Sinalização celular e a resposta a estrogénios do epitélio mamário

Estrogens, such as 17β-estradiol (E2) are essential for normal growth and differentiation of the mammary gland. There are two estrogen receptors (ERs), ERα and ERβ which are ligand activated transcription factors. ERα stimulates proliferation and is the single most powerful predictor of breast cancer prognosis and since 70% of breast cancers express ERα, strategies to block this receptor are the primary breast cancer treatment. Unlike ERα, the role of ERβ in breast cancer and its potential as alternative therapeutic target remains controversial, mainly due to the lack of correlation between results obtained in vitro and epidemiological studies. The aim of this thesis was to increase our understanding of the molecular and cellular mechanisms of estrogen signaling in normal and cancerous cells, in different cellular contexts and with focus on ERβ. In Paper I we characterized the effect of the flavone PD098059 - which is a commonly used MEK1 inhibitor - on activation of transcription by ERα and ERβ. We found that the estrogenic effect of PD098059 is dose dependent in concentrations ranging from 1 – 10 μM and that activation of transcription by ER is suppressed by the inhibitory effect of PD98059 on MEK1 at concentrations above 50 μM. In agreement with its flavone nature, PD098059 had a much stronger effect on ERβ than on ERα transcriptional activity. Therefore, use of this compound for the study of signalling events in cells expressing ER should be carefully considered. In Paper II we assessed the effect of ERβ agonists in vivo and administered under different conditions in vitro. In basal conditions, ERβ induced apoptosis; however, in vivo ERβ agonists stimulated proliferation and inhibited apoptosis. In vivo effects were reproduced in culture, by activation of MAPK/ERK½ pathway with epidermal growth factor or basement membrane extract. In addition, insulin signalling and PI3-K/AKT activation was necessary for stimulation of proliferation. These results suggest that the cellular context modulates ERβ activity. Manuscript presents preliminary work aimed at the set-up of a methodological strategy to isolate ERs and to identify interacting proteins in different cellular contexts and which could modulate the bi-phased effects of ERβ in cell growth. In conclusion, the studies presented in this thesis contribute to clarify the apparent contradictory information regarding ERβ function in normal and cancerous mammary epithelium and suggest that the cellular context should be considered when ERβ effects are studied.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Mitochondrial dysfunction in fatty acid β-oxidation disorders

Mitochondria are central organelles for cell survival with particular relevance in energy production and signalling, being mitochondrial fatty acid β–oxidation (FAO) one of the metabolic pathways harboured in this organelle. FAO disorders (FAOD) are among the most well studied inborn errors of metabolism, mainly due to their impact in health. Nevertheless, some questions remain unsolved, as their prevalence in certain European regions and how pathophysiological determinants combine towards the phenotype. Analysis of data from newborn screening programs from Portugal and Spain allowed the estimation of the birth prevalence of FAOD revealing that this group of disorders presents in Iberia (and particularly in Portugal) one of the highest European birth prevalence, mainly due to the high birth prevalence of medium chain acyl-CoA dehydrogenase deficiency. These results highlight the impact of this group of genetic disorders in this European region. The characterization of mitochondrial proteome, from patients fibroblasts with FAOD, namely multiple acyl-CoA dehydrogenase deficiency (MADD) and long chain acyl-CoA dehydrogenase deficiency (LCHADD), provided a global perspective of the mitochondrial proteome plasticity in these disorders and highlights the main molecular pathways involved in their pathogenesis. Severe MADD forms show an overexpression of chaperones, antioxidant enzymes (MnSOD), and apoptotic proteins. An overexpression of glycolytic enzymes, which reflects cellular adaptation to energy deficiency due to FAO blockage, was also observed. When LCHADD fibroblasts were analysed a metabolic switching to glycolysis was also observed with overexpression of apoptotic proteins and modulation of the antioxidant defence system. Severe LCHADD present increased ROS alongside with up regulation of MnSOD while moderate forms have lower ROS and down-regulation of MnSOD. This probably reflects the role of MnSOD in buffering cellular ROS, maintain them at levels that allow cells to avoid damage and start a cellular response towards survival. When ROS levels are very high cells have to overexpress MnSOD for detoxifying proposes. When severe forms of MADD were compared to moderate forms no major differences were noticed, most probably because ROS levels in moderate MADD are high enough to trigger a response similar to that observed in severe forms. Our data highlights, for the first time, the differences in the modulation of antioxidant defence among FAOD spectrum. Overall, the data reveals the main pathways modulated in FAOD and the importance of ROS levels and antioxidant defence system modulation for disease severity. These results highlight the complex interaction between phenotypic determinants in FAOD that include genetic, epigenetic and environmental factors. The development of future better treatment approaches is dependent on the knowledge on how all these determinants interact towards phenotype.!

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

Caracterização química e bioquímica dos frutos de ARBUTUS UNEDO L. e de RUBUS FRUTICOSUS AGG.: contribuição para a sua valorização

Dissertação mest., Agricultura sustentável, Universidade do Algarve, 2007

Purificação e caracterização de proteínas Gla nos modelos peixe e mamífero

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2010

Dissection of the early steps in the assembly of the 20S proteasome in Saccharomyces cerevisiae

Tese de dout., Bioquímica (Biologia Celular e Molecular), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Cloning, expression and purification of pneumococcal proteins = clonagem, expressão e purificação de proteínas de pneumococcus

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2008

Produção e purificação da proteína rica em gla (grp) humana recombinante

Dissertação de mest., Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Estudo da reversão das alteracções funcionais da actina após processo de oxidação/redução

Relatório de estágio da licenciatura, Bioquímica, Faculdade de Ciência e Tecnologia da Universidade do Algarve, 2007

Estudo de refolding oxidativo de proteínas

O folding oxidativo de proteínas consiste na formação de pontes dissulfureto intramoleculares envolvendo a oxidação de grupos tiol no sentido da criação de uma ligação entre duas cisteínas. Esta modificação postransducional é essencial para a estabilidade das proteínas, principalmente em proteínas secretadas para o meio extracelular. In vivo, o folding oxidativo ocorre no retículo endoplasmático e é assistido por uma série de proteínas que atuam como catalisadores. Estas reações em cadeia necessitam da presença de um aceitador final de eletrões. No presente trabalho foram estudadas duas vias que atuam no reticulo endoplasmático para o refolding oxidativo da proteína modelo Ribonuclease A: Uma via envolve a interação entre duas proteínas, a Endoplasmic Recticulum Oxireductase 1 (Ero1) e a Protein Disulfide Isomerase (PDI); A outra via envolve a interação da PDI com a Peroxiredoxin IV (PRDX4). Foi igualmente estudado o refolding oxidativo com uma enzima homóloga da PRDX4, a PRDX2, no sentido de compreender se existe especificidade na interação entre a PRDX4 e a PDI. O estudo do refolding oxidativo da Ribonuclease foi realizado in vitro e avaliado em géis SDS-PAGE-Tricina com o objetivo de verificar a diferença de mobilidades entre a Ribonuclease reduzida e oxidada no gel. Na via da PRDX4/PDI e PRDX2/PDI é necessária a introdução de Glucose e Glucose Oxidase, responsáveis pela produção de peróxido de hidrogénio que atua como aceitador final de eletrões desta via. Em todas as vias foi observado refolding oxidativo da RNase. Na via da Ero1/PDI este foi substancialmente mais rápido e ocorre, embora em muito menor grau, mesmo na ausência da PDI. Na via da PRDX4/PDI o refolding é mais lento e foi constatado que não existe especificidade da PRDX4 para a PDI visto que, na presença da PRDX2, os resultados foram semelhantes aos resultados obtidos com a PRDX4.

The effect of sucrose on protein stability and fibrillation of prion and S6 protein

Aggregation and fibrillation of proteins have a great importance in medicine and industry. Misfolding and aggregation are the basis of many neurodegenerative diseases like Alzheimer and Parkinson. Osmolytes are molecules that can accumulate within cells and act as protective agents and they can inclusively act as protein stabilizers when cells are exposed to stress conditions. Osmolytes can also act as protein stabilizers in vitro. In this work, two different proteins were studied, the ribosomal protein from Thermus thermophilus and the mouse prion protein. The existence of an unstructured N-terminal on the prion protein does not affect its stability. The effect of the osmolyte sucrose on the fibrillation and stabilization of these two proteins was studied through kinectic and equilibrium measurements. It was shown that sucrose is able to compact the native structure of S6 protein in fibrillization conditions. Sucrose affects also folding and unfolding kinetic of S6 protein, delaying unfolding and increasing folding rate constants. The mechanism of stabilization by sucrose is non-specific because it is distributed for all protein structure, as it was demonstrated by a protein engineering approach. Sucrose delays the process of formation and elongation of S6 and prion protein from mouse. This delay is the result of the compaction of the native structure refered above. However, cellular toxicity studies have shown that fibrils formed in the presence of sucrose are more toxic to neuronal cells.

Extração e estudo das proteínas da goma da alfarroba com vista à sua clarificação

O objectivo primordial deste trabalho foi efectuar a extracao e o estudo das proteinas da Goma da Alfarroba ou Locust Bean Gum (LBG) com vista a sua clarificacao e purificacao. Para atingir este objectivo em primeiro lugar estudou-se o teor de etanol que se deveria utilizar numa suspensao aquosa de LBG, uma vez que a LBG e um polissacarido que hidrata facilmente com agua e consequentemente origina solucoes altamente viscosas que impossibilitam a realizacao de tecnicas de extracao de proteinas. Para isso estudou-se o comportamento da LBG em suspensoes aquosas com 10% (p/v) de LBG e diferentes concentracoes de etanol, designadamente 10% (v/v), 20% (v/v), 30% (v/v) e 40% (v/v). Com este estudo concluiu-se que seria necessario utilizar uma concentracao de 40% (v/v) de etanol a 96% em cada suspensao de LBG a 10% (p/v) para conseguir evitar a sua hidratacao excessiva. Assim, todas as extracoes neste trabalho foram realizadas de acordo com estas condicoes. Posteriormente com vista a extracao das proteinas da LBG testaram-se diferentes tratamentos, designadamente tratamentos alcalinos, acidos e com detergentes. Segundo a bibliografia consultada para os tratamentos alcalinos, realizaram-se estas extracoes com hidroxido de sodio (NaOH) com concentracoes de 0,025 M, 0,05M, 0,1M e 0,2M. Estes tratamentos foram iniciados com uma concentracao de NaOH a 0,2M, contudo apos analise dos resultados verificou-se que esta originou uma LBG com uma coloracao mais amarela do que a LBG bruta (inicial) apresentava. Sendo a ideia final a clarificacao da goma este nao foi um resultado desejavel e como tal testaram-se concentracoes inferiores de NaOH, acima referidas. No final concluiu-se que todas estas extracoes efetuadas com NaOH originavam uma LBG mais amarela do que a LBG bruta e que assim este nao seria o tratamento mais adequado para o objectivo pretendido. Como tal prosseguiram-se os estudos com um tratamento acido, que atraves de consulta bibliografica se iniciou com acido sulfurico (H2SO4). O tratamento acido iniciou-se com uma concentracao de H2SO4 de 0,1M, testando-se posteriormente tambem uma extracao com H2SO4 com concentracao de 0,05M. No final destas extracoes verificou-se que a LBG obtida em ambas apresentava uma coloracao mais clara do que a LBG bruta, o que representava, numa primeira analise, um bom resultado. Por fim efectuaram-se extracoes solido-liquido com diferentes detergentes, designadamente com Tween 20, Tween 40, Tween 60, Tween 65, Tween 80, Tween 85, Triton X-100, Triton X-114 e SDS todos a uma concentracao de 0,1% (v/v) a excepcao do Tween 65 em que foi utilizada uma concentracao de 0,1% (p/v), porque a temperatura ambiente este detergente e solido. No final destas extracoes concluiu-se que na maioria dos casos a LBG apresentava uma coloracao mais clara do que a LBG bruta. Para quantificar as proteinas extraidas atraves de cada um dos tratamentos acima mencionados utilizaram-se dois metodos, o metodo de Bradford para quantificar as proteinas presentes nos sobrenadantes das extracoes e o metodo de Kjeldahl para quantificar as proteinas ainda presentes na LBG apos as extracoes. Analisando os resultados obtidos por estes metodos concluiu-se que a extracao efetuada com Tween 80, em que se efectuou uma lavagem adicional a LBG durante a filtracao com uma solucao de agua destilada e etanol a 40% (v/v), foi a que apresentou melhores resultados. Assim, foi com esta amostra proteica que se prosseguiram os estudos as proteinas. Para realizar os estudos as proteinas extraidas efectuou-se uma eletroforese em SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) com o intuito de verificar o estado da amostra. Analisando os geles obtidos constatou-se que a amostra se encontrava em boas condicoes. Deste modo realizou-se uma eletroforese bidimensional (2 – D) das proteinas extraidas da LBG. Nesta tecnica, como se desconheciam que tipo de proteinas se encontravam presentes na amostra utilizou-se uma banda de pH para a Focagem Isoelectrica (1a Dimensao) entre 3 e 10 e a separacao por Peso Molecular (2a Dimensao) foi efetuada em SDS-PAGE. Analisando os geles obtidos concluiu-se que a maioria das proteinas presentes nesta amostra apresentam pontos isoelectricos na gama de pH entre 5 e 6 e tem pesos moleculares entre 40 e 60 kDa. Pode-se entao concluir que o objectivo deste trabalho foi apenas parcialmente atingido uma vez que se conseguiu extrair e estudar algumas das caracteristicas das proteinas presentes na LBG, mas nao se alcancou a sua clarificacao.

Deteção de proteínas virais com nanossensores óticos

O desenvolvimento de tecnologias para a deteção de vírus é um tema de grande interesse em medicina de diagnóstico. Neste contexto, os biossensores têm-se revelado importantes ferramentas bioanalíticas, na medida em que permitem efetuar deteções moleculares de forma rápida, específica, reprodutível e com baixo custo associado. No âmbito desta dissertação, estabeleceram-se metodologias para a deteção fluorescente do fator de infetividade viral (Vif) do VIH através de um nanossensor ótico funcionalizado com anticorpos recombinantes anti-Vif, fragmentos variáveis de cadeia simples 4BL. Quantum dots carboxílicos conjugados a proteínas Vif são excitados a um comprimento de onda de 405 nm, transmitindo luz fluorescente a 605 nm a um fotodetetor de silício amorfo hidrogenado com um filtro de fluorescência integrado. Para implementar um sistema de deteção sensível e eficiente, estudaram-se estratégias de ativação de superfície em chips de vidro. A variabilidade das condições experimentais de um protocolo de silanização com (3-mercaptopropil)-trimetoxisilano (MPTS) foi avaliada através da análise de ângulos de contacto e densidades moleculares de fluoresceína funcionalizada com maleimida. Estabeleceu-se que, uma incubação dos substratos por 4 h com 5% (w/v) MPTS, seguida de cura a 110 ºC por 2 h, e redução com 10 mM ditiotreitol por 30 min, promove a formação de camadas de organosilanos de qualidade com grupos tiol reativos bem orientados. Estratégias de imobilização do elemento recetor foram testadas em substratos de vidro ativados e em sistemas de microfluídica vidro/PDMS, tomando por base o tag de afinidade, glutationa S-transferase (GST), do anticorpo recombinante. Vidros funcionalizados com glutationa e anti-GST mostraram-se igualmente reativos ao anticorpo GST-4BL, resultando uma capacidade de ligação do antigénio de aproximadamente 4.76 x 1010 moléculas/cm2 de QD-Vif, o que corresponde a 15% da cobertura máxima de superfície. Dos ensaios em sistemas de microfluídica resultaram sinais fracos e poucos reprodutíveis, destacando a necessidade de desenvolver metodologias de funcionalização mais apropriadas.

Nanopartículas poliméricas com aplicação na administracão pulmunar de proteínas

O interesse da indústria farmacêutica na administração de biomoléculas não é recente, uma vez que desde que a insulina foi comercializada pela primeira vez em 1923, temos vindo a assistir ao aumento do número de trabalhos relacionados com as aplicações terapêuticas deste tipo de moléculas. No entanto, a aplicação terapêutica destas moléculas regista várias limitações devidas essencialmente à sua estrutura e propriedades físico-químicas, bem como à sua estabilidade. Com o aparecimento e evolução da nanomedicina, tornou-se possível o desenvolvimento e funcionalização de nanopartículas transportadoras de biomoléculas, dotando este sistema de um enorme potencial para várias terapêuticas. As vantagens deste tipo de sistema de administração incluem a proteção das moléculas ativas encapsuladas, proporcionando um aumento da biodisponibilidade, e tornam também possível a administração das biomoléculas por vias menos invasivas. Esta dissertação tem por objetivo explorar as potencialidades da via pulmonar para a administração de biomoléculas. Neste sentido, serão focadas os vários tipos de nanopartículas de polímeros naturais e sintéticos como sistema de administração com aplicação na administração por esta via.