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.

Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

Amyotrophic Lateral Sclerosis (ALS) is a non-cell-autonomous disease. In early 2000, seminal and concordant studies demonstrated that glia modulates disease progression in a mouse model expressing a mutant form of SOD1. This effect has not been validated yet in a transgenic mouse expressing TDP-43Q331K, so we selectively deleted the transgenic TDP-43 in astrocytes and observed an improvement of motor symptoms and a rescue of the cognitive impairment. We performed RNA sequencing of astrocytes purified from this mouse model, observing an increased expression of genes related to proliferation and de-differentiation at the early symptomatic stage. Additionally, we confirmed a relative increase in glial cell proliferation by Ki67 staining in vivo and by EdU staining in primary astrocytic cultures derived from this mouse model. By interrogating the genomes of 23.475 ALS patients versus 12.577 healthy controls, we revealed the enrichment of SNPs in the responsive elements of transcription factors linked to proliferation with a hub around c-myc. No differences were observed in c-Myc RNA and protein expression and stability in primary TDP-43Q331K astrocytes, while phosphorylated (active) c-Myc form was found to be increased by immunostaining. We observed this increase in several ALS models, including primary astrocytes expressing SOD1G93A, iNPC-derived astrocytes obtained from C9orf72 and sporadic ALS patients, and HEK293 cells overexpressing TDP-43. Interestingly, phosphorylated c-Myc form was found at higher levels than controls also in iPSCderived motoneurons expressing C9orf72, TDP-43M337V, SOD1I114T or derived from sporadic patients. c-Myc plays many functions in the cell, among which it enhances the release of extracellular vesicles (EVs), nanolipid structures shown to impact the non-cell autonomous degeneration in vitro. Our in vitro model showed a significant increase in the production of small EVs derived from ALS astrocytes. We observed that WT glia-derived EVs play a prosurvival effect on receiving neurons; this effect was lost when neurons were treated with gliaderived EVs from TDP-43Q331K cultures or WT cultures overexpressing c-Myc. Proteomic analysis revealed that ALS EVs and EVs derived from c-Myc-overexpressing cells were deficient in extracellular matrix proteins and integrins. Interestingly, the EVs isolated from TDP-43Q331K primary astrocytes were also less prone to enter recipient neurons. In addition, we notice that the pro-survival effect induced by WT glial EVs was lost on transgenic neurons, suggesting that receiving neurons carrying the mutation feature surface or endocytic alterations that prevent the transmission of the EV-induced prosurvival effect. To further characterize this hypothesis, we showed that HEK293 cells over-expressing TDP-43 feature impaired EV uptake and that iPSC-derived TDP-43M337V, SOD1I114T, and sporadic motoneurons feature altered endocytic pathways. All these data highlight alterations in the glial-to-neuron communication in ALS and suggest that the expression and function of integrins and extracellular matrix proteins should be further investigated in ALS, specifically focusing on rescuing neuronal endocytic pathways to achieve a functional treatment.

Frontotemporal Dementia with parkinsonism linked to chromosome 17 (FTDP-17) (OMIM # 600274) is a tauopathy caused by mutations in the MAPT gene. This gene encodes for Tau protein and its alternative splicing normally produces 6 different isoforms with three (3R) or four (4R) repeats of microtubule-binding domains, originated from the alternative splicing of exon 10 in the MAPT transcript. In normal adult brain, neurons and glia cells contain both 3R and 4R splicing isoforms in a 1:1 ratio. Several mutations in the MAPT gene impair exon 10 splicing, causing unbalance between 4R and 3R Tau isoforms (4R > 3R), leading to Tau 4R protein accumulation as insoluble neuronal deposits. Therapeutical correction of MAPT splicing isoforms balance is, in principle, possible using either exon-specific siRNAs, which degrade exon-10-containing mRNA in the cytoplasm, or splice-switching antisense RNAs, that induce skipping of exon 10 during the splicing of MAPT pre-mRNA in the nucleus. Both approaches have been explored in the Laboratory of RNA Biology and Biotechnology at CIBIO (University of Trento) using splicing reporters. Subsequently, several siRNAs and antisense RNAs have been demonstrated to efficiently engage their target (pre-)mRNA and restore 4R:3R balance in neuroblastoma cell lines in culture. Aim of the present work is to obtain pre-clinical evidence of the efficiency of the two approaches, in order to move towards clinical studies. To this purpose, I set up a disease model consisting in hiPSCs-derived neurons carrying a mutation in intron 10, where a C is substituted with a T in position 16 (MAPT IVS10+16; EBiSC, depositor Sigma-Aldrich SIGi001-A-12) and compared them with the appropriate isogenic healthy control (EBiSC; depositor Sigma-Aldrich SIGi001-A-1). 3R and 4R Tau mRNA and protein levels were evaluated at various days of differentiation and neuronal maturation. In my hands, IVS10+16 neurons showed increase 4R Tau mRNA expression at 120 days of differentiation, resembling the patient’s phenotype. The unbalance 4R:3R is reflected in the Tau protein, as assessed by Western blotting . I am presently evaluating other outcome measures of disease in this cellular model, such as synaptic impairment and electrophysiology . The Laboratory of RNA Biology and Biotechnology has reported that microRNAs (miRNAs) can be used as biomarkers of Frontotemporal Dementia (FTD). In particular, we recently reported that miR-92a-3p, miR-320a and miR-320b are misregulated in the plasma of FTD patients in comparison to healthy individuals (manuscript under review). I set out to measure these miRNAs in d120 IVS10+16 and isogenic neurons and in their conditioned medium. I found that all three miRNAs of interest were significantly increased in IVS10+16 samples compared to WT neurons, therefore representing a useful measure of therapeutical efficacy in our protocols. With the use of fluorescently labelled siRNAs, I then tackled the problem of delivering siRNA molecules to mature neurons and set up a protocol for their efficient delivery. Consequently, day120 IVS10+16 and WT neurons were transfected with different concentration of scramble and therapeutic siRNAs and the restoration of the 4R:3R Tau balance was assessed. My results suggest a promising potential for the use of isoform-specific siRNAs in FTDP-17 and possibly in other tauopathies. At the same time, I intended to validate in the same hiPSC-derived neuronal disease model, U1 and U7 chimeric splice-switching antisense RNAs that had been previously tested by plasmid transfection in neuroblastoma cell lines. To overcome the limitation represented by poor plasmid transfection efficiency in mature neurons, I encapsulated them into recombinant adeno-associated viruses (rAAVs). After having optimized the production of rAAVs and set the transduction conditions, IVS10+16 mature neurons were transduced with AAV expressing chimeric splice-switching antisense RNAs. Although neurons successfully got transduced and the cassette transcribed, there was no therapeutic effect when viruses were tested in d130 IVS10+16 neurons. I am presently exploring different experimental paradigms, to test the hypothesis that the 4R:3R unbalance can be prevented in mature neurons.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.

Understanding the relationships among biomolecules and how these relationships change between healthy and disease states is an important question in modern biology and medicine. The advances in high-throughput techniques has led to the explosion of biological data available for analysis, allowing researchers to investigate multiple molecular layers (i.e. omics data) together. The classical statistical methods could not address the challenges of combining multiple data types, leading to the development of ad hoc methodologies, which however depend on several factors. Among those, it is important to consider whether “prior knowledge” on the inter-omics relationships is available for integration. To address this issue, we thus focused on different approaches to perform three-omics integration: supervised (prior knowledge is available), unsupervised and semi-supervised. With the supervised integration of DNA methylation, gene expression and protein levels from adipocytes we observed coordinated significant changes across the three omics in the last phase of adipogenesis. However, in most cases, interactions between different molecular layers are complex and unknown: we explored unsupervised integration methods, showing that their results are influenced by method choice, pre-processing, number of integrated data types and experimental design. The strength of the inter-omics signal and the presence of noise are also proven as relevant factors. Since the inclusion of prior knowledge can highlight the former while decreasing the influence of the latter, we proposed a semi-supervised approach, showing that the inclusion of knowledge about inter-omics interactions increases the accuracy of unsupervised methods when solving the problem of sample classification.

During the past three years, we assisted to the rise of a new pathogen that afflicted the world with a global pandemic. Working in an era of rapid change has posed important challenges and the focus of research has shifted more and more toward topics of greater social utility. However, this period has also brought a new role for wastewater highlighting how it can provide insight into the health of a community. This is the approach of Wastewater-based Epidemiology (WBE). The work presented here aimed to deepen this approach not only at the theoretical level but also contributing with an ongoing monitoring of about 30 months. The main objectives were (i) to collect information on the recently discovered SARS-CoV-2 virus, its biology, transmission mechanism, and role in wastewater treatment plants (WWTP); (ii) set up a surveillance system that would allow to monitor SARS-CoV-2 infections over time, obtaining early information on its spread among the population to support the Health Authority. Starting from a detailed study of the shedding mechanisms of SARS-CoV-2 in the feces of infected patients, we moved on to the evaluation of the viral concentrations in the sewage system and the wastewater entering the WWTP. The possibility of a faecal-oral transmission route of the virus was investigated by evaluating the data about viability and infectivity in wastewater. The natural processes of decay of the virus in wastewater and the reduction of its concentration in the different treatment stages of WWTPs were explored in literature and with experimental data. At the same time, we developed a SARS-CoV-2 surveillance system in wastewater by applying different detection methods. Some practical and scientific aspects of the analysis protocol have been studied in depth such as the choice of the type of sample, the storage temperatures, and the pre-heat treatments aimed at making the analysis safer for the operator. The choice of the concentration method was evaluated to comply with the low concentration of the viral titer and therefore the crucial importance of this phase of the protocol. During the monitoring campaign, we further investigated aspects related to data processing and developed normalization approaches. Samples from WWTPs in the province of Trento were analysed weekly and sampling frequencies and curve smoothing methods deriving from those data were evaluated. The trend curves thus obtained were compared with those deriving from clinical data provided by the local Health Authority and signals of early warnings of virus diffusion trends in the population were highlighted. With the alternation of the different variants of the virus and the evidence of their importance in the development of new waves of infection, a PCR based genotyping method has been devised to rapidly identify the already known variants. In conclusion, this research project addressed a broad spectrum of aspects related to the WBE approach in contrasting the COVID-19 emergency and confirmed that wastewater could be a valuable source of information and management support for this and other emerging pathogens or micropollutants.

Nowadays, both gene mutations detection and function investigation are expected to assume a key role in diseases understanding and in many other biotechnological fields. In fact, gene mutations are often cause of genetic diseases and gene function analysis itself can help to have a broader vision on cells health status. Traditionally, gene mutations detection is carried out at pre-translational/sequence level (transcriptomic approach). On the other hand, the function of innumerable sequenced genes can be investigated by delivering them into cells through transfection methods and observing their expression result at post-translational level (proteomic approach). In this context, Micro-ElectroMechanical Systems (MEMSs) offer the intrinsic advantages of miniaturization: low sample and reagent consumption, reduction of costs, shorter analysis time and higher sensitivity. Their applications range from the whole cell assays to molecular biology investigations. On this subject, the thesis deals with two different tools for gene analysis: a Lab-on-Cell and cantilever-based sensors for in-vitro cell transfection and label-free Single Nucleotide Poly-morphisms (SNPs) detection, respectively. Regarding the first topic, an enhanced platform for single-site electroporation and controlled transfectants delivery has been presented. The device consists of a gold MicroElectrode Array (MEA) with multiple cell compartments, integrated microfluidics based on independent channels and nanostructured titanium dioxide (ns-TiO2) functionalized electrodes. Different activities have been reported, from the study of the microfabrication substrates bioaffinity and device development to the electroporation results. The functional characterization of the system has been carried out by electroporating HeLa cells with a small fluorescent dye and then, in order to validate the approach for gene delivery, with plasmid for the enhanced expression of the Green Fluorescent Protein (pEGFP-N1). The second research activity has been focused on a detection module aimed at the integration in a Lab-on-Chip (LOC) for the early screening of autoimmune diseases. The proposed approach consists of piezoresistive SOI-MEMS cantilever arrays operating in static mode. Their gold surface (aimed at the binding of specific thiolated DNA probes) has been deeply analyzed by means of Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) revealing an evident gold non-uniformity and low content together with oxygen and carbon contaminations. Different technological and cleaning solutions have been chosen in order to optimize the system. However, other improvements will be required. Moreover, the feasibility of the spotting technique has been demonstrated by verifying microcantilever mechanical resistance and good surface coverage without cross-contaminations. Finally, as future perspective, possible biological protocols and procedures have been also proposed and discussed starting from literature.