GENERATION OF AUTHENTIC HUMAN NEOCORTICAL NEURONS FROM INDUCED PLURIPOTENT STEM CELLS TO INVESTIGATE 7Q11.23 GENE DOSAGE IMBALANCES

Questo lavoro di tesi ha avuto lo scopo di studiare lo sviluppo della neocorteccia umana ed i meccanisimi alla base della sua compromissione che risultano nell\u2019insorgenza di patologie del neurosviluppo mediante un\u2019analisi dei profili trascrizionali e della morfologia di neuroni neocorticali umani generati a partire da cellule staminali pluripotenti indotte (iPSCs). Data l\u2019importanza di basarsi su un paradigma di neurogenesi in vitro riproducibile e affidabile nel generare neuroni neocoritcali umani autentici, prima di adottare questo sistema modello per lo studio di patologie del neurosviluppo, nella prima fase di questa ricerca abbiamo eseguito un\u2019ampia caratterizzazione trascrizionale, molecolare e funzionale del protocollo di differenziamento. Le dinamiche trascrizionali che regolano il neurosviluppo in vitro sono state studiate effettuando esperimenti di RNA-sequencing sia a livello di popolazione che di singola cellula. In combinazione con diverse analisi bioinformatiche tra cui l\u2019analisi delle componeti principali (PCA), l\u2019analisi dei geni differenzialemtne espressi e l\u2019analisi WGCNA. L\u2019analisi dei profili trascrizionali \ue8 stata accompagnata da un\u2019ampia analisi di d\u2019immunocitochimica che ha permesso di confermare l\u2019identit\ue0 e lo stadio di sviluppo delle cellule in coltura. Inoltre, la maturit\ue0 funzionale dei neuroni derivati da iPSCs \ue8 stata ulteriormente confermata dalla loro capacit\ue0 di generare potenziali d\u2019azione, sostenere pattern di scarica complessi e sviluppare attivit\ue0 sinaptica spontanea eccitatoria ed inibitoria. Complessivamente, i risultati ottenuti da questo ampio e diversificato pannello di analisi hanno permesso di stabilitre la riproducibilit\ue0 del protocollo di differenziamento e la sua competenza nel generare con elevata efficienza principalmente neuroni neocorticali autentici. Successivamente abbiamo applicato questo protocollo di differenziamento neocorticale come sistema modello per studiare due patologie del neurosviluppo dovute alla delezione e duplicazione di una regione comprendente circa 1.5 - 1.8 Mb (megabasi) collacata sul braccio lungo (q) del cormosoma 7 nella banda 11.23. Duplicazioni e delezioni di questa regione sono di particolare interesse in quanto le due sindromi che ne risultano, rispettivamente la sindrome di Willams (WS) e la sindrome da duplicazione 7q11.23 (7q11DUP), presentano fenotipi cognitivi e comportamentali caratterizzati da profili simili e tratti simmetricamente opposti. La frequente comorbidit\ue0 della sindrome da duplicazione 7q11.23 con altre patologie del neurosviluppo come l\u2019autismo e la schizofrenia in contrasto con la sindrome di Williams che \ue8 una sindrome ben caraterizzata non associata ad altre patologie del neurosviluppo, rende lo studio dell\u2019 alterato dosaggio genico del locus 7q11.23 estremamente interessante per identificare con precisione i meccanismi molecolari caratteristici di ciascuna condizione clinica, condivisi da entrabme le sindromi e comuni anche ad altre patologie del neurosviluppo. A questo scopo, abbiamo generato diverse linee di iPS a partire da un ampio gruppo di individui, comprendente individui sani e pazienti affetti dalla sindrome di Williams (WS) e dalla sindrome di duplizazione 7q11.23, che sono poi state differenziate in neuroni neocorticali applicando il protocollo precedentemetne caraterizzato. Confermata l\u2019identit\ue0 e l\u2019autenticit\ue0 dei neuroni neocorticali generati da iPSCs, stiamo attualmente identificando i geni ed i meccanismi molecolari disregolati in specifici sottotipi di neuroni che abbiano la maggior rilvenza clinica. Inoltre, l\u2019analisi morfologica dei neuroni neocorticali umani ottenuti da pazienti WS e soggetti sani ha permesso di confermare nell\u2019uomo molte alterazioni morfologiche dei neuroni neocorticali osservate in un modello murino knockout per Dnajc30, un gene ancora funzionalmente non caraterizzato compreso nel locus 7q11.23.This research project has been aimed to investigate human neocortical development in healthy and diseased subjects by analyzing and comparing the transcriptional profiles and cellular morphologies of human neocortical cells derived from induced pluripotent stem cells (iPSCs). Given the importance to rely on a solid and highly reproducible iPSCs-based differentiation protocol that generates authentic neocortical neurons in vitro with high efficiency before applying it as a model system of human neurodevelopmental disorders, in the first phase of this study we performed a comprehensive transcriptional, cellular and physiological characterization of the in vitro neurodevelopmental paradigm. The transcriptional dynamics regulating in vitro neocortical development have been investigated by performing RNA-sequencing (RNA-seq) at both population and single- cell level in combination with several bioinformatics analyses including principal component analysis (PCA), differential gene expression analysis and weighted gene co-expression network analysis (WGCNA). The transcriptional results were corroborated by the widespread positivity for a selected panel of informative cell-fate and cell-stage specific markers detected through immunocytochemistry and the physiological maturity of our iPSCs-derived neocortical neurons was further confirmed by their ability to generate action potentials, develop complex firing patterns and sustain excitatory and inhibitory spontaneous synaptic activity. Overall, these results fully validated the reproducibility of the differentiation protocol and its efficiency and reliability in generating physiologically mature authentic neocortical neurons. Subsequently, we applied this extensively characterized neocortical differentiation paradigm to model in vitro two human neurodevelopmental disorders caused by symmetrical copy number variations (CNVs) of the Williams-Beuren syndrome chromosome region (WBSCR) located on the long arm (q) of chromosome 7 at position 11.23 (7q.11.23 locus). 7q11.23 CNVs are of special interest as the two disorders resulting from the deletion (Williams syndrome, WS) and duplication (7q.11.23 duplication syndrome, 7q11DUP) of this region exhibit cognitive and behavioral phenotypes marked by both similar features and symmetrically opposite traits. The association of 7q11DUP to complex neurodevelopmental disorders such as autism spectrum disorder and schizophrenia, while WS is a well-characterized syndrome without clear overlap to complex neurodevelopmental disorders make the study of this locus extremely interesting to identify the molecular mechanisms unique to each clinical condition, common to both syndromes and shared with other complex neurodevelopmental disorders. To this aim, we generated several iPSCs lines from a large cohort comprising WS individuals, 7q11DUP patients and healthy subjects and differentiated them into neocortical neurons by applying the previously in-depth characterized protocol. Having assessed the quality of our iPSCs-derived neocortical neurons, we are currently identifying neuronal subtypes specific genes and gene networks having the most statistically significant relationship to these disorders through single cell RNA-sequencing analysis. Furthermore, morphometric analysis of WS and control iPSCs-derived neocortical neurons has confirmed in humans many neuronal morphological abnormalities observed in a mouse knockout for Dnajc30, a previously uncharacterized gene contained in the 7q11.23 locus

Long walk to genomics : history and current approaches to genome sequencing and assembly

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes

Introduction to M Theory and AdS/CFT Duality

An introductory survey of some of the developments that have taken place in superstring theory in the past few years is presented. The main focus is on three particular dualities. The first one is the appearance of an 11th dimension in the strong coupling limit of the type IIA theory, which give rise to M theory. The second one is the duality between the type IIB theory compactified on a circle and M theory on a two-torus. The final topic is an introduction to the recently proposed duality between superstring theory or M theory on certain anti de Sitter space backgrounds and conformally invariant quantum field theories.Comment: 26 pages; To be published in the Proceedings of a conference held in Corfu, Greece in September 1998. v2: reference adde

Prostaglandin E2 stimulates the expansion of regulatory hematopoietic stem and progenitor cells in type 1 diabetes

Hematopoietic stem and progenitor cells (HSPCs) are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D) and remains the only "experimental therapy" that has achieved a satisfactory rate of remission (nearly 60%) in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes

Measurement of the azimuthal anisotropy of Y(1S) and Y(2S) mesons in PbPb collisions at √S^{S}NN = 5.02 TeV

The second-order Fourier coefficients (υ$_{2}$) characterizing the azimuthal distributions of Υ(1S) and Υ(2S) mesons produced in PbPb collisions at $\sqrt{s_{NN}}$ = 5.02 TeV are studied. The Υmesons are reconstructed in their dimuon decay channel, as measured by the CMS detector. The collected data set corresponds to an integrated luminosity of 1.7 nb$^{-1}$. The scalar product method is used to extract the υ$_{2}$ coefficients of the azimuthal distributions. Results are reported for the rapidity range |y| < 2.4, in the transverse momentum interval 0 < p$_{T}$ < 50 GeV/c, and in three centrality ranges of 10–30%, 30–50% and 50–90%. In contrast to the J/ψ mesons, the measured υ$_{2}$ values for the Υ mesons are found to be consistent with zero

Performance of the CMS Level-1 trigger in proton-proton collisions at √s = 13 TeV

At the start of Run 2 in 2015, the LHC delivered proton-proton collisions at a center-of-mass energy of 13\TeV. During Run 2 (years 2015–2018) the LHC eventually reached a luminosity of 2.1× 10$^{34}$ cm$^{-2}$s$^{-1}$, almost three times that reached during Run 1 (2009–2013) and a factor of two larger than the LHC design value, leading to events with up to a mean of about 50 simultaneous inelastic proton-proton collisions per bunch crossing (pileup). The CMS Level-1 trigger was upgraded prior to 2016 to improve the selection of physics events in the challenging conditions posed by the second run of the LHC. This paper describes the performance of the CMS Level-1 trigger upgrade during the data taking period of 2016–2018. The upgraded trigger implements pattern recognition and boosted decision tree regression techniques for muon reconstruction, includes pileup subtraction for jets and energy sums, and incorporates pileup-dependent isolation requirements for electrons and tau leptons. In addition, the new trigger calculates high-level quantities such as the invariant mass of pairs of reconstructed particles. The upgrade reduces the trigger rate from background processes and improves the trigger efficiency for a wide variety of physics signals

Measurement of the Y(1S) pair production cross section and search for resonances decaying to Y(1S)μ⁺μ⁻ in proton-proton collisions at √s = 13 TeV

The fiducial cross section for Y(1S)pair production in proton-proton collisions at a center-of-mass energy of 13TeVin the region where both Y(1S)mesons have an absolute rapidity below 2.0 is measured to be 79 ± 11 (stat) ±6 (syst) ±3 (B)pbassuming the mesons are produced unpolarized. The last uncertainty corresponds to the uncertainty in the Y(1S)meson dimuon branching fraction. The measurement is performed in the final state with four muons using proton-proton collision data collected in 2016 by the CMS experiment at the LHC, corresponding to an integrated luminosity of 35.9fb$^{-1}$. This process serves as a standard model reference in a search for narrow resonances decaying to Y(1S)μ$^{+}$μ$^{-}$ in the same final state. Such a resonance could indicate the existence of a tetraquark that is a bound state of two bquarks and two b̅ antiquarks. The tetraquark search is performed for masses in the vicinity of four times the bottom quark mass, between 17.5 and 19.5GeV, while a generic search for other resonances is performed for masses between 16.5 and 27GeV. No significant excess of events compatible with a narrow resonance is observed in the data. Limits on the production cross section times branching fraction to four muons via an intermediate Y(1S)resonance are set as a function of the resonance mass

Pileup mitigation at CMS in 13 TeV data

With increasing instantaneous luminosity at the LHC come additional reconstruction challenges. At high luminosity, many collisions occur simultaneously within one proton-proton bunch crossing. The isolation of an interesting collision from the additional "pileup" collisions is needed for effective physics performance. In the CMS Collaboration, several techniques capable of mitigating the impact of these pileup collisions have been developed. Such methods include charged-hadron subtraction, pileup jet identification, isospin-based neutral particle "δβ" correction, and, most recently, pileup per particle identification. This paper surveys the performance of these techniques for jet and missing transverse momentum reconstruction, as well as muon isolation. The analysis makes use of data corresponding to 35.9 fb$^{-1}$ collected with the CMS experiment in 2016 at a center-of-mass energy of 13 TeV. The performance of each algorithm is discussed for up to 70 simultaneous collisions per bunch crossing. Significant improvements are found in the identification of pileup jets, the jet energy, mass, and angular resolution, missing transverse momentum resolution, and muon isolation when using pileup per particle identification

Identification of heavy, energetic, hadronically decaying particles using machine-learning techniques

Machine-learning (ML) techniques are explored to identify and classify hadronic decays of highly Lorentz-boosted W/Z/Higgs bosons and top quarks. Techniques without ML have also been evaluated and are included for comparison. The identification performances of a variety of algorithms are characterized in simulated events and directly compared with data. The algorithms are validated using proton-proton collision data at √s = 13TeV, corresponding to an integrated luminosity of 35.9 fb−1. Systematic uncertainties are assessed by comparing the results obtained using simulation and collision data. The new techniques studied in this paper provide significant performance improvements over non-ML techniques, reducing the background rate by up to an order of magnitude at the same signal efficiency

Observation of electroweak production of Wγ with two jets in proton-proton collisions at √s = 13 TeV

A first observation is presented for the electroweak production of a W boson, a photon, and two jets in proton-proton collisions. The W boson decays are selected by requiring one identified electron or muon and an imbalance in transverse momentum. The two jets are required to have a high dijet mass and a large separation in pseudorapidity. The measurement is based on data collected with the CMS detector at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb$^{-1}$. The observed (expected) significance for this process is 4.9 (4.6) standard deviations. After combining with previously reported CMS results at 8 TeV, the observed (expected) significance is 5.3 (4.8) standard deviations. The cross section for the electroweak W$_{γjj}$ production in a restricted fiducial region is measured as 20.4 +/- 4.5 fb and the total cross section for W$_{γ}$ production in association with 2 jets in the same fiducial region is 108 +/- 16 fb. All results are in good agreement with recent theoretical predictions. Constraints are placed on anomalous quartic gauge couplings in terms of dimension-8 effective field theory operators