Barcelona, Catalonia, Spain
- High throughput screening system specialist: experiment design, development of new assays - Microscopes and Robots manager: mostly imaging and maintenance - Histology and imaging specialist: from the tissue to the image
- Study director for two R&D projects in oncology and for other services. - Responsible person for an automated imaging system. - Performing experiments and develop new laboratory techniques.
Pulmonary fibrosis (PF) results from wound healing repair impairment, characterised by increased matrix production and altered ECM and tissue rigidity. There are no treatment to revert the disease. microRNAs represent a novel strategy for the cure of organ fibrosis, with more than 40 molecules in phase II clinical trial. microRNA based therapies either aim to increase expression of a microRNA to repress a target mRNA (mirmimics), or decrease microRNA expression to upregulate a target mRNA (antagomir). Analysis of microRNA expression in the Bleomycin mouse model of lung fibrosis identified more than 20 microRNAs that were significantly modified in their expression during fibrosis induction and progression. Using the Bleomycin model, I identified 4 microRNAs (miR-21a-5p, miR-34a- 5p, miR-29a-3p and miR-378-5p) as potential pro-fibrotic microRNAs due to progressively increased expression correlated with progression of fibrotic features. Results from primary mouse fibroblast cultures showed that overexpression of miR-29a-3p results in a reduction of Col1a1 expression, suggesting an antifibrotic role for this microRNA. miR-21a-5p expression decreased Smad7 expression in both primary mouse lung fibroblasts and in the Bleomycin model of lung fibrosis. Treatment with a miR-21a mirmimic resulted in increased lung fibrosis in the Bleomycin mouse model, while treatment with a miR-21a antagomir resulted in reduced lung fibrosis. My results identify miR-21a repression as a potential novel strategy to treat IPF. In lung fibrosis the increase in ECM causes mechanical stretch impairment due to the increase in tissue stiffness. Using an ad hoc in vitro system, I found that collagen as culture substrate and age were increasing ECM gene expression, while mechanical stretch was reducing same gene expression, in both primary and immortalised lung cells (epithelial and mesenchymal).
In April, I have started a post-graduated internship in Cardiff working on Peritoneal Dialysis (PD) in Dr Matthias Eberl team. The aim of my project is to clarify: if conventional solutions are more harmful than new biocompatible solutions in PD patients, through the relative quantification of a set of microRNAs which we are going to employ as inflammation biomarker. Obviously, this project is exploiting patient’s effluent samples, who were treated with the two different conditions. For this scope I am improving my skills in molecular techniques as RNA extraction and qRT-PCR, using new kits, specific for microRNAs. Moreover, I have learned fundamental techniques as the staining/FACS Canto, which has a plethora of application in Biology and are well-known by the member of Eberl group. Another fundamental feature of this internship is the possibility to participate in Seminars and Lectures with Scientist from all around the world.
I investigated whether treatment with specific synthetic agonists for cannabinoid receptor 1 (CB1) triggers up-regulation of four different microRNAs (miR-146a, miR-155, let-7b and miR-20a) that are known to be involved in inflammatory pathways. To first establish an up-regulation in microRNA expression, I studied the effect of CB1 triggering on the protein level of three regulators of the microRNAs biogenesis. To this end, I measured protein levels of Drosha, Dicer and AgoII by Western Blot in UMG-u87 cells, a stable cell line from glioblastoma. These experiments demonstrated that all three proteins were up-regulated after one day of treatment with a peak around the second day and suggested that cannabinoid agonists may lead to the induction of microRNA expression. microRNAs act mostly in their transcriptional inhibition through a RNA induced silencing complex (RISC), in which single strand microRNAs bind a complementary sequence in the 3' UTR of mRNA targets. Therefore, in order to evaluate the specific activation of the four microRNAs, I developed four different PCAT modified vectors for each microRNA that contained a specific recognition sequence for endogenous microRNAs at the 3' UTR end of the reporter gene chloramphenicol acetyltransferase. Using this approach, any induction of microRNA expression upon CB1 stimulation would be detectable via the indirect down-regulation of chloramphenicol acetyltransferase, as detected using sandwich ELISA. To demonstrate if cannabinoid treatment induces specific microRNA expression, I tested samples in both treated and untreated conditions in triplicate, compared to cells transfected with a non-modified PCAT vector serving as negative control. As expected the level adsorbance revealed in treated samples suggest an strong induction of all the four microRNAs after a CB1 treatment, confirming the existence of a cross-talk between: inflammation and pain.
From March to July 2011, as all students who wished to graduate in the Bachelor of Medical Biotechnology, I had my first training in a laboratory. In particular, I started it at Department of Clinical Molecular Biology in Clinic University campus of Naples (DBBM) under the supervision of Professor Esposito and Dr De Falco, following a diagnostic project in a family with a dead-child affected by Harlequin Ichthyosis. The project aimed at the identification, through a linkage analysis, of informative repeat sequences, as SNPs, STRs and VNTRs, which surround ABCA12 gene. However, I followed the work of Dr De Falco, who worked in both molecular diagnostic and research. This training educated me in Bioinformatics and it gave me basic knowledge in Molecular Biology techniques such as pipetting, DNA extraction from blood, agarose and acrylamide gel run, and microscope.