Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

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.

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Biochemistry, 2004, 43 (46), pp 14566–14576 DOI: 10.1021/bi0485833

C-reactive protein in early risk assessment in acute pancreatitis

ABSTRACT:C-reactive protein (CRP) has been widely used in the early risk assessment of patients with acute pancreatitis (AP), but unclear aspects about its prognostic accuracy in this setting persist. This project evaluated first CRP prognostic accuracy for severity, pancreatic necrosis (PNec), and in-hospital mortality (IM) in AP in terms of the best timing for CRP measurement and the optimal CRP cutoff points. Secondly it was evaluated the CRP measured at approximately 24 hours after hospital admission (CRP24) prognostic accuracy for IM in AP individually and in a combined model with a recent developed tool for the early risk assessment of patients with AP, the Bedside Index for Severity in AP (BISAP). Two single-centre retrospective cohort studies were held. The first study included 379 patients and the second study included 134 patients. Statistical methods such as the Hosmer-Lemeshow goodness-of-fit test, the area under the receiver-operating characteristic curve, the net reclassification improvement, and the integrated discrimination improvement were used. It was found that CRP measured at approximately 48 hours after hospital admission (CRP48) had a prognostic accuracy for severity, PNec, and IM in AP better than CRP measured at any other timing. It was observed that the optimal CRP48 cutoff points for severity, PNec, and IM in AP varied from 170mg/l to 190mg/l, values greater than the one most often recommended in the literature – 150mg/l. It was found that CRP24 had a good prognostic accuracy for IM in AP and that the cutoff point of 60mg/l had a negative predictive value of 100%. Finally it was observed that the prognostic accuracy of a combined model including BISAP and CRP24 for IM in AP could perform better than the BISAP alone model. These results might have a direct impact on the early risk assessment of patients with AP in the daily clinical practice.--------- RESUMO: A proteina c-reactiva (CRP) tem sido largamente usada na avaliação precoce do risco em doentes com pancreatite aguda (AP), mas aspectos duvidosos acerca do seu valor prognóstico neste contexto persistem. Este projecto avaliou primeiro o valor prognóstico da CRP para a gravidade, a necrose pancreática (PNec) e a mortalidade intra-hospitalar (IM) na AP em termos do melhor momento para efectuar a sua medição e dos seus pontos-de-corte óptimos. Em segundo lugar foi avaliado o valor prognóstico da proteína c-reactiva medida aproximadamente às 24 horas após a admissão hospitalar (CRP24) para a IM na AP isoladamente e num modelo combinado, que incluiu uma ferramenta de avaliação precoce do risco em doentes com AP recentemente desenvolvida, o Bedside Index for Severity in Acute Pancreatitis (BISAP). Dois estudos unicêntricos de coorte retrospectivo foram realizados. O primeiro estudo incluiu 379 doentes e o segundo estudo incluiu 134 doentes. Metodologias estatísticas como o teste de Hosmer-Lemeshow goodness-of-fit, a area under the receiver-operating characteristic curve, o net reclassification improvement e o integrated discrimination improvement foram usadas. Verificou-se que a CRP medida às 48 horas após a admissão hospitalar (CRP48) teve um valor prognóstico para a gravidade, a PNec e a IM na AP melhor do que a CRP medida em qualquer outro momento. Observou-se que os pontos de corte óptimos da CRP48 para a gravidade, a PNec e a IM na AP variaram entre 170mg/l e 190mg/l, valores acima do valor mais frequentemente recomendado na literatura – 150mg/l. Verificou-se que a CRP medida aproximadamente às 24 horas após a admissão hospitalar (CRP24) teve um bom valor prognóstico para a IM na AP e que o ponto de corte 60mg/l teve um valor preditivo negativo de 100%. Finalmente observou-se que o valor prognóstico de um modelo combinado incluindo o BISAP e a CRP24 para a IM na AP pode ter um desempenho melhor do que o do BISAP isoladamente. Estes resultados podem ter um impacto directo na avaliação precoce do risco em doentes com AP na prática clínica diária.

Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)