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.

Evaluating Dynamic Signage for Emergency Evacuation using an Immersive Video Environment

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

Avatar modeling: a telepresence study with natural user interface

Dissertação para obtenção do Grau de Mestre em Engenharia Electrotécnica e de Computadores

Application of geographic information system and modelling in health impact assessment for urban road mobility in Vietnam

Transport is an essential sector in modern societies. It connects economic sectors and industries. Next to its contribution to economic development and social interconnection, it also causes adverse impacts on the environment and results in health hazards. Transport is a major source of ground air pollution, especially in urban areas, and therefore contributing to the health problems, such as cardiovascular and respiratory diseases, cancer, and physical injuries. This thesis presents the results of a health risk assessment that quantifies the mortality and the diseases associated with particulate matter pollution resulting from urban road transport in Hai Phong City, Vietnam. The focus is on the integration of modelling and GIS approaches in the exposure analysis to increase the accuracy of the assessment and to produce timely and consistent assessment results. The modelling was done to estimate traffic conditions and concentrations of particulate matters based on geo-references data. A simplified health risk assessment was also done for Ha Noi based on monitoring data that allows a comparison of the results between the two cases. The results of the case studies show that health risk assessment based on modelling data can provide a much more detail results and allows assessing health impacts of different mobility development options at micro level. The use of modeling and GIS as a common platform for the integration of different assessments (environmental, health, socio-economic, etc.) provides various strengths, especially in capitalising on the available data stored in different units and forms and allows handling large amount of data. The use of models and GIS in a health risk assessment, from a decision making point of view, can reduce the processing/waiting time while providing a view at different scales: from micro scale (sections of a city) to a macro scale. It also helps visualising the links between air quality and health outcomes which is useful discussing different development options. However, a number of improvements can be made to further advance the integration. An improved integration programme of the data will facilitate the application of integrated models in policy-making. Data on mobility survey, environmental monitoring and measuring must be standardised and legalised. Various traffic models, together with emission and dispersion models, should be tested and more attention should be given to their uncertainty and sensitivity

Experimental and numerical approaches for structural assessment in new footbridge designs (SFRSCC–GFPR hybrid structure)

Within the civil engineering field, the use of the Finite Element Method has acquired a significant importance, since numerical simulations have been employed in a broad field, which encloses the design, analysis and prediction of the structural behaviour of constructions and infrastructures. Nevertheless, these mathematical simulations can only be useful if all the mechanical properties of the materials, boundary conditions and damages are properly modelled. Therefore, it is required not only experimental data (static and/or dynamic tests) to provide references parameters, but also robust calibration methods able to model damage or other special structural conditions. The present paper addresses the model calibration of a footbridge bridge tested with static loads and ambient vibrations. Damage assessment was also carried out based on a hybrid numerical procedure, which combines discrete damage functions with sets of piecewise linear damage functions. Results from the model calibration shows that the model reproduces with good accuracy the experimental behaviour of the bridge.

A integração de técnicas BIM (Building Information Modeling) no projeto e na construção de estruturas de madeira

Dissertação de mestrado integrado em Engenharia Civil

Vascular disease modeling using induced pluripotent stem cells: Focus in Hutchinson-Gilford Progeria Syndrome

Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.014

Climate change effects on animal and plant phylogenetic diversity in southern Africa

Much attention has been paid to the effects of climate change on species' range reductions and extinctions. There is however surprisingly little information on how climate change driven threat may impact the tree of life and result in loss of phylogenetic diversity (PD). Some plant families and mammalian orders reveal nonrandom extinction patterns, but many other plant families do not. Do these discrepancies reflect different speciation histories and does climate induced extinction result in the same discrepancies among different groups? Answers to these questions require representative taxon sampling. Here, we combine phylogenetic analyses, species distribution modeling, and climate change projections on two of the largest plant families in the Cape Floristic Region (Proteaceae and Restionaceae), as well as the second most diverse mammalian order in Southern Africa (Chiroptera), and an herbivorous insect genus (Platypleura) in the family Cicadidae to answer this question. We model current and future species distributions to assess species threat levels over the next 70years, and then compare projected with random PD survival. Results for these animal and plant clades reveal congruence. PD losses are not significantly higher under predicted extinction than under random extinction simulations. So far the evidence suggests that focusing resources on climate threatened species alone may not result in disproportionate benefits for the preservation of evolutionary history.

Term Structure Dynamics, Macro-Finance Factors and Model Uncertainty

This paper extends the Nelson-Siegel linear factor model by developing a flexible macro-finance framework for modeling and forecasting the term structure of US interest rates. Our approach is robust to parameter uncertainty and structural change, as we consider instabilities in parameters and volatilities, and our model averaging method allows for investors' model uncertainty over time. Our time-varying parameter Nelson-Siegel Dynamic Model Averaging (NS-DMA) predicts yields better than standard benchmarks and successfully captures plausible time-varying term premia in real time. The proposed model has significant in-sample and out-of-sample predictability for excess bond returns, and the predictability is of economic value.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Wellposedness of a nonlinear, logarithmic Schrödinger equation of Doebner-Goldin type modeling quantum dissipation

This paper is concerned with the modeling and analysis of quantum dissipation phenomena in the Schrödinger picture. More precisely, we do investigate in detail a dissipative, nonlinear Schrödinger equation somehow accounting for quantum Fokker–Planck effects, and how it is drastically reduced to a simpler logarithmic equation via a nonlinear gauge transformation in such a way that the physics underlying both problems keeps unaltered. From a mathematical viewpoint, this allows for a more achievable analysis regarding the local wellposedness of the initial–boundary value problem. This simplification requires the performance of the polar (modulus–argument) decomposition of the wavefunction, which is rigorously attained (for the first time to the best of our knowledge) under quite reasonable assumptions.

Microfluidic processor allows rapid HER2 immunohistochemistry of breast carcinomas and significantly reduces ambiguous (2+) read-outs.

Biomarker analysis is playing an essential role in cancer diagnosis, prognosis, and prediction. Quantitative assessment of immunohistochemical biomarker expression on tumor tissues is of clinical relevance when deciding targeted treatments for cancer patients. Here, we report a microfluidic tissue processor that permits accurate quantification of the expression of biomarkers on tissue sections, enabled by the ultra-rapid and uniform fluidic exchange of the device. An important clinical biomarker for invasive breast cancer is human epidermal growth factor receptor 2 [(HER2), also known as neu], a transmembrane tyrosine kinase that connotes adverse prognostic information for the patients concerned and serves as a target for personalized treatment using the humanized antibody trastuzumab. Unfortunately, when using state-of-the-art methods, the intensity of an immunohistochemical signal is not proportional to the extent of biomarker expression, causing ambiguous outcomes. Using our device, we performed tests on 76 invasive breast carcinoma cases expressing various levels of HER2. We eliminated more than 90% of the ambiguous results (n = 27), correctly assigning cases to the amplification status as assessed by in situ hybridization controls, whereas the concordance for HER2-negative (n = 31) and -positive (n = 18) cases was 100%. Our results demonstrate the clinical potential of microfluidics for accurate biomarker expression analysis. We anticipate our technique will be a diagnostic tool that will provide better and more reliable data, onto which future treatment regimes can be based.

A flow cytometry based oligotrophic pollutant exposure test to detect bacterial growth inhibition and cell injury.

Toxicity of chemical pollutants in aquatic environments is often addressed by assays that inquire reproductive inhibition of test microorganisms, such as algae or bacteria. Those tests, however, assess growth of populations as a whole via macroscopic methods such as culture turbidity or colony-forming units. Here we use flow cytometry to interrogate the fate of individual cells in low-density populations of the bacterium Pseudomonas fluorescens SV3 exposed or not under oligotrophic conditions to a number of common pollutants, some of which derive from oil contamination. Cells were stained at regular time intervals during the exposure assay with fluorescent dyes that detect membrane injury (i.e., live-dead assay). Reduction of population growth rates was observed upon toxicant insult and depended on the type of toxicant. Modeling and cell staining indicate that population growth rate decrease is a combined effect of an increased number of injured cells that may or may not multiply, and live cells dividing at normal growth rates. The oligotrophic assay concept presented here could be a useful complement for existing biomarker assays in compliance with new regulations on chemical effect studies or, more specifically, for judging recovery after exposure to fluctuating toxicant conditions.

Characterization and clinical evaluation of CD10+ stroma cells in the breast cancer microenvironment.

PURPOSE: There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10(+) stromal cells, we aimed to better characterize this cell population and its clinical relevance. EXPERIMENTAL DESIGN: We developed a CD10(+) stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10(+) cells by coculture experiments of representative breast cancer cell lines with the different CD10(+) stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. RESULTS: This 12-gene CD10(+) stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10(+) cell types contribute to the CD10(+) stroma signature, although mesenchymal stem cells have the highest CD10(+) stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2(+) subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. CONCLUSIONS: Our results highlight the importance of CD10(+) cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2(+) breast cancer disease.