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.

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.