Protein glycosylation of exosomes from ovarian carcinoma cells: structures and biological roles

Dissertation presented to obtain the Ph.D. degree in BiologyExosomes are small membrane vesicles that are secreted by several cell types including tumour cells. They are formed intracellularly by an inward budding of the membrane of endosomal compartments which are converted to multivesicular bodies. Exosomes are then released into the extracellular environment after fusion of the multivesicular bodies with the plasma membrane. Upon internalization by other cells they may transfer proteins and RNA among cells. Tumour-derived exosomes can promote angiogenesis, cell proliferation, tumour cell invasion and immune evasion. These vesicles have been found in biological fluids such as malignant ascites and blood and can therefore be used not only to identify potential biomarkers of disease but also in vaccination.(...)Fundação para a Ciência e Tecnologia (FCT) and Fundo Social Europeu (FSE) for financial support (SFRH/BD/30622/2006)

o novo papel do Estado; análise da perspectiva da evolução recente do Direito Administrativo; o exemplo do sector da energia

Over the last years, we have been witnessing vast transformations in Administrative Law, most of them driven by a change in the role of the State in the economy and contemporary society. This change, instead of entailing a reduction in State’s functions, has been responsible for the development of a new model of State: the Regulatory State. This thesis departs from the analysis of this new model of State, in the broad context of liberalization and privatization of public utilities, particularly network industries. It then questions, through the frame of the modern legal theory of regulation, in what way the new functions taken over by the State lead to an alteration of concepts and legal instruments used by Administrative Law. This theoretical analysis is complemented by an examination of a paradigmatic case study, which is provided by the Portuguese energy sector (electricity and natural gas).Nos últimos anos tem-se assistido a importantes transformações no âmbito do Direito Administrativo, grande parte das quais motivada por uma alteração do papel do Estado na economia e na sociedade contemporâneas. Essa alteração, ao invés de significar uma redução das funções do Estado, deu lugar ao aparecimento de um novo modelo de Estado: o Estado Regulador. A presente dissertação parte da análise desse novo modelo de Estado, no contexto dos movimentos de liberalização e de privatização dos tradicionais serviços públicos económicos prestados em rede, para, no quadro da moderna teoria jurídica da regulação, questionar de que forma a assunção de novas funções pelo Estado implica uma alteração dos conceitos, bem como dos instrumentos jurídicos usados pelo Direito Administrativo. A análise teórica é complementada pelo exame de um caso de estudo paradigmático fornecido pelo sector da energia português (electricidade e gás natural)

Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB

Funding: This study was supported by Fundaçã o para a Ciência e Tecnologia (FCT): C.E. was supported by a post-doctoral fellowship (SFRH/BPD/78491/2011), L.B.-L. by a PhD fellowship (SFRH/BD/131938/2017) and D.C.B. by the FCT Investigator Program (IF/00501/2014/CP1252/CT0001). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). This article was supported by the LYSOCIL project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 811087. Deposited in PMC for immediate release.Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.publishersversionpublishe

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.EU Joint Programme JPND/0003/2011, FCT grant: Pest-OE/EQB/LA0004/2011, FCT PhD fellowship

Redeployment e contrato psicológico : estudo de caso em multinacional do sector químico em Portugal

Mestrado em Gestão de Recursos HumanosO presente estudo tem como objetivo principal definir qual o tipo de Contrato Psicológico vigente num grupo de colaboradores de uma empresa multinacional do sector químico, que após um processo de downsizing recorreu ao redeployment como estratégia de reter alguns dos seus talentos possibilitando a sua recolocação em outras suas subsidiárias. Recorreu-se a uma metodologia mista, através de análise qualitativa de duas entrevistas e de análise quantitativa, através de análise de dados, após a sua recolha, através de um questionário online. Analisou-se o Contrato Psicológico em sete dimensões de cumprimento por parte do funcionário, cinco dimensões de cumprimento por parte da organização e três dimensões relativas à relação da organização com o funcionário, as transições no Contrato Psicológico. Estas dimensões agrupam-se em quatro tipos de contrato: Relacional, Transacional, Transitório e Equilibrado. Também se avaliou a quebra e a violação do Contrato Psicológico, através de escalas também já existentes e testadas para esse âmbito. Os resultados indicam a prevalência do contrato Relacional, composto pelas dimensões Estabilidade e Lealdade do funcionário e Lealdade da organização. Revelaram ainda que não existiu quebra ou rutura do Contrato Psicológico e que o tipo Transitório, que poderia ter obtido maiores valores dada a mudança que existiu para estes colaboradores, foi por sua vez, o mais baixo.This study aimed to analyze the type of Psychological Contract present in a group of workers from a multinational company of the chemical sector which after a downsizing process, resorted redeployment as a retention strategy for some employees, relocating them in some other subsidiaries of the group. A mixed methodology was used, through qualitative analysis of two interviews and quantitative analysis, through data analysis, after collection, across an online questionnaire. The Psychological Contract was analyzed in seven dimensions of compliance by the employee, five dimensions of compliance by the organization and three dimensions on the organization's relationship with the employee, the transitions in the Psychological Contract. These dimensions are grouped into four types of contract: Relational, Transactional, Transitional and Balanced. It was also assessed the breach and violation of the Psychological Contract through scales already tested for this issue. The results revealed a highlight for the Relational Contract, composed of the dimensions? stability and loyalty of the employee and loyalty of the organization. The results revealed that there was no breach or disruption of the Psychological Contract and the Transitional type, which could have obtained higher values, given the change that existed for these employees, was in turn, the lowest.info:eu-repo/semantics/publishedVersio

Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells

PURPOSE: To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover. METHODS: Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting. RESULTS: Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression. CONCLUSIONS: Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy

Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction

PURPOSE: We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS: We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS: AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS: We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation

Formation of lipofuscin-like autofluorescent granules in the retinal pigment epithelium requires lysosome dysfunction

Funding Information: Supported by Funda??o para a Ciência e Tecnologia (FCT) ? Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union?s Horizon 2020 programme under grant agreement No. 811087. Funding Information: Supported by Fundação para a Ciência e Tecnologia (FCT) – Portugal co-funded by FEDER under the PT2020 Partnership Agreement (to MCS, including project PTDC/MED-PAT/30385/2017, iNOVA4Health-UIDB/04462/2020, research infrastructure PPBI-POCI-01-0145-FEDER-022122, M-ERA.NET 2/0005/2016), Boehringer Ingelheim (to MCS), Fight for Sight UK (to MCS), Wellcome Trust grant number 212216/Z/18/Z/ (to CEF). MJH was funded by Moor-fields Eye Charity with the Bill Brown 1989 Charitable Trust PhD studentship 538158, MLS was funded by FCT-CEECIND/01536/2018, ACF was funded by FCT PhD studentship (PD/BD/135503/2018). This work was developed with the support from the research infrastructure PPBI-POCI-01-0145-FEDER-022122, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund) and this article is supported by the LYSOCIL project funded by the European Union’s Horizon 2020 programme under grant agreement No. 811087. Publisher Copyright: Copyright 2021 The AuthorsPURPOSE. We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. METHODS. We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. RESULTS. AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. CONCLUSIONS. We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.publishersversionpublishe

Interaction and uptake of exosomes by ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells.</p> <p>Methods</p> <p>SKOV3 exosomes were labelled with carboxyfluoresceine diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts.</p> <p>Results</p> <p>In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide β-lactose reduced exosomes uptake to a comparable extent as the control D-glucose.</p> <p>Conclusions</p> <p>In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.</p

Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing

Exosomes are nanosized (30–100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines.Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA.For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We found that most abundant exosomal RNA species are the fragments of 28S and 18S rRNA subunits. This limits the number of reads from other RNAs. To increase the number of detectable transcripts and improve the accuracy of their expression level the protocols allowing depletion of fragmented rRNA should be utilized in the future RNA-seq analyses on exosomes. Present data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers