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CRISPR nucleases are efficient tools to edit cellular genomes in a variety of organisms. However, the in vivo application of this technology is still severely limited by unwanted genomic cleavages, that are further increased by long-term expression of the nuclease and can lead to unpredictable results. To address this limitation, we developed a yeast-based assay which allows to simultaneously evaluate the on- and off-target activity towards two engineered genomic targets in order to select optimized Streptococcus pyogenes Cas9 (SpCas9) variants. The screening of SpCas9 variants obtained by random mutagenesis of the Rec1-II domain allowed the identification of hits with increased on/off ratios. Through the combination of the identified mutations within a single variant we isolated the best performing nuclease, that we named evoCas9 (evolved Cas9). Side by side analyses with recently reported rationally designed variants demonstrated a significant improvement in fidelity of our evoCas9. In addition, to control Cas9 persistence into cells over time, we developed a Self-Limiting Cas9 circuitry for Enhanced Safety (SLiCES) which consists of an expression unit for SpCas9, a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. This self-limiting circuit, by controlling Cas9 levels, results in increased genome editing specificity. For its in vivo utilization, we integrated SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Following its delivery into target cells, the lentiSLiCES circuit is switched on to edit the intended genomic locus while simultaneously promoting its own neutralization through SpCas9 inactivation. The two strategies here developed represent complementary approaches to address a major issue in the genome editing field. On one hand, by preserving target cells from residual nuclease activity, our hit and go SLiCES system increases the safety margins for genome engineering. On the other, if compared to published structure-guided protein engineering approaches, our in vivo screening increases the likelihood to identify the best combination of amino acid substitutions for the generation of novel, error-free SpCas9 and could represent a valid strategy to enhance the specificity of other RNA-guided nucleases.

BACKGROUND: Objective of this thesis has been the analysis of the sequence specific transcription factor p53, a critical tumor suppressor protein, specifically, the crosstalk (or functional interactions) with other transcription factors, namely, the estrogen receptors, and the modeling in reconstituted assays of the interaction of p53 with positive and negative cofactors (e.g. MDM4 and 53BP1) and the impact of small molecules, including chemotherapeutic drugs, on such interactions. Previous reports have revealed a complex, often negative, crosstalk between p53 and estrogen receptors (ERs) related in part to the physical interaction between the two proteins. An example of transcriptional cooperation mediated by cognate, non-canonical cis-elements was instead discovered for the angiogenesis related VEGFR1, FLT1 promoter. MAIN TASK: Transcriptional cooperation between p53 and ERs was sought out on a global scale using the human breast adenocarcinoma MCF7 cells as a model and transcriptome analyses. Cells were subjected to single or combinatorial treatments with the chemotherapeutic agent doxorubicin (able to induce p53 protein stabilization) and the ER ligand 17β-estradiol (E2). 201 differentially expressed genes, that showed limited responsiveness to either doxorubicin treatment or ER ligand alone, but were up-regulated in a greater than additive manner following combined treatment were identified. Among sixteen genes chosen for validation using quantitative real-time PCR (qPCR), seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in stable MCF7 clone cells silenced for p53, or cooperative targets of p53 and ER. Based on exposure to 5-fuorouracil (another genotoxic drug) and nutlin-3a (a non-genotoxic p53-specific activator), the combined response identified genes that were consistently regulated, although with different kinetics (e.g. INPP5D, CDH26, KRT15), while others (e.g. TLR5, SOX9) were treatment selective. Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes were also performed to interrogate a direct, cis-mediated p53 and ERs regulation. While these analyses confirmed the identification of novel direct p53 targets, the important contribution of ER in their transcriptional modulation and the role of non-canonical response elements, the correlation between occupancy levels and gene expression varied. SECONDARY TASK: Using a newly developed miniaturized yeast-based assay, functional interactions between p53 and its regulators MDM4 and 53BP1 was investigated. MDM4 was confirmed as a p53 negative regulator and the impact of nutlin-3a or RITA (apoptosis inducer through p53 binding) on the p53-MDM4 interaction was explored. Instead, no stimulatory effect of the p53 co-activator 53BP1 was detected. CONCLUSIONS: Collectively, the results indicate that combinatorial activation of p53 and ER can induce novel gene expression programs which have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments. The yeast-based assay represents a versatile tool to study p53 interactions with cofactors.

The acquired immune deficiency syndrome (AIDS) has caused over 60 million deaths since the etiological agent, human immunodeficiency virus type 1 (HIV-1), was first discovered in 1981. Over 6000 new HIV-1 infections are reported every day, predominantly in economically deprived regions of sub-Saharan Africa. Despite impressive developments in antiretroviral therapy, current medical intervention is unable to prevent or cure HIV-1 infection, necessitating expensive life-long treatment. Difficulties in establishing a vaccine or cure, arise from its capacity to cause life-long latent infection, and its extraordinary ability to evolve resistance to therapeutic intervention. Capsid uncoating is the process by which p24CA proteins (CA) disassemble from the viral ribonucleoprotein during the early phase of the HIV-1 lifecycle. Despite intensive investigation, much is yet unknown about the spatial and temporal occurrence of this process within the cell, and the viral or host cellular factors involved. However, studies investigating p24CA mutations which alter the stability, and consequently the kinetics of capsid uncoating, have shown that timely capsid uncoating is crucial for efficient HIV-1 infection. Recent advancements in microscopic techniques have enabled high resolution analysis of cellular protein interactions, including the in situ localisation and dynamics of these events. We have developed three imaging techniques for the analysis of different aspects of HIV-1 capsid uncoating: 1) a dual-fluorescently labelled virus 2) a fluorescently labelled antibody targeting a capsid internalised repeat peptide array, and 3) a split-luciferase system tagging an internalised component of the capsid core. Fluorescent labelling of both the CA and IN proteins enabled the sensitive and specific analysis of uncoating in response to both restriction factors and CA mutations, and the visualisation of CA colocalised pre-integration complexes (PICs) within the nucleus. This system is ideally suited for studying the longer-term kinetics of uncoating, and the in-situ visualisation of protein interactions. The use of the repeat peptide array in conjunction with fluorescently labelled antibodies, reinforced reports of an initial uncoating event early after viral fusion. This system enabled the rapid and reproducible imaging of uncoating events in real-time, within the same cell sample population. Finally, the split-luciferase system added further weight to a primary early uncoating stage, and showed capsid disassembly responses specific to mutations within the p24CA that affect the stability of the viral core. Put together, these three assays support a model of uncoating involving an initial early phase of uncoating, followed by a more gradual disassembly of CA from the PICs during cytoplasmic trafficking towards the nucleus. The colocalisation of these components within the nucleus suggests incomplete uncoating at the time of nuclear docking. The user-friendliness of the split-luciferase system, along with its capacity for high-throughput, real-time analysis, support great potential for its use as a screening assay for testing antiviral compounds targeting capsid uncoating events.

p53 and NF-kB families of Transcription Factors (TFs) are among the most studied proteins in tumour biology, typically known to function as antagonists, although recent studies identified example of positive, even cooperative interactions. p53 and NF-kB act as dimer or dimer of dimers, bind cis regulatory elements (referred herein as Response Elements REs) of which multiple versions exist in the genome, and coordinate very large networks of target genes, through highly regulated transactivation specificities. TAp53 is a tumour suppressor activated upon genotoxic and physiological stress and involved primarily in deciding senescence, cell cycle arrest or apoptosis as cell fate; the NF-kB proteins are involved in cell survival, proliferation and innate immunity responses. In our study we tried to elucidate in detail the intrinsic nucleotide preferences of p53 and NF-kB as sequence specific TFs and their impact on transactivation specificity. Selecting various in vivo validated cognate REs and testing various ad-hoc sequence permutations, we evaluated the role of identity and positioning of nucleotides in transactivation potential and specificity. To this aim different transcription assays were used, starting from a defined assay in yeast where p53 or NF-kB protein levels and the sequence of the RE are the only variables. With human wild type p53, I tested various REs used in co-crystallization studies probing nucleotide positions that are not directly contacted by the p53 DNA binding domain, to explore the effect of DNA conformational shifts on transactivation. Also, we investigated the effect on strength and direction of p53-induced transcription of changes in the nucleotides flanking an RE, selected based on torsional flexibility measurements. For the NF-kB family, relA/p65 and NFkB1/p50 were tested as single proteins or when co-expressed using a panel of REs selected based on different DNA binding affinities. The correlation between DNA binding and transactivation potential was examined. Further, the negative modulator IkB-alpha was co-expressed and its impact measured as a function of NF-kB protein type, expression level, or RE being tested. Both for p53 and NF-kB studies, we confirmed that the hierarchical organization of nucleotides within REs observed with yeast was reasonably well conserved in A549, H1299 or MCF7 human cells using transient transfection and/or treatments to activate endogenous p53 or NF-kB. Finally, I contributed to an ongoing study focusing on the interplay between p53 and NF-kB at the transcriptional level. Using microarrays and quantitative PCR we had observed highly synergistic expression of a group of genes involved potentially in metastasis, cell growth and proliferation upon combined treatment of MCF7 cells with doxorubicin, a chemotherapeutic agent, and TNF-alpha, an inflammatory cytokine. I have studied regions of the promoters of several such synergistic genes carrying putative p53 and NF-kB binding sites to study cis-mediated regulation of gene expression. This led to the investigation of cell type specific effects and the contribution of cofactors in the transcriptional synergy. Thus the main goals of my thesis work have been: 1. To evaluate the hierarchy of nucleotides in the DNA code read by p53 and NF-kB as sequence-specific transcription factors. 2. Investigate the conservation of transactivation capacity and specificity between yeast and mammalian systems for the tested panel of REs. 3. Estimate the contribution of physical properties of DNA (torsional rigidity or flexibility) contiguous to a p53 RE in influencing the strength and the direction of transcription. 4. Explore the molecular mechanisms underlying transcriptional synergy in response to Doxorubicin and TNF alpha treatment.

Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide, is one of the major factors regulating cell metabolism and NAMPT is considered a promising target for treating cancer. The NAMPT inhibitor (FK866) has exhibited anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we showed that FK866 induced translation arrest through the activation of 5’AMP-activated protein kinase (AMPK), inhibition of mTOR/4EBP1 signaling, and phosphorylation of EIF2a in leukemia cells. Cancer drug resistance continues to be a major challenge in medical oncology. Deregulated cellular metabolism is linked to such cell resistance. Indeed, both components of the glycolytic and mitochondrial pathways are involved in altered metabolism conferring chemoresistance in several cancers. In this study, we developed FK866-resistant models in T- lymphoblastic leukemia (CCRF-CEM) and breast cancer (MDA-MB231) cell lines to investigate the molecular mechanism of pharmacoresistance to NAMPT inhibitor (FK866). Our resistant cells were not inhibited at the translational level by FK866 and the drug-induced metabolic adaptations of the resistant cells conferred an advantage to counteract FK866 toxicity. We reveal a molecular mechanism by which FK866 resistant CCRF-CEM cells utilize alternative sources for NAD production to fuel cell metabolism, and metabolic reprogramming was associated to the drug resistance. Importantly, the FK866- induced metabolic alteration was overcome by the co-administration of FK866 with compounds targeting metabolism, thereby rendering a synergistic outcome and restoring cell susceptibility towards FK866. We highlighted a molecular target that favors acquiring of resistance in leukemia and breast cancer cells. In conclusion, targeting metabolic alterations associated with drug resistance to FK866 may open up unexplored opportunities for the development of new therapeutic strategies as a combinatorial treatment for cancer.

Life-like behaviors such as fission, fusion and movement can be artificially re-created exploiting highly simplified protocell systems. This thesis is mainly focused on chemotaxis protocell systems and their integration with biological systems in order to show potential future applications. 1-Decanol droplets, formed in an aqueous medium containing decanoate at high pH, become chemotactic when a chemical gradient is placed in the external aqueous environment. We investigated the behavior of these droplets, their ability to transport and deposit living and non-living objects and to interface them with biofilms. To make the artificial system compatible with natural living systems we developed a partially hydrophobic alginate capsule as a protective unit that can be precisely embedded in a droplet, transported along chemical gradients and deposited. We developed a system that was able to transport: Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. Both bacteria survived the transport. However, yeast survived but not in a consistent and repeatable way. Next, we evolved the system to transport human cell lines. We found that A549 cells survive encapsulation but not the transport. A549 cells are in fact very sensitive to toxic 1-decanol. We however found out that this cell line secretes compounds able to decrease the surface tension and to increase the capsule-droplet affinity. Finally we discuss future solutions for the effective transport of human cells.

Of 25000 genes encoded from genome, more than 90% are subject to alternative splicing or other post-transcriptional modifications. All these events produce a high number of different proteins that form the basis for the high variety of cells. The RNAbinding proteins (RBPs) play crucial roles in this variability by regulating many steps of biological processes regarding RNA metabolism. The heterogeneous nuclear ribonucleoproteins (hnRNPs) belong to big family of RBPs involved in many aspects of RNA metabolism including RNA stability, intracellular transport and translation. More recently, RALY, a RNA-binding protein associated with the lethal yellow mutation in mouse, has been identified as new member of the hnRNP family even if, its biological function remains still elusive. My PhD project aimed to characterize human RALY and to assess its function in mammalian cells. Initially I dentified the expression pattern of this protein into the cell and I characterized the functional nuclear localization sequence that localizes RALY protein into the nuclear compartment. In order to better understand the role of RALY in the cells, I identified the proteins component of RALY-containing complexes using a new assay named iBioPQ (in vivo-Biotinylation-Pulldown-Quant assay). I also performed polyribosome profiling assay to check the resence of RALY in translating mRNAs. Moreover, a microarray assay was performed in order to identify potential mRNAs whose metabolism appears dependent on RALY expression. Taken together, the results that I obtained suggest that RALY is involved in mRNA metabolism. Unfortunately more studies remain to do before shedding some light on the biological role of RALY in mammals

Aberrant activation of metabolic pathways has emerged as an hallmark of proliferating cancer cells and pharmaceutical approaches targeting cell metabolism hold great potential for cancer treatment. A critical factor in cellular metabolism is nicotinamide adenine dinucleotide (NAD+) and cancer cells highly rely on it to face increased metabolic demands and proliferation rates. Intracellular NAD+ is a key metabolite involved in several cellular processes, acting either as a coenzyme in redox reactions or as a substrate for NAD+-degrading enzymes such as poly (ADP-ribose) polymerases (PARPs), CD38, and sirtuins, regulating processes that undergo fundamental changes during malignant transformation. Although NAD+ can be generated de novo from tryptophan precursor, the major route of biosynthesis is through a nicotinamidesalvage process. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide in mammalian cells. A number of cancers present an increased expression of NAMPT, and high NAMPT levels have been shown to be essential to support cancer cell growth, survival and EMT transition and to correlate with adverse prognosis. NAMPT is therefore a key factor regulating tumor cell metabolism and is thus considered a promising anti-cancer target. FK866 is a specific NAMPT inhibitor that lowers NAD+ concentration in cancer cells, reducing the activity of NAD+-dependent enzymes, impacting on ATP production and promoting cell death. NAMPT inhibition was proven to be highly effective in both lymphoid and myeloid-derived hematological malignancies in preclinical studies without affecting healthy cells, such as hematopoietic stem cells. FK866 has completed a phase I trial in oncology with advanced solid tumors. Thrombocytopenia was the dose-limiting toxicity, suggesting that this drug is a good candidate for clinical applications. We investigated the mechanism of action of FK866 in T-ALL derived cell lines as well as in primary leukemia cells. FK866-induced metabolic stress and NAMPT ablation elicited a strong arrest of protein synthesis as early cell response. FK866 induced activation of the AMP-activated protein kinase (AMPK), which subsequently drove the inhibition of the mTOR/4EBP1 signaling cascade and of the major initiation factor EIF2A, impairing protein synthesis. Furthermore, FK866-induced stress reduced the levels of the anti-apoptotic protein MCL1 and impacted on the endoplasmic reticulum homeostasis. In addition, we established and characterized an FK866-resistant model derived from the T-ALL cell line Jurkat. Target-specific acquired resistance has been described after several therapies and can be modeled in vitro by growing cells in presence of increasing concentrations of drug. In our resistant cells, FK866 treatment only partially impacted on NAD+ content, whereas ATP levels were recovered and protein translation resumed. Notably, during in vitro acquisition of drug resistance, mutations in the NAMPT gene have not occurred. In the last years, many NAMPT inhibitors have been synthesized and characterized. The obtained results provide new insight into the role of the NAMPT-mediated NAD+ salvage pathway in cancer cell metabolism and the molecular mechanisms of FK866, which will be useful to formulate specific and effective combinatorial drug therapies.

Nef is an HIV -1 accessory protein with a fundamental role for virus replication in vivo and for the development of AIDS. Among its several activities, Nef is essential for full HIV-1 infectivity, a function highly prominent in lymphoid cells. So far, the mechanism by which Nef promotes HIV-1 infectivity has remained elusive. Over the course of 3 years, my PhD research activity has led to the identification of the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as potent inhibitors of HIV-1 infectivity counteracted by the viral protein Nef [Rosa et al., 2015]. SERINC5 is predominantly localized on the plasma membrane where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef relocalizes SERINC5 to an endosomal compartment preventing its incorporation into HIV-1 particles. The ability to counteract SERINC5 is conserved in Nef proteins encoded by different primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus (MLV). These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 in the interaction of the host with retroviral pathogens. Remarkably, SERINC5 potently inhibits HIV-1 even in the presence of Nef in a dose-dependent manner, suggesting that this cellular factor might be exploited as an anti-HIV-1 therapeutic gene.