Recurrent disruptive mutations in chromodomain helicase DNA-binding protein 2 and 8 (CHD2 and CHD8) are emerging as prominent risk factors for Epilepsy and ASD, respectively. While both CHD2 and CHD8 play important roles in chromatin regulation and transcription, not fully dissected are the molecular consequences of the inactivating mutations described in patients. Here, we first investigated how chromatin reacts to CHD8 suppression by analysing a panel of histone modifications in human induced pluripotent stem cell-derived neural progenitors (hiNPC). CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affected highly expressed, CHD8-bound genes. Strikingly, the levels of transcription in cells presenting reduced H3K36me3 in the gene body appeared unchanged, however the process of alternative splicing was instead significantly affected. In particular, we have found aberrant alternative splicing patterns – mainly affecting alternative first and exon skipping events - of ~ 2000 protein coding genes implicated in “RNA splicing”, “mitotic cell cycle phase transition” and “mRNA processing”. Mechanistically, mass-spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator Heterogeneous Nuclear Ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain CHD8-suppression splicing phenotype, partially implicating SETD2, H3K36me3 methyltransferase. Most of the mutations in CHD2 and CHD8 genes are disruptive, leading to protein haploinsufficiency. Thus, any molecular manipulation eliciting an increase in CHD2/CHD8 proteins, could prove beneficial for therapeutic development. Here, we intended to provide a Proof-of-Principle of how SINEUP - recently described class of non-coding RNAs able to augment, in a specific and controlled way, the expression of target proteins - can increase the translation of CHD2/CHD8 proteins and possibly rescue haploinsufficiency-associated phenotypes. With this purpose, we designed and cloned SINEUP targeting human CHD2/CHD8 isoforms and tested their efficacy in human induced pluripotent stem (iPS), induced neural progenitor cells (hiNPC) expressing normal and reduced levels of the target proteins and in patients’ fibroblasts bearing CHD8 heterozygous loss-of-function mutations. While stimulation by different CHD2/CHD8-SINEUP molecules didn’t elicit any effect in wild-type cells with physiologic levels of CHD2/CHD8, CHD2/CHD8-SINEUP were fully effective in haploinsufficient conditions, when reduced levels of the target proteins were expressed. Functionally, CHD8-SINEUP were able to revert molecular phenotypes associated to CHD8-suppression, i.e. the transcriptional dysregulation of the ASD-related genes, MBD3 and SHANK3, and restore the genome-wide reduction of H3K36me3 enrichment. Strikingly, chd8-SINEUP injection in vivo, in chd8-morpholino treated, developing zebrafish embryos, confirmed that stimulation of internal methionine could rescue chd8-suppression induced macrocephaly phenotype. In conclusion, CHD2/CHD8-SINEUP molecules represent a Proof-of-Concept towards the development of an RNA-based therapy for neurodevelopmental syndromes, with implications for and beyond ASD and epilepsy, surely relevant to a large repertory of presently incurable brain genetic diseases.

This work regards the use of high performance computing (HPC) methods for a new bioinformatics challenge: the analysis of Terabyte-size data generated by the new ultra high throughput sequencing (UHTS) technology. As in microarray or mass spectrometry cases, public repositories are growing to store data from the next generation studies produced in laboratories around the world. These can be used to access to a large number of samples from experiments with different individuals, populations and sequencing platforms. Also experimental data of scientific articles are published in these stores, enabling to repeat and verify their results (reproducibility). An automatic downloader and analyzer system (D-Daemons architecture) is proposed to interface to a public repository of sequence reads, select all the experiments that match some research parameters, defined by a user, download them and apply an analysis pipeline to evidence their similarity or variability. A software pipeline based on this architecture and operating in a HPC environment has been developed to analyze the downloaded UHTS files in the shortest time possible. A case study of the system on “Colorectal Cancer (CRC) cell line†datasets and an aligner selection in a SNP discovery task on three RNA-Seq datasets (Human Breast tissue and of BT474 & MCF7 cell lines) are presented.

Single Nucleotide Polymorphisms (SNPs) represent the most abundant type of genetic variation and they are a valuable tool for several biological applications like linkage mapping, integration of genetic and physical maps, population genetics as well as evolutionary and protein structure-function studies. SNP genotyping by mapping DNA reads produced via Next generation sequencing (NGS) technologies on a reference genome is a very common and convenient approach in our days, but still prone to a significant error rate. The need of defining in silico true genetic variants in genomic and transcriptomic sequences is prompted by the high costs of the experimental validation through re-sequencing or SNP arrays, not only in terms of money but also time and sample availability. Several open-source tools have been recently developed to identify small variants in whole-genome data, but still the candidate variants, provided in the VCF output format, present a high false positive calling rate. Goal of this thesis work is the development of a bioinformatic method that classifies variant calling outputs in order to reduce the number of false positive calls. With the aim to dissect the molecular bases of grape acidity (Vitis vinifera L.), this tool has been then used to select SNPs in two grapevine varieties, which show very different content of organic acids in the berry. The VCF parameters have been used to train a Support Vector Machine (SVM) that classifies the VCF records in true and false positive variants, cleaning the output from the most likely false positive results. The SVM approach has been implemented in a new software, called VerySNP, and applied to model and non-model organisms. In both cases, the machine learning method efficiently recognized true positive from false positive variants in both genomic and transcriptomic sequences. In the second part of the thesis, VerySNP was applied to identify true SNPs in RNA-seq data of the grapevine variety Gora Chirine, characterized by low acidity, and Sultanine, a normal acidity variety closely related to Gora. The comparative transcriptomic analysis crossed with the SNP information lead to discover non-synonymous polymorphisms inside coding regions and, thus, provided a list of candidate genes potentially affecting acidity in grapevine.

The human genome is characterized by an extent of functions that act further than its genetic role. Indeed, the genome can also affect cellular processes by nongenetic means through its physical and structural properties, specifically by exerting mechanical forces that shape nuclear morphology and architecture. The balancing between two chromatin compartments with antagonist functions, namely Transcriptional and Polycomb condensates, is required for preserving nuclear mechanical properties and its perturbation is causative of the pathogenic condition Kabuki syndrome (KS) (Fasciani et al., 2020). KS is a rare monogenic disease caused by the haploinsufficiency in the KMT2D gene encoding for MLL4, a H3K4-specific methyltransferase important for the regulation of gene expression. By interrogating the effect of KMT2D haploinsufficiency in Mesenchymal Stem Cells (MSCs) we discovered that MLL4 loss of function (LoF) impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture and the cell mechanoresponsiveness during differentiation (Fasciani et al., 2020). These results suggest that altered nuclear mechanics rely on chromatin architecture and could potentially lead to changes in cell responses to external mechanical stimuli. In the present work, we investigated the role of Transcriptional and Polycomb condensates in tuning nuclear responses to different external mechano-physical conditions. To affect nuclear mechanics, we employed the use of several mechanical devices (e. g. substrate stiffness, microchannels with constrictions, and cell confinement). We found that Polycomb and Transcriptional condensates are modulated by changes in substrate rigidity in healthy conditions and that MLL4 LoF impairs the MSCs nuclear condensates-driven mechanical response. Furthermore, we observed that MLL4 LoF impacts nuclear adaptation to confined spaces by incrementing susceptibility to nuclear envelope rupture. We also showed that the increased nuclear fragility in MLL4 LoF is accompanied by an alteration of cell migratory capacity and survival rate. Altogether these findings suggest that MLL4 LoF impairs cell responses to external mechanical stimuli, shedding light on the pathological connection between the altered cell mechanoresponsiveness during differentiation and KS phenotype in terms of skeletal and cartilage anomalies.

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.

Recall-by-genotype (RbG) strategies are bottom-up approaches to conducting targeted follow-up studies or substudies with eligible participants. They use specific genetic information derived from pre-vious genome-wide association studies or whole-genome sequenc-ing enabled by next-generation sequencing. Genetic information may be partially disclosed when certain partici-pants are recalled for RbG studies, and information on the study design and eligibility criteria is provided. These distinguishing pecu-liarities of RbG approaches have ethical, legal, and social/societal implications (ELSI). In this thesis, we present and discuss the results of research on the ELSI aspects of RbG approaches and within the Cooperative Health Research in South Tyrol (CHRIS) studies (RbG1, RbG2) on genetic risk factors of Parkinson's disease (PD). We used various qualitative and quantitative methods, including in-terviews, surveys and focus group discussions (FGD). Thereby, we sought to address the need for qualitative data from diverse stake-holders, including critical voices in the CHRIS research ecosystem, such as participants, researchers, ethics board members, and study assistants, to develop effective recall and communication strategies through a collaborative approach refining the CHRIS RbG policy. The exploration began with a literature review revealing the explicit and implicit ELSI of RbG study designs. It uncovered a consensus on the significant ethical challenges RbG poses while highlighting the diversity in consent models and Return of Research Results (RoRR) policies employed in different research and biobanking con-texts. Then, a secondary analysis of interviews and surveys from a mixed-methods study with CHRIS RbG participants from the RbG pilot study (RbG1) followed. Alongside the second follow-up RbG study (RbG2) study, we then designed a survey, informed by the results of RbG1, to gather further perspectives on their experience of an RbG study, and other fundamental considerations pertinent to en-gagement and communication in RbG studies. Then, to explore the operational and practical aspects of RbG studies, we identified the relevant stakeholders who shape and decide on RbG study designs. Consequently, we designed and conducted FGD to examine stake-holder perspectives on the RbG study design, communication, and disclosure strategies. Further, we collected feedback and views from CHRIS study personnel and coordinators who accompanied the RbG1 and 2 study process. Finally, we conducted a large-scale sur-vey with CHRIS participants to strengthen the conclusions of previ-ous empirical research. This collaborative approach aims to refine the CHRIS RbG policy, develop effective recruitment and commu-nication strategies, and promote transparency. The study's findings underscore the value of personalised engage-ment and sensitive communication through tailored disclosure and communication strategies. Stakeholder views on ELSI in RbG stud-ies reveal diversity, highlighting the need for adaptable approaches aligned with study contexts. Overall, the results suggest that partici-pants are highly interested in receiving information on carrier status on the genetic variations investigated by the RbG study, but views and motivations were heterogeneous. This adds to the complexity of integrating these insights into communication strategies and disclo-sure policies. More research is necessary to investigate the effects of various disclosure strategies, the impact of disclosure on awareness, and how framing affects participants' reception of study-specific information.

Finding one’s way back to a safe refuge or recalling the best place to find food is essential to all animals including human beings. We engage in future actions based on past events. So how does our brain compute such important cognitive tasks? Is it an innate ability we have from birth that is hardwired into the blueprint of our brains? And what happens if for some reason we realize that we are unable to perform these cognitive abilities in old age or due to a neurological disorder? The hippocampus is the main area of the brain involved in memory and learning. Animal studies show evidence of its role in spatial navigation and memory. The complex network of spatial cells in the hippocampus, all participate in constructing a cognitive map in the brain, where an animal stores information about the external environment and uses it to engage in future actions. However, despite the importance in its function, the hippocampus is also one of the first areas of the brain to be affected by aging and other neurological disorders. The present thesis used the help of various animal models to answer three questions: first whether hippocampal function is present immediately at birth, second whether genes can regulate hippocampal activity and third whether a sensitive task such as reorientation can highlight hippocampal alteration caused by age. To answer the first question, we used the domestic chick that has the advantage of being tested after hatching. We show evidence that a change in environmental shape can alter hippocampal activity in naïve chicks, suggesting that hippocampal function is present already in early stages of life. Furthermore, we investigated if genes regulate hippocampal activity. We used a mouse model that carried one half of the Williams syndrome deletion, a disorder known for its hippocampal deficit. We show evidence that genes on the proximal deletion of Williams syndrome deletion, can alter reorientation and episodic memory, two hippocampal related functions. Finally, we aimed to find an appropriate task to highlight the allocentric difficulty that arises in age. We used aged animals of two species (mice and rats) and tested them in the reorientation paradigm. We show that this simple task has potential to be a better suited assay to evaluate hippocampal behavior.

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.

The human gut microbiota plays a crucial role in the functioning of the gastrointestinal tract and its alteration can lead to gastrointestinal abnormalities and inflammation. Additionally, the gut microbiota modulates central nervous system (CNS) activities affecting several aspect of host physiology. Motivated by the increasing evidences of the role of the gut microbiota in the complex set of interactions connecting the gut and the CNS, known as gut-brain axis, in this Ph.D. thesis we asked whether the gastrointestinal abnormalities and inflammation commonly associated with neurological disorders such as Rett syndrome (RTT) and Autism could be related to alterations of the bacterial and fungal intestinal microbiota. First, since only few reports have explored the fungal component of the gut microbiota in health and disease, we characterized the gut mycobiota in a cohort of healthy individuals, in order to reduce the gap of knowledge concerning factors influencing the intestinal microbial communities. Next, we compared the gut microbiota of three cohorts of healthy, RTT and autistic subjects to investigate if these neurological disorders harbour alterations of the gut microbiota. Culture-based and metataxonomics analysis of the faecal fungal populations of healthy volunteers revealed that the gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. Different fungal species were isolated showing phenotypic adaptation to the intestinal environment. High frequency of azoles resistance was also found, with potential clinical significance. It was further observed that autistic subjects are characterized by a reduced incidence of Bacteroidetes and that Collinsella, Corynebacterium, Dorea and Lactobacillus were the taxa predominating in the gut microbiota of autistic subjects. Constipation has been associated with different bacterial patterns in autistic and neurotypical subjects, with constipated autistic individuals characterized by higher levels of Escherichia/Shigella and Clostridium cluster XVIII than constipated neurotypical subjects. RTT is a neurological disorder caused by loss-of-function mutations of MeCP2 and it is commonly associated with gastrointestinal dysfunctions and constipation. We showed that RTT subjects harbour bacterial and fungal microbiota altered from those of healthy controls, with a reduced microbial richness and dominated by Bifidobacterium, different Clostridia and Candida. The alterations of the gut microbiota observed did not depend on the constipation status of RTT subjects while this microbiota produced altered SCFAs profiles potentially contributing to the constipation itself. Phenotypical and immunological characterizations of faecal fungal isolates from RTT subjects showed Candida parapsilosis as the most abundant species isolated in RTT, genetically unrelated to healthy controls’ isolates and with elevated resistance to azoles. Furthermore these isolates induced high levels of IL-10 suggesting increased tolerance and persistence within the host. Finally, the importance of multiple sequence alignment (MSA) accuracy in microbiome research was investigated comparing three implementations of the widely used NAST algorithm. By now, different implementations of NAST have been developed but no one tested the performances and the accuracy of the MSAs generated with these implementations. We showed that micca, a new bioinformatics pipeline for metataxonomics data improves the quality of NAST alignments by using a fast and memory efficient reimplementation of the NAST algorithm.

Background. Infections caused by Pseudomonas aeruginosa are the main cause of morbidity and mortality in Cystic Fibrosis (CF) patients and occur via primary colonisation of the airway followed by the accumulation of pathoadaptive mutations in the bacterial genome which increase fitness in the lung environment and result in chronicization. A better understanding of i) the evolutionary dynamics occurring during chronic airway infections in CF patients and ii) the genetic adaptation of strains to the CF lung environment, might give further clues for preventive measures or novel therapies to control CF infections in the future. In this work, we obtained genomic sequences of 40 P. aeruginosa isolates from a single CF patient collected over an eight-year period (2007-2014) and analysed the population in terms of clonality of the isolates, phylogenetic relationships, and presence of polymorphisms and variants between the strains. Population structure and microevolution. In silico Multilocus Sequence Typing (MLST) analysis revealed a characteristic single clonal population dominated by a previously characterized sequence type (ST390) and a small number of new, closely related ST variants (ST1863, ST1864, ST1923). EBURST analysis of the sequence types revealed that all members of this population belong to the same clonal lineage and likely evolved from a single ancestral colonizing strain. Furthermore, the phylogenetic analysis based on SNPs also divided the population into two subpopulations derived from the evolution of the first infecting strain. The annotation of SNPs allowed us to identify mutations with moderate or high impact. Genes with high impact variants encoded respiratory nitrate reductase subunit gamma nail, polyprotein signal peptidase lspA, the ABC transporter-binding protein aaltP, the copper resistance protein A precursor pcoAin, and four hypothetical proteins. The evolution of strains in the CF airway is characterized by the loss of many virulence traits, including motility and protease secretion, along with the acquisition of multidrug resistance. Functional phenotypic assays of the collection, including motility and secretion of proteases, showed a decrease over time in the persistent isolates. We also determined the antibiotic susceptibility profile of the collection; while early isolates were found to be susceptible to almost all these antibiotics, resistant phenotypes dramatically increased over time in the population. Functional studies on specific strains. To identify additional functional variations related to pathoadaptive mutations occurring in the course of chronic infection in CF, we then selected three isolates for further characterization: one early CF isolate (TNCF_23 isolated in 2007); one clonal late CF isolate (TNCF_175 isolated in 2014); one clinical isolate (VrPa97) from a non-CF patient belonging to the same sequence type (ST390) as the former isolates. With this approach, we aimed to identify additional phenotypic and functional variations between isolates with a very homogeneous genomic background, in an attempt to find out new pathoadaptive mutations occurring in the course of chronic infection in CF. Specifically, the following traits were investigated: killing of C. elegans and G. mellonella (in vivo virulence); immunomodulatory properties (IL-8 ELISA assay); competitive growth in Artificial Sputum Medium (ASM); functionality of Type Six Secretion System (T6SS). Despite their close genetic relatedness, considerable variations were observed between the three isolates, among which the late isolate TNCF_175 showed several alterations 7 putatively resulting from the adaptation process to the CF lung. TNCF_175 presented a mutation in tssK3, part of H3-T6SS; this mutation (C958T) was therefore introduced in the reference strains PAO1 and PA14, and mutated strains were subsequently complemented; killing rate on C. elegans and growth rate in ASM in mutant and complemented strains were evaluated. Conclusions. A rare feature of this strain collection is the consistent number of clonal isolates obtained from a single patient over a rather long period of 8 eight years, thus providing a model to look at microevolutionary trends within a highly homogenous bacterial population, and avoiding potential biases due to the host genetic background and clinical history. In spite of the close genomic relatedness of all isolates, a surprisingly high diversity was observed for the majority of tested phenotypes. Investigating the competitive ability of early versus late strains we propose a role for T6SS in the adaptation process to the CF lung environment. Our data suggest that once persistence has been established, a strain no longer requires its T6SS, allowing loss of function mutations to occur. Conversely, acute and early CF strains still carry a number of virulence factors, including T6SS that potentially provide an advantage in outcompeting other microorganisms in the initial stage of CF infection.