Browsing by Issue Date, starting with "2019-09-18"
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- Studying and targeting the functions of p53 mRNA in carcinogenesisPublication . Damasceno, Beatriz; Candeias, Marco Marques; Dias, DeodáliaThe majority of mRNA translation is ensured by the canonical cap-dependent translation, which represents the cellular process with the biggest expense of energy and resources. Thus, in response to various adverse conditions such as hypoxia, viral infection, nutrients and growth factors starvation, endoplasmic-reticulum stress (ER-stress), among others, the canonical cap-dependent translation mechanism is supressed. However, in these adverse situations the expression of certain proteins that mediate adaptation and damage repair is of extreme importance. Under these limiting conditions some key mRNAs ensure the translation of these pivotal proteins through alternative non-canonical translation initiation mechanisms. One well-known example of a regulatory protein that is expressed even in very unfavourable conditions is the tumour suppressor p53. The TP53 gene, containing three promoters and together with alternative internal initiation and alternative splicing, expresses at least fifteen reported isoforms. The p53 isoforms have been shown to have unique transcription targets, often linked to non-redundant functions, and demonstrate responsiveness to different specific stress signals. These findings help to better understand how p53 integrates such a variety of pathways including DNA repair, cell cycle regulation, apoptosis, metabolism and even aging. Having such a wide range of functions that deeply impact the cellular survival, p53 expression is regulated through a variety of mechanisms that include the Hdm2 (murine double minute 2 human homolog) mediated poly-ubiquitination and 26S-mediated proteasomal degradation. However, despite its tight regulation, p53 proteins are frequently reported to grant cells with carcinogenic-like features, causing particularly aggressive and treatment-resistant tumours. Indeed, TP53 gene is the most commonly mutated gene in cancer, representing a strategic target for transformed cells, as the aberrant expression of the p53 protein isoforms offers the means to overcome the extremely challenging microenvironments, characteristic in cancer onset and tumour development. Our team's research has been greatly focused on the Δ160p53 isoform, specifically the study of the recently identified Δ160p53 IRES, as this isoform promotes, to a greater extent than other p53 isoforms, nefarious carcinogenic-like functions, namely invasion and cellular proliferation. One approach we propose to better understand the Δ160p53 IRES-mediated translation initiation mechanism is the identification of small drug compounds that inhibit Δ160p53 IRES. For this purpose, we started by establishing the optimal conditions, cell line and the system for this drug screening. Simultaneously, we cloned neomycin resistant plasmid constructs containing a bicistronic system with two reporter genes (Renilla Luciferase and Firefly Luciferase), using already available Δ160p53 IRES containing sequences, in order to select stable eukaryotic cells for the drug screening assays. Even though at the time of this thesis' experiments we did not have the chance to test the cloned plasmids and evaluate their performance in the drug screening assays, we present the best cloning strategy out of the two approaches used. In parallel, our laboratory has invested in the identification of other cancer-related IRES-translated mRNAs specifically regulated by Hdm2, as it is known that Hdm2 can act as an ITAF, positively regulating commonly deregulated proteins in cancer. With this in mind, we modified the Hdm2 sequence using site directed mutagenesis to create two missense mutations in the 395th codon. The 395th codon has a Serine that is phosphorylated upon DNA damage, triggering the Hdm2's activity as an ITAF. Thus, we altered the Serine to an Alanine, which cannot be phosphorylated, therefore it will not be able to bind to mRNAs. The second mutagenesis aimed the modification of the Serine to Aspartic acid. The aspartic acid mimics the phosphorylated Serine, thereby permanently inducing the Hdm2's activity as an ITAF. These sequences were sent to the partner laboratory of the Department of Human Genetics in Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), in Lisbon, Portugal, to perform co-immunoprecipitation (co-IP) assays of Hdm2-bound mRNAs.For a long time, researchers focused on the p53 regulation at the protein level however, attentions started to turn to the p53 mRNA as more evidences of mRNA's regulatory functions, beyond the well studied role in translation, appeared. Indeed, it was demonstrated that p53 mRNA has a protective function over p53 protein by blocking its degradation under cellular stress, resembling a regulatory "noncoding"-like function. Furthermore, recent findings have correlated the mammalian mRNA post-transcriptional modification on adenine bases by a methyl group in the nitrogen-6 position (m6A) with cap-independent translation initiation. We were intrigued by reports that pointed to the existence of m6A modifications located in the vicinities of IRESs, suggesting a potential correlation of this alteration with IRES-mediated translation initiation. Therefore, we proposed to determine if the adenine of the 213th codon, a commonly mutated codon of the Δ160p53 isoform, bears the m6A modification. To identify the methylation on Δ160p53 mRNA at one-nucleotide resolution we optimized hybrid-DNA probe binding and T3 ligase experiments, using total RNA extracted from HeLa (Human cervical cancer-derived cell line) and A549 (Human lung carcinoma-derived cell line). Two sets of probes were custom made for the Δ160p53 segment of interest containing the 213th codon, and also for both MALAT1 positive control and Δ160p53 sequence containing AUG160, used as negative control. The obtained products were amplified through PCR, and separated by agarose gel separation. Even though it was not possible to determine if the adenine of the 213th codon is methylated, we were able to demonstrate the designed probes are binding to the complementary sequences, and also that the T3 ligase is successfully joining the left and right probes. With the fulfilment of the objectives defined for the present thesis, we seek to discern which are the best approaches for the drug screening, targeted to the inhibition of the Δ160p53 IRES, as well as offer essential tools for the Hdm2-coIP assays and drug screening. Additionally, by undertaking a new method in our laboratory to determine the existence of the still poorly understood m6A mRNA modification in Δ160p53, we ambition to lay the foundations for further studies in regard to this modification's function in non-canonical mRNA translation. Ultimately, with the present thesis we hope to assist the valuable investigation regarding the mechanisms of IRES-mediated translation, and to provide new insights that support the development of new therapeutic strategies.
- Role of Lactobacillus crispatus in vaginal infections: insights from metagenomics and metabolomics studiesPublication . Gaspar, C.; Rolo, J.; Vieira, Luís; Donder, G.; Martinez-de-Oliveira, J.; Palmeira-de-Oliveira, A.Objective: Lactobacillus spp. constitute the most prevalent bacterial microorganism in the vaginal milleu. Several probiotic mechanisms have been associated with Lactobacillus, but the most relevant one is lactate production – resulting in a low pH value, typical of the healthy vagina. We aim to elucidate the role of a native vaginal microorganism, Lactobacillus crispatus, in vaginal infections. Methods: Twenty-four vaginal washes have been collected from women (mean 38±14 years of age) attending a gynecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling. The distribution of bacterial species, and their prevalence was assessed by next-generation sequencing of the 16S V4 region. In addition, lactate was quantified in all washes by a commercial kit. Results: L. crispatus was dominant (>70%) in 11 out of 24 samples – diagnosed for vaginal atrophy (VA, 3), cytolytic vaginosis (CV, 2), lactobacillosis (LB, 2) and vulvovaginal candidosis (VVC, 1). For three samples, diagnostic was inconclusive. Lactate was increased in CV, LB and VA cases only. One sample, diagnosed with VVC had 23% dominance of L. crispatus; and lower lactate metabolite. L. crispatus was not detected in the remaining 12 cases (6 VVC, 4 VA, 1 healthy and 1 inconclusive). Of these, only 5 samples had moderate lactate metabolite. Conclusions: L. crispatus dominance, associated with increased lactate production, was observed in CV, LB and VA cases. These results indicate that this microorganism might have a role in dysbiosis of the vagina associated with these specific pathologies.
- Regulatory RNA genes are targeted by Copy Number Variation in Autism Spectrum DisorderPublication . Marques, Ana RitaAutism Spectrum Disorder (ASD) is a clinically heterogeneous neurodevelopmental disorder. Genetic factors are estimated to account for 50 to 80% of the familial ASD risk, but most of the genetic determinants are still not known and a role for epigenetic factors is likely. In this study we explored the potential role of noncoding RNAs in ASD by comparing the frequency of Copy Number Variants (CNVs) targeting microRNA (miRNA) or long noncoding (lncRNA) genes in ASD patients with control subjects. We found 34 miRNA genes associated with ASD that are predicted to regulate genes enriched by 6 significant pathways, 2 of which, the PI3K-Akt and MAPK signaling pathways, have been implicated in ASD. We also found 700 lncRNA genes associated with ASD. These results support our hypothesis that genetic variants targeting noncoding regulatory RNAs are involved in ASD pathophysiology. This systems biology integrative strategy will provide a better understanding of the biological processes underlying ASD, and contribute to biomarker and drug target discovery.