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- Internal Ribosome Entry Site-Dependent Translation Dysregulation-Related DiseasesPublication . Marques, Rita; Lacerda, Rafaela; Romão, LuísaInternal ribosome entry site (IRES)-mediated translation is an alternative mechanism of translation initiation, known for maintaining protein synthesis when canonical translation is impaired. During a stress response, it contributes to cell reprogramming and adaptation to the new environment.
- LipNanoCar Technology – A Versatile and Scalable Technology for the Production of Lipid NanoparticlesPublication . Esgueira, Vera L.R.; Lopes, Clara P.A.; dos Santos, Ana Catarina A.; Pinto, Fátima; Sousa, Silvia A.; de Barros, Dragana P.C.; Leitão, Jorge H.; Fonseca, Luis P.The extensive knowledge in the miniemulsion technique used in biocatalysis applications by the authors allowed the development of drug delivery systems that constitutes the LipNanoCar technology core for the production of lipid nanoemulsions and solid lipid nanoparticles. The LipNanoCar technology, together with adequate formulations of different oils, fatty acids, surfactants, and temperature, allows the entrapment of several bioactive and therapeutic compounds in lipid nanoparticles for cosmetic, nutrition, and pharmaceutical applications. The LIpNanoCar technology allowed lipid nanoparticles production with average sizes ranging from 100 to 300 nm and Zeta potentials between −55 and −20 mV. Concomitantly, high entrapment or encapsulation efficiencies (%EE) were achieved, as illustrated in this work for β-carotene and vitamins derivatives (>85%) for cosmetic application, and for antibiotics currently used in chemotherapy, like rifampicin (69–85%) and pyrazinamide (14–29%) against Mycobacterium tuberculosis (TB), and ciprofloxacin (>65%) and tobramycin (~100%) in Cystic Fibrosis (CF) respiratory infections therapy. Ciprofloxacin presented, for example, a quick-release from the lipid nanoparticles using a dialysis tubing (96% in the first 7 h), but slower than the free antibiotic (95% in the first 3 h). This result suggests that ciprofloxacin is loaded near the external surface of the lipid nanoparticles. The toxicity and validation of entrapment of antibiotics in lipid nanoparticles for Cystic Fibrosis therapy were assessed using Caenorhabditis elegans as an animal model of bacterial infection. Fluorescence microscopy of an entrapped fluorescent dye (DiOC) confirmed the uptake of the lipid nanoparticles by ingestion, and their efficacy was successfully tested in C. elegans. Burkholderia contaminans IST408 and Burkholderia enocepacia K56–2 infections were tested as model bacterial pathogens difficult to eradicate in Cystic Fibrosis respiratory diseases.
- Dermal Delivery of Lipid Nanoparticles: Effects on Skin and Assessment of Absorption and SafetyPublication . Pinto, Fátima; Fonseca, Luis P.; de Barros, Dragana P.C.During the recent decades, dermal delivery has achieved visible popularity mainly due to the increase of chronic skin diseases and the demand for targeted delivery and patient compliance. Dermal delivery provides an attractive alternative to oral drug delivery, promoting the drug application directly at the site of action, resulting in higher localized drug concentration with reduced systemic drug exposure. Among several types of drug delivery systems used in dermal delivery are the lipid nanoparticles, which include solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs). These lipid nanocarriers have attracted great interest and have been intensively studied for their use in dermal applications. Lipid nanoparticles increase the transport of active compounds through the skin by improving drug solubilization in the formulation, drug partitioning into the skin, and fluidizing skin lipids. Moreover, these nanocarriers are composed of biologically active and biodegradable lipids that show less toxicity and offer many favorable attributes such as adhesiveness, occlusion, skin hydration, lubrication, smoothness, skin penetration enhancement, modified release, improvement of formulation appearance providing a whitening effect, and offering protection of actives against degradation. This chapter focuses on the effects of lipid nanoparticles in dermal delivery, on the types of active compounds that are used in their formulation and application, some aspects related to their possible toxicity, and a description of the most commonly used techniques for the evaluation of drug absorption on the skin.
- New “Omics” Approaches as Tools to Explore Mechanistic NanotoxicologyPublication . Ventura, Célia; Torres, Vukosava; Vieira, Luís; Gomes, Bruno; Rodrigues, António Sebastião; Rueff, José; Penque, Deborah; Silva, Maria JoãoIn the last years, “omics” approaches have been applied to study the toxicity of nanomaterials (NM) with the aim of obtaining insightful information on their biological effects. One of the most developed “omics” field, transcriptomics, expects to find unique profiles of differentially expressed genes after exposure to NM that, besides providing evidence of their mechanistic mode of action, may also be used as biomarkers for biomonitoring purposes. Moreover, several NM have been associated with epigenetic alterations, i.e., changes in the regulation of gene expression caused by differential DNA methylation, histone tail modification and microRNA expression. Epigenomics research focusing on DNA methylation is increasingly common and the role of microRNAs is being better understood, either promoting or suppressing biological pathways. Moreover, the proteome is a highly dynamic system that changes constantly in response to a stimulus. Therefore, proteomics can identify changes in protein abundance and/ or variability that lead to a better understanding of the underlying mechanisms of action of NM while discovering biomarkers. As to genomics, it is still not well developed in nanotoxicology. Nevertheless, the individual susceptibility to NM mediated by constitutive or acquired genomic variants represents an important component in understanding the variations in the biological response to NM exposure and, consequently, a key factor to evaluate possible adverse effects in exposed individuals. By elucidating the molecular changes that are involved NM toxicity, the new “omics” studies are expected to contribute to exclude or reduce the handling of hazardous NM in the workplace and support the implementation of regulation to protect human health.
- Overview of Adverse Outcome Pathways and Current Applications on NanomaterialsPublication . Rolo, Dora; Tavares, Ana; Vital, Nádia; Silva, Maria João; Louro, HenriquetaNanomaterials (NMs) have important and useful applications in chemical industry, electronics, pharmaceuticals, food and others. Their rapid proliferation presents a dilemma to regulators regarding hazard identification and increased concerns for public health. The Adverse Outcome Pathways (AOPs) are innovative central elements of a toxicological knowledge framework, developed for supporting chemical risk assessment based on mechanistic reasoning. AOPs describe a sequence of causally linked events at different levels of biological organisation, triggered by exposure to a stressor (like chemicals or NMs) leading to an adverse health effect in humans or wildlife. The integrative analysis of the cellular and molecular mechanisms of nanotoxicity towards the identification of connected adverse outcomes drives a sequential line – an AOP landscape definition. Each defined AOP is available for crossing data, linking known and unknown landscapes, reducing the reliance on animal studies, associated costs and ethical issues. NMs have unique properties, with specific associated toxicological challenges, which may represent unknown AOP landscapes. In this chapter, an overview of AOPs as important novel strategic tools in nanotoxicology is presented, highlighting the current applications in hazard identification and human health risk assessment.
- Cellular and Molecular Mechanisms of Toxicity of Ingested Titanium Dioxide NanomaterialsPublication . Vieira, Adriana; Gramacho, Ana; Rolo, Dora; Vital, Nádia; Silva, Maria João; Louro, HenriquetaAn exponential increase in products containing titanium dioxide nanomaterials (TiO2), in agriculture, food and feed industry, lead to increased oral exposure to these nanomaterials (NMs). Thus, the gastrointestinal tract (GIT) emerges as a possible route of exposure that may drive systemic exposure, if the intestinal barrier is surpassed. NMs have been suggested to produce adverse outcomes, such as genotoxic effects, that are associated with increased risk of cancer, leading to a concern for public health. However, to date, the differences in the physicochemical characteristics of the NMs studied and other variables in the test systems have generated contradictory results in the literature. Processes like human digestion may change the NMs characteristics, inducing unexpected toxic effects in the intestine. Using TiO2 as case-study, this chapter provides a review of the works addressing the interactions of NMs with biological systems in the context of intestinal tract and digestion processes, at cellular and molecular level. The knowledge gaps identified suggest that the incorporation of a simulated digestion process for in vitro studies has the potential to improve the model for elucidating key events elicited by these NMs, advancing the nanosafety studies towards the development of an adverse outcome pathway for intestinal effects.
- Hazard Assessment of Benchmark Metal-Based Nanomaterials Through a Set of In Vitro Genotoxicity AssaysPublication . Vital, Nádia; Pinhão, Mariana; Yamani, Naouale El; Rundén-Pran, Elise; Louro, Henriqueta; Dušinská, Maria; Silva, Maria JoãoFor safety assessment of nanomaterials (NMs), in vitro genotoxicity data based on welldesigned experiments is required. Metal-based NMs are amongst the most used in consumer products. In this chapter, we report results for three metal-based NMs, titanium dioxide (NM- 100), cerium dioxide (NM-212) and silver (NM-302) in V79 cells, using a set of in vitro genotoxicity assays covering different endpoints: the medium-throughput comet assay and its modified version (with the enzyme formamidopyrimidine DNA glycosylase, Fpg), measuring DNA strand beaks (SBs) and oxidized purines, respectively; the micronucleus (MN) assay, assessing chromosomal damage; and the Hprt gene mutation test. The results generated by this test battery showed that all NMs displayed genotoxic potential. NM-100 induced DNA breaks, DNA oxidation damage and point mutations but not chromosome instability. NM-212 increased the level of DNA oxidation damage, point mutations and increased the MN frequency at the highest concentration tested. NM-302 was moderately cytotoxic and induced gene mutations, but not DNA or chromosome damage. In conclusion, the presented in vitro genotoxicity testing strategy allowed the identification of genotoxic effects caused by three different metal-based NMs, raising concern as to their impact on human health. The results support the use of this in vitro test battery for the genotoxicity assessment of NMs, reducing the use of more expensive, time-consuming and ethically demanding in vivo assays, in compliance with the 3 R’s.
- Inflammatory Tumor MicroenvironmentPublication . Matos, PauloThe development of tumors requires an initiator event, usually exposure to DNA damaging agentes that cause genetic alterations such as gene mutations or chromosomal abnormalities, leading to deregulated cell proliferation. Although the mere stochastic accumulation of further mutations may cause tumor progression, it is now clear that an inflammatory microenvironment has a major tumor promoting influence on initiated cells, in particular when a chronic inflammatory reaction already existed before the initiated tumor cell was formed. Moreover, inflammatory cells become mobilized in response to signals emanating from tumor cells. In both cases, the microenvironment provides signals that initiated tumor cells perceive by membrane receptors and transduce via downstream kinase cascades to modulate multiple cellular processes and respond with changes in cell gene expression, metabolism, and morphology. Cytokines, chemokines, and growth factors are examples of major signals secreted by immune cells, fibroblast, and endothelial cells and mediate an intricate cell-cell crosstalk in an inflammatory microenvironment, which contributes to increased cancer cell survival, phenotypic plasticity and adaptation to surrounding tissue conditions. Eventually, consequente changes in extracellular matrix stiffness and architecture, coupled with additional genetic alterations, further fortify the malignant progression of tumor cells, priming them for invasion and metastasis.
- Alternative SplicingPublication . Jordan, PeterAlternative splicing (AS) is a critical post-transcriptional regulatory mechanism used by more than 95% of transcribed human genes and responsible for structural transcript variation and proteome diversity.
- Translational regulation by upstream open reading frames and its relevance to human genetic diseasePublication . Fernandes, Rafael; Romão, LuísaUpstream open reading frames (uORFs) are cis‐acting elements, located before or overlapped with the main coding ORF (mORF), that regulate cap‐dependent translation efficiency in a transcript‐specific manner. More than half of the human transcripts bear at least one uORF. In addition, it has been recently revealed that many of these uORFs initiate at non‐AUG codons, which significantly increases the complexity and diversity of the human translatome. These regulons are considered repressors of downstream translation but, in some biological contexts, they induce mORF expression. There are several the mechanisms by which AUG and non‐AUG uORFs regulate gene expression, allowing the cell to control transcript‐specific translation according to its needs. Also, we describe several examples of uORF genetic variants associated with human genetic diseases. Studying these cases and understanding the resultant abnormal mechanisms of uORF‐mediated translational control is of extreme importance for the development of new therapeutic strategies.