DNA fragility in the context of neural stem cell fate : a multi-method integrative exploration of genome dynamics

Recent advances in mapping the complex genetic architecture underlying various debilitating brain disorders have enabled identification of several genetic risk variants. However, these risk variants only explain part of the heritability and vulnerability to these disorders in early development. Moreover, de novo somatic mutations have been detected in subsets of brain cells, which might account for a significant portion of the missing heritability. However, it remains unclear where these mutations come from and at what developmental stage they might occur. Genome fragility is subject to the functional activity and spatial chromatin organization characteristic of a distinct cell identity. Under physiological conditions, cells regulate their chromatin structure and organization to express necessary genes. DNA topoisomerases are a key player in all of these processes and in replication. Through generation of transient breaks in the DNA, topoisomerases are able to resolve topological problems and thereby activation of particular sections of the genome. Beyond topoisomerases, the genome is subject to perpetual challenges with DNA double-strand breaks (DSBs) being among the most deleterious. Each cell is estimated to suffer numerous transient DSBs per day, most of which are repaired. Incorrectly repaired DSBs however, pose a major threat to genome stability through formation of mutations or potential genomic rearrangements. Although the exact relationship of DNA damage to differentiation is still unclear, a recent investigation into neural specification demonstrated that loss of DNA repair sensors leads to centrosome amplification, thereby resulting in defective mitosis and chromosomal instability. Ensuing excessive stem cell proliferation and replication stress also happen to be a hallmark of neurodevelopmental disorders (NDDs). Despite the emerging evidence linking endogenous DSBs to NDDs, there has been a lack of genome-wide maps of DSBs spontaneously arising at different stages of human neurogenesis. This thesis brings together (I) a correlative genomics study describing endogenous DSBs genome-wide during neural differentiation in a cell-type specific manner, and (II) a mechanistic study into the regulatory role of Topoisomerase 1 (TOP1) in transcription and proliferation. In paper I, we mapped the genomic DSB landscape of cells at various stages of neural differentiation and correlated our maps with genomic and epigenomic features. In so doing, we provide clues on how DSB formation and their incorrect repair might contribute to the pathogenesis of NDDs. The current view is that transcription-associated DSBs seem to be the main driver of de novo mutations. Indeed, we found that DSBs preferentially form around the transcription start site (TSS) of transcriptionally active genes, as well as at chromatin loop anchors in proximity of highly transcribed genes. This follows from the accumulation of DNA torsional stress and topoisomerase activity in these regions. Interestingly, hotspots of endogenous DSBs were detected around the TSS of highly transcribed genes involved in general cellular processes and along the gene body of long, neural-specific genes whose human orthologues had been previously implicated in NDDs. Through our integrative multimethod approach we corroborate previous findings regarding DSB-fragile loci at TSSs and loop anchors, and find a unique distribution pattern for this fragility in post-mitotic neurons. We show a cell type-specific preference for DSB accumulation in specific NDD genes and begin to describe the relation of DSB fragility and chromatin conformation. In paper II, we investigated the role of Topoisomerase I (TOP1) in relation to transcription in the context of replication stress across mitosis and as subject of interruption of interphase chromatin conformation. In particular, we investigated different stages of the cell cycle for transcription patterns and transcriptional spiking by RNA polymerase II (RNAPII) in human colon carcinoma cells. TOP1 relieves torsional stress in actively transcribed DNA and facilitates the expression of long genes, many of which are important for neural functions. However, TOP1 also plays a direct role in transcriptional control through interaction with RNAPII Carboxy-Terminal Domain (CTD). We investigated control cells and a knock-in (KI) clone lacking TOP1 exon4, the phosphor-CTD-binding site for RNAPII. We found that in early mitosis TOP1 clears RNAPII during transcriptional elongation. When the TOP1 CTD-binding domain is disrupted, we detected replication stress and delay in mitotic exit. In this case, chromatin becomes topologically stressed, increasing the need for TOP2A cleavage resulting in DSBs. However, we did not detect substantial changes in DSB markers gamma- H2AX and 53BP1 when comparing WT and KI cells across different stages of the cell cycle. Therefore, we conclude that the observed delay in mitotic exit is most likely due to the deregulation of gene expression, rather than to the activation of DNA repair pathways. Acute depletion of TOP1 through the auxin-degron system resulted in absence of RNAPII spiking at the TSS. Efficient removal of RNAPII from chromosomes by TOP1 in early mitosis is both a prerequisite for the timely spike of RNAPII at TSSs in mid mitosis and might affect cellular memory. Indeed, we found that when mitotic transcription is poorly regulated, individual proliferating cells have a greater variance in transcriptional levels and thus could lead to loss of cell identity. Concluding from these findings, we demonstrate that endogenous DSBs are distributed differentially in a cell type-specific manner. Through our integrative multi-method approach, we corroborate previous findings regarding DSB-fragile loci and discovered a unique distribution pattern for DSBs in post-mitotic neurons. We show a preference for specific NDDs genes and begin to describe the relation of DSB fragility and chromatin conformation in a developmental context. We assessed the role of TOP1 in a model for replication stress and found that outside of its canonical torsional stress function, the direct interaction with RNAPII across the cell cycle is crucial in maintaining transcriptional memory and could feed into loss of cell identity. While not exhaustive, the findings described in these papers begin to elucidate a complex mystery of human NDDs and provide valuable datasets for further investigation of genome fragility. Taken together, these findings contribute to a better understanding of how neural genome dynamics affect high transcriptional or replicative burden during neurodevelopment

Internal migration, family formation and social stratification in Europe. A life course approach.

Le traiettorie di mobilità geografica generalmente intersecano altri percorsi di corsi di vita come l’istruzione, la carriera lavorativa e le scelte familiari. L’approccio del corso di vita pone l’attenzione sul fatto che eventi diversi non sono esperienze separate ma collegate tra loro. Concentrandosi su 11 paesi europei (Austria, Repubblica Ceca, Danimarca, Francia, Germania, Grecia, Italia, Polonia, Spagna, Svezia e Svizzera), questa analisi ha tre obiettivi principali: descrivere i modelli di vita sperimentati dai migranti interni, analizzare il processo di selezionesulle diverse traiettorie di migrazioni e identificare l’associazione con specifici risultati lavorativi e con le tratiettorie di mobilità sociale. Per raggiungere questi obiettivi, abbiamo applicato una Sequence and Cluster Analysis ai dati SHARELIFE (2008-09 e 2017). I risultati preliminari rivelano che i diversi percorsi migratori risultano caratterizzati da una marcata selettività (basata principalmente sull’istruzione), particolarmente nei contesti dove la migrazione funziona bene come strategia di ascesa sociale. Inoltre, alcune specifiche traiettorie di vita risultano associate a migliori opportunità in campo lavorativo.Geographical mobility trajectories generally intersects other life course patterns such as student career, job experiences and family choices. Life course approach emphasizes that different events are not separate experiences, but are linked to each other. Focusing on 11 European countries (Austria, Czech Republic, Denmark, France, Germany, Greece, Italy, Poland, Spain, Sweden, and Switzerland), this analysis has three main aims: describing the life patterns experienced by internal migrants, analyzing the selection into different migration trajectories, and identifying the association with different occupational achievements and social mobility pathways. To achieve these goals, we applied Sequence and Cluster Analysis to SHARELIFE data (2008-09 and 2017). Preliminary results reveal that different migration patterns are characterized by a marked selectivity of movers (mainly based on education), particularly in contexts where migration works well as an escalator strategy. In addition, specific life trajectories are associated to better occupational returns

RI-MACS : an innovative approach for future automation systems

he European funded Radically Innovative Mechatronics and Advanced Control Systems (RI-MACS) research project was initiated to address some of the challenges in the future automation systems. Its mission is to bring the innovation offered by the Information Technology (IT) domain into the production life cycle, improving some of the existing inflexibilities in typical manufacturing automation plants. In this paper, some of the key aspects of automation systems engineering that require changes to ensure future competitiveness are highlighted. An overview of current approaches to automation are reviewed in relation to wired and wireless networking, automation architectures, and design environments

Cáncer de Mama: Correlación de Hallazgos entre RNM y pieza Operatoria de Mastectomía

1 p.Del analisis global surge una alta tasa de sospecha por RMN (29/30: 96,6%), con una manifiesta incidencia de lesiones multifocales y multicéntricas (55%). Sin embargo, al anlizar la correlación entre los focos de carcinoma detectados por la AP y la información de la RNM, observamos un incremento de la discordancia directamente proporcional al número de focos. La discordancia global entre RNM y AP fue del 32,6% con el 14,5% de falsos positivos y el 18,1% de falsos negativos.Fil: Valfré, Roberto. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Cátedra de Informática Médica; Argentina.Fil: Bosco, Shirley. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital Nacional de Clínicas. Cátedra de Anatomía Normal; Argentina.Fil: Del Castiilo, Andrés. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. II Cátedra de Clínica Ginecológica; Argentina.Fil: Del Castillo, Andrés. Provincia de Córdoba. Hospital Provincial Rawson. Cátedra de Ginecología y Mastología; Argentina.Fil: Del Castillo, Andrés. Instituto Modelo de Ginecología y Obstetricia (IMGO); Argentina.Fil: Del Castillo, Soledad: Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital Universitario de Maternidad y Neonatología; Argentina.Fil: Del Castillo, Soledad: Provincia de Córdoba. Hospital Provincial Rawson. Cátedra de Ginecología; Argentina.Fil: Del Castillo, Soledad. Instituto Modelo de Ginecología y Obstetricia (IMGO); Argentina.Fil: Garello, Néstor César. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital Nacional Maternidad y Neonatología. II Cátedra de Clínica Ginecológica; ArgentinaFil: Martelloto, Gladys. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital Nacional de Clínicas. Cátedra de Anatomía Patológica; Argentina.Fil: Barujel, Gisela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Escuela de Graduados. Cátedra de Patología Mamaria. Argentina.Fil: Ballarino, Lucrecia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. II Cátedra de Ginecología; Argentina.Fil: Del Castillo, René. Universidad Nacional de La Rioja. Departamento Académico de Ciencias de la Salud y de la Educación. Cátedra de Clínica Ginecológica; Argentina.Fil: Del Castillo, René. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas. Hospital Nacional de Clínicas. II Cátedra de Clínica Ginecológica: Argentina.Fil: Del Castillo, René. Instituto Modelo de Ginecología y Obstetricia (IMGO); Argentina.Obstetricia y Ginecologí

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile

Clinical characteristics, management and in-hospital mortality of patients with COVID-19 In Genoa, Italy

To describe clinical characteristics, management and outcome of COVID-19 patients; and to evaluate risk factors for all-cause in-hospital mortality

Impatto a breve termine dell'inquinamento dell'aria nelle citt\ue0 coperte dalla sorveglianza epidemiologica EpiAir2

OBIETTIVO: stimare l\u2019impatto a breve termine dell\u2019inquinamento atmosferico sulla popolazione adulta di 23 citt\ue0 italiane nel periodo 2006-2009 nell\u2019ambito del progetto EpiAir2. DISEGNO, MATERIALI E METODI: per ogni citt\ue0 inclusa nello studio \ue8 stato calcolato l\u2019impatto dell\u2019effetto a breve termine dell\u2019inquinamento atmosferico sulla mortalit\ue0. In particolare, sono stati calcolati i decessi attribuibili a concentrazioni delle polveri (PM10 e PM2.5) superiori a soglie differenti definite dalla legislazione europea o nell\u2019ambito delle linee guida dell\u2019Organizzazione mondiale della sanit\ue0 (per il PM10: 20 e 40 \u3bcg/m3, riduzione del 20% ad arrivare a 20 \u3bcg/m3 e superamento del limite di 35 giorni con concentrazioni medie di 50 \u3bcg/m3; per il PM2.5: 10, 18 e 25 \u3bcg/m3, riduzione del 20% ad arrivare a 18 \u3bcg/m3). La stima di impatto \ue8 stata ottenuta combinando la stima di effetto delle polveri, il livello di mortalit\ue0 osservato e i livelli di concentrazione degli inquinanti misurati dalle reti di monitoraggio urbane. Per quanto riguarda le stime di effetto, sono state utilizzate le distribuzioni a posteriori specifiche per citt\ue0 risultanti da una metanalisi bayesiana. L\u2019incertezza sulle stime di impatto \ue8 stata calcolata con metodi Monte Carlo. RISULTATI: nell\u2019insieme delle 23 citt\ue0 valutate nel presente studio il numero di decessi attribuibili agli effetti a breve termine delle concentrazioni di PM10 superiori a 20 \u3bcg/m3 e di PM2.5 superiori a 10 \u3bcg/m3 nel periodo 2006-2009 \ue8 risultato rispettivamente pari allo 0,9% (assumendo indipendenza tra citt\ue0 l\u2019intervallo di credibilit\ue0 all\u201980% \ue8 0,4-1,4) e allo 0,8% (ICr80% 0,2-1,3) della mortalit\ue0 naturale. L\u2019impatto delle concentrazioni di polveri PM10 e PM2.5 \ue8 risultato concentrato nelle citt\ue0 della Pianura Padana, della Piana fiorentina, e nelle grandi realt\ue0 metropolitane di Roma, Napoli e Palermo: per il PM10 la percentuale sui decessi \ue8 risultata 1,0% (ICr80% 0,4-1,5) contro 0,4% (ICr80% 0,2-0,7) nelle altre citt\ue0 analizzate. Se i livelli di concentrazione delle polveri fossero stati inferiori del 20%, complessivamente l\u2019impatto si sarebbe ridotto del 42% per il PM10 e del 51% per il PM2.5. CONCLUSIONI: i livelli di inquinamento osservati nel periodo in studio sono stati responsabili di un numero importante di decessi nelle citt\ue0 analizzate. Politiche di contenimento basate sulla diminuzione percentuale delle concentrazioni annuali di polveri interesserebbero tutte le citt\ue0 coperte dallo studio e potrebbero ridurre in modo importante l\u2019impatto dell\u2019inquinamento sulla salute