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- Evaluation of the role of miR-9 and miR-29 in amyloid pathway of Alzheimer's diseasePublication . Riscado, Micaela Sofia Ribeiro; Sousa, Fani Pereira de; Pereira, Patrícia Alexandra NunesAlzheimer’s disease (AD) is a neurodegenerative disorder which occupies the 3rd place of the diseases that cause disability and death in the elderly, but their causes are yet in need of a better understanding. Also, the search of the etiopathological path leading from a healthy state to full-blown dementia could help with the establishment of more effective treatments. MicroRNAs (miRs or miRNAs) are small regulatory non-coding RNAs (sncRNA), which are involved in post-transcriptional gene expression regulation. Diverse studies have shown that the expression levels of several miRNAs are decreased in AD patients and, there is also evidence that certain miRNAs can control the pathologic hallmarks related to the biomarkers of AD. The main biomarkers of AD are extracellular deposits of amyloid-ß peptide (Aß) along with an accumulation of intraneuronal hyperphosphorylated form of the microtubule-associated protein Tau. The aim of this dissertation is to study the role of two under-expressed miRNAs in AD, miR-29 and miR-9, that are related to the regulation of messenger RNA (mRNAs) that encode proteins involved in various pathological pathways of AD. The selection of miRNAs with decreased levels was based on the possibility of performing its delivery into the cells, in order to contradict the pathological environment of AD. To accomplish this issue, synthetic miRNAs and precursors of miRNAs (pre-miRNAs) produced by two different methods (chemical and enzymatic synthesis, respectively) were used, in order to evaluate if the preparation method had influence in the biological activity. Thus, the pre-miRNAs were produced in the laboratory through in vitro transcription using a plasmid DNA (pDNA) as a template and were further subjected to a purification step. To evaluate the biological activity of the miRNAs, N2a695 cells (mutated APP expressing neuroblastoma mouse cell line that mimics the pathological pathway of AD) were used as in vitro model. Two different methods were also used for cells transfection, Lipofectamine and chitosan-nanoparticles for RNA encapsulation and delivery. After RNA extraction, the mRNA expression levels were evaluated for the APP, BACE1 and PS1 proteins. The findings demonstrated a silencing effect on the expression of APP mRNA by pre-miR-9 and of the BACE mRNA by miR-29b and pre-miR-29b. In turn, PS1 mRNA levels were decreased after cells transfection with all types of miRNAs/pre-miRNAs used in this study, being the most prominent result obtained by RT-qPCR. Western blot assays were also performed, in order to quantify the expression levels of the proteins in study. In conclusion, a better understanding of how miRNAs act and what are their specific targets may have a major impact on the pharmaceutical industry, because being regulators in the pathologic pathways in certain diseases, such as AD, they can be used as new therapeutic approaches to regulate altered proteins expression in this pathological environments.
- Non-coding RNAs: Emerging from the discovery to therapeutic applicationsPublication . Baptista, Bruno; Riscado, Micaela; Queiroz, João; Pichon, Chantal; Sousa, F.The knowledge about non-coding RNAs (ncRNAs) is rapidly increasing with new data continuously emerging, regarding their diverse types, applications, and roles. Particular attention has been given to ncRNA with regulatory functions, which may have a critical role both in biological and pathological conditions. As a result of the diversity of ncRNAs and their ubiquitous involvement in several biologic processes, ncRNA started to be considered in the biomedical field, with immense potential to be exploited either as biomarkers or as therapeutic agents in certain pathologies. Indeed, ncRNA-based therapeutics have been proposed in many disorders and some even reached clinical trials. However, to prepare an RNA product suitable for pharmacological applications, certain criteria must be fulfilled, and it has to be guaranteed RNA purity, stability, and bioactivity. So, in this review, the different types of ncRNAs are identified and characterized, by describing their biogenesis, functions, and applications. A perspective on the main challenges and innovative approaches for the future and broad therapeutic application of RNA is also presented.
- Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 DetectionPublication . Carvalho, Josué; Nunes, J. Lopes; Figueiredo, Joana; Santos, Tiago; Miranda, André; Riscado, Micaela; Sousa, Fani; Duarte, A. P.; Socorro, Sílvia; Tomaz, Cândida; Felgueiras, Mafalda; Teixeira, Rui; Faria, Conceição; Cruz, CarlaThe fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.