Medulloblastoma (MB) is the most common pediatric brain tumor with high morbidity and mortality. Among the four major MB groups, patients with Group3 MB currently have the worst outcome and nearly 50% are metastatic at the time of diagnosis. However, the molecular mechanisms underlying Group 3 MB are still unknown. Exploiting in vivo transfection of mouse cerebellar cells, we tested different patient- specific combinations of genes for their ability to induce Group 3 MB in mice. We identified Otx2 and cMYC as strong inducers of tumors faithfully recapitulating human Group 3 MB. To identify a druggable signaling pathway, we analyzed sequencing data of human patients harboring Otx2/cMYC overexpression. Among all the putative oncosuppressors, SMARCA4 is the most frequently mutated. Indeed, co-expression of SMARCA4 with Otx2 and cMYC is able to block tumorigenesis, through regulation of CDKN2B and CRABP1 expression. Interestingly, patient-specific SMARCA4T910M mutant is able to block wild- type SMARCA4 effects in a dominant-negative manner. Since SMARCA4 has an antagonistic relationship with histone methyltransferase EZH2, we used EZH2-inhibitors to recapitulate SMARCA4 antitumorigenic effects. Little is known about Group 3 MB developmental origin and several embryonic mouse cerebellar progenitors have been proposed as possible cell of origin. Here, we found that in postnatal mouse cerebellum, the S100b+ cells are competent to induce medulloblastoma while Math1, Sox2 and Ascl1 positive cells seem to be unresponsive to oncogenic insults. Taken together, our data suggest that the competency of different cells to develop MB could change during brain development, depending on their developmental stage and cellular identity.

This thesis aimed to explore eukaryotic cellular processes upon the virulent attack of low doses of a well-known pore forming toxin (staphylococcal α-hemolysin (αHL)) and to develop a new biotech application using the same protein.

Atrial fibrillation (AF) is the most common sustained arrhythmia worldwide and a frequent cause of hospitalization. Moreover, it represents one of the most frequent complication following cardiac surgery with an incidence of around 30% and an important predictor of patient morbidity. The exact pathophysiological mechanisms responsible for the onset and perpetuation of AF are not completely understood. However, clinical and experimental insights on the factors causing AF have suggested that atrial fibrillation is a multi-factorial phenomenon. Atrial fibrillation is characterized by a highly complex and irregular electrical activation of the atrial tissue, which is the manifestation of diverse abnormalities (electrical, structural, metabolic, neurohormonal, and/or molecular alterations) in diverse pathological conditions. In particular, it has been shown that fibrosis, a phenomenon in which extracellular matrix (ECM) components, mainly fibrillar collagen, accumulate between cardiomyocytes, leads to the inhomogeneous atrial electrical conduction typical of fibrillation. Recent studies have suggested that the deregulation of gene expression may act as a molecular mechanism of arrhythmogenesis. In particular, miRNAs, a new class of non-coding RNAs have rapidly emerged as one of the key players in the gene expression regulatory network, so variations in their expression levels may constitute a pathway for the arrhythmia-induced atrial remodeling. The present study aims to investigate the structural and molecular features of atrial tissue, with particular attention to fibrosis, which may be involved in the formation of a pro-arrhythmic substrate. By using both histological and advanced microscopy techniques, intramural fibrotic content and 3D collagen network properties were determined in atrial samples, collected during cardiac surgery in patients who developed or not AF. The quantitative analysis indicated a general decrease of collagen content from the outer (the epicardium) to the inner (the endocardium) myocardial wall, in the overall patient population. However, AF patients presented higher fibrotic values compared to sinus rhythm (SR) patients in the deeper myocardial layers, thus supporting the hypothesis that an accumulation of fibrotic tissue within the myocardial wall may represent an important structural contributor in the pathophysiology of AF. In addition to a quantitative assessment, collagen properties such as fibers orientation (degree and anisotropy) and scale dimension, were determined by non-linear optical microscopy techniques. The analysis revealed that in SR patients collagen network showed a fine architecture characterized by thin fibrils with changing angles and directions compared to AF, where fibers tended to pack-up in larger bundles of defined directions. A quantitative analysis of the 3D collagen network features, throughout the atrial wall, revealed that fibers orientation and scale dimension changed along tissue depth in both SR and AF patients, with larger values of orientation and fiber changes in AF tissues. These results highlight the spatial rearrangement and thickening of the 3D collagen network in AF patients, suggesting its possible role in the maintenance of the arrhythmia. Numerous evidence indicated that also an altered regulation of gene expression may play an important role in the mechanisms of atrial remodeling which underlie AF. In this perspective, the expression pattern of some miRNAs known to target different genes involved in diverse mechanisms that underlie AF was evaluated. A panel of miRNAs (miR-1, miR-133a/b, miR-30c, miR-29a/b, miR-208a/b, miR-328, miR-499, miR-590 and miR-21), principally involved in the formation of a pro-arrhythmic substrate, was selected after an accurate review of the literature and analyzed by RT-qPCR, in AF patients versus SR individuals. To accurately determine the levels of analyzed miRNAs, their expression data are usually normalized relatively to endogenous and/or exogenous reference genes. To date, no general agreement between different normalization strategies has been found, in particular in cardiac tissue, for the study of AF. For these reasons, a preliminary study aiming to establish the best endogenous reference genes for miRNAs data normalization was performed. Specifically, different well-established analysis tools such as NormFinder, GeNorm, BestKeeper and ∆Ct method, were applied on five commonly used endogenous reference transcripts such as 5S, U6, SNORD48, SNORD44 and miR-16. The suitable reference gene obtained, SNORD48, was applied for miRNAs data normalization. Our findings revealed that miRNAs expression levels were different in AF compared to SR patients. MiR-208a and miR-208b displayed statistically significant differences between the two populations. To investigate possible relationships between miRNAs expression levels and the fibrotic content a correlation measurement was also performed. Our analysis revealed that miR-21 and miR-208b were close to a significant correlation with fibrosis. In conclusion, this work introduced new techniques and implemented new methods of analysis for the study of the substrate of AF. In particular, the results obtained with this multiscale approach, from structural to molecular level, exacerbated the role of fibrosis as a critical contributor in the formation of a pro-arrhythmic substrate. Nonetheless, further studies are needed for a better understanding of the ways in which structural, molecular and also cellular remodeling may alter the impulse propagation in the myocardium.

Background and rationale Translation is a fundamental biological process occurring in cells, carried out by ribosomes simultaneously bound to an mRNA molecule (polyribosomes). It has been exhaustively demonstrated that dysregulation of translation is implicated in a wide collection of pathologies including tumours and neurological disorders. Latest findings reveal the existence of translational regulatory mechanisms acting in cis or trans with respect to the mRNAs and governing the movement and the position of ribosomes along transcripts or directly impacting on the ribosome catalogue of its constituent proteins. For this reason, translational controls also account for widespread uncoupling between transcript and protein abundances in cells. To explain the poor correlation between transcripts and protein levels, many computational models of translation have been developed. Usually, these approaches aim at predicting protein abundances in cells starting from the mRNA abundance. Despite the efforts of these modelling studies, a consensus model remains elusive, drawing to contradictory conclusions concerning the role of mRNA regulatory elements such as the usage of codons (codon usage bias) and slowdown mechanism at the beginning of the coding sequence (ramp). More recently, following the rapid and widespread diffusion of ribosome footprinting assays (RiboSeq), which enables the dissection of translation at single nucleotide resolution, a number of computational pipelines dedicated to the analysis of RiboSeq data have been proposed. These tools are typically designed for extracting gene expression alterations at the translational level, while the positional information describing fluxes and positions of ribosomes along the transcript is still underutilized. Therefore, the polysome organization, in term of number and position of ribosomes along the transcript and the translational controls directed in shaping cellular phenotypes is still open to breakthrough discoveries. Broad objectives The aim of my thesis is the development of mathematical and computational tools integrated with experimental data for a comprehensive understanding of translation regulation and polysome organization rules governing the number of ribosomes per polysome and the ribosome position along transcripts. Project design and methods With this purpose, I developed riboWaves, an integrated bioinformatics suite divided in two branches. riboWaves includes in the first branch two modeling modules: riboAbacus, predicting the number of ribosomes per transcript, and riboSim, predicting ribosome localization along mRNAs. In the second branch, riboWaves provides two pipelines, riboWaltz and riboScan, for detailed analyses of ribosome profiling data aimed at providing meaningful and yet unexplored ribosome positional information. The models and the pipelines are implemented in C and R, respectively. riboAbacus and riboWaltz are available on GitHub. Results To predict the number of ribosomes per transcript and the position of ribosomes on mRNAs, I applied riboAbacus and riboSim, respectively, to transcriptomes of different organisms (yeast, mouse, human) for understanding the role of translational regulatory elements in tuning polysome in different organisms. First, I trained and validated performances of riboAbacus taking advantage of Atomic Force Microscopy images of polysomes, while performances of riboSim were assessed employing ribosome profiling data. Predictions provided by riboAbacus and riboSim were evaluated in parallel. I showed that the average number of ribosomes translating a molecule of mRNA can be well explained by the deterministic model, riboAbacus, that includes as features the mRNA levels, the mRNA sequences, the codon usage bias and a slowdown mechanism at the beginning of the CDS (ramp hypothesis). The predictions of ribosome localization by riboSim that used as features the mRNA sequence, the codon usage and the ramp, were run for yeast, mouse and human. I observed a good similarity between the predicted and experimental positions of ribosomes along transcripts in yeast, while poor similarity was obtained between predicted and experimental ribosome positions in the two mammals, suggesting the presence of more elaborate controls that tune ribosomes movement in higher eukaryotes than in simple species. After having developed two tools for the analyses of RiboSeq data and extraction of positional information on ribosome localization along transcripts, I applied both riboWaltz and riboScan in a case study. The aim was to dissect possible defects in ribosome localization in tissues of a mouse model of Spinal Muscular Atrophy (SMA). SMA is a neurodegenerative disorder caused by low levels of the Survival of Motor Neuron protein (SMN) in which translational impairments are recently emerging as possible cause of the disease. I analysed ribosome profiling data obtained from three different types of RiboSeq variants in healthy and SMA-affected mouse brains at the early-symptomatic stage of the disease. I observed i) a significant drop-off of translating ribosomes along the coding sequence in the SMA condition (using riboWaltz); ii) in SMA-affected mice, the possible accumulation of ribosomes along the 3' UTR in neuro-related mRNAs (using riboScan); iii) the involvement of SMN-specialized ribosomes in playing a very intimate role with the elongation stage of translation of the first codons of transcripts (riboWaltz), iv) the loss of ribosomes at the 3rd codon in SMA in transcripts bound by SMN-specialized ribosomes and v) a remarkable connection between SMN and the down-regulation of genes in SMA-affected mice. Overall, these findings confirmed previous observation about possible SMN-related dysregulations of local protein synthesis in neurons. More importantly, they unravel a completely new role of SMN in tuning translation at multiple levels (initiation, elongation and the recycling of terminating ribosomes), opening new hypotheses and scenarios for explaining the most devastating genetic disease, leading cause worldwide of infant mortality. Conclusions The present work provides a new comprehensive and integrated scenario for better understanding translation and demonstrates that this approach is a very powerful strategy to pave the way for new understanding of fine alteration in polysome organization and functional control in both physiological and pathological conditions.

Glioblastoma (GBM) represents the most frequent and lethal cancer affecting the central nervous system for which no cure is currently available. The presence of Glioma Stem Cells (GSCs) has been proposed to be at the root of therapeutic failures due to their intrinsic abilities of escaping common treatments and relapsing the pathology. Thus, advances in therapeutic options may derive from the manipulation of mechanisms controlling the GSCs self-renewal, survival and functions. RE1-Silencing Transcription Factor (REST) is a master repressor of neuronal developmental programme in non-neuronal lineages, recently described as a main actor in the maintenance of the GSCs’ tumorigenic competence as its knockdown strongly impairs GSCs stemness both in vitro and in vivo. However, REST is critical for restraining neuronal cellular identity in various tissues, so that a targeted therapy to this transcriptional repressor is likely to present numerous side effects. Here, by taking advantage of a Tet-on system for the manipulation of REST expression in both human GSCs and Neural Stem Cell lines (hNSCs), we performed a transcriptomic profiling analysis in order to identify novel tumour-specific REST-regulated functions and molecular targets. Our analyses confirmed the previously reported roles of REST in neural tissues and enlightened novel REST functions in hGSCs, including the regulation of alternative hGSCs identity/state. Finally, analysis of hGSC-specific REST-regulated genes in GBM patients’ dataset revealed an inverse correlation with glioma aggressiveness, thus establishing a hGSC REST score that might provide a useful prognostic tool.

More than 300 human conditions, ranging from cancer predisposition to developmental and neurological mendelian disorders, are caused by haploinsufficiency (HI), a genetic condition by which mutational inactivation of a single allele leads to reduced protein levels and is enough to produce the disease phenotype. Therefore, translational enhancement of the spare allele could exert a therapeutic effect. Here we propose a novel approach for the potential rescue of haploinsufficiency disease loci based on the insertion of specific single nucleotide changes in the Kozak sequence. Since this sequence controls translation by regulating start codon recognition, we aimed at identifying and introducing specific nucleotide variations to enhance translation and rescue haploinsufficiency. To do so, we used CRISPR-Cas base editors, able to generate single nucleotide changes in genomic DNA without the need of a donor DNA and without creating double-strand breaks. We performed a high-throughput screening to evaluate the strength of the Kozak sequences of 231 haploinsufficient genes. We compared the translational efficiency of each wild-type sequence to that of several variants using FACS-seq, which combines fluorescence-activated cell sorting and high-throughput DNA sequencing. We thus selected 5 candidate genes (PPARGC1B, FKBP6, GALR1, NRXN1, and NCF1) and several nucleotide variations able to up-regulate translation. Finally, we used CRISPR-Cas base editors to reproduce the most efficient variants of NCF1 in a cell model relevant for the associated haploinsufficient disease and verified the increase of protein levels. This study proposes a novel therapeutic strategy to rescue haploinsufficiency and sheds new insights into the regulatory mechanisms underlying the translational process. On a broader level, the possibility of modulating gene expression by acting exclusively on translation expands the CRISPR-Cas genome editing applications.

Lipids were once thought to only be the building blocks of cell membranes and to serve as energy reserves. With time however, it became increasingly clear that they are actually involved in many more roles. Not surprisingly, the comprehensive characterisation of lipids in cells and tissues has experienced a growing interest worldwide, to the point that the term "lipidomics" was coined. This field is a subset of metabolomics, and the interesting point about these two sciences is that they are closest to the phenotype as compared to their "omics" counterparts (genomics, trascriptomics, ...), because metabolites and lipids are the end products of the –omics cascade. We have investigated mass spectrometry-based lipidomics from different perspectives: first of all, we have devised a targeted approach in which we have focused on sphingolipids and their perturbations. We started by working on neuronal cell cultures where we inhibited GBA, a key enzyme of the sphingolipid metabolism known to be one of the risk factors for Parkinson's disease. We found a significant sphingolipid unbalance characterised by an accumulation of glycosyl-ceramides. We then moved on by investigating the effects that LRRK2, an important and complex protein known to be related to autosomal-dominant forms of the disease, has on sphingolipids. We worked on mouse models, and we compared the sphingolipid profiles of wild-type (Lrrk2+/+) and knock-out (Lrrk2–/–) mice, finding a marked increase in ceramide levels and, more in general, in all lipids downstream of GBA. Such results hint to a possible interaction between LRRK2 and GBA, with LRRK2 playing a role in GBA regulation. In a second lipidomics investigation, we tried to understand whether or not anti-cancer treatments affect the lipid composition of tumours. Specifically, we concentrated on a common anti-angiogenic drug, whose aim is to starve cancer cells by inhibiting angiogenesis, a process required by the tumours to grow. We considered four different adenocarcinoma cell lines, which were subcutaneously inoculated into mice; the "control" animals received no treatment, whereas the "treated" ones were periodically given the drug. Interestingly, we found the treatment to have significant effects on the cancer lipidome, although the different lines responded unequally to the drug. Such results may reflect the huge heterogeneity of cancers and of individual responses to the treatment. Finally, we developed an informatics algorithm that deals with labelling experiments. The key point is that mass spectrometry measures isotopic patterns of analytes, which depend on the isotopic distribution of the elements; consequently, if an analyte incorporates the stable isotope employed in a labelling experiment, it will show a modified isotopic pattern. Our algorithm analyses such pattern, estimating the abundance of the incorporated label; we first tested it over carefully planned samples, and then we used it in a biochemical application where we wished to establish whether the rate of de novo lipogenesis is influenced by diet. This was accomplished by designing an experiment where mice were given partially deuterated water, while being fed different diets; we were able to ascertain that diet does indeed affect de novo lipogenesis, with the lowest rates occurring on fat-rich diets. We are confident that our tool may find useful applications, considering that stable isotope-based labelling experiments are becoming more and more popular.

Transcriptome analysis by total mRNA profiling provides a measurement of the degree of variation for the amount of each single mRNA species after a physiological or pathological transition of cell state. It has become a general notion that variations in protein levels do not necessarily correlate with variations in total mRNA levels, for the presence of post-transcriptional controls which influence the fate of cytoplasmic mRNAs and affect their translational fitness. Nevertheless, the extent of this phenomenon and the rules, if any, governing it are still generally unknown. To address this issue we took advantage of a number of studies performed using polysomal mRNA profiling in combination with classical total mRNA profiling in different mammalian and yeast systems. A normalization of the raw data coming from these datasets and a statistical meta-analysis aimed at maximizing uniformity in data processing have been performed. From the comparison of the results an extensive uncoupling between transcriptome and translatome variations of mRNA levels emerges, measured by a significant difference between steady state and polysomal fold changes induced by a cellular physiological or pathological transition. It seems clear that virtually the majority of significant changes in cytoplasmic mRNA steady-state levels are subjected to a further elaboration by a post-transcriptional decision program, leading either to a widespread buffering of the cytoplasmic changes which transfers only a small fraction of them to translation, either to the creation of new changes which cannot be detected at the transcriptional level, yet capable of heavily influencing protein synthesis rates. An explanatory model characterized by a cytoplasmic mRNA storage compartments is proposed and the involvement of P-bodies and the miRNA pathway in post-transcriptional reprogramming of gene expression has been experimentally tested in the biological model of EGF induction, in order to explain how a change in translational fitness can counteract or magnify a parallel change in citoplasmic mRNA availability. To investigate the role of specific cellular mechanism in generating uncoupling between transcriptome and translatome changes, the experimental model has been altered through silencing of three key genes involved in post-transcriptional regulation pathways: 4E-T, Xrn1 and Dicer.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked neuromuscular disorder characterized by the progressive dysfunction and loss of lower motor neurons. SBMA is caused by the expansion of a CAG tandem repeat encoding a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which includes eight other neurological diseases caused by the same mutation in unrelated genes. PolyQ diseases share common features, such as that polyQ proteins are typically expressed throughout the body, yet they cause specific neuronal loss. It remains to be clarified why specific sub-populations of neurons degenerate in each polyQ disease. The well-known structure and function of AR make SBMA a good model to investigate polyQ disease pathogenesis. Androgen binding to AR results in its nuclear translocation and binding to androgen-responsive elements (AREs) to regulate gene expression. Moreover, AR is highly phosphorylated. Recently, we obtained evidence that phosphorylation of polyQ-AR by cyclin-dependent kinase 2 (CDK2) at serine 96 increases toxicity. This post-translational modification was enriched in neurons. Therefore, we hypothesized that phosphorylation of polyQ-AR at serine 96 modulates its function in response to activation of neuronal activity, a level of regulation altered in SBMA. We carried out a microarray analysis in resting and stimulated neurons in which AR was activated by androgens. Our preliminary results suggest that AR activation drives a differential gene expression program in stimulated neurons. In order to analyze the role of CDK2 and serine 96 phosphorylation in vivo, we deleted one or both CDK2 alleles in SBMA mice. Modulation of CDK2 expression reduced polyQ-AR phosphorylation at serine 96, decreased polyQ-AR accumulation in neurons, and attenuated disease manifestations in SBMA mice. Finally, we carried out an unbiased high-throughput screening of phosphatase and kinase inhibitors. As read-out, we analyzed polyQ-AR nuclear translocation induced by testosterone, in order to identify compounds to lower polyQ-AR toxicity. We isolated 6 phosphatase and 17 kinase inhibitors as modifiers of polyQ-AR nuclear shuttling. Among them, we found two compounds targeting Cdc25, a known activator of CDK2. Cdc25 modulation altered serine 96 phosphorylation, toxicity and transcriptional activity of polyQ-AR in cells. Our results support the idea that Cdc25 represents a potential candidate to develop new therapeutic strategies for SBMA. In summary, our findings show that serine 96 phosphorylation modifies AR physiological functions in neurons and polyQ-AR toxicity in SBMA.

Bacterial Outer Membrane Vesicles (OMVs) are naturally produced by all Gram-negative bacteria and play a key role in their biology and pathogenesis. Over the last few years, OMVs have become an increasingly attractive vaccine platform for three main reasons. First, they contain several Microbe-Associated-Molecular Patterns (MAMPs), crucial for stimulating innate immunity and promoting adaptive immune responses. Second, they can be easily purified from the culture supernatant, thus making their production process inexpensive and scalable. Third, OMVs can be engineered with foreign antigens. However, the OMV platform requires some optimization for a full-blown exploitation. First, OMVs carry a number of endogenous proteins that would be useful to eliminate to avoid possible interference of immune responses toward the vaccine antigens. Second, OMVs carry abundant quantities of lipopolysaccharide (LPS). LPS is a potent stimulator of the immune system, therefore is essential for OMV adjuvaticity, but such adjuvanticity has to be modulated to avoid reactogenicity. In this study, we have addressed the two issues by creating a strain releasing OMVs with a minimal amount of endogenous proteins and containing a detoxified LPS. In particular, we first developed a CRISPR/Cas9-based genome editing tool which allows the inactivation of any “dispensable†gene in two working days. The efficacy and robustness of this tool was validated on 78 “dispensable genes†. Using our CRISPR/Cas9 protocol, an OMV proteome-minimized E. coli strain, named E. coli BL21(DE3)Δ58, deprived of 58 OMV associated proteins was created. We demonstrated that E. coli BL21(DE3)Δ58 had growth kinetics similar to the progenitor strain and featured a remarkable increase in OMV production. Two additional genes involved in the LPS biosynthetic pathway (msbB and pagP) were subsequently inactivated creating E. coli BL21(DE3)Δ60 which released OMVs with a substantially reduced reactogenicity. The exploitation of the two strains in vaccine applications was finally validated. We successfully engineered E. coli BL21(DE3)Δ58 and E. coli BL21(DE3)Δ60 with several different antigens, demonstrating that such antigens compartmentalized with high efficiency in the OMVs. We also demonstrated that the engineered OMVs from E. coli BL21(DE3)Δ58 and E. coli BL21(DE3)Δ60-derived OMVs elicited high antigen-specific antibody and T cell responses.