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Browsing by Author "Silva, Bruno"

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  • Active Flexible Films for Food Packaging: A Review
    Publication . Azevedo, Ana G.; Barros, Carolina; Miranda, Sónia; Machado, Ana Vera; Castro, Olga; Silva, Bruno; Saraiva, Margarida; Silva, Ana Sanches; Pastrana, Lorenzo; Carneiro, Olga Sousa; Cerqueira, Miguel A.
    Active food packaging is a dynamic area where the scientific community and industry have been trying to find new strategies to produce innovative packaging that is economically viable and compatible with conventional production processes. The materials used to develop active packaging can be organized into scavenging and emitting materials, and based on organic and inorganic materials. However, the incorporation of these materials in polymer-based flexible packaging is not always straightforward. The challenges to be faced are mainly related to active agents' sensitivity to high temperatures or difficulties in dispersing them in the high viscosity polymer matrix. This review provides an overview of methodologies and processes used in the production of active packaging, particularly for the production of active flexible films at the industrial level. The direct incorporation of active agents in polymer films is presented, focusing on the processing conditions and their effect on the active agent, and final application of the packaging material. Moreover, the incorporation of active agents by coating technologies and supercritical impregnation are presented. Finally, the use of carriers to help the incorporation of active agents and several methodologies is discussed. This review aims to guide academic and industrial researchers in the development of active flexible packaging, namely in the selection of the materials, methodologies, and process conditions.
    2022-06-16Journal article Open access Show more
  • Alternative polyadenylation and nonsense-mediated decay coordinately regulate the human HFE mRNA levels
    Publication . Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa
    Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 39 untranslated region (UTR), along with exon seven. Therefore, this 39 UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 39-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements.
    2012-04Journal article Open access Show more
  • An overview of molecular basis of iron metabolism regulation and the associated pathologies
    Publication . Silva, Bruno; Faustino, Paula
    Iron is essential for several vital biological processes. Its deficiency or overload drive to the development of several pathologies. To maintain iron homeostasis, the organism controls the dietary iron absorption by enterocytes, its recycling by macrophages and storage in hepatocytes. These processes are mainly controlled by hepcidin, a liver-derived hormone which synthesis is regulated by iron levels, inflammation, infection, anemia and erythropoiesis. Besides the systemic regulation of iron metabolism mediated by hepcidin, cellular regulatory processes also occur. Cells are able to regulate themselves the expression of the iron metabolism-related genes through different post-transcriptional mechanisms, as the alternative splicing, microRNAs, the IRP/IREs system and the proteolytic cleavage. Whenever those mechanisms are disturbed, due to genetic or environmental factors, iron homeostasis is disrupted and iron related pathologies may arise.
    2015-07Journal article Restricted access Show more
  • Base molecular da hemocromatose hereditária não-clássica em Portugal
    Publication . Faria, Ricardo; Silva, Bruno; Silva, Catarina; Loureiro, Pedro; Queiroz, Ana; Esteves, Jorge; Mendes, Diana; Fleming, Rita; Vieira, Luís; Gonçalves, João; Lavinha, João; Faustino, Paula
    A Hemocromatose Hereditária (HH) é uma doença autossómica recessiva caracterizada pela absorção excessiva de ferro a nível intestinal e sua acumulação em órgãos vitais, podendo originar cardiomiopatia, cirrose e carcinoma hepatocelular. O correspondente diagnóstico molecular é obtido pela associação com genótipos específicos no gene HFE (homozigotia para p.Cys282Tyr ou heterozigotia composta p.Cys282Tyr/p.His63Asp). Contudo, nos países do sul da Europa, cerca de um terço dos doentes com diagnóstico clínico de HH não apresenta os referidos genótipos. Para identificar a base molecular da HH não-clássica em Portugal usaram-se metodologias de pesquisa geral de variantes genéticas (SSCP e dHPLC), Next-Generation Sequencing (NGS) e sequenciação de Sanger, cobrindo seis genes relacionados com o metabolismo do ferro em 303 doentes. Identificaram-se 69 variantes diferentes e de vários tipos, por ex. missense, nonsense, de splicing, que perturbam a transcrição do gene ou a regulação da tradução do mRNA. Seguidamente, realizaram-se estudos in silico e in vitro para esclarecer o significado etiológico de algumas das novas variantes. Concluiu-se que a base molecular desta patologia é bastante heterogénea e que a NGS é uma ferramenta adequada para efetuar a análise simultânea dos vários genes num grande número de amostras. Contudo, o estabelecimento da relevância clínica de algumas variantes requer a realização de estudos funcionais.
    2016-05-12Journal article Open access Show more
  • Crosstalk between genetics, gene expression and biochemical markers supports systemic iron homeostasis dysregulation in alzheimer disease
    Publication . Crespo, Ângela C.; Silva, Bruno; Ferreira, Catarina; Marques, Liliana; Marcelino, Erica; Maruta, Carolina; Costa, Sónia; Timóteo, Ângela; Vilares, Arminda; Simões, Frederico; Faustino, Paula; Correia, Ana Paula; Verdelho, Ana; Porto, Graça; Guerreiro, Manuela; Herrero, Ana; Costa, Cristina; de Mendonça, Alexandre; Costa, Luciana; Martins, Madalena
    2014-06Conference object Open access Show more
  • Decrease in APP and CP mRNA expression supports impairment of iron export in Alzheimer's disease patients
    Publication . Guerreiro, Cláudia; Silva, Bruno; Crespo, Ângela; Marques, Liliana; Costa, Sónia; Timóteo, Ângela; Marcelino, Erica; Maruta, Carolina; Vilares, Arminda; Matos, Mafalda; Couto, Frederico S.; Faustino, Paula; Verdelho, Ana; Guerreiro, Manuela; Herrero, Ana; Costa, Cristina; de Mendonça, Alexandre; Martins, Madalena; Costa, Luciana
    Alzheimer's disease (AD) is a neurodegenerative disorder of still unknown etiology and the leading cause of dementia worldwide. Besides its main neuropathological hallmarks, a dysfunctional homeostasis of transition metals has been reported to play a pivotal role in the pathogenesis of this disease. Dysregulation of iron (Fe) metabolism in AD has been suggested, particularly at the level of cellular iron efflux. Herein, we intended to further clarify the molecular mechanisms underlying Fe homeostasis in AD. In order to achieve this goal, the expression of specific Fe metabolism-related genes directly involved in Fe regulation and export was assessed in peripheral blood mononuclear cells (PBMCs) from 73AD patients and 74 controls by quantitative PCR. The results obtained showed a significant decrease in the expression of aconitase 1 (ACO1; P=0.007); ceruloplasmin (CP; P<0.001) and amyloid-beta precursor protein (APP; P=0.006) genes in AD patients compared with healthy volunteers. These observations point out to a significant downregulation in the expression of genes associated with ferroportin-mediated cellular Fe export in PBMCs from AD patients, when compared to controls. Taken together, these findings support previous studies suggesting impairment of Fe homeostasis in AD, which may lead to cellular Fe retention and oxidative stress, a typical feature of this disease
    2015-10Journal article Restricted access Show more
  • Decrease of β-amyloid peptide precursor and ceruloplasmin mRNA levels in patients with Alzheimer’s disease support impairment of cellular iron efflux in this pathology
    Publication . Silva, Bruno; Guerreiro, Cláudia; C. Crespo, Ângela; Marques, Liliana; Marcelino, Erica; Maruta, Carolina; Costa, Sónia; Timóteo, Ângela; Vilares, Arminda; Simões Couto, Frederico; Faustino, Paula; Verdelho, Ana; Guerreiro, Manuela; Herrero, Ana; Costa, Cristina; de Mendonça, Alexandre; Martins, Madalena; Costa, Luciana
    Introduction: Alzheimer’s disease (AD) is the most common neurodegenerative disorder and the leading cause of dementia worldwide. Because of the clinical interest in its early diagnosis and prediction of patient evolution and prognosis, the identification of AD biomarkers is of crucial importance. Several lines of evidence implicate an imbalance of the redox-active biometals in AD. Metal-catalyzed hydroxyl radicals are potent mediators of cellular injury and are central to the oxidative injury hypothesis of AD pathogenesis [1,2]. Importantly, previous genetic and biochemical studies in AD patients support a concerted systemic iron metabolism dysregulation, namely at the level of cellular iron efflux [3]. Herein, we intended to further understand the molecular mechanisms underlying iron homeostasis in this pathology. In order to achieve this goal, the expression of specific iron metabolism-related genes directly involved in iron metabolism regulation and cellular iron export was measured in peripheral blood mononuclear cells from AD patients and healthy controls. Also, serum ceruloplasmin (CP) concentration and its oxidase activity were measured in all participants in the study, given the known role of this oxidase in facilitating iron exit from cells. Through this study we expect to further clarify the contribution of iron metabolism disruption to the etiopathogenesis of AD.
    2014-09Conference object Restricted access Show more
  • Differential HFE Gene Expression is Regulated by Alternative Splicing in Human Tissues
    Publication . Martins, Rute; Silva, Bruno; Proença, Daniela; Faustino, Paula
    Background - The pathophysiology of HFE-derived Hereditary Hemochromatosis and the function of HFE protein in iron homeostasis remain uncertain. Also, the role of alternative splicing in HFE gene expression regulation and the possible function of the corresponding protein isoforms are still unknown. The aim of this study was to gain insights into the physiological significance of these alternative HFE variants. Methodology/Principal Findings - Alternatively spliced HFE transcripts in diverse human tissues were identified by RT-PCR, cloning and sequencing. Total HFE transcripts, as well as two alternative splicing transcripts were quantified using a real-time PCR methodology. Intracellular localization, trafficking and protein association of GFP-tagged HFE protein variants were analysed in transiently transfected HepG2 cells by immunoprecipitation and immunofluorescence assays. Alternatively spliced HFE transcripts present both level- and tissue-specificity. Concerning the exon 2 skipping and intron 4 inclusion transcripts, the liver presents the lowest relative level, while duodenum presents one of the highest amounts. The protein resulting from exon 2 skipping transcript is unable to associate with β2M and TfR1 and reveals an ER retention. Conversely, the intron 4 inclusion transcript gives rise to a truncated, soluble protein (sHFE) that is mostly secreted by cells to the medium in association with β2M. Conclusions/Significance - HFE gene post-transcriptional regulation is clearly affected by a tissue-dependent alternative splicing mechanism. Among the corresponding proteins, a sHFE isoform stands out, which upon being secreted into the bloodstream, may act in remote tissues. It could be either an agonist or antagonist of the full length HFE, through hepcidin expression regulation in the liver or by controlling dietary iron absorption in the duodenum.
    2011-03-03Journal article Open access Show more
  • Duodenal Cytochrome B and Hephaestin Expression is Regulated by the Soluble HFE Isoform
    Publication . Silva, Bruno; Martins, Rute; Proença, Daniela; Faustino, Paula
    INTRODUCTION: Hereditary Hemochromatosis is an autosomal recessive disorder characterized by excessive intestinal iron absorption and iron deposition in organs such as liver, heart and pancreas, potentially leading to cirrhosis, hepatocelular carcinoma, diabetes, cardiac failure and arthritis. This disorder is mainly due to mutations in HFE gene. HFE protein associates with beta-2 microglobulin (B2M) for trafficking to the cell surface. However, the HFE’s role on iron homeostasis is not completely cleared. It may regulate hepcidin expression in the liver and iron trafficking in the duodenum. Several HFE alternative splicing transcripts have been reported, but their structural and functional characterization have been poorly studied. MATERIALS AND METHODS: Aiming to investigate the putative biological role of an alternative HFE transcript originated by the intron 4 inclusion, we measured its expression level in several human tissues by quantitative Real-Time PCR. Also, we produced the corresponding GFP-tagged HFE variant. HepG2 cells were transfected with this construct and protein cellular location analyzed by immunofluorescence, using B2M, TfR1 and calnexin antibodies. In parallel, immunoprecipitation was performed. Finally the intron 4 inclusion variant was over-expressed in a human duodenum adenocarcinoma cell line (Hutu-80) under normal and iron overload conditions and the expression of several iron metabolism genes (TFR1, DMT1, DCYTB, SLC40A1 and HEPH) evaluated by quantitative Real-Time PCR. RESULTS: We have found that the intron 4 inclusion transcript has an ubiquitous expression in the analyzed tissues, being its relative expression higher in duodenum and lower in the liver. Also, we found that this variant gives rise to a truncated protein (sHFE) that is secreted by the cells and is able to maintain its interaction with B2M. Its overexpression in HuTu-80 cells showed that sHFE down-regulates the duodenal cytochrome b (CYBRD1) expression in about 20% independently of cellular iron status, as it happens with the HFE_full length protein. Also, sHFE seems to be involved in the down-regulation of hephaestin (HEPH) expression, being its effect higher in the presence of iron overload (reduction of ~40 and ~50%, respectively). CONCLUSIONS: Through this study we might have unveiled the contribution of the HFE’s intron 4 inclusion splice variant to the maintenance of iron homeostasis. sHFE may be secreted into the bloodstream and act in remote tissues such as the duodenum, down-regulating the expression of some of the iron metabolism related genes, as CYBRD1 and HEPH, and consequently reducing dietary iron absorption. Also we are currently exploring the hypothesis of a possible effect of sHFE in the expression of other iron metabolism related genes in hepatic cells and macrophages.
    2013-04Conference object Open access Show more
  • Expression of iron metabolism-related genes is altered in Familial Hypercholesterolemia patients
    Publication . Mateus, Ana; Silva, Bruno; Marques, Liliana; Faustino, Paula; Bourbon, Mafalda; Costa, Luciana
    Atherosclerosis (ATH) is the major cause of cardiovascular diseases (CVD) causing great morbidity and mortality. Typically, ATH develops and progresses silently for years, without symptoms. Familial Hypercholesterolemia (FH) is an autosomal genetic disorder characterized by high levels of total and LDL cholesterol, a familial history of hypercholesterolemia and premature ATH. Atherogenesis is characterized by an early deposition of LDL in the artery walls, leading to the recruitment and activation of peripheral blood mononuclear cells (PBMC) to the plaque. In addition, monocyte-derived macrophages accumulate oxidized LDL and differentiate into foam cells, leading to the development of complex and unstable lesions. The oxidative modification of LDL, accelerated, or even initiated by transition metals as iron (Fe) has been implicated as an early step in the formation of atheromatous lesions.
    2013-06Conference object Open access Show more