais excepto de imobilizado. Como principais conclusões da investigação, em consequência da criação dos centros hospitalares, são de referir os ganhos de escala na fusão de hospitais de reduzida dimensão e com mais serviços complementares. --------ABSTRACT - Driven by the current pressure on resources induced by budgetary cuts, the Portuguese Ministry of Health is imposing changes in the management model and organization of NHS hospitals. The most recent change is based on the creation of Hospital Centres that are a result of administrative mergers of existing hospitals. In less than 10 years the number of hospitals passed from around 90 to around 50, only due to the mergers and without any change in the existing number of physical institutions. According to the political discourse, one of the main goals expected from this measure is the creation of synergies and more efficiency in the use of available resources. However, the merger of the hospitals has been a political decision without support or evaluation of the first experiments. The aim of this study is to measure the results of this policy by looking at economies of scale namely through reductions in the expenditures, as expected and sought by the MoH. Data used covers 7 years (2003‐2009) and 75 hospitals, number that has been reduced my the enoumerous mergers during the last decade. This work uses a stochastic frontier analysis through the translog cost function to examine the gains from mergers, which were decomposed into technical efficiency and economies of scale. It was analised these effects by the creation of three specific hospital centers, using a longitudinal approach to compare the period pre‐merger (2003‐2006) with the post‐merger period (2007‐09). To measure changes in inpatient hospital production volume and length of stay are going to be considered as done by Vita (1990) and Schuffham et al. (1996). For outpatient services the number of consultations and emergencies are going to be considered (Vita, 1990; Fournier e Mitchell, 1992; Carreira, 1999). Total variable cost is considered as the dependent variable explained the aforementioned ones. After a review of the literature results expected point to benefits from the mergers, namely a reduction in total expenditures and in the number of duplicated services. Results extracted from our data point in the same direction, and thus for the existence of some economies of scale only for small hospitals.

Crystallographic studies on molybdopterin-dependent enzymes

Dissertação para obtenção do Grau de Doutor em Bioquímica, Especialidade Bioquímica Estrutural

Cilia motility studies in zebrafish embryos

A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and Biomedicine

Information flow analysis using data-dependent logical propositions

Dissertação para obtenção do Grau de Mestre em Engenharia Informática

The mechanism of formate oxidation by metal-dependent formate dehydrogenases

J Biol Inorg Chem (2011) 16:1255–1268 DOI 10.1007/s00775-011-0813-8

Modelling HIV/AIDS length of stay in Portugal

Tese apresentada como requisito parcial para obtenção do grau de Doutor em Estatística e Gestão de Informação pelo Instituto Superior de Estatística e Gestão de Informação da Universidade Nova de Lisboa

Financial crisis spillovers to the corporate sector: Bank-dependent borrowers versus bond market paticipants

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

Welfare analysis of regulating mobile termination rates in the UK with an application to the Orange/T-Mobile merger

We present a calibrated model of the UK mobile telephony market with four mobile networks; calls to and from the fixed network; network-based price discrimination; and call externalities. Our results show that reducing mobile termination rates broadly in line with the recent European Commission Recommendation to either pure long-run incremental cost ; reciprocal termination charges with fixed networks; or Bill & Keep (i.e. zero termination rates), increases social welfare, consumer surplus and networks profits. Depending on the strength of call externalities, social welfare may increase by as much as £ 990 million to £ 4.5 billion per year, with Bill & Keep leading to the highest increase in welfare. We also apply the model to estimate the welfare effects of the 2010 merger between Orange and T-Mobile under different scenarios concerning MTRs, and predict that consumer surplus decreases strongly.

Trans-Synaptic Signaling by Activity-Dependent Cleavage of Neuroligin-1

Throughout the brain, patterns of activity in postsynaptic neurons influence the properties of synaptic inputs. Such feedback regulation is central to neural network stability that underlies proper information processing and feature representation in the central nervous system. At the cellular level, tight coupling of presynaptic and postsynaptic function is fundamental to neural computation and synaptic plasticity. The cohort of protein complexes at the pre and postsynaptic membrane allows for tight synapse-specific segregation and integration of diverse molecular and electrical signals.(...)

Avaliação epidemiológica dos internamentos através de um serviço de urgência de segunda linha (SII)

RESUMO: Introdução: A Comissão Nacional para a reestruturação dos Serviços de Saúde Mental em 2004, fez uma proposta de âmbito regional, ao nível da região de Saúde do Norte, levando a uma alteração da rede de referenciação hospitalar dos internamentos em psiquiatria. Métodos: Realizou-se um estudo epidemiológico longitudinal para avaliar factores preditivos de internamento através de um serviço de Urgência de Segunda Linha (SII), que de algum modo reflectisse o funcionamento dos serviços de internamento na região de Saúde do Norte, ao longo de 12 anos, nomeadamente relacionando com factores organizacionais contemplados no Plano Nacional de Saúde Mental e na reorganização da rede de referenciação hospitalar. Resultados: Durante os 12 anos do estudo, verificou-se um aumento estatisticamente significativo do número e duração de internamentos através do SII, com ponto de partida no ano 2008-2009, e de novo a partir do ano 2010-2011 (nº de internamentos), para o qual contribuiu a alteração da rede hospitalar na região de saúde do Norte, nomeadamente pelo facto do HMLemos, assumir a responsabilidade de novo, dos internamentos das áreas de Famalicão, Gondomar e Santa Maria da Feira. Em relação ao número de internamentos, e na análise exploratória, encontramos nas áreas hospitalares fora da área de influência do HMLemos, uma contribuição positiva significativa para o aumento do nº de internamentos ao longo dos anos com os Dx (290, 296, 297, 291, 309). Em relação à área do HMLemos restrita (PVC, STT, Matosinhos, Porto Ocidental), de referir a contribuição positiva significativa dos Dx 309 e 301, para o aumento do número de internamentos ao longo do tempo, sendo que a prevalência maior se mantém relacionada às Psicoses (Dx 295, 296 e 297). Não se concluiu por uma contribuição estatisticamente significativa ( positiva ou negativa), das variáveis independentes idade, sexo ou natureza do internamento em relação à variável dependente ( duração de internamentos/ano). Em relação á variável dependente (nº de internamentos/ano), relativamente aos doentes fora de área de influência do HMLemos, concluiu-se uma contribuição positiva estatisticamente significativa da variável independente idade. Conclusões: Através da análise exploratória foi possível perceber o esforço realizado pelos hospitais no sentido de melhorar a equidade e acessibilidade dos doentes à Saúde Mental, a par da reorganização da rede hospitalar. De destacar a necessidade de encontrar alternativas às situações de internamento, com menos critérios de gravidade diagnóstica, nomeadamente reforçar a importância da criação de consultas de crises nos respectivos Hospitais de Dia dos DPSM.----------------ABSTRACT:Introduction : The National Commission for the restructuring of mental health services in 2004 , has proposed at a regional level ( North Health Region), a change in the network of hospital referrals of admissions in psychiatry. Methods: We conducted a longitudinal epidemiological study to assess predictors of hospitalization through a Second Line Emergency Service ( SII) , that somehow reflect the operation of inpatient services in North Health Region, over 12 years, particularly relating to organizational factors included in the National Mental Health Plan and reorganization of the hospital referral network. Results: During the period of the study, there was a statistically significant increase in the number and duration of hospitalizations through the SII, with starting point in the year 2008-2009 and again from 2010-2011 (number of admissions) , for which counted the change of the hospital network referral in Northern health region , in particular because Hospital Magalhães Lemos (HMLemos) , took the new responsibility of admissions from areas of Famalicão, Gondomar and Santa Maria da Feira . Regarding the number of hospitalizations, in the exploratory analysis , we found in hospital areas outside the area of influence of HMLemos , a significant positive contribution to the increase in number of admissions over the years with Diagnosis of 290, 296, 297, 291 , 309 in the ICD-9. With respect to the restricted area of HMLemos (PVC, STT , Porto Ocidental and Matosinhos) , we found a significant positive contribution of Diagnosis 309 and 301, to increase the number of hospitalizations over time, with higher prevalence rates remaining the psychoses ( Dx 295, 296 and 297 ) . Did not conclude for any statistically significant contribution (positive or negative) of the independent variables age, sex and nature of admission to the dependent variable (duration of hospitalization / year). In relation to the dependent variable (number of admissions / year) relative to patients outside the area of influence of HMLemos, it was found a statistically significant positive contribution of the independent variable age . Conclusions: Through the exploratory analysis, it was possible to see the efforts made by hospitals to improve the accessibility of patients to Mental Health, throughout the hospital network reorganization. Its important to highlight the need to find alternatives to inpatient admissions in those with less gravity diagnostic criteria, reinforcing the importance of creating specific crisis consultations in Day Hospital regime.

Evaluation of the welfare impact of regulating mobile termination rates in Portugal

Following the European Commission’s 2009 Recommendation on the Regulatory Treatment of Fixed and Mobile Termination Rates in the EU, the Portuguese regulatory authority (ANACOM) decided to reduce termination prices in mobile networks to their long-run incremental cost (LRIC). Nevertheless, no serious quantitative assessment of the potential effects of this decision was carried out. In this paper, we adapt and calibrate the Harbord and Hoernig (2014) model of the UK mobile telephony market to the Portuguese reality, and simulate the likely impact on consumer surplus, profits and welfare of four different regulatory approaches: pure LRIC, reciprocal termination charges with fixed networks, “bill & keep”, and asymmetric termination rates. Our results show that reducing MTRs does increase social welfare, profits and consumer surplus in the fixed market, but mobile subscribers are seriously harmed by this decision.

A logical foundation for session-based concurrent computation

Linear logic has long been heralded for its potential of providing a logical basis for concurrency. While over the years many research attempts were made in this regard, a Curry-Howard correspondence between linear logic and concurrent computation was only found recently, bridging the proof theory of linear logic and session-typed process calculus. Building upon this work, we have developed a theory of intuitionistic linear logic as a logical foundation for session-based concurrent computation, exploring several concurrency related phenomena such as value-dependent session types and polymorphic sessions within our logical framework in an arguably clean and elegant way, establishing with relative ease strong typing guarantees due to the logical basis, which ensure the fundamental properties of type preservation and global progress, entailing the absence of deadlocks in communication. We develop a general purpose concurrent programming language based on the logical interpretation, combining functional programming with a concurrent, session-based process layer through the form of a contextual monad, preserving our strong typing guarantees of type preservation and deadlock-freedom in the presence of general recursion and higher-order process communication. We introduce a notion of linear logical relations for session typed concurrent processes, developing an arguably uniform technique for reasoning about sophisticated properties of session-based concurrent computation such as termination or equivalence based on our logical approach, further supporting our goal of establishing intuitionistic linear logic as a logical foundation for sessionbased concurrency.

A type system for value-dependent information flow analysis

Information systems are widespread and used by anyone with computing devices as well as corporations and governments. It is often the case that security leaks are introduced during the development of an application. Reasons for these security bugs are multiple but among them one can easily identify that it is very hard to define and enforce relevant security policies in modern software. This is because modern applications often rely on container sharing and multi-tenancy where, for instance, data can be stored in the same physical space but is logically mapped into different security compartments or data structures. In turn, these security compartments, to which data is classified into in security policies, can also be dynamic and depend on runtime data. In this thesis we introduce and develop the novel notion of dependent information flow types, and focus on the problem of ensuring data confidentiality in data-centric software. Dependent information flow types fit within the standard framework of dependent type theory, but, unlike usual dependent types, crucially allow the security level of a type, rather than just the structural data type itself, to depend on runtime values. Our dependent function and dependent sum information flow types provide a direct, natural and elegant way to express and enforce fine grained security policies on programs. Namely programs that manipulate structured data types in which the security level of a structure field may depend on values dynamically stored in other fields The main contribution of this work is an efficient analysis that allows programmers to verify, during the development phase, whether programs have information leaks, that is, it verifies whether programs protect the confidentiality of the information they manipulate. As such, we also implemented a prototype typechecker that can be found at http://ctp.di.fct.unl.pt/DIFTprototype/.