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.

TDP43 is an ubiquitously expressed RNA-binding protein implicated in several aspects of RNA metabolism. It can shuttle between the nucleus and the cytoplasm; however, when it is mutated in some familial Amyotrophic Lateral Sclerosis (ALS) cases, it undergoes nuclear clearance and cytoplasmic accumulation, driving neuronal degeneration. The same phenotype is present in patients bearing ALS-inducing mutations in other genes and ALS sporadic patients, defining TDP43 proteinopathy as a common feature in this pathology. Why does it cause specific motor neuron death? Our quantitative proteomics analysis of the TDP43 interactome revealed the interaction with components of the mRNA surveillance pathway, suggesting a still undiscovered function in nonsense-mediated decay. We demonstrated that TDP43 acts translation- and SMG1-dependently as a mRNA decay enhancer of specific transcripts by binding their 3’UTR. In particular, it leads to the down-regulation of transcripts with a long 3’UTR. From our sequencing data of spinal cords from TDP43Q331K transgenic mouse model and of motor neuron-like NSC-34 cells silenced for TDP43 emerged that TDP43 plays another striking role in the 3’UTR, modulating mRNA alternative polyadenylation and promoting the generation of shorter transcripts. This finding is supported by the direct interaction of TDP43 with the cleavage stimulation factor, a core component of the polyadenylation machinery. These results broaden our knowledge of the role of TDP43 in the post-transcriptional gene expression regulation. The impairment of these two biological processes by TDP43 proteinopathy could have implications in ALS pathogenesis, representing possible new targets for therapeutic approaches.

Basal breast cancer is a heterogeneous disease whose unfavourable outcome is determined by a high risk of tumour relapse and metastasis formation. The potential of a cancer cell to adapt to foreign environments is favoured by oncogenic cell plasticity, which is supported by epigenetic reprogramming. It was previously demonstrated that MYC acts as an oncogenic reprogramming factor by inducing epigenetic rewiring at enhancers (Poli et al., 2018). This causes the activation of oncogenic pathways and pro-metastatic transcription factors such as SOX9, but scant pieces of evidence support a causal link between epigenetic alteration of oncogenic enhancers and cell plasticity. In the present work, we investigated the establishment of an alternative epigenetic program during tumorigenesis in a basal breast cancer xenograft derived model. We found that tumorigenic cells, primary tumour derived cells and metastasis derived cells showed intrinsically different phenotypic and epigenetic signatures, and that metastatic derived cells were characterized by the acquisition of pro-metastatic features, such as migration and invasion, that may increase their metastatic potential. Specifically, we provided data supporting the notion that changes of the chromatin landscape during tumour progression increased the responsiveness of cancer cells to environmental cues that they may encounter during dissemination and colonization of distant organs. We focused on investigating the role played by putative regulatory elements localized around the SOX9 locus, whose chromatin accessibility and interaction with the SOX9 promoter were increased in metastatic cells. We observed that SOX9 expression was responsive to the activation of the retinoic acid (ATRA) pathway, and our data suggests that this response may be strengthened by transcriptional memory priming SOX9 regulatory elements after a first exposure, so that the response is faster and more robust after the second one. SOX9 transcription modulation and ATRA response were also shown to be linked to the activation of a quiescence program specific of metastatic cells, which we hypothesise may favour cells during the dissemination steps of the metastatic cascade.

The presence of networks of correlation between gray matter volumes of brain regions - as measured across subjects in a group of individuals - has been consistently described in several human studies, an approach termed structural covariance MRI (scMRI). Complementary to prevalent brain connectivity modalities like functional and diffusion-weighted imaging, this approach can provide valuable insight into the mutual influence of regional trophic and plastic processes occurring between brain regions. Previous investigations highlighted coordinated growth of these regions within specific structural networks in healthy populations and described their derangement in pathological states. However, a number of fundamental questions about the origin and significance of these couplings remains open and the mechanisms behind the formation of scMRI networks are still poorly understood. To investigate whether analogous scMRI networks are present in lower mammal species amenable to genetic and experimental manipulation such as the laboratory mouse, I coupled high resolution morpho-anatomical MRI with network-based approaches on a large cohort of genetically-homogeneous wild-type mice (C57Bl6/J). To this purpose, I first developed a semi-automated pipeline enabling reliable Voxel Based Morphometry (VBM) of gray matter volumes in the mouse. To validate this approach and its ability to detect plastic changes in brain structures, I applied it to a cohort of aged mice treated with omega-3 polyunsaturated fatty acids (n3-PUFA). This study revealed that treatment with n3PUFA, but not isocaloric olive oil preserved gray matter volume of the hippocampus and frontal cortices, an effect coincident with amelioration of hippocampal-based spatial memory functions. I next employed VBM to investigate scMRI networks in inbred mice using a seed-based approach. In striking resemblance with human findings, I observed the presence of homotopic (i.e. bilateral) architecture in several scMRI cortical and subcortical networks, a finding corroborated by Independent Component Analyses. Subcortical structures also showed highly symmetric inter-hemispheric correlations, with evidence of distributed antero-posterior networks in diencephalic regions of the thalamus and hypothalamus. Hierarchical cluster analysis revealed six identifiable clusters of cortical and sub-cortical regions corresponding to previously described neuroanatomical systems. This work documents for the first time the presence of homotopic cortical and subcortical scMRI networks in the mouse brain, and is poised to pave the way to translational use of this species to investigate the elusive biological and neuroanatomical underpinnings of scMRI network development and its derangement in neuropathological states.

Cell-free transcription and translation reactions lie at the heart of the rising field known as in vitro synthetic biology and their existence is fundamental for the reconstitution of artificial cells. While researchers are exploring different ways to create such reactions, the common feature that they share is the use of a template DNA to carry the information for the specific function that the reaction is required to perform. The scope of this thesis is to elucidate the relationship between the genotype and the phenotype in such reactions, investigating both transcription and translation using state of the art fluorescence spectroscopy.

Lateralization of the nervous system enhances optimization of neural circuitry and parallel processing in individual organisms. Over groups of individuals, brain-behavioural asymmetries might present a direction in the occurrence of the bias (the majority of the individuals showing the same direction at the population level) that has been mathematically demonstrated to be an evolutionarily stable strategy in social groups, thus optimizing coordination and cooperation. The superfamily Apoidea represents a group in which both the study of the appearance of population-level asymmetries and advantages in individual organisms (e.g., in the A. mellifera model) can be exploited. Here I described a study on olfactory lateralization in a primitively eusocial species of Apoidea, B. terrestris. I reported here that this species showed a direction in the behavioural asymmetry of short-term odour memory, but only individual-level differences in odour detection at the periphery of the nervous system. Moreover, B. terrestris showed a morphological difference at the level of the population in the number of structures where olfactory neurons are housed. In the same subfamily Apoidea, the perennial eusocial honeybee, A. mellifera, is a good candidate for assessing neural correlates of odour asymmetries. Lateralization in olfactory memory was reported in this species in the past; here I performed for the first time a study of anatomical and functional asymmetries within the brain, in the first olfactory neuropils, the antennal lobes. I measured a subset of glomeruli in naïve individuals and found symmetrical volumes between the sides for those glomeruli that are mainly activated by odours that show lateralization in behvaiour. Furthermore, I performed single-antenna recall tests, conditioning bees to extend their proboscis (in the so-called PER paradigm) in association with those odours that more strongly activated functional responses in the selected glomerular subset. The behavioural tests showed an odour dependency in the capacity of bees to recall compounds with the two antennae. A broader subset of glomeruli was measured after long-term memory formation and symmetrical volumes were confirmed in all glomerular classes revealing also memory-dependent shrinkage effect. At the functional level, I performed in vivo calcium imaging data of the bee antennal lobes. Odor-evoked activity maps were recorded with two-photon microscopy allowing for better spatial and temporal resolution compared to conventional fluorescence microscopy. A first comparison between sides from wide-field fluorescence microscopy data showed a left/right difference in distance between odour representations and different mixture interactions within each lobe. In the same social species, A.mellifera, I reported the results of experiments measuring social interactions between pairs of bees with only one antenna in use, revealing that animals tested with only their right antenna in use exhibited better social context-dependent behaviours. Overall, these results provide new evidence for the occurrence of behavioural lateralizations at the population level, and identify some of their possible anatomical and functional correlates. Finally, in relation to previous studies these results tighten the link between the occurrence of population-level asymmetries and their evolution in a social context.

The sequence-specific transcription factor p53 is considered a master gene of cellular responses to homeostasis changes. It is also a prominent tumor suppressor gene with the title of “guardian of the genome”. The increasing number of transcriptome analyses in cell lines treated with different agents activating p53, continues to add complexity to the vast transcriptional networks p53 regulates. To investigate mRNA translational control as an additional dimension of p53-directed gene expression responses, we performed translatome analyses upon its activation either by different agents or cellular contexts. Considered as a proxy for the proteome, the translatome allows us to characterize the translational status of each mRNA, independently from transcriptional modulations, and to evaluate the implications or correlations of changes in relative mRNA translation efficiencies with the phenotypic outcome. We first performed treatment-specific translatome profiling in MCF7 cells upon Doxorubicin and Nutlin-3a treatments. Among translated genes, we detected the presence of translationally enhanced mRNAs with a virtually absent transcriptional modulation; those genes were enriched for apoptotic functions, suggesting that the apoptotic phenotype might be controlled not only at the transcriptional, but also at the translational level. Seeking mechanisms underlying the mRNAs translational rate upon p53 activation, we identified the modulation of six RNA-binding proteins, where hnRNPD (AUF1) and CPEB4 are direct p53 targets, whereas SRSF1, DDX17, YBX1 and TARDBP are indirect targets, modulated at the translational level in a p53-dependent manner. In detail, we demonstrated the contribution of at least two p53-dependent translational mechanisms related to YBX1 translational repression, suggesting the presence of a controlled regulon at the crossroad of YBX1 mRNA translation. Given our finding that apoptotic genes appear to be controlled by p53 also at the translational level, we decided to explore whether mRNAs translational control mechanisms are indeed an additional checkpoint to the phenotype. To this aim, we performed a cell-type specific translatome study upon Nutlin-3a treatment, a drug with evident therapeutic prospective. SJSA1, HCT116 and MCF7 cells were chosen as they exhibit different cellular responses to Nutlin-3A (cell cycle arrest, apoptosis, or both, respectively). Our preliminary data suggests that translational modulation can affect the complex process of cell fate choice upon p53 activation. Indeed, a lack of overlap among genes differentially modulated at the translational level was evident. Motif search analysis at the 5’- and 3’-UTR of those genes highlighted the presence of different motifs in the three cell lines and the specific correlation of a C-rich motif with the apoptotic phenotype. Preliminary data on this motif will be presented and discussed. Two independent projects will be presented as appendixes, both of them related to the general idea that more than one factor may determine the p53 response. Starting from the analysis of possible p53 interactions with other transcriptional co-factors, we investigated the cooperative interaction between p53 and NFκB. For the second project, combining data previously obtained by means of yeast-based p53 transactivation assays, we developed an algorithm, p53retriever, to scan DNA sequences and thus identify p53 response elements and classify them based on their transactivation potential.

During the last decades public health policy makers have been increasingly turning to mathematical modeling to support their decisions. This trend has been calling for the introduction of a new class of models that not only are capable to explain qualitatively the dynamics of infectious diseases, but also have the capability to provide quantitatively reliable and accurate results. To this aim models are becoming more and more detailed and informed with data. However, there is still much to be done in order to capture the individual and population features that shape the spread of infectious diseases. This thesis addresses some issues in epidemiological modeling that warrant further investigation. In Chapter 1 we introduce an age-structured individual-based stochastic model of Varicella Zoster Virus (VZV) transmission, whose main novelty is the inclusion of realistic population dynamics over the last century. This chapter represents an attempt to answer the need pointed out by recent studies for a better understanding of the role of demographic processes in shaping the circulation of infectious diseases. In Chapter 2 we use the model for VZV transmission developed in Chapter 1 to evaluate the effectiveness of varicella and HZ vaccination programs in Italy. With a view to the support of public health decisions, the epidemiological model is coupled with a cost-effectiveness analysis. To the best of our knowledge, this work represents the first attempt to evaluate the post-vaccination trends in varicella and HZ, both from an epidemiological and economic perspective, in light of the underlying effect of demographic processes. Another novelty of this study is that we take into account the uncertainty regarding the mechanism of VZV reactivation, by comparing results obtained using two different modeling assumptions on exogenous boosting. In Chapter 3 we retrospectively analyze the spatiotemporal dynamics of the 2009 H1N1 influenza pandemic in England, by using a spatially-explicit model of influenza transmission, accounting for socio-demographic and disease natural history data. The aim of this work is to investigate whether the observed spatiotemporal dynamics of the epidemic was shaped by a spontaneous behavioral response to the pandemic threat. This chapter, represents an attempt to contribute to the challenge of understanding and quantifying the effect of human behavioral changes on the spread of epidemics. In Chapter 4 we investigate the current epidemiology of measles in Italy, by using a detailed computational model for measles transmission, informed with regional heterogeneities in the age-specific seroprevalence profiles. The analysis performed in this chapter tries to fill some of the existing gaps in the knowledge of the epidemiological features of vaccine preventable diseases in frameworks characterized by a low circulation of the virus.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. The search for new effective chemotherapeutic agents to treat GBM has proven challenging throughout the last few decades. As a result, very limited pharmacological treatment is currently available. GBM aggressiveness is associated with its glioblastoma stem cells (GSCs) component, which is responsible for resistance to therapy. Therefore, new specific pharmacological approaches directed to eradicate GSCs are endowed with a great therapeutic potential. GSCs have been shown to rely on mitochondrial respiration for their high energy demand. In order to have a functional mitochondrial respiration process, the five complexes forming the oxidative phosphorylation (OXPHOS) chain have to be built by the coordinate assembly of proteins translated by either the cytosolic or the mitochondrial ribosomes. Given their endosymbiotic origin and despite the evolutionary changes occurred the mitochondrial ribosomes (mitoribosomes) still share structural and functional similarities with the bacterial ones, particularly considering the functional ribosomal core. In the light of these similarities, we hypothesized that antibiotics targeting bacterial ribosomes could be exploited to inhibit mitoribosomes, affecting mitochondrial translation and OXPHOS assembly, and hence leading to detrimental effect on GSCs viability. We performed a high-content imaging driven screening of several bacterial ribosome targeting antibiotics and identified Drug A as the most promising compound due to its cytotoxic and mitotoxic effects on GSCs. We demonstrated that Drug A effectively prevents GSCs expansion, resulting to be over an order of magnitude more effective in GSCs growth inhibition than temozolomide, the only drug used in first line GBM therapy. We then investigated the mechanism of action of Drug A, proving that it inhibits mitochondrial translation and, as a consequence, it decreases the functionality of the OXPHOS complexes reducing mitochondrial respiration capacity. Moreover, we obtained the structure of this compound bound to the human mitoribosome using cryo-electron microscopy, which provides the basis for further development of more potent analogs. Finally we proved the efficacy of Drug A in vivo using a xenograft mouse model of GBM. Our results suggest that mitochondrial translation represents a therapeutic target for GBM and show that Drug A, acting via inhibition of mitochondrial translation, is extremely effective against GSCs. Given the urgent medical need for novel therapeutic approaches in GBM treatment, Drug A represents a promising therapeutic solution that is worth further preclinical and clinical investigations.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive loss of striatal and cortical neurons. HD is caused by an abnormal polyglutamine (polyQ) expansion in Huntingtin protein (HTT). HTT controls vesicular trafficking along axons in neurons through interaction with components of the molecular motor machinery. Arginine methylation is one of the most abundant post-translational modifications (PTMs) and is catalyzed by protein arginine methyltransferases (PRMTs). Recent evidence supports a key role for arginine methylation in neurodegeneration and particularly in polyglutamine diseases. However, whether HTT is methylated at arginine residues has not been investigated yet and the role of arginine methylation in HD pathogenesis remains to be fully elucidated. In this thesis, I show that vesicle-associated HTT is methylated in vivo at two evolutionarily conserved arginine residues, namely R101 and R118. Methylation of HTT at R118 is catalyzed by Protein Arginine Methyltransferase 6 (PRMT6), which localizes on vesicles together with HTT, whereas further analyses are required to identify the enzyme(s) responsible for R101 methylation. Interestingly, loss of PRMT6-mediated R118 methylation reduces the association of HTT with vesicles, impairs anterograde axonal transport and exacerbates polyQ-expanded HTT toxicity. Conversely, PRMT6 overexpression improves the global efficiency of anterograde axonal transport and rescues cell death in neurons expressing polyQ-expanded HTT. These findings establish a crucial role of arginine methylation as a modulator of both normal HTT function and polyQ-expanded HTT toxicity and identify PRMT6 as a novel modifier of HD pathogenesis. Importantly, defects in HTT methylation may contribute to neurodegeneration in HD and promoting arginine methylation of HTT might represent a new therapeutic strategy for HD.