Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: from high-content screens to in vivo assays

The CRISPR-Cas9 system has revolutionized genome engineering, allowing precise modification of DNA in various organisms. The most popular method for conducting CRISPR-based functional screens involves the use of pooled lentiviral libraries in selection screens coupled with next-generation sequencing. Screens employing genome-scale pooled small guide RNA (sgRNA) libraries are demanding, particularly when complex assays are used. Furthermore, pooled libraries are not suitable for microscopy-based high-content screens or for systematic interrogation of protein function. To overcome these limitations and exploit CRISPR-based technologies to comprehensively investigate epigenetic mechanisms, we have generated a focused sgRNA library targeting 450 epigenetic regulators with multiple sgRNAs in human cells. The lentiviral library is available both in an arrayed and pooled format and allows temporally-controlled induction of gene knock-out. Characterization of the library showed high editing activity of most sgRNAs and efficient knock-out at the protein level in polyclonal populations. The sgRNA library can be used for both selection and high-content screens, as well as for targeted investigation of selected proteins without requiring isolation of knock-out clones. Using a variety of functional assays we show that the library is suitable for both in vitro and in vivo applications, representing a unique resource to study epigenetic mechanisms in physiological and pathological conditions

Innovative Observers for Induction Motor Control

In this thesis the problem of designing observers and controllers for induction motors has been studied and thoroughly discussed using tools from the nonlinear system theory, both for analysis and control purposes. Useful techniques have been used to identify the machine parameters off-line. In the Introduction was dealt the design a control system for a drive of asynchronous machines that uses a voltage source inverter to generate the currents and voltages which carry the drive by making use of an observer of the state rotor variables. The used control algorithms is the \textit{field oriented control} (FOC). The model of the asynchronous induction motor is described starting from the mechanical model, the electrical model through the Steinmetz equivalent electrical circuit that is the IEEE recommended equivalent circuit, the State Model of Asynchronous Motor and the mathematical model, completing with the discretization of the last model, realizing the Discrete time mathematical model. In Chapter 1 several observer are taken in account in order to be able to compare their strengths and weaknesses. At first it's developed a (FOLO) Full Order Luemberger Observer and its Reduced Order version. Other observer that was considered it's the Sliding one. Then a version of Non linear Flux observer was synthesized in order to consider the effects of saturation which introduce a nonlinear effects. At last the (EKF) Extended Kalman Filter was considered, both in complex (ECKF) and the (RAKF) Robust Adaptive Kalman Filter version. In Chapter 2 have been presented experimental and simulation results obtained by testing each of the previous algorithms. Excellent results were obtained by use of observer based on Kalman Filter, and in particular, the Extended Complex Kalman Filter, because no matrix inversion is required. In fact the operation of matrix inversion that is necessary in the classic Extended Kalman Filter is therefore translated in the inverse of a real number in the proposed Extended Complex Kalman Filter. In conclusion, a set of tools present in control theory have been applied successfully in motion control systems with induction motors. This work is certainly not a complete treatment, since many basic parts are omitted (only the references are given), and for this reasons is to be understood as completion of studies related to the subject matter

Hydrodynamic Electronic Transport

The “flow” of electric currents and heat in standard metals is diffusive with electronic motion randomized by impurities. However, for ultraclean metals, electrons can flow like water with their flow being described by the equations of hydrodynamics. While theoretically postulated, this situation was highly elusive for decades. In the past decade, several experimental groups have found strong indications for this type of flow, especially in graphene-based devices. In this review, we give an overview of some of the recent key developments, on both the theoretical and experimental sides

Integrable and Chaotic Dynamics of Spins Coupled to an Optical Cavity

We show that a class of random all-to-all spin models, realizable in systems of atoms coupled to an optical cavity, gives rise to a rich dynamical phase diagram due to the pairwise separable nature of the couplings. By controlling the experimental parameters, one can tune between integrable and chaotic dynamics on the one hand and between classical and quantum regimes on the other hand. For two special values of a spin-anisotropy parameter, the model exhibits rational Gaudin-type integrability, and it is characterized by an extensive set of spin-bilinear integrals of motion, independent of the spin size. More generically, we find a novel integrable structure with conserved charges that are not purely bilinear. Instead, they develop "dressing tails" of higher-body terms, reminiscent of the dressed local integrals of motion found in many-body localized phases. Surprisingly, this new type of integrable dynamics found in finite-size spin-1/2 systems disappears in the large-S limit, giving way to classical chaos. We identify parameter regimes for characterizing these different dynamical behaviors in realistic experiments, in view of the limitations set by cavity dissipation

Robust Satellite Techniques for Volcanic and Seismic Hazards Monitoring

Several satellite techniques have been proposed to monitor events related to seismic and volcanic activity. A selfadaptive approach (RAT, Robust AVHRR Techniques) has recently been proposed which seems able to recognise space-time anomalies, differently related to such events, also in the presence of highly variable contributions from atmospheric (transmittance), surface (emissivity and morphology) and observational (time/season, but also solar and satellite zenithal angles) conditions. On the basis of NOAA-AVHRR data, the RAT aprroach has already been applied to Mount Etna volcanic ash cloud monitoring in daytime, and to seismic area monitoring in Southern Italy. This paper presents the theoretical basis for the extension of RAT approach also to nighttime volcanic ash cloud detection, together with its possible implementation to lava flow monitoring. One example of successful forecasting (few days before) of a new lava vent opening during the Mount Etna eruption of July 2001 will be discussed in some detail. Progress on the use of the same approach on seismically active area monitoring will be discussed by comparison with previous results achieved on the Irpinia-Basilicata earthquake (MS = 6.9), which occurred on November 23rd 1980 in Southern Italy

A Minimal Model of Signaling Network Elucidates Cell-to-Cell Stochastic Variability in Apoptosis

Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli. We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways.Comment: 9 pages, 6 figure

Target-Specific Precision of CRISPR-Mediated Genome Editing

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms

Anisomycin activates JNK and sensitises DU 145 prostate carcinoma cells to Fas mediated apoptosis

Treatment of the hormone refractory prostate cancer cell line DU 145 with sublethal concentrations of chemotherapeutic drugs has been reported to sensitise these cells to Fas mediated apoptosis. However, the mechanism by which this occurs has not been determined. Our group has shown that inhibition of JNK activity completely abrogates the effects of chemotherapeutic drugs. Using anisomycin, a potent JNK agonist, we have demonstrated a role for JNK in Fas mediated apoptosis in DU 145 cells. Inhibition of Caspase 8 and Caspase 9 completely inhibits this process which suggests that DU 145 cells require mitochondrial amplification of the Fas apoptotic signal. Furthermore, we have shown that inhibition of Fas mediated apoptosis is an early event in DU 145 cells, occurring upstream of Caspase 8 cleavage. It is hoped that identifying the target of JNK will allow novel therapies to be developed for the treatment of hormone refractory prostate cancer. Such therapies are especially important because no single or combined treatment to date has significantly prolonged survival in patients with hormone refractory prostate cancer

The linker histone H1.0 generates epigenetic and functional intratumor heterogeneity

Tumors comprise functionally diverse subpopulations of cells with distinct proliferative potential. Here, we show that dynamic epigenetic states defined by the linker histone H1.0 determine which cells within a tumor can sustain the long-term cancer growth. Numerous cancer types exhibit high inter- and intratumor heterogeneity of H1.0, with H1.0 levels correlating with tumor differentiation status, patient survival, and, at the single-cell level, cancer stem cell markers. Silencing of H1.0 promotes maintenance of self-renewing cells by inducing derepression of megabase-sized gene domains harboring downstream effectors of oncogenic pathways. Self-renewing epigenetic states are not stable, and reexpression of H1.0 in subsets of tumor cells establishes transcriptional programs that restrict cancer cells’ long-term proliferative potential and drive their differentiation. Our results uncover epigenetic determinants of tumor-maintaining cells