Molecular interactions are wired in a fascinating way resulting in complex behavior of bio-logical systems. Theoretical modeling provides us a useful framework for understanding the dynamics and the function of such networks. The complexity of the biological systems calls for conceptual tools that manage the combinatorial explosion of the set of possible interac-tions. A suitable conceptual tool to attack complexity is compositionality, already success-fully used in the process algebra field to model computer systems. We rely on the BlenX programming language, originated by the beta-binders process calculus, to specify and si-mulate high-level descriptions of biological circuits. Gillespie’s stochastic simulation algo-rithm applied for BlenX simulations requires the decomposition of phenomenological func-tions into basic elementary reactions. Systematic unpacking of complex reaction mechanisms into BlenX templates is shown. The estimation/derivation of missing parameters and the challenges emerging from compositional model building in stochastic process algebras are discussed. A biological example on circadian clock is presented as a case study of modeling.

Autism spectrum disorders (ASD) comprise a genetically heterogeneous group of neurodevelopmental disabilities characterized by repetitive behaviors as well as deficits in communication and social/emotional interactions and behaviors. Defects in GABA transmission have been hypothesized to underlie the symptoms of ASD (Ben-Ari, Khalilov, Kahle, & Cherubini, 2012). Engrailed-2 (En2) is a homeodomain transcription factor involved in regionalization and patterning of the midbrain and hindbrain regions (Joyner, 1996; Gherbassi and Simon, 2006). En2 is expressed in the developing and adult mouse midbrain/hindbrain region (Joyner, 1996), as well as the adult mouse forebrain (Tripathi et al., 2009). Genome wide association studies have shown that two intronic single-nucleotide polymorphisms in the human En2 gene are significantly associated with ASD (Benayed et al., 2009). En2-/- mice lack the homeodomain of En2 (En2hd/hd mice; Joyner et al., 1991, referred to as En2-/-) and display neuropathological and behavioral changes relevant to ASD. We first investigated the distribution of different interneuron subtypes in the cerebral cortex and hippocampus of En2-/- mice. Results showed a significant loss of parvalbumin, somatostatin and neuropetide Y positive interneurons, in both regions of En2-/- brains. Brain-derived neurotrophic factor (BDNF) is a crucial factor for the postnatal development of forebrain GABAergic neurons, namely PV interneurons. We therefore investigated whether altered BDNF expression may be related to the altered development of GABAergic interneurons, described in En2-/- mice. Considering the important role of BDNF in forebrain GABAergic interneuron development, we decided to determine whether interneuron loss in En2-/- forebrain might be related to altered expression of BDNF and its signaling receptors. We evaluated the expression of different BDNF mRNA and protein isoforms in various brain areas from wild-type (WT) and En2-/- mice. Quantitative RT-PCR indicated an alteration of various splicing variants of BDNF mRNA in the cerebral cortex but not hippocampus and cerebellum of adult En2-/- mice, as compared to WT controls. Immunoblot analyses instead revealed increased levels of mature BDNF and reduced levels of truncated- and pro-BDNF isoforms in the hippocampus of En2-/- vs. WT mice. These data indicate a role of BDNF in the anatomical phenotype of En2-/- mice, suggesting that BDNF might be involved in the developmental defects of the GABAergic system in this mouse model of ASD. We next investigated the effect of BDNF on GABAergic and glutamatergic neuron connectivity using primary cultures of hippocampal neurons from WT and En2-/- mice. Results showed that BDNF is able to increase the number of GABAergic and glutamatergic synaptic contacts in En2-/- hippocampal neurons. Taken together, our results suggest a beneficial role of BDNF in rescuing synaptic defects in En2-/- mice.

MicroRNAs (miRNAs) are small non coding RNAs of 18-25 nt, capable of regulating mRNA translation and gene expression at post-transcriptional level. Alteration of miRNAs expression is often associated with human diseases, such as cancers and neurodegenerative pathologies. The main objective of this study is an analysis of the post-transcriptional regulation played by miRNAs of two important genes, MAPT and GRN, involved in Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17). This is one of the major degenerative dementia syndromes, characterized by atrophy of the prefrontal and anterior temporal lobes. Several studies identified 44 pathogenic mutations in MAPT, which encodes for microtubule associated tau protein. In the brain tau has important functions in the assembly and stability of microtubules, that are fundamental for neuronal integrity and function. Disruption of tau role due to tau aggregations cause devastating effects that trigger neurodegeneration. To date 69 different mutations were found in GRN in the presence of FTD. GRN encodes a secreted precursor protein called progranulin that is expressed in neurons, microglia and represents an important growth factor involved in the regulation of multiple processes. The study identified miRNAs that can bind to GRN and MAPT mRNAs, affecting the translation efficiency of these transcripts, with a consequently reduction of the protein production. The project combined standard bioinformatic tools with a novel capture affinity assay, called miR-CATCH, in order to identify the best putative miRNAs. The assay was performed with specific biotinylated anti-sense oligonucleotides for the pull-down of GRN and MAPT mRNAs in different neuroblastoma cell lines. The binding of selected miRNAs on the 3’UTR regions of these genes, was validated using over-expressing vectors and reporter constructs to perform luciferase assays. Regulation of selected miRNAs over-expression on progranulin and tau proteins was investigated in a neuroblastoma cell line, using western blot. This effect was further analyzed with Real Time PCR reactions and polysomal analysis to understand if the selected miRNAs control progranulin and tau expression through a mechanism of translational repression or transcripts degradation. Two of the analyzed candidates, miR-659-3p and miR-608 were found important for the regulation of both genes, GRN and MAPT implicated in the disease, with a subsequent control on the endogenous progranulin and tau production in neuroblastoma cell line. Whereas miR-939-5p and miR-615-5p are only involved in the regulation of GRN expression. In addition we identified several SNPs located on miRNAs binding sites that are predicted to increase or decrease miRNAs binding. Since neurological disorders are strongly influenced by common genetic variability, SNPs or variations that overlapped miRNA-binding sites, could represent potential and important risks factors for generation of FTD.

N6-methyladenosine (m6A) is one of the most common reversible RNA modifications, conserved in all eukaryotes from yeast to humans. It is mainly observed in the 3’UTR, the 5’UTR and near the Transcription Start Sites (TSS), inside a consensus sequence identified as [G/A/U][A/G][m6A-C][U/A/C. The m6A modification is involved in the regulation of many different biological phenomena, starting from development, stemness, adipogenesis, spermatogenesis and many others. Its misregulation has been found to be the driving force of different pathologies, most importantly has been widely described as having a major role in carcinogenesis. The methylation machinery is composed of methyltransferases, erasers and readers, the latter recognizing the modification upon target RNAs and guiding them to different fates. The proteins that act as readers of the methylation belong to the YTH-domain family, with four cytoplasmic isoforms (YTHDF1-3;YTHDC2), involved in the regulation of translation and decay of m6A-containing mRNAs, and one nuclear isoform (YTHDC1), involved in alternative splicing. The YTH domain proteins have been identified to have major roles in cancer development, mainly causing misregulation of cancer-related RNA targets. However, its role in the development of the pediatric tumor Neuroblastoma was still largely unknown. In my PhD project I focused on characterizing the role of the YTHDF1 protein in Neuroblastoma development, reporting that YTHDF1 supports Neuroblastoma aggressiveness by enhancing its proliferation, tumour-sphere forming ability and its invasiveness. To address whether modulating the YTHDF1-mediated regulation of m6A transcripts signaling through YTHDF1 could represent a new pharmacological strategy target in NB, I performed for the first time an High Throughput Screening aiming to interfere with the RNA binding ability of the YTH domain, testing a 2000 compound library of small molecules. Among others, only one molecule was identified for as a strong binder, and consequently demonstrated its direct interaction with the binding pocket of the YTHD by different biochemical approaches. For the first time has been proposed an encouraging proof-of-concept of m6A readers as potential pharmacological targets for the treatment of NB, that led to the repurposing of an already FDA approved drug as an inhibitor for this novel target.

HuR, the ubiquitously expressed member of the ELAV (embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds to AREs (AU-rich elements) and mainly stabilizes ARE-containing mRNAs, e.g. TNFα, VEGF, c-FOS, favoring specific protein translation. TNFα mRNA is one of the most important target mRNA of HuR since the protein encoded by this gene mediates the inflammatory response and its overexpression is correlated with autoimmune diseases and cancer-related inflammation. Specific drugs are already available that can inhibit TNFα protein but cause important side-effects, as insurgence of tumoral pathologies, due to high immunodepression. Therefore, inhibition of TNFα mRNA translation by specific inhibitors targeting HuR, only in those cells undergoing pathological anomalies, is an alternative, intriguing novel therapeutic approach that deserves investigation. By REMSA and AlphaScreen assays we identified a family of low molecular weight inhibitors, called Tanshinones, among which DHTS-I (Dihydrotanshinone – I) was the most potent. Tanshinones are well known in the traditional Chinese Medicine Practice, and these anti-inflammatory agents possess the ability to prevent HuR-RNA complex formation in vitro. We further identified structural determinants of HuR and DHTS interaction using RRM1&RRM2 tandem domains. EMSA and AlphaScreen experiments, with truncated ΔRRM1 and mutants revealed that DHTS is a competitive binder of HuR with respect of target RNA. To ameliorate the solubility of DHTS, we synthesized a number of DHTS analogs, of which the most potent and soluble compound was named MFM49. We evaluated the anti-inflammatory potential of DHTS and DHTS analogs and the HuR-dependent mechanism of action, revealing that, at least in part, DHTS and DHTS analogs rely on HuR to exert their mechanism of action. Influence on NF-kB activation by DHTS and MFM49 upon LPS co-stimulation was not seen in immunofluorescence studies. So here, we disclose a previously unrecognized molecular mechanism of action exerted by DHTS, and anti-inflammatory potential of DHTS analogs opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer like anomalies.

During the development of the nervous system, axons grow and generate a complex network of interconnected neurons. To establish these connections, the tip of the growing axon, the growth cone, is guided by chemotropic cues en route to its target with exquisite precision. Axons must sometimes navigate a significant distance before reaching their final destination. As an alternative to energy-expensive protein transport from distant cell bodies, seminal studies have revealed that growth cones rely on local mRNA translation to generate certain proteins acutely on demand. These cue-induced newly synthesized proteins contribute to fuel growth cone steering. Several groups reported the presence of Dicer at growth cones, and I observe the presence of endogenous Dicer in RGC axons of FLAG-HA2-Dicer transgenic mice. These observations raise the intriguing possibility that not only proteins but also miRNAs could be produced locally in this compartment. In my work, I have therefore explored whether miRNA biogenesis occurs locally within growth cone and if this is important for growth cone steering, using Xenopus laevis retinal ganglion cell (RGC) axons as a model. Specific precursor microRNAs (pre-miRNAs) are detected in pure Xenopus RGC axonal preparations by miRNA-seq and PCR, and endogenous pre-miR-181a-1 is actively tracked to distal axons by hitchhiking on vesicles. Upon exposure to Sema3A, but not Slit-2, pre-miR-181a-1/a-2 are processed within axons by Dicer into newly generated miRNAs (NGmiRNAs). In contrast, pre-miR-182 remains unprocessed upon Sema3A exposure, highlighting a mechanism that is not only cue-, but also pre-miRNA molecule specific. Inhibiting NGmiRNAs in axons abolishes growth cone responsiveness to cues ex vivo. miRNAs are thus locally produced and these newly generated miRNAs mediate cue-induced growth cone steering. To deepen mechanistic insights, I assess whether newly generated miRNAs silence the translation of specific mRNAs in response to cues using FRAP analysis with a Venus reporter. I observe that APP and TUBB3 are locally translated in axons in basal conditions and that are both silenced in response to Sema3A. I uncover that this cue-induced silencing of TUBB3 is mediated by newly generated miRNAs specifically in axons ex vivo and in vivo. Taken together, these results indicate that newly generated miRNAs gate cue-induced silencing of a specific subset of mRNAs in time and space, thereby regulating growth cone behavior. Local biogenesis of miRNAs in axons constitute an important additional regulatory layer in the complex mechanism of axon targeting.

Chemotherapy still represents the most common and sometimes the only possible therapeutic option for advanced breast cancer. Its efficacy is profoundly threatened by intrinsic or acquired chemoresistance, which, in some cases, can be unexpectedly promoted by the chemotherapeutic drugs used. ETV7, a poorly characterized ETS factor with no established roles in breast cancer so far, is reported here to be activated at the transcriptional level by chemotherapy and be able to promote breast cancer cells chemoresistance. This project proposes a novel drug resistance circuitry in breast cancer cells, specifically to Doxorubicin, governed by the ETV7 repressive action on DNAJC15, a gene whose low expression was previously associated with drug resistance in breast and ovarian cancer. Moreover, the impact of ETV7 in causing drug resistance is proved here to extend also to another type of drug, 5-Fluorouracil (5-FU), a chemotherapeutic agent commonly used in combination with Doxorubicin for breast cancer treatment. In this case, additional novel ETV7 targets (DPYD and DPEP1) were established and can represent important mediators for ETV7-mediated resistance to 5-FU. Additional relevant data, relative to biological implications for ETV7 in breast cancer progression, is provided here by the ETV7 ChIP-seq analysis, which reveals the first reported in vivo mapping of ETV7 occupancy. Through ChIP-seq novel ETV7 direct targets have been identified, some of them yet unexplored in breast cancer; notably, their expression revealed to be capable of predicting breast cancer patients outcome. Furthermore, a possible control exerted by ETV7 on the TGF-Î2 pathway, discovered by enrichment analysis on ChIP-seq ETV7 targets, suggests a different and opposing role for ETV7 activation in either advanced stage cancers or normal and early stage breast cancers. Taken collectively, the results from this project propose an important key role for ETV7 in triggering breast cancer multi-drug resistance phenotype in response to chemotherapy, by controlling the expression of specific targets.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) form a large family of RNA-binding proteins (RBPs) that exert numerous functions in RNA metabolism. For example, soluble hnRNPs bind to RNAs to mediate their maturation, processing, and shuttling from the nuclear compartment to the cytoplasm. Additionally, hnRNPs might interact with chromatin to regulate the transcription and the post-transcriptional modification of nascent transcripts. RALY is a member of the hnRNP family that binds poly-U rich elements within several RNAs and regulates the expression of specific transcripts. RALY is upregulated in different types of cancer and its downregulation has been shown to impair cell proliferation. In my PhD project, I characterized RALY to interact with transcriptionally active chromatin in a transcription-dependent manner and to cause a global decrease of RNA Polymerase II (RNAPII)-mediated transcription when downregulated, without affecting RNAPII elongation rate. Through microarray analysis of RALY-downregulated HeLa cells, I detected an altered expression of numerous genes involved in transcription promotion and cell cycle regulation, including the E2F transcription factors family. Due to its relevant role in regulating the cell cycle, I focused on the proliferation-promoting factor E2F1. I demonstrated that the stability of E2F1 mRNA is reduced in cells lacking RALY expression, with a resulting reduction of E2F1 protein levels. As a consequence of RALY knock-out, HeLa cells present a slower cell proliferation compared to control cells. Finally, by crossing the list of RALY targets with the list of genes affected by RALY downregulation, I propose a positive role of RALY in regulating the fate of specific transcripts. Taken together, my results highlight the importance of RALY expression for transcription and cell proliferation.

Il mar Mediterraneo è un bacino acquifero peculiare per la recente colonizzazione di specie aliene, per l’evento geologico legato alla Crisi di salinità del Messiniano e per l’ampio range di salinità. L’individuazione dei meccanismi di colonizzazione si è incentrata sullo studio morfologico, istologico e molecolare delle specie Asperarca nodulosa ed Anadara demiri (Arcidae-Bivalvia-Mollusca). La ricerca si è basata sulla caratterizzazione morfologica, con utilizzo del microscopio elettronico a scansione, al fine di individuare il tipo di sviluppo larvale. Successivamente i dati rilevati al S.E.M. sono stati supportati dall’indagine istologica che ha evidenziato la presenza di gonadi a sessi distinti e la non incubazione larvale. L’ulteriore analisi filogenetica ha permesso di evidenziare la netta suddivisione tra le tre popolazioni studiate, indagine effettuata tramite marcatori arbitrari (RAPDs) e nucleari specifici (ITS). I risultati ottenuti trovano supporto da quanto noto su base morfologica. I dati, nel complesso, mostrano una perdita delle capacità di diffusione della specie tramite sviluppo larvale plantotrofico a favore di quello lecitotrofico o diretto; tale tesi è ulteriormente supportata dai dati molecolari che mostrano una netta separazione delle popolazioni prese in esame ed un conseguente isolamento tra individui appartenenti a zone di profondità del Mediterraneo (sub-bacini abissali). La ricerca ha, inoltre,esaminato i meccanismi di introduzione attuali nel bacino acquifero che è soggetto ad una nuova invasione da parte specie aliene dovuta all’apertura del canale di Suez. L’analisi si è focalizzata sullo studio per l’ individuazione dell’origine della specie aliena A. demiri , di presunta derivazione Indo-Pacifica, ma rivelatasi, nei dati preliminari, di origine Atlantica.

No description available.