The clonal distribution of BRAFV600E in papillary thyroid carcinoma (PTC) has been recently debated. No information is currently available about precursor lesions of PTCs. My first aim was to establish whether the BRAFV600E mutation occurs as a subclonal event in PTCs. My second aim was to screen BRAF mutations in histologically benign tissue of cases with BRAFV600E or BRAFwt PTCs in order to identify putative precursor lesions of PTCs. Highly sensitive semi-quantitative methods were used: Allele Specific LNA quantitative PCR (ASLNAqPCR) and 454 Next-Generation Sequencing (NGS). For the first aim 155 consecutive formalin-fixed and paraffin-embedded (FFPE) specimens of PTCs were analyzed. The percentage of mutated cells obtained was normalized to the estimated number of neoplastic cells. Three groups of tumors were identified: a first had a percentage of BRAF mutated neoplastic cells > 80%; a second group showed a number of BRAF mutated neoplastic cells < 30%; a third group had a distribution of BRAFV600E between 30-80%. The large presence of BRAFV600E mutated neoplastic cell sub-populations suggests that BRAFV600E may be acquired early during tumorigenesis: therefore, BRAFV600E can be heterogeneously distributed in PTC. For the second aim, two groups were studied: one consisted of 20 cases with BRAFV600E mutated PTC, the other of 9 BRAFwt PTCs. Seventy-five and 23 histologically benign FFPE thyroid specimens were analyzed from the BRAFV600E mutated and BRAFwt PTC groups, respectively. The screening of BRAF mutations identified BRAFV600E in “atypical” cell foci from both groups of patients. “Unusual” BRAF substitutions were observed in histologically benign thyroid associated with BRAFV600E PTCs. These mutations were very uncommon in the group with BRAFwt PTCs and in BRAFV600E PTCs. Therefore, lesions carrying BRAF mutations may represent “abortive” attempts at cancer development: only BRAFV600E boosts neoplastic transformation to PTC. BRAFV600E mutated “atypical foci” may represent precursor lesions of BRAFV600E mutated PTCs.

Strategies to promote active aging and counteract the development of age-related diseases are among the most challenging researches in the framework of Horizon 2020, accordingly with the World Health Organization's declaration that "increased longevity without quality of life is an empty prize”. The aim of this thesis is to investigate the role of blood circulating microRNAs (miRs) and their expression profile characterizing aging and longevity trajectories, and particularly to distinguish between healthy and unhealthy longevity. To this purpose two experimental designs were defined, the former applied the advanced technology of smallRNA-sequencing (Illumina platform) to screen circulating miRs in a small cohort of different aged people, the latter was based on selected miRs analyzed on a larger cohort. The protocol for sequencing analysis, including library preparation, was optimized and applied on 12 donors, i.e. 3 young healthy donors, 3 old healthy donors, 3 healthy centenarians and 3 unhealthy centenarians. Significant miRs identified by sequencing, i.e. miR-30a-5p, -766-3p, -598-3p, were measured on a larger cohort of 48 subjects. Aging-related miRs previously described, i.e. miR-133a-3p, -206, -16, were analyzed in the same cohort of 48 donors. Circulating miR-206 and miR-16 levels described significant trajectories of aging, while miR-598-3p and miR-133a-3p levels characterized longevity trajectories. All these miRs are involved in the PI3K-Akt signaling, a central pathway for aging process. Finally, blood circulating molecules able to distinguish between healthy and unhealthy were obtained by joining the identified miRs and hemato-biochemical parameters, opening the possibility for further studies on therapeutic approaches.

Urban airborne particulate matter (PM) is known to increase morbidity and mortality due to cardiopulmonary diseases related to inflammatory processes and genotoxic effects. The aim of this thesis is to highlight the toxic and carcinogenic potential of airborne particulate matters collected during different seasons at a site that is located in the northern area of the city of Bologna by using alternative in vitro tests, such as the cell transformation assay with BALB/c 3T3 A31-1-1 and Bhas 42 cells. The purpose is also to evaluate the lifetime cancer risks associated with air inhalation in different sites (rural and urban), by using the relative potency of compounds belonging to the same chemical class (PAHs and nitro-PAHs) and the specific unit of carcinogenic risk. None of the organic or inorganic extracts of PM2.5 and PM1 induced a significant increase in the average number of transformed foci/plate or in the transformation frequency of BALB/c 3T3 A311-1 cells, whereas the results obtained by Bhas 42 cell transformation showed a significant increase in the average number of transformed foci/plate. All the analyzed organic extracts showed promoting effects in Bhas 42 cells. The application of the UR cancer risk to the transformed value of B(a)P equivalents in the winter– autumn campaigns leads to estimate an increase in the cancer risk similar to that defined in the literature (1 x 10-4 for exposure to 1 ng/m3). The calculated cancer risk was about one order of magnitude lower in the summer campaigns. In conclusion, the proposed approach, based on the integration of the data derived from in vitro testing and cancer risk assessment, could represent a reliable model for investigating environmental mixtures and predicting their effects on toxicological relevant endpoints.

Alzheimer's disease (AD) is a multifactorial and progressive form of dementia with a senile onset that affects specific areas of the brain. Recent genome wide association (GWA) studies reported that the allele 4 of apolipoprotein E (APOE) and single nucleotide polymorphisms (SNPs) in other genes that regulate inflammatory pathways, such as the gene coding for clusterin (CLU), are associated with AD. The hypothesis is that all of these genes may be involved in different mechanisms mediated by herpes viruses and we argued that the concomitant presence of SNPs in these genes in the same individual may represent a genetic signature predisposing to AD. The present study is focused on SNPs in CLU, interferon (IFN)-λ3/IL-28B, Med23 and the transcription factor IRF7, which are genes involved in antiviral responses and their association with AD and cognitive deterioration. Moreover, the effects of IL-28B, Med23 and IRF7 genotypes upon the presence of epstein-barr virus (EBV) and human herpes virus 6 (HHV-6) in the peripheral blood of AD and controls (CTR) have been also investigated. The activation of the innate immune system has a key role as a promoting factor for AD and in AD patients activated microglia release cytokines that induce neuro-inflammation. In this thesis gene variants and different expression of genes involved in the innate immune response in case-control population studies and in a mouse model of AD were investigated. Results from these experiments suggest that individuals with a particular genetic makeup in defensive mechanisms of the innate immunity may be at risk of defective immune responses. Impaired immunity against persistent viruses such as those of herpes family, might result in chronic and inappropriate activation of microglia, abnormal Aβ production and increased amyloid deposition. Cycles of virus latency and infections may therefore contribute to neurodegeneration associated with AD in genetically predisposed elderly.

Cell competition describes the result of a mechanism of fitness comparison undertaken by cells inhabiting the same tissue, that leads to the elimination of the weakest cells and, in the physiology, to the formation of a homogeneous organ. Over the years, many molecules have been identified that are involved in cell competition and among them MYC oncoprotein: from Drosophila to mammals, cell populations characterised by higher expression of MYC induce apoptotic death of the neighbours, allowing the fittest to acquire an advantage in space occupancy. My work defined the presence of markers of MYC-mediated cell competition in primary and secondary human carcinomas and demonstrated through experiments in human cancer cell lines that MYC modulation is per se sufficient to induce competitive behaviours in both genetically distant and identical cells. Noteworthy, MYC under-regulation in the fittest cell line is sufficient to undermine its competitive status, suggesting a role for MYC-mediated cell competition in the selective growth of tumour clones and, as a consequence, in cancer evolution. In addition, I was able to demonstrate a functional cooperation between MYC and p53 in this phenomenon. The data obtained in the Drosophila model, where MYC over-expressing and MYC knock-down clones have been induced within a growing tumour, suggest that MYC-mediated cell competition is normally at work in these malignant cells, and it shapes cancer evolution through the elimination of the less fit cells (with lower levels of MYC) and the expansion of the most performant ones (with higher levels of MYC), demonstrating an evolutionary role played in defining the composition and the size of the final mass.

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

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