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The coordinated action of transcriptional and post-transcriptional machineries shapes gene expression programs at steady state and determines their concerted response to perturbations. We have developed Nanodynamo, an experimental and computational workflow for quantifying the kinetic rates of nuclear and cytoplasmic steps of the RNA life cycle. Nanodynamo is based on mathematical modelling following sequencing of native RNA from cellular fractions and polysomes. We have applied this workflow to triple-negative breast cancer cells, revealing widespread post-transcriptional RNA processing that is mutually exclusive with its co-transcriptional counterpart. We used Nanodynamo to unravel the coupling between transcription, processing, export, decay and translation machineries. We have identified a number of coupling interactions within and between the nucleus and cytoplasm that largely contribute to coordinating how cells respond to perturbations that affect gene expression programs. Nanodynamo will be instrumental in unravelling the determinants and regulatory processes involved in the coordination of gene expression responses.

Drug-induced liver injury (DILI) remains the main reason for drug development attritions largely due to poor mechanistic understanding. Toxicogenomic to interrogate the mechanism of DILI has been broadly performed. Gene coregulation network-based transcriptome analysis is a bioinformatics approach that potentially contributes to improve mechanistic interpretation of toxicogenomic data. Here we performed an extensive concentration time course response-toxicogenomic study in the HepG2 cell line exposed to 20 DILI compounds, 7 reference compounds for stress response pathways, and 10 agonists for cytokines and growth factor receptors. We performed whole transcriptome targeted RNA sequencing to more than 500 conditions and applied weighted gene coregulated network analysis to the transcriptomics data followed by the identification of gene coregulated networks (modules) that were strongly modulated upon the exposure of DILI compounds. Preservation analysis on the module responses of HepG2 and PHH demonstrated highly preserved adaptive stress response gene coregulated networks. We correlated gene coregulated networks with cell death onset and causal relationships of 67 critical target genes of these modules with the onset of cell death was evaluated using RNA interference screening. We identified GTPBP2, HSPA1B, IRF1, SIRT1, and TSC22D3 as essential modulators of DILI compound-induced cell death. These genes were also induced by DILI compounds in PHH. Altogether, we demonstrate the application of large transcriptome datasets combined with network-based analysis and biological validation to uncover the candidate determinants of DILI.

L'organizzazione tridimensionale (3D) della cromatina all'interno del nucleo cellulare determina la separazione dei compartimenti della cromatina attiva e inattiva. Questi sono guidati dalla concentrazione locale di domini genomici con attività trascrizionale e caratteristiche epigenetiche simili, nonché dall’arricchimento locale delle proteine associate che portano alla formazione di strutture subnucleari distinte. Le tecniche genomiche di riferimento per mappare l’architettura 3D della cromatina si basano sulla quantificazione delle frequenze di contatto tra i loci genomici, che non rilevano la rilocazione dei compartimenti della cromatina attraverso le regioni subnucleari. I domini di cromatina situati spazialmente nello stesso quartiere nucleare 3D sono esposti allo stesso ambiente biochimico, condividendo così una solubilità simile. Sulla base di questa logica, abbiamo sviluppato una nuova tecnica sperimentale, la 4fSAMMY-seq, abbinata a metodi di analisi dei dati bioinformatici su misura per mappare l'accessibilità della cromatina, partendo dalle proprietà biochimiche dei domini genomici e utilizzando appena 10.000 cellule. Possiamo estrarre informazioni sulla segmentazione lineare delle regioni genomiche eucromatiche ed eterocromatiche, nonché sulla loro segregazione 3D in compartimenti cromatinici attivi e inattivi. Con 4fSAMMY-seq possiamo ricapitolare in un singolo esperimento le proprietà caratteristiche dei distinti stati della cromatina, incluso il compartimento Polycomb altamente dinamico. La struttura tridimensionale della cromatina è fondamentale per la corretta regolazione della funzione del genoma. Le lamine nucleari sono attori chiave del ripiegamento del genoma e partecipano al mantenimento della struttura della cromatina. Le lamine si dividono in tipo A e tipo B, e hanno una pletora di funzioni nei nuclei, dalle proprietà meccaniche, come resistenza alle forze e alla meccanotrasduzione, all'organizzazione radiale della cromatina nel nu, Chromatin three-dimensional (3D) organization inside the cell nucleus results in the separation of active and inactive chromatin compartments. These are driven by the local concentration of genomic domains with similar transcriptional activity and epigenetic features, as well as the local enrichment of their associated proteins leading to the formation of distinct subnuclear structures. The reference genomics techniques to map chromatin 3D architecture are based on the quantification of contact frequencies between genomic loci, that doesn’t’ detect chromatin compartments relocation across subnuclear regions. Chromatin domains spatially located in the same 3D nuclear neighbourhood are exposed to the same biochemical milieu, thus sharing similar solubility. Based on this rationale, we developed a new experimental technique, the 4fSAMMY-seq, paired with tailored bioinformatic data analysis methods to map chromatin accessibility, starting from the biochemical properties of genomic domains and using as little as 10,000 cells. We can extract information on the linear segmentation of euchromatic and heterochromatic genomic regions, as well as on their 3D segregation in active and inactive chromatin compartments. With 4fSAMMY-seq we can recapitulate in a single experiment the characteristic properties of the distinct chromatin states, including the highly dynamic Polycomb compartment. The tridimensional chromatin structure is fundamental for the correct regulation of the genome function. Nuclear lamins are key players of the genome folding and participate in the maintenance of the chromatin structure. Lamins are divided in A-type and B-types, and have a plethora of functions in the nuclei, from mechanical properties, as resistance to forces and mechanotransduction, to the radial organization of chromatin in the nucleus. Lamin A and B interact with chromatin in specific heterochromatic regions called lamina associated domains (LADs). These domains are positioned in the perip

Le cellule B svolgono un ruolo multifunzionale nel microambiente tumorale, influenzando la patogenesi del cancro. Essendo la seconda popolazione di cellule immunitarie più abbondante in molti tipi di cancro, il loro impatto varia a seconda del contesto tumorale unico. Tuttavia, le indagini sul ruolo delle cellule B nel cancro finora hanno prodotto risultati contraddittori, con alcuni studi che indicano un ruolo favorevole alla crescita tumorale e altri che suggeriscono attività anticancro. Oltre al loro ruolo nella produzione di anticorpi, le cellule B specifiche per il cancro naturalmente presenti possono contribuire direttamente alla soppressione tumorale attraverso i seguenti meccanismi: i) uccisione diretta delle cellule tumorali mediante l'espressione di granzima B, TRAIL e FasL e ii) induzione di una robusta immunità delle cellule T attraverso l'attivazione di risposte infiammatorie e la loro funzione di cellule presentanti antigeni professionali (APC). Le cellule B che infiltrano il tumore spesso formano compartimenti immunitari noti come strutture linfoidi terziarie (TLS), e la loro presenza nei tumori umani è associata a una risposta favorevole all'immunoterapia. Studi preclinici hanno dimostrato che il trapianto adottivo di cellule B specifiche per il tumore, raccolte dai linfonodi di drenaggio del tumore (TDLN) o isolate mediante separazione con dextrameri specifici per l’ antigene, può ridurre la progressione tumorale e indurre l'immunità delle cellule T specifiche per il tumore. Tuttavia, le cellule B specifiche per il tumore naturalmente presenti sono rare e difficili da selezionare ed espandere dalla vasta repertorio policlonale delle cellule B dei pazienti. Questo studio ha utilizzato la riparazione diretta per omologia (HDR) per riorientare i recettori delle cellule B (BCR) per mirare agli antigeni associati al tumore, con un focus particolare sul recettore del fattore di crescita epidermico umano 2 (hHER2). Attraverso l'ottimizzazione delle c, As the second most abundant immune cell population in many cancer types, their impact varies depending on the unique tumor context. Yet, investigations on the role of B cells in cancer have thus far yielded contradictory results, with some studies indicating a tumor-promoting role and others suggesting anticancer activity. In addition to their role in antibody production, naturally occurring cancer-specific B cells can directly contribute to tumor suppression by i) directly killing tumor cells via the expression of granzyme B, TRAIL, and FasL, and ii) by triggering robust T cell immunity through the activation of inflammatory responses and their professional antigen-presenting cell (APC) function. Tumor-infiltrating B cells often form immune compartments known as tertiary lymphoid structures (TLS), and their presence in human tumors is associated with a favorable response to immunotherapy. Preclinical studies have shown that the adoptive transplant of tumor-specific B cells, collected from tumor-draining lymph nodes (TDLN) or isolated by antigen dextramer sorting, can reduce tumor progression and induce tumor-specific T cell immunity. However, naturally occurring tumor-specific B cells are rare and challenging to select and expand from the extensive polyclonal repertoire of patients' B cells. This study utilized Homology direct repair (HDR) gene editing to reprogram B cell receptors (BCRs) to target tumor-associated antigens, with a particular focus on the human epidermal growth factor receptor 2 (hHER2). Through the optimization of stimulation cocktails and culture conditions, we developed an editing protocol achieving a 50% editing efficiency in primary murine B cells. The edited B cells were confirmed to express hHER2-BCR, differentiate into a germinal center (GC) phenotype in long-term cultures, and effectively present antigens to induce T-cell proliferation in in vitro assays. Using in vivo breast cancer and melanoma models expressing hHER2 antigen, the infusio

La fibrodisplasia ossificante progressiva (FOP) è una rara malattia congenita che provoca l'ossificazione eterotopica (HO) nei muscoli scheletrici. Deriva da una mutazione con guadagno di funzione (R206H) nel gene Acvr1 che codifica per il recettore dell'activina di tipo I, che porta all'attivazione aberrante delle proteine morfogenetiche ossee e delle vie di segnalazione dell'activina A. I pazienti sperimentano riacutizzazioni infiammatorie episodiche nei muscoli scheletrici che innescano l'HO. Il ruolo preciso dei macrofagi nei tessuti affetti da HO rimane poco chiaro e richiede ulteriori chiarimenti. Per modellare FOP abbiamo utilizzato il ceppo murino transgenico condizionale Acvr1R206Hlox/lox; Gt(ROSA26)SorCreERT2. La tomografia computerizzata (TC) ha rivelato che dopo l'espressione del recettore mutato, i topi FOP sviluppano osso ectopico dopo aver ricevuto danno muscolare a 14 e 21 giorni. Al fine di esplorare il coinvolgimento del sistema immunitario innato nell'inizio e nella progressione dell'HO, abbiamo diminuito selettivamente i monociti circolanti tramite quattro iniezioni endovenose di liposomi contenenti clodronato in topi FOP. Le scansioni TC hanno rivelato una formazione ossea ectopica significativamente ridotta nei topi FOP impoveriti di macrofagi rispetto ai controlli a 14 e 21 giorni dopo il danno. Per ottenere ulteriori informazioni sulla segnalazione precoce che porta all'HO, il sequenziamento dell'RNA a singola cellula è stato eseguito sui muscoli di topi FOP a 5 e 7 giorni dopo il danno. L'analisi bioinformatica ha rivelato che i progenitori fibro-adipogenici (FAP) sono arricchiti in vie di segnalazione correlate alla condro/osteogenesi e all'ipossia nei topi FOP. Inoltre, i macrofagi FOP hanno mostrato una maggiore espressione dei marcatori di differenziazione degli osteoclasti e hanno mostrato un profilo pro-infiammatorio sovraregolato. In sintesi, questi risultati affermano che i topi FOP replicano fedelmente le caratteristiche osservate, Fibrodysplasia Ossificans Progressiva (FOP) is a rare congenital disease that results in heterotopic ossification (HO) in skeletal muscles. It arises from a gain-of-function mutation (R206H) in the Acvr1 gene encoding for the activin type I receptor, which leads to the aberrant activation of the bone morphogenetic proteins and activin A signalling pathways. Patients experience episodic inflammatory flare-ups in skeletal muscles that trigger HO. The precise role of macrophages in the tissues affected by HO remains unclear and requires further elucidation. To model FOP we used the Acvr1R206Hlox/lox;Gt(ROSA26)SorCreERT2 conditional transgenic mouse strain. Computerized tomography (CT) revealed that upon expression of the mutated receptor, FOP mice develop ectopic bone after receiving muscle injury at 14 and 21 days. In order to explore the involvement of the innate immune system in the initiation and progression of HO, we selectively depleted circulating monocytes via four intravenous injections of clodronate liposomes in FOP mice. CT scans revealed significantly reduced ectopic bone formation in macrophage-depleted FOP mice compared to controls at 14 and 21 days post-injury. To gain further insights into the early signalling leading to HO, single-cell RNA sequencing was performed on muscles of FOP mice 5 and 7 days after pinch injury. Bioinformatic analysis revealed that fibro-adipogenic progenitors (FAPs) were enriched in pathways related to chondro/osteogenesis and hypoxia in FOP mice. Furthermore, FOP macrophages exhibited heightened expression of osteoclast differentiation markers and displayed an upregulated pro-inflammatory profile. In summary, these findings affirm that FOP mice faithfully replicate the features observed in patients and underscore the critical role of macrophages in HO. Importantly, single-cell transcriptomics suggests that macrophages and FAPs collaborate to form a cellular environment that promotes and sustains bone formation, even in the ear

Liquid-liquid phase separation (LLPS) is pivotal in forming biomolecular condensates, which are crucial in several biological processes. Intrinsically disordered regions (IDRs) are typically responsible for driving LLPS due to their multivalency and high content of charged residues that enable the establishment of electrostatic interactions. In our study, we examined the role of charge distribution in the condensation of the disordered N-terminal domain of human topoisomerase I (hNTD). hNTD is densely charged with oppositely charged residues evenly distributed along the sequence. Its LLPS behavior was compared with that of charge permutants exhibiting varying degrees of charge segregation. At low salt concentrations, hNTD undergoes LLPS. However, LLPS is inhibited by high concentrations of salt and RNA, disrupting electrostatic interactions. Our findings show that, in hNTD, moderate charge segregation promotes the formation of liquid condensates that are sensitive to salt and RNA, whereas marked charge segregation results in the formation of aberrant condensates. Although our study is based on a limited set of protein variants, it supports the applicability of the “stickers-and-spacers” model to biomolecular condensates involving highly charged IDRs. These results may help generate reliable models of the overall LLPS behavior of supercharged polypeptides.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

N6-methyladenosine (m6A) is the most abundant internal eukaryotic mRNA modification, and is involved in the regulation of various biological processes. Direct Nanopore sequencing of native RNA (dRNA-seq) emerged as a leading approach for its identification. Several software were published for m6A detection and there is a strong need for independent studies benchmarking their performance on data from different species, and against various reference datasets. Moreover, a computational workflow is needed to streamline the execution of tools whose installation and execution remains complicated. We developed NanOlympicsMod, a Nextflow pipeline exploiting containerized technology for comparing 14 tools for m6A detection on dRNA-seq data. NanOlympicsMod was tested on dRNA-seq data generated from in vitro (un)modified synthetic oligos. The m6A hits returned by each tool were compared to the m6A position known by design of the oligos. In addition, NanOlympicsMod was used on dRNA-seq datasets from wild-type and m6A-depleted yeast, mouse and human, and each tool’s hits were compared to reference m6A sets generated by leading orthogonal methods. The performance of the tools markedly differed across datasets, and methods adopting different approaches showed different preferences in terms of precision and recall. Changing the stringency cut-offs allowed for tuning the precision-recall trade-off towards user preferences. Finally, we determined that precision and recall of tools are markedly influenced by sequencing depth, and that additional sequencing would likely reveal additional m6A sites. Thanks to the possibility of including novel tools, NanOlympicsMod will streamline the benchmarking of m6A detection tools on dRNA-seq data, improving future RNA modification characterization.

Abbiamo recentemente identificato una nuova funzione degli elementi trasponibili LINE1 nella regolazione della quiescenza e dell'esaurimento delle cellule CD4+ umane. Abbiamo scoperto che gli elementi LINE1 vengono trascritti e inclusi in nuovi esoni di varianti di splicing di geni di attivazione delle cellule CD4+, qui definiti come trascritti LINE1. È interessante notare che questi trascritti sono espressi nelle cellule CD4+ naive, ma vengono rapidamente down-regolati dopo l'attivazione cellulare. Insieme a KAP1 e NUCLEOLINA, i trascritti LINE1 formano un complesso regolatorio, indicato come complesso LINE1-trascritto/KAP1/NUCLEOLINA (L1TXC), che sembra imporre la quiescenza cellulare, influenzando l'espressione di vari geni bersaglio, compresi quelli dai quali i trascritti LINE1 sono originariamente trascritti. Ipotizziamo che il L1TXC eserciti un meccanismo regolatorio complesso per preservare la quiescenza cellulare, influenzando possibilmente la topologia nucleare delle cellule CD4+ naive in quiescenza. Qui, stiamo sfruttando una strategia computazionale di multi-omici per ottenere una visione integrata di come questo complesso regoli l'organizzazione della cromatina dello stato quiescente nelle cellule CD4+ naive. Il nostro workflow può essere suddiviso in tre parti principali: (1) innanzitutto, intendiamo identificare i siti genomici direttamente occupati dal L1TXC; (2) in seguito, ci aspettiamo di definire quali regioni si trovino in prossimità spaziale con i siti L1TXC; (3) infine, proponiamo di effettuare una caratterizzazione multidimensionale di queste regioni, esaminando le loro funzioni biologiche, così come il loro ambiente epigenetico e le relazioni con la topologia nucleare complessiva. Il nostro lavoro è destinato a far luce su un meccanismo ancora non caratterizzato, fornendo nuovi principi generali dell'organizzazione del genoma e descrivendo come gli elementi LINE1 e il L1TXC regolino l'epigenoma dei linfociti T CD4+ umani per sostenere la, We have recently identified a novel function for LINE1 transposable elements in regulating human CD4+ T cell quiescence and exhaustion. We found that LINE1 elements are transcribed and included in novel exons of splicing variants of CD4+ T cell activation genes, hereafter defined LINE1-transcripts. Interestingly, these transcripts are expressed in naïve CD4+ T cells, but are rapidly downregulated following cell activation. Together with KAP1 and NUCLEOLIN, LINE1-transcripts form a regulatory complex, indicated as LINE1-transcript/KAP1/NUCLEOLIN complex (L1TXC), that appears to enforce cell quiescence, influencing the expression of various target genes, including those from which LINE1-transcripts are originally transcribed. We hypothesize that the L1TXC exerts an intricate regulatory mechanism to preserve cell quiescence, possibly shaping the nuclear topology of quiescent naïve CD4+ T cells. Here, we are exploiting a multi-omics computational strategy to get an integrated view of how this complex regulates the chromatin organization of the quiescent state in naïve CD4+ T cells. Our workflow can be divided into three main parts: (1) first, we intend to identify the genomic sites directly occupied by the L1TXC; (2) afterwards, we expect to define what regions are found in spatial proximity with the L1TXC-sites; (3) to conclude, we propose to perform a multidimensional characterization of these regions, surveying their biological functions, as well as their epigenetic environment and relationships with the overall nuclear topology. Our work is expected to shed light on a still uncharacterized mechanism, providing novel general principles of genome organization and describing how LINE1 elements and L1TXC regulate the epigenome of human CD4+ T lymphocytes to sustain cell quiescence.

L'obiettivo principale di questo lavoro di ricerca è di cercare di aumentare la comprensione dei meccanismi che determinano l'immunità cellulare e umorale durante l'infezione da SARS-CoV-2 in pazienti mild e severe, tenendo in considerazione lo status iniziale del paziente (Notarbartolo et al., 2021). Inoltre, è stato dimostrato che la patologia da COVID-19 risulta essere grave e pericolosa per la vita di individui con patologie preesistenti perchè possono influenzare l'elicitazione di una risposta immunitaria protettiva. Nel mio lavoro pubblicato (Favalli et al, 2022) abbiamo fornito un quadro completo delle risposte immunitarie umorali e cellulari nei pazienti affetti da malattie reumatiche muscoloscheletriche (RMD) che presentano risposte immunitarie compromesse a causa del loro sistema immunitario e a causa dell'uso prolungato di farmaci disease-modifying anti-rheumatic drugs (DMARD). A seguito della vasta campagna di vaccinazione con i nuovi vaccini a RNA, è stato fondamentale caratterizzare a fondo i meccanismi che determinano l'immunità ibrida in individui vaccinati sani durante la schedule vaccinale BNT162b2 (Galeota et al., submitted). Inoltre, l'intento è stato anche quello di valutare la risposta immunitaria in pazienti oncologici sottoposti a diversi trattamenti farmacologici, noti per interferire con la risposta immunitaria alle vaccinazioni o durante l’infezione da SARS-CoV-2 (manoscritto in preparazione)., The primary goal of this research is to increase our understanding of immunity determinants during the course of the SARS-CoV-2 infection in mild and severe patients, especially given its initial status as an unfamiliar and unknown infection (Notarbartolo et al., 2021). Moreover, COVID-19 has been demonstrated to be severe and life threatening in individuals with pre-existing medical conditions that can affect the elicitation of a protective immune response. In my published work (Favalli et al, 2022) we provided a complete picture of humoral and cellular immune responses in rheumatic musculoskeletal disease (RMD) who have impaired immune responses due to their compromised immune system and due to prolonged use of disease-modifying anti-rheumatic drugs (DMARDs). Following the extensive vaccination campaign using the new RNA vaccines, it was crucial to thoroughly characterize the mechanisms determining hybrid immunity in vaccinated individuals at different times after the BNT162b2 vaccine (Galeota et al., submitted). Moreover, the intent was also to evaluate the immune response in another category of patients, oncologic ones, and how various treatments, known to modify the immune response in opposite directions, might potentially interfere with the immune response to SARS-CoV-2 vaccinations in some way (manuscript in preparation).

Le immunoterapie per il trattamento del cancro sono state oggetto di ricerca per molto tempo, ma le basi molecolari che causano l’exhaustion, la progressiva perdita di funzionalità e la ridotta capacità proliferativa delle cellule T infiltranti il tumore (TILs) sono ancora in gran parte sconosciute. Si sa che gli RNA non codificanti regolano funzioni cellulari e plasticità in diversi contesti, compreso il differenziamento delle cellule T. Tuttavia, si sa poco circa i meccanismi epigenetici per il mantenimento della quiescenza delle cellule T e se possano essere responsabili dell'exhaustion delle cellule T nel microambiente tumorale. In questo contesto, è stato investigato il ruolo degli elementi trasponibili (TEs) nella regolazione dell'espressione genica nelle cellule del sistema immunitario, ma una chiara comprensione dei meccanismi con cui i TEs influenzano la progressione del tumore e il differenziamento delle cellule immunitarie non è ancora chiaro, e svelare la complessità nascosta dei TEs in diversi tipi cellulari potrebbe aprire nuove opportunità terapeutiche nel cancro e in altre malattie multifattoriali. Infatti, la difficoltà nello studio dei TEs nei dati omici, e in particolare nella trascrittomica, ci porta a interrogarci se importanti molecole di RNA non codificanti derivate dai TEs, ancora non rilevate, potrebbero agire come modulatori epigenetici della plasticità dei linfociti in individui sani o in pazienti oncologici. In questa tesi, esploro il panorama degli RNA derivati dai TEs nei nuclei delle cellule T circolanti prima e dopo l'attivazione del recettore del TCR, e nelle cellule T infiltranti il microambiente tumorale. La scoperta degli RNA derivati dai TEs come regolatori epigenetici dell'identità e della differenziazione delle cellule T potrebbe ispirare e motivare lo studio delle dinamiche di trascrizione dei TEs attraverso le numerose sottopopolazioni di TILs, e svelare se gli RNA derivati dai TEs giocano un ruolo nell'exhaustion p, Immunotherapies for cancer treatment have been researched for a long time, but the molecular basis that cause exhaustion, progressive loss of functionality and reduced proliferative capability of TILs are still largely unknown. Non-coding RNAs have been known to regulate cellular functions and plasticity in several context, including T cell differentiation. However, little is known about the epigenetic mechanisms for the maintenance of T cell quiescence, and whether they could be responsible of T cell exhaustion in the tumor microenvironment. In this context, the role of TEs in the regulation of gene expression in immune cells have been investigated, but a clear picture of the mechanisms by which TEs affect tumour progression and immune cells differentiation still eludes us, and unveiling the hidden complexity of TEs in different cell types may open new therapeutic opportunities in cancer and other multifactorial diseases. Indeed, the difficulty of studying TEs in omics data, and in particular in transcriptomics, leads to question whether important TE-derived non-coding and still undetected tissue-specific RNA molecules could act as epigenetic modulators of lymphocytes plasticity in healthy individuals or in cancer patients. In this thesis, I explore the landscape of TE RNAs in the nuclei of circulating T cells before and after TCR activation, and in T cells infiltrating the tumor microenvironment. The discovery of TE RNAs as epigenetic regulators of T cells identity and differentiation could inspire and motivate the study of the transcriptional dynamics of TEs across the numerous subsets of TILs, and unveil whether TE RNAs play a role in the progressive exhaustion of T cells in the tumor microenvironment. As detecting the TEs activity in NGS data analysis is notoriously challenging, an important portion of the work depicted in this thesis concerns the development of novel computational approaches to dissect the TE signal in omics data for which there is a lack of s

A comprehensive understanding of molecular changes during brain aging is essential to mitigate cognitive decline and delay neurodegenerative diseases. The interpretation of mRNA alterations during brain aging is influenced by the health and age of the animal cohorts studied. Here, we carefully consider these factors and provide an in-depth investigation of mRNA splicing and dynamics in the aging mouse brain, combining short- and long-read sequencing technologies with extensive bioinformatic analyses. Our findings encompass a spectrum of age-related changes, including differences in isoform usage, decreased mRNA dynamics and a module showing increased expression of neuronal genes. Notably, our results indicate a reduced abundance of mRNA isoforms leading to nonsense-mediated RNA decay and suggest a regulatory role for RNA-binding proteins, indicating that their regulation may be altered leading to the reshaping of the aged brain transcriptome. Collectively, our study highlights the importance of studying mRNA splicing events during brain aging.

Motor neuron (MN) demise is a hallmark of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Post-transcriptional gene regulation can control RNA's fate, and defects in RNA processing are critical determinants of MN degeneration. N6-methyladenosine (m6A) is a post-transcriptional RNA modification that controls diverse aspects of RNA metabolism. To assess the m6A requirement in MNs, we depleted the m6A methyltransferase-like 3 (METTL3) in cells and mice. METTL3 depletion in embryonic stem cell-derived MNs has profound and selective effects on survival and neurite outgrowth. Mice with cholinergic neuron-specific METTL3 depletion display a progressive decline in motor behavior, accompanied by MN loss and muscle denervation, culminating in paralysis and death. Reader proteins convey m6A effects, and their silencing phenocopies METTL3 depletion. Among the m6A targets, we identified transactive response DNA-binding protein 43 (TDP-43) and discovered that its expression is under epitranscriptomic control. Thus, impaired m6A signaling disrupts MN homeostasis and triggers neurodegeneration conceivably through TDP-43 deregulation.

Mitochondria have been the focus of extensive research for decades since their dysfunction is linked to more than 150 distinct human disorders. Despite considerable efforts, researchers have only been able to skim the surface of the mitochondrial social complexity and the impact of inter-organelle and inter-organ communication alterations on human health. While some progress has been made in deciphering connections among mitochondria and other cytoplasmic organelles through direct (i.e., contact sites) or indirect (i.e., inter-organelle trafficking) crosstalk, most of these efforts have been restricted to a limited number of proteins involved in specific physiological pathways or disease states. This research bottleneck is further narrowed by our incomplete understanding of the cellular alteration timeline in a specific pathology, which prevents the distinction between a primary organelle dysfunction and the defects occurring due to the disruption of the organelle’s interconnectivity. In this perspective, we will (i) summarize the current knowledge on the mitochondrial crosstalk within cell(s) or tissue(s) in health and disease, with a particular focus on neurodegenerative disorders, (ii) discuss how different large-scale and targeted approaches could be used to characterize the different levels of mitochondrial social complexity, and (iii) consider how investigating the different expression patterns of mitochondrial proteins in different cell types/tissues could represent an important step forward in depicting the distinctive architecture of inter-organelle communication.

Accumulation of mutations and chromatin conformation changes in hematopoietic stem cells (HSCs) lead to disease development such as Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). MDS and AML are age-associated clonal disorders characterized by hematopoietic dysfunctions and impaired HSCs differentiation. MDS commonly progresses to AML, which is an aggressive type of leukemia. STAT3 and STAT5 are important cellular regulators and their constitutive activation has been linked to various solid and hematologic malignancies, including MDS and AML, through dysregulated transcription. To elucidate the role of STAT3 and STAT5 in leukemic transformation, MDS and AML have been utilized to model pre-leukemia and leukemia respectively. Transcriptome changes have been identified by STAT3, STAT5A or STAT5B knockdowns (KDs) in MDS and AML cells followed by mRNA-sequencing. Genome-wide STAT3, STAT5A and STAT5B DNA binding has been explored through CUT&Tag. Differential gene expression and binding profiles in MDS and AML determined the direct target gene networks of each factor in both conditions, revealing a distinct function including pathways involved in the cell cycle, apoptosis and differentiation. STAT3 and STAT5A differentially regulated genes between MDS and AML have been identified, providing novel targets for biomarker development and therapeutic intervention. Also, a possible interplay between STAT3 and STAT5 activation in AML has been uncovered through increased phosphorylation of STAT5 and STAT3 in STAT3 and STAT5 KDs respectively, and thus via the regulation of common downstream targets. These findings describe a functional overlap between STAT3 and STAT5 in AML for the regulation of shared targets essential for important cellular functions in AML, such as leukemic metabolism. ATAC-sequencing has provided insights into the chromatin conformation states and detected higher accessibility in MDS compared to AML. This finding has unveiled potential change, Accumulation of mutations and chromatin conformation changes in hematopoietic stem cells (HSCs) lead to disease development such as Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). MDS and AML are age-associated clonal disorders characterized by hematopoietic dysfunctions and impaired HSCs differentiation. MDS commonly progresses to AML, which is an aggressive type of leukemia. STAT3 and STAT5 are important cellular regulators and their constitutive activation has been linked to various solid and hematologic malignancies, including MDS and AML, through dysregulated transcription. To elucidate the role of STAT3 and STAT5 in leukemic transformation, MDS and AML have been utilized to model pre-leukemia and leukemia respectively. Transcriptome changes have been identified by STAT3, STAT5A or STAT5B knockdowns (KDs) in MDS and AML cells followed by mRNA-sequencing. Genome-wide STAT3, STAT5A and STAT5B DNA binding has been explored through CUT&Tag. Differential gene expression and binding profiles in MDS and AML determined the direct target gene networks of each factor in both conditions, revealing a distinct function including pathways involved in the cell cycle, apoptosis and differentiation. STAT3 and STAT5A differentially regulated genes between MDS and AML have been identified, providing novel targets for biomarker development and therapeutic intervention. Also, a possible interplay between STAT3 and STAT5 activation in AML has been uncovered through increased phosphorylation of STAT5 and STAT3 in STAT3 and STAT5 KDs respectively, and thus via the regulation of common downstream targets. These findings describe a functional overlap between STAT3 and STAT5 in AML for the regulation of shared targets essential for important cellular functions in AML, such as leukemic metabolism. ATAC-sequencing has provided insights into the chromatin conformation states and detected higher accessibility in MDS compared to AML. This finding has unveiled potential change

DNA double-strand breaks (DSBs) are a significant threat to cell viability due to the induction of genome instability and the potential loss of genetic information. One of the key players for early DNA damage response is the conserved Mre11/Rad50 Nbs1/Xrs2 (MRN/X) complex, which is quickly recruited to the DNA’s ruptured ends and is required for their tethering and their subsequent repair via different pathways. The MRN/X complex associates with several other proteins to exert its functions, but it also exploits sophisticated internal dynamic properties to orchestrate the several steps required to address the damage. In this review, we summarize the intrinsic molecular features of the MRN/X complex through biophysical, structural, and computational analyses in order to describe the conformational transitions that allow for this complex to accomplish its multiple functions.

Quercetin (QUER) is a natural polyphenolic compound endowed with beneficial properties for human health, with anti-aging effects. However, although this flavonoid is commercially available as a nutraceutical, target molecules/pathways underlying its pro-longevity potential have yet to be fully clarified. Here, we investigated QUER activity in yeast chronological aging, the established model for simulating the aging of postmitotic quiescent mammalian cells. We found that QUER supplementation at the onset of chronological aging, namely at the diauxic shift, significantly increases chronological lifespan (CLS). Consistent with the antioxidant properties of QUER, this extension takes place in concert with a decrease in oxidative stress. In addition, QUER triggers substantial changes in carbon metabolism. Specifically, it promotes an enhancement of a pro-longevity anabolic metabolism toward gluconeogenesis due to improved catabolism of C2 by-products of yeast fermentation and glycerol. The former is attributable to the Sir2-dependent activity of phosphoenolpyruvate carboxykinase and the latter to the L-glycerol 3-phosphate pathway. Such a combined increased supply of gluconeogenesis leads to an increase in the reserve carbohydrate trehalose, ensuring CLS extension. Moreover, QUER supplementation to chronologically aging cells in water alone amplifies their long-lived phenotype. This is associated with intracellular glycerol catabolism and trehalose increase, further indicating a QUER-specific influence on carbon metabolism that results in CLS extension.

Introduction: Aging is associated with a defective immune response. Both the innate and adaptive arms of the immune system are affected with age, causing an overall decline of immunocompetences. With polychromatic flow cytometry (PFC), the project aimed to look deeper at the immune system to identify age and sex-related changes. Designing Panels: Multicolour panel design for profiling young and old lymphocytes obtained from peripheral blood has been completed. To do this, markers of interest were selected to address the aims of the study and then moved to carefully match the markers with the ideal fluorophores, considering the Flow Cytometer available (BD LSR Fortessa X20 – Blue, Red, Violet, Ultraviolet, Yellow/Green). Antibody titrations, Spreading error assessments and machine standardisation experiments were done. Testing was critical and allowed the review of controls necessary for proper analysis of the data. The analysis of optimisation experiments, tests and cohort runs were done by using FlowJo 10th edition. Results: We show that out of fifty four healthy donors with age have an overall weakened T cell immune compartment in both sexes, and display several alterations mostly involving CD8+ T cell subsets. We also define changes in markers of exhaustion (PD1 and TIGIT) and senescence (CD57) with age. Meanwhile, we define an increase with age amongst a very rare T cell subset that has been recently discovered amongst CD8+ TSCM memory cells (CCR7+, CD45RO-, CD95+, TIGIT+, PD1+). In B cell subsets we assess transcription factors PAX5 and XBP1 and for the first time, we report a decrease in the frequencies of XBP1 with age. We also measure mitochondrial superoxide indicators and show changes with age in T cells, but also in B and NK cells, which specify an impaired redox balance with age. NK cells display a decrease of CD56BRIGHT subset mainly driven my males. Conclusion: Overall, the standardized PFC assays can detect multiple biological phenomena and can provid, Introduction: Aging is associated with a defective immune response. Both the innate and adaptive arms of the immune system are affected with age, causing an overall decline of immunocompetences. With polychromatic flow cytometry (PFC), the project aimed to look deeper at the immune system to identify age and sex-related changes. Designing Panels: Multicolour panel design for profiling young and old lymphocytes obtained from peripheral blood has been completed. To do this, markers of interest were selected to address the aims of the study and then moved to carefully match the markers with the ideal fluorophores, considering the Flow Cytometer available (BD LSR Fortessa X20 – Blue, Red, Violet, Ultraviolet, Yellow/Green). Antibody titrations, Spreading error assessments and machine standardisation experiments were done. Testing was critical and allowed the review of controls necessary for proper analysis of the data. The analysis of optimisation experiments, tests and cohort runs were done by using FlowJo 10th edition. Results: We show that out of fifty four healthy donors with age have an overall weakened T cell immune compartment in both sexes, and display several alterations mostly involving CD8+ T cell subsets. We also define changes in markers of exhaustion (PD1 and TIGIT) and senescence (CD57) with age. Meanwhile, we define an increase with age amongst a very rare T cell subset that has been recently discovered amongst CD8+ TSCM memory cells (CCR7+, CD45RO-, CD95+, TIGIT+, PD1+). In B cell subsets we assess transcription factors PAX5 and XBP1 and for the first time, we report a decrease in the frequencies of XBP1 with age. We also measure mitochondrial superoxide indicators and show changes with age in T cells, but also in B and NK cells, which specify an impaired redox balance with age. NK cells display a decrease of CD56BRIGHT subset mainly driven my males. Conclusion: Overall, the standardized PFC assays can detect multiple biological phenomena and can provid

DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors, or by post-translational modifications of histones, thus increasing chromatin flexibility and the accessibility of repair enzymes to the DNA. Here, we review histone post-translational modifications occurring around a DSB in the yeast Saccharomyces cerevisiae and their role in DSB repair, with particular attention to DSB repair pathway choice.

È noto che la corretta organizzazione tridimensionale del genoma influenza l'espressione spazio-temporale dei geni specifici del “lignaggio” durante i processi di differenziamento e invecchiamento delle cellule staminali. Abbiamo messo a punto una nuova versione della tecnica SAMMY-seq per mappare con precisione regioni genomiche separate per proprietà biochimiche. Questa tecnica consente di identificare i domini eterocromatici ed eucromatici e la loro compartimentalizzazione nel nucleo. I vantaggi pratici cruciali di questo metodo includono: la sua applicabilità su appena 10mila cellule; costi ridotti; poche fasi di manipolazione e tempi di esecuzione brevi. Nelle cellule staminali muscolari postnatali (MuSC) abbiamo osservato una distribuzione riproducibile dei domini genomici eucromatici ed eterocromatici, in linea con le modificazioni epigenetiche note. I nostri risultati evidenziano come le MuSCs nel corso della vita presentino un'organizzazione cromatinica globale stabile, accompagnata da cambiamenti di solubilità che favoriscono processi come l'attivazione delle MuSCs, ma che possono diventare ostacoli durante l'invecchiamento per il corretto mantenimento del pool. Inoltre, descriviamo le alterazioni ambientali della nicchia muscolare, sottolineando una popolazione di supporto alle MuSCs, i progenitori fibroadipogenici (FAPs), che presentano drammatiche alterazioni trascrizionali durante l'invecchiamento. La nostra ampia caratterizzazione dell'ambiente e dell'organizzazione cromatinica delle MuSCs amplia la nostra conoscenza dei processi di quiescenza, attivazione e invecchiamento, ponendo le basi per lo studio del ruolo dell'epigenoma in condizioni patologiche., The correct 3D organization of the genome is known to influence the spatiotemporal expression of lineage-specific genes during stem cell differentiation and aging processes. We introduce a novel evolution of the SAMMY-seq technique to precisely map genomic regions separated by their biochemical properties. This single-handedly technique enables the identification of heterochromatic and euchromatic domains and their compartmentalization in the nuclear space. Crucial practical advantages of this method include: its applicability on as little as 10K cells; reduced costs; few manipulation steps and short execution time. In postnatal Muscle Stem Cells (MuSCs) we observed a reproducible distribution of euchromatic and heterochromatic genomic domains, in line with known epigenetic signatures. Our findings highlight how MuSCs over life exhibit a global steady chromatin organization, accompanied by solubility changes that favour processes such as MuSCs activation but may become obstacles during aging for proper pool maintenance. Still, we describe environmental alterations of the muscle niche, emphasizing a supportive population of MuSCs, namely Fibroadipogenic progenitors (FAPs), which exhibit dramatic transcriptional alterations during aging. Our extensive characterization of the environment and chromatin organization in MuSCs expands our understanding of quiescence, activation and aging processes, laying the groundwork for the study of the role of the epigenome in pathological conditions.

Il misfolding proteico e l’aggregazione di proteine sono problemi essenziali alla base di diversi disordini neurodegenerativi. Tali malattie sono caratterizzate dalla presenza di inclusioni di proteine misfoldate che possono interferire con la corretta proteostasi, portando alla perdita dell’attività neuronale cerebrale. Ciononostante, la formazione di aggregati proteici è un meccanismo messo in atto anche da cellule eucariote sane per proteggere il proprio proteoma. Tra tali siti di deposito, i Dendritic cells Aggresome-Like Induced Structures (DALISs) sono stati recentemente identificati in cellule dendritiche. Essi rappresentano dei depositi transienti per proteine difettive ubiquitinate ed antigeni, che si formano in risposta a diversi stress. La loro funzione non è ancora completamente compresa, specialmente dopo essere stati identificati anche in cellule non appartenenti al sistema immunitario. Ad oggi, nessuna indagine è stata condotta in altre cellule eucariote ma, fungendo da siti di deposito per proteine ubiquitinate, ci si aspetta di incontrarli anche in altri modelli cellulari. Il lievito Saccharomyces cerevisiae viene ampiamente utilizzato per la ricerca sugli aggregati cellulari, poiché presenta un ampio range di inclusioni dinamiche che sono stati dimostrate essere conservati tra diverse specie, nonostante il maggior grado di complessità nella composizione proteica e nelle funzionalità negli eucarioti multicellulari. Abbiamo identificato quattro diverse condizioni di stress in cui il lievito S. cerevisiae manifesta la formazione di corpi citoplasmatici: shock termico a 42°C, stress acuto da etanolo, deprivazione di azoto e carenza di glucosio. Abbiamo quindi sottoposto S. cerevisiae a questi stimoli di stress e investigato la formazione di inclusioni insolubili, concentrandoci sugli aggregati ubiquitinati. A questo fine, abbiamo deciso di includere anche il trattamento con puromicina come condizione di stress a cui esporre le cellule di lievito, Protein misfolding and aggregation are major issues in neurodegenerative disorders. Such diseases are characterized by the presence of large inclusions of misfolded proteins which can interfere with the proper proteostasis, possibly leading to loss of neuronal activity. Nevertheless, the formation of protein aggregates is a conserved mechanism engaged also by healthy eukaryotic cells in response to external stimuli that alter their physiological environment. In this context, protein aggregates function as well-organized deposition sites which help the cell to protect its proteome in the waiting for the restoration of the physiological condition. Among the organized cytoplasmic deposition sites for proteins, Dendritic cell Aggresome-Like Induced Structures (DALISs) has recently been identified in dendritic cells. They represent transient deposits for ubiquitinated defective proteins and antigens, which are formed in response to several stresses. Their function is still not completely understood, especially since they were later characterized in non-immune cells as well. No investigation has ever been conducted in other eukaryotic cells, but since they act as deposition sites for ubiquitinated misfolded proteins, we can expect to identify them in other cellular models as well. The yeast Saccharomyces cerevisiae is widely used for the research on cellular aggregates, as it displays a range of different dynamic deposition sites that are demonstrated to be conserved between different species, despite the increased complexity in protein composition and function in multicellular eukaryotes. We identified four main stress conditions under which the yeast Saccharomyces cerevisiae manifests the formation of large cytoplasmic bodies, namely heat shock temperatures at 42°C, severe ethanol stress, nitrogen depletion and glucose starvation. Therefore, we subjected yeast cells to these stress conditions and investigated the formation of insoluble inclusions, with a particular focu

Il ruolo principale delle biotecnologie industriali è quello di fornire soluzioni innovative per alcune delle più grandi sfide del mondo. Nonostante il potenziale e le tecniche innovative applicate, i processi microbiologici bio-based necessitano ancora di ulteriori studi per diventare pervasivi e quindi sostituire i processi di produzione tradizionali. Per rendere i processi microbici economicamente fattibili e rispettosi dell'ambiente, uno dei fattori chiave risiede nella scelta della biomassa di partenza. In una logica di bioeconomia circolare, i sottoprodotti e le biomasse residue devono essere considerati come materie prime di partenza del processo. L'uso di queste biomasse non solleva questioni etiche e allo stesso tempo è economicamente vantaggioso e orientato all'ambiente. La maggior parte di queste biomasse residue sono residui agricoli e forestali, una famiglia di biomasse caratterizzate da una struttura lignocellulosica. Il problema legato al loro utilizzo nelle bioraffinerie a base microbica è quello di trovare un pretrattamento efficiente per convertirli in zuccheri fermentabili e altri nutrienti, riducendo al minimo il rilascio di inibitori della crescita microbica. Parlando di bioraffinerie microbiche, ci sono due aspetti principali da tenere a mente durante la progettazione del processo: la biomassa di partenza e l'ospite microbico. L’host finale può essere scelto seguendo due approcci complementari: i) sfruttare la biodiversità microbica già presente in natura, scegliendo l'ospite finale in base alle sue caratteristiche innate, particolarmente vantaggiose in uno specifico processo produttivo; ii) lavorare su una cell factory già nota, customizzandola secondo le necessità. Nel Capitolo 2 è stata valutata una specifica classe di lieviti non convenzionali, denominata lieviti oleaginosi, per ottenere oli microbici (SCOs) per la produzione di biodiesel a partire da scarti dell'industria della barbabietola da zucchero. Lipomyces starkeyi è stat, The role of industrial biotechnology is to provide game-changing solutions for some of the world’s greatest challenges. From climate change to alternative energy sources and to sustainable productions, industrial biotechnology is fighting to find new sustainable solutions. Despite the promising potential and the innovative techniques applied, bio-based biological processes still need further studies for becoming pervasive and therefore substituting the traditional processes of production. To make microbial processes economically feasible and environmentally friendly, one of the key factors resides in the choice of the starting biomass. In a logic of circular bioeconomy, by-products and residual biomasses have to be considered as starting feedstocks of the process. The use of these biomasses does not raise ethical issues and at the same time is economically advantageous and environment oriented. Indeed, they do not compete with the food industry, as they are usually production waste. Most of these residual biomasses are agricultural and forest residues, a family of biomasses characterised by a lignocellulosic structure. The problem related to their use in microbial-based biorefineries is to find an efficient pretreatment to convert them into fermentable sugars and other nutrients, while reducing to a minimum the release of inhibitors of microbial growth. Talking about microbial-based biorefinery as a substitute to petrol-based refinery, there are two main topics to keep in mind during the process design: the starting biomass and the microbial host. The chassis which will be involved in the final production process can be chosen following two complementary approaches: i) exploiting microbial biodiversity already present in nature by picking the final host depending on its innate characteristics, particularly advantageous in a specific production process; ii) working on a well-known cell factory by customising it as needed. In this thesis both principles were followed.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.

Calcium homeostasis and signaling processes in Saccharomyces cerevisiae, as well as in any eukaryotic organism, depend on various transporters and channels located on both the plasma and intracellular membranes. The activity of these proteins is regulated by a number of feedback mechanisms that act through the calmodulin-calcineurin pathway. When exposed to hypotonic shock (HTS), yeast cells respond with an increased cytosolic calcium transient, which seems to be conditioned by the opening of stretch-activated channels. To better understand the role of each channel and transporter involved in the generation and recovery of the calcium transient—and of their feedback regulations—we defined and analyzed a mathematical model of the calcium signaling response to HTS in yeast cells. The model was validated by comparing the simulation outcomes with calcium concentration variations before and during the HTS response, which were observed experimentally in both wild-type and mutant strains. Our results show that calcium normally enters the cell through the High Affinity Calcium influx System and mechanosensitive channels. The increase of the plasma membrane tension, caused by HTS, boosts the opening probability of mechanosensitive channels. This event causes a sudden calcium pulse that is rapidly dissipated by the activity of the vacuolar transporter Pmc1. According to model simulations, the role of another vacuolar transporter, Vcx1, is instead marginal, unless calcineurin is inhibited or removed. Our results also suggest that the mechanosensitive channels are subject to a calcium-dependent feedback inhibition, possibly involving calmodulin. Noteworthy, the model predictions are in accordance with literature results concerning some aspects of calcium homeostasis and signaling that were not specifically addressed within the model itself, suggesting that it actually depicts all the main cellular components and interactions that constitute the HTS calcium pathway, and thus can

Motivation: Approaches such as chromatin immunoprecipitation followed by sequencing (ChIP-seq) represent the standard for the identification of binding sites of DNA-associated proteins, including transcription factors and histone marks. Public repositories of omics data contain a huge number of experimental ChIP-seq data, but their reuse and integrative analysis across multiple conditions remain a daunting task. Results: We present the Combinatorial and Semantic Analysis of Functional Elements (CombSAFE), an efficient computational method able to integrate and take advantage of the valuable and numerous, but heterogeneous, ChIP-seq data publicly available in big data repositories. Leveraging natural language processing techniques, it integrates omics data samples with semantic annotations from selected biomedical ontologies; then, using hidden Markov models, it identifies combinations of static and dynamic functional elements throughout the genome for the corresponding samples. CombSAFE allows analyzing the whole genome, by clustering patterns of regions with similar functional elements and through enrichment analyses to discover ontological terms significantly associated with them. Moreover, it allows comparing functional states of a specific genomic region to analyze their different behavior throughout the various semantic annotations. Such findings can provide novel insights by identifying unexpected combinations of functional elements in different biological conditions.

Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 30 of mRNA and drive the release of the polypeptide chain.However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

I linfociti infiltranti il tumore (TIL) sono i componenti principali del microambiente tumorale e svolgono un ruolo centrale nell'immunità antitumorale. Durante l'immunoediting del cancro, i TIL diventano disfunzionali, un fenotipo associato alla compromissione delle funzioni effettrici, alla ridotta crescita cellulare e alla ridotta capacità di uccisione. Infatti, i trattamenti di immunoterapie contro il cancro cercano di ripristinare lo stato disfunzionale dei TIL al fine di promuovere la clearance del tumore. Le immunoterapie rappresentano oggigiorno la nuova frontiera nella lotta contro il cancro, ma ancora poco si sa per quanto riguarda i modulatori epigenetici responsabili delle proprietà dei TIL; la scoperta di nuove molecole e meccanismi di azione possibili potrebbe migliorare sostanzialmente le conoscenze relative alle immunoterapie e alla reattività del paziente ad esse. Per questo motivo, stiamo applicando nuovi approcci e tecnologie in questo campo, vale a dire lo studio delle funzioni degli elementi trasponibili (TE) come nuovi attori epigenetici nell'identità, nella plasticità e nell'adattabilità dei TIL all'ambiente tumorale. I TE sono sequenze di DNA ripetute che coprono il 40-45% del genoma umano e diversi studi suggeriscono che i TE esercitano una funzione cruciale nella regolazione epigenetica sia in cis che in trans, essendo una fonte di RNA regolatori non codificanti e partecipando al ripiegamento della cromatina. Tra i TE, siamo interessati alle possibili funzioni epigenetiche degli elementi LINE1, che rappresentano il 18% del genoma umano, considerati come nuove molecole chiave coinvolte nella regolazione epigenetica dell'identità cellulare. Fino ad oggi il ruolo e la dinamica degli RNA derivati dai TEs sono stati studiati solo nelle cellule staminali embrionali o durante lo sviluppo dell'organismo, mentre non c'era evidenza delle loro possibili funzioni in cellule completamente differenziate derivate da tessuti adulti, come i linfocit, Tumor infiltrating lymphocytes (TILs) are the principal components of the tumor microenvironment, and play a central role in antitumor immunity. During cancer immunoediting, TILs became dysfunctional, a phenotype associated to effector functions impairment, reduced cell growth and decreased killing capability. Indeed, cancer immunotherapies treatments try to revert TILs dysfunctional state in order to promote tumor clearance. Immunotherapies nowadays represent the novel frontier in fighting cancer but still little is known regarding the epigenetic modulators responsible for TILs properties; the discovery of novel, possible targetable molecules and mechanisms could substantially improve knowledges regarding immunotherapies and patient responsiveness to them. For this reason, we are applying novel approaches and technologies in this filed, namely the investigation of transposable elements (TEs) functions as novel epigenetic players in TILs identity, plasticity and adaptability to the environmental cancer driven milieu. TEs are interspersed repetitive DNA sequences that cover 40 - 45% of the human genome and growing evidence suggests that TEs exert a crucial function in epigenetic regulation both in cis and in trans, being a source of non-coding regulatory RNAs and participating to chromatin folding. Among TEs, we are interested in the possible epigenetic functions of LINE1 elements, that represents 18% of the human genome, considered as novel key molecules involved in epigenetic regulation of cell identity. Until today the role and the dynamics of TEs-derived RNAs were investigated only in embryonic stem cells or during organism development, while there was no evidence of their possible functions in fully differentiated cells derived from adult tissues, as human Naïve T lymphocytes, that are plastic cells able to adapt and differentiate to diverse effector cells based on the cytokine milieu. We demonstrate that, among T cells subsets, there is a specific enrichment f

Il carcinoma prostatico (PCa) è il secondo tumore maschile più ricorrente, spesso caratterizzato da un esito sfavorevole a causa della sua elevata eterogeneità clinica e molecolare e della sua frequente multifocalità. Sempre più grandi sforzi della ricerca epigenetica si concentrano nel rilevamento di nuovi biomarcatori in grado di migliorare la diagnosi e la prognosi del PCa. Tra i livelli epigenetici, l'architettura nucleare della cromatina rispetta regole precise che garantiscono il corretto funzionamento del genoma e il mantenimento dell'identità cellulare, e il suo rimodellamento è emerso come un nuovo attore cruciale durante la trasformazione maligna. Tuttavia, l'occorrenza e le implicazioni funzionali dei cambiamenti strutturali della cromatina nel cancro rimangono scarsamente caratterizzate. Abbiamo recentemente sviluppato una tecnica basata sul sequenziamento ad alto rendimento, denominata 4fSAMMY-seq (4 fractions Sequential Analysis of MacroMolecules accessibilitY), consistente nel frazionamento sequenziale della cromatina in base al suo stato di accessibilità e solubilità che consente di isolare e caratterizzare sia l'eterocromatina che l'eucromatina. Un vantaggio chiave della nostra tecnologia è che richiede solo poche migliaia di cellule viventi, rendendola adatta per analisi su materiali limitati come campioni bioptici derivati dalla pratica clinica. In questo lavoro, abbiamo applicato 4fSAMMY-seq su campioni bioptici freschi di pazienti sottoposti a mappatura esplorativa della prostata per la diagnosi di cancro alla prostata. Abbiamo esaminato la conformazione del genoma e la sua associazione con il profilo trascrizionale nelle biopsie di controllo prive di tumore e in quelle del cancro alla prostata. Nei tessuti sani “bulk”, contenenti un mix di cellule epiteliali, leucociti e stroma, 4fSAMMY-seq ha consentito l’identificazione di un pattern conservato di regioni di eucromatina ed eterocromatina associate alle rispettive signatures epigenet, Prostate cancer (PCa) is the second most recurrent male tumor, often characterized by an unfavorable outcome due to its high clinical and molecular heterogeneity and its frequent multifocality. Increasingly growing epigenetic research efforts are implicated in the detection of novel biomarkers improving PCa diagnosis and prognosis. Among the epigenetic layers, the chromatin nuclear architecture abides tight rules that ensure proper genome functions and cell identity maintenance, and its remodeling has emerged as a novel crucial player during malignant transformation. Yet, the occurrence and the functional implications of chromatin structural changes in cancer remains poorly characterized. We have recently developed a high-throughput sequencing based technique, named 4fSAMMY-seq (4 fractions Sequential Analysis of MacroMolecules accessibilitY), consisting of the sequential fractionation of the chromatin according to its accessibility and solubility status to isolate and characterize both heterochromatin and euchromatin. A key advantage of our method is that it only requires few thousands of living cells, making it suitable for analyses on limited materials as biopsy samples derived from clinical practice. In this study, we applied 4fSAMMY-seq on fresh biopsy specimens from patients undergoing explorative prostate mapping biopsy for prostate cancer diagnosis. We examined the genome conformation and its association with transcriptional profile in control tumor-free biopsies and prostate cancer biopsies. In bulk healthy tissues, containing a mix of epithelial cells, leukocytes and stroma, 4fSAMMY-seq allowed the detection of a conserved pattern of euchromatin and heterochromatin regions associated with the respective epigenetic signatures. We also found a quantitative correlation between chromatin solubility and gene expression. On the other hand, cancer biopsies are characterized by different degrees of genome architectural and transcriptome alterations, reflecting the

Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of mechanisms-revealing data, but interpretative analysis tools specific for the testing systems (e.g. hepatocytes) are lacking. In this study, we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/), an R-Shiny-based implementation of weighted gene co-expression network analysis (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. The 398 gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, were perturbed by specific stressors and showed preservation in rat systems (rat primary hepatocytes and rat in vivo liver), with the exception of DNA damage and oxidative stress responses. A subset of 87 well-annotated and preserved modules was used to evaluate mechanisms of toxicity of endoplasmic reticulum (ER) stress and oxidative stress inducers, including cyclosporine A, tunicamycin and acetaminophen. In addition, module responses can be calculated from external datasets obtained with different hepatocyte cells and platforms, including targeted RNA-seq data, therefore, imputing biological responses from a limited gene set. As another application, donors’ sensitivity towards tunicamycin was investigated with the TXG-MAPr, identifying higher basal level of intrinsic immune response in donors with pre-existing liver pathology. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data.

eTRANSAFE is a research project funded within the Innovative Medicines Initiative (IMI), which aims at developing integrated databases and computational tools (the eTRANSAFE ToxHub) that support the translational safety assessment of new drugs by using legacy data provided by the pharmaceutical companies that participate in the project. The project objectives include the development of databases containing preclinical and clinical data, computational systems for translational analysis including tools for data query, analysis and visualization, as well as computational models to explain and predict drug safety events.

Ras oncoproteins play a crucial role in the onset, maintenance, and progression of the most common and deadly human cancers. Despite extensive research efforts, only a few mutant-specific Ras inhibitors have been reported. We show that cmp4-previously identified as a water-soluble Ras inhibitor- targets multiple steps in the activation and downstream signaling of different Ras mutants and isoforms. Binding of this pan-Ras inhibitor to an extended Switch II pocket on HRas and KRas proteins induces a conformational change that down-regulates intrinsic and GEF-mediated nucleotide dissociation and exchange and effector binding. A mathematical model of the Ras activation cycle predicts that the inhibitor severely reduces the proliferation of different Ras-driven cancer cells, effectively cooperating with Cetuximab to reduce proliferation even of Cetuximab-resistant cancer cell lines. Experimental data confirm the model prediction, indicating that the pan-Ras inhibitor is an appropriate candidate for medicinal chemistry efforts tailored at improving its currently unsatisfactory affinity.

The field of transcriptional regulation generally assumes that changes in transcripts levels reflect changes in transcriptional status of the corresponding gene. While this assumption might hold true for a large population of transcripts, a considerable and still unrecognized fraction of the variation might involve other steps of the RNA lifecycle, that is the processing of the premature RNA, and degradation of the mature RNA. Discrimination between these layers requires complementary experimental techniques, such as RNA metabolic labeling or block of transcription experiments. Nonetheless, the analysis of the premature and mature RNA, derived from intronic and exonic read counts in RNA-seq data, allows distinguishing between transcriptionally and post-transcriptionally regulated genes, although not recognizing the specific step involved in the post-transcriptional response, that is processing, degradation, or a combination of the two. We illustrate how the INSPEcT R/Bioconductor package could be used to infer post-transcriptional regulation in TCGA RNA-seq samples for Hepatocellular Carcinoma.

Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization remains unknown. To decouple these two molecular processes, we used cytosolic long noncoding RNAs (lncRNAs) as models for endogenous transcripts that are not translated. We show that, despite interacting with the miRNA-loaded RNA-induced silencing complex, the steady-state abundance and decay rates of these transcripts are minimally affected by miRNA loss. To further validate the apparent requirement of translation for miRNA-dependent decay, we fused two lncRNA candidates to the 3’-end of a protein-coding gene reporter and found this results in their miRNA-dependent destabilization. Further analysis revealed that the few natural lncRNAs whose levels are regulated by miRNAs in mESCs tend to associate with translating ribosomes, and possibly represent misannotated micropeptides, further substantiating the necessity of target translation for miRNA-dependent transcript decay. In summary, our analyses suggest that translation is required for miRNA-dependent transcript destabilization, and demonstrate that the levels of coding and noncoding transcripts are differently affected by miRNAs.

Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene's RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene's ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

The covalent modification of RNA molecules is a pervasive feature of all classes of RNAs and has fundamental roles in the regulation of several cellular processes. Mapping the location of RNA modifications transcriptome-wide is key to unveiling their role and dynamic behaviour, but technical limitations have often hampered these efforts. Nanopore direct RNA sequencing is a third-generation sequencing technology that allows the sequencing of native RNA molecules, thus providing a direct way to detect modifications at single-molecule resolution. Despite recent advances, the analysis of nanopore sequencing data for RNA modification detection is still a complex task that presents many challenges. Many works have addressed this task using different approaches, resulting in a large number of tools with different features and performances. Here we review the diverse approaches proposed so far and outline the principles underlying currently available algorithms.