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 da fosfatase String/Cdc25 como um regulador da neurotoxicidade de Tau em Drosophila

As tauopatias, grupo onde se inclui a doença de Alzheimer (AD), são caracterizadas pela deposição intracelular de emaranhados neurofibrilares (NFTs), compostos principalmente por formas hiperfosforiladas da proteína Tau, uma proteína que se associa aos microtúbulos. Os mecanismos moleculares subjacentes à neurotoxicidade induzida por Tau não são ainda claros. Drosophila melanogaster tem sido usada para modelar diversas doenças neurodegenerativas humanas, incluindo as tauopatias. Neste trabalho foi usado o sistema visual de Drosophila como modelo para identificar os passos que podem levar à acumulação de Tau em Tauopatias. Durante o desenvolvimento do olho de Drosophila, a expressão ectópica de hTau induz um olho rugoso, em consequência da neurotoxicidade, e que pode ser utilizado para identificar modificadores do fenótipo. A fosfatase codificada por string /cdc25 (stg), um regulador universal da transição G2/M, foi previamente identificada como um supressor da neurotoxicidade associada à expressão da proteina Tau. No entanto, os mecanismos moleculares que estão na base desta interação genética nunca foram estudados, desconhecendo-se também se a atividade fosfatase de Stg/Cdc25 é essencial para modular os níveis de fosforilação de Tau. O objetivo deste projeto consistiu em elucidar os mecanismos que se encontram na base da interação Stg-Tau. Para alcançar este objectivo, usou-se uma abordagem genética e bioquímica. Os resultados obtidos sugerem que Stg é um possível modulador da neurotoxicidade de Tau.

The role of cellular prion protein in Alzheimer’s disease

Alzheimer’s disease (AD) is the most prevalent age-related neurodegenerative disease that leads to cognitive impairment and dementia. The major defined pathological hallmark of AD is the accumulation of amyloid beta (Aβ), a neurotoxic peptide, derived from beta and gamma-secretase cleavage of the amyloid precursor protein (APP). It has been described that cellular prion protein (PrPC) plays a role in the pathogenesis of Alzheimer disease. Although, the role of PrPC is still unclear, previous studies showed contradictious results. To elucidate this issue, the main objective of the present study is to investigate the influence of a knockout of the PRNP gene in 5XFAD mice, 5xFAD mice exhibited 5 mutations related to familial Alzheimer disease. These mice show an Aβ1-42 accumulation and an increased neuronal loss during aging. To create a bi-transgenic 5xFAD mice were crossed with Prnp0/0 Zurich 1 mice (prion protein knockout mice). We subjected two transgenic mice (5xFAD and Prnp0/05xFAD) at different ages (3, 9 and 12 months of age) to a battery of task to evaluate cognitive and motoric deficits and a biochemical analysis (ELISA, western blot and immunohistochemistry) to investigate the regulation and potential involvement of downstream signaling proteins in the Aβ induced toxicity process dependent of the PrPC concentration. The study revealed that the deficits induced by Aβ mediated toxicity appeared earlier in 5xFAD mice (9 months of age) than in Prnp0/05xFAD (12 months of age). Investigating the amount of amyloid beta in 5xFAD mice we observed a PrPC dependent regulation in 9 month-old animals of Aβ1−40 but not of the toxic form Aβ1−42. We did not found in Prnp0/05xFAD mice the up-regulation of P-Fyn, Fyn or Cav-1 as we found in 5xFAD mice. This suggests an important role of PrPC in Alzheimer’s disease as a promoter of toxic effect of Aβ oligomers. Our results may suggest the loss of PrPC delays the toxicity of amyloid beta. In conclusion, our data support a role of PrPC as a mediator of Aβ toxicity in AD by promoting early onset of disease.