Browsing by Author "Marinho, H. Susana"
Now showing 1 - 10 of 18
Results Per Page
Sort Options
- Antagonist G-targeted liposomes for improved delivery of anticancer drugs in small cell lung carcinomaPublication . Carvalheiro, Manuela; Ferreira-Silva, Margarida; Holovanchuk, Denys; Marinho, H. Susana; Moreira, João Nuno; Soares, Helena; Corvo, M. Luisa; Cruz, Maria Eugénia M.Ligand-mediated targeted liposomes have the potential to increase the therapeutic efficacy of anticancer drugs. This work aimed to evaluate the ability of antagonist G, a peptide targeting agent capable of blocking the action of multiple neuropeptides, to selectivity improve targeting and internalization of liposomal formulations (long-circulating liposomes, LCL, and stabilized antisense lipid particles containing ionizable amino lipid, SALP) to H69 and H82 small cell lung carcinoma (SCLC) cell lines. Antagonist G-targeted LCL and SALP were prepared by two different methods (either by direct covalent linkage at activated PEG grafted onto the liposomal surface or by post-insertion of DSPE-PEG-antagonist-G-conjugates into pre-formed liposomes). Association of the liposomal formulations with target SCLC cells was studied by fluorescence microscopy using fluorescence-labeled liposomes and confirmed quantitatively with [3H]-CHE-labelled liposomes. An antisense oligodeoxynucleotide against the overexpressed oncogene c-myc(as(c-myc)) was efficiently loaded into SALP formulations, the encapsulation efficiency decreased due to the inclusion of the targeting ligand. Also, liposome size was affected by as(c-myc) physical-chemical properties. The amount of antagonist G linked to the surface of the liposomal formulations was dependent on the coupling method and lipid composition used. Covalent attachment of antagonist G increased liposomes' cellular association and internalization via receptor-mediated and clathrin-dependent endocytosis, as assessed in SCLC cell lines. Biodistribution studies in healthy mice revealed a preferential lung accumulation of antagonist G-targeted SALP as compared to the non-targeted counterpart. Lung levels of the former were up to 3-fold higher 24 h after administration, highlighting their potential to be used as delivery vectors for SCLC treatment.
- Cellular polarity in aging: role of redox regulation and nutritionPublication . Soares, Helena; Marinho, H. Susana; Real, Carla; Antunes, FernandoCellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.
- Centrosome positioning and development of ciliopathies: role of the human centrosomal protein TBCCD1Publication . Carmona, Bruno; Camelo, Carolina; Mehraz, Manon; Lemullois, Michel; Ferreira, David C.; Nolasco, Sofia; Lince-Faria, Mariana; Marinho, H. Susana; Bettencourt-Dias, Mónica; Tassin, Anne-Marie; Soares, HelenaAims/Context: Primary cilia are specialized microtubule-based signaling organelles that convey extracellular signaling and cellular polarity into a cellular response. Defects in primary cilia assembly/function cause severe diseases known as ciliopathies, typified by clinical manifestations, like infertility, obesity, brain problems, blindness, and kidney cysts. Primary cilia assembly entails centrosome migration to the plasma membrane where a centriole docks, maturates into a basal body (BB), and assembles the cilia axoneme. The human centrosomal TBCCD1 is a critical factor in centrosome positioning previously identified by us. Our aim is to discover the mechanisms/signals required for the correct positioning of the centrosome during cilia assembly, and how these mechanisms, when compromised, are related to ciliopathies. Methods: The proximity-dependent identification (BioID) assay was used to screen for TBCCD1 interactors. Immunofluorescent and super-resolution microscopy, as well as Western blot, were used to study the levels and cellular localization of the identified TBCCD1 interactors in human RPE1 cells overexpressing or depleted of TBCCD1. To study the impact of TBCCD1 knockdown in motile cilia the ciliate Paramecium, containing ∼3,000 motile cilia, was used. Results: Our BioID screen for TBCCD1 interactors identified several well-known proteins encoded by ciliopathy genes, e.g. the centrosomal protein OFD1 involved in the Orofacial-Digital Syndrome. We show that TBCCD1 knockdown and overexpression in RPE1 cells affects OFD1 distribution. Super-resolution microscopy shows TBCCD1 is localized at the distal region of the centrosome and that its depletion dramatically affects the centrosome subdistal protein CEP170, a component of cilia basal feet. In Paramecium, the TBCCD1 knockdown causes abnormal BB-associated structures organization and anomalous BB positioning/anchoring defects. Conclusions: Our data support a role for TBCCD1 in the maintenance of centrosome structure and in BB anchoring at the cell membrane during ciliogenesis. TBCCD1 is emerging as a novel protein with a role in human ciliopathies.
- Functional interactions of the ciliopathy-involved protein KIAA0753 with the regulator of centrosome positioning TBCCD1Publication . Ferreira, David C.; Carmona, Bruno; Nolasco, Sofia; Marinho, H. Susana; Soares, Helena
- Gene silencing using siRNA for preventing liver ischaemia-reperfusion injuryPublication . Marinho, H. Susana; Marcelino, Paulo; Soares, Helena; Corvo, Maria LuísaIschaemia-reperfusion injury (IRI), a major complication occurring during organ transplantation, involves an initial ischemia insult, due to loss of blood supply, followed by an inflammation-mediated reperfusion injury. A variety of molecular targets and pathways involved in liver IRI have been identified. Gene silencing through RNA interference (RNAi) by means of small interference RNA (siRNA) targeting mediators of IRI is a promising therapeutic approach. Objective: This study aims at reviewing the use of siRNAs as therapeutic agents to prevent IRI during liver transplantation. Method: We review the crucial choice of siRNA targets and the advantages and problems of the use of siRNAs. Results: We propose possible targets for siRNA therapy during liver IRI. Moreover, we discuss how drug delivery systems, namely liposomes, may improve siRNA therapy by increasing siRNA stability in vivo and avoiding siRNA off-target effects. Conclusion: siRNA therapeutic potential to preclude liver IRI can be improved by a better knowledge of what molecules to target and by using more efficient delivery strategies.
- “Healthy Life”: interaction of polyphenols with lipid bilayers and their effects in human cellsPublication . Filipe, Hugo A. L.; Peneda, Catarina; Marquês, Joaquim T.; Machuqueiro, Miguel; Ramos, João C.; Santos, Maria da Soledade; Marinho, H. Susana; Viana, Ana S.; Soares, Helena; Almeida, Rodrigo F. M. deThis work concerns the transversal project of the CQB thematic line: “Healthy Life: Molecular Interventions and Regulation Mechanisms”. Biologically active plant phytochemicals have a broad range of pharmacological effects including anticarcinogenic, antimicrobial, antioxidant, and anti-inflammatory activity. [1] Notwithstanding the possibility of having a specific target, phytochemicals must interact and permeate through cell membranes in the body. Indeed, it was suggested that those molecules insert into the membranes and thereby may have a promiscuous activity by changing structural properties of lipid bilayers. [2] Some well-known phenolic acids such as caffeic (CA), rosmarinic (RA) and chlorogenic (CGA) acids, whose identification in plant extracts has been achieved by CQB research groups, were selected to be addressed in first place. All the phenolic acids studied have low lipophilicity and among them, RA was the only one with a partition to biological membrane models measurable by fluorescence spectroscopy, as opposed to CA and CGA. Cyclic voltammetry measurements using an electrode modified with a supported lipid bilayer, also indicated a higher affinity of RA to lipid membranes. In addition, oxidation/reduction of the phenolic acids displayed higher reversibility in the lipid milieu than in the aqueous bulk. Indeed, the reduced form of phenolic acids was unstable in aqueous solution. In particular, in DMEM/F-12 cell culture media, a colour change observed after incubation with each compound could be reverted by the addition of a reducing agent. The higher reversibility of phenolic acids oxidation/reduction, once they were inserted in the lipid membrane, may contribute to the stability of the compounds and prevent the formation of degradation products. Molecular dynamics (MD) simulations are being performed to probe the location and orientation of these and other selected compounds in lipid bilayers. The influence of the phenolic acids in the cytoskeleton organization, both actin filaments and microtubules, of a human retinal pigment epithelial cell line (RPE1) was also investigated. All compounds induced concentration and time dependent effects, translated in structural alterations mainly at the cell periphery, and also in the perturbation of cell division. Moreover, it was not evident that these compounds induce apoptosis under the conditions tested. RA seemed to induce evident effects at earlier times and at lower concentrations, as compared to CA and CGA. This higher sensibility of RPE1 cells to RA correlates with the higher affinity of this compound to the lipid bilayer.
- Hydrogen peroxide as a key player in bystander effectsPublication . Pinto, V.; Carmona, Bruno; Antunes, F.; Marinho, H. Susana; Vieira, Lina Oliveira; Soares, Helena
- Hydrogen peroxide sensing, signaling and regulation of transcription factorsPublication . Marinho, H. Susana; Real, Carla; Cyrne, Luísa; Soares, Helena; Antunes, FernandoThe regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and, (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M-1s−1 and ≥ 1.3 × 103 M-1s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.
- Noncoding RNAs as critical players in regulatory accuracy, redox signaling, and immune cell functionsPublication . Gomes, Anita Quintal; Real, C.; Antunes, F.; Marinho, H. Susana; Nolasco, Sofia; Soares, HelenaThe transcriptome of multicellular organisms is much more complex than initially thought because it includes a large number of noncoding RNAs (ncRNAs). Data regarding ncRNAs suggest that organism complexity better correlates with the percentage of each genome that is transcribed into these molecules. The most studied classes of ncRNAs are short interfering RNAs, microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and long noncoding RNAs (lncRNAs). In this chapter, we review the biogenesis pathways and general functions of miRNAs, piRNAs, and lncRNAs. We focus on the roles of miRNAs and lncRNAs in gene expression regulation, centering on redox signaling and immune cell development, and highlight some implications for human pathologies. Finally, we analyze current knowledge concerning the use of ncRNAs in diagnosis, prognosis, and therapeutics, and discuss their role in the development of the immune system and the regulatory functions of H2O2 during the course of metazoan evolution.
- Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiaePublication . Camelo, Carolina; Vilas-Boas, Filipe; Cepeda, Andreia Pereira; Real, Carla; Barros-Martins, Joana; Pinto, Francisco; Soares, Helena; Marinho, H. Susana; Cyrne, LuisaDuring exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2. Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2, GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells.