High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2

A novel high-content immunofluorescence assay as a tool to identify at the single cell level γ-globin inducing compounds

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ-and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathie

The Future role of inorganic crystal scintillators in dark matter investigations

Crystal scintillators and in particular inorganic scintillators play an important role in the investigation of Dark Matter (DM) and other rare processes. The investigation of a DM signature, as the annual modulation, or the directionality technique requires the use of highly radiopure detectors able to explore the very low energy region maintaining a high stability of the running conditions. In this paper, the cases of NaI(Tl), ZnWO4 and SrI2(Eu) crystal scintillators are described in the framework of our activities at the Gran Sasso National Laboratory of the INFN. Their role, the obtained results in DM investigation, as well as their potential and perspectives for the future are reviewed

Investigation on rare nuclear processes in Hf nuclides

In this work, a review of recent studies concerning rare nuclear processes in Hf isotopes is presented. In particular, the investigations using HP-Ge spectrometry and Hf-based crystal scintillators are focused; the potentiality and the results of the "source = detector" approach are underlined. In addition, a short introduction concerning the impact of such kind of research in the context of astroparticle and nuclear physics is pointed out. In particular, the study of α decay and double beta decay of 174Hf, 176Hf, 177Hf, 178Hf, 179Hf, 180Hf isotopes either to the ground state or to the lower bounded levels have been discussed. The observation of α decay of 174Hf isotope to the ground state with a T1/2=7.0(1.2)×1016 y is reported and discussed. No decay was detected for α decay of 174Hf isotope at the first excited level of daughter and of 176Hf, 177Hf, 178Hf, 179Hf, 180Hf isotopes either to the ground state or to the lower bounded levels. The T1/2 lower limits for these decays are at the level of 1016–1020 y. Nevertheless, the T1/2 lower limits for the transitions of 176Hf→172Yb (0+→0+) and 177Hf→173Yb (7/2−→5/2−) are near to the theoretical predictions, giving hope to their observation in the near future. All the other experimental limits (∼1016–1020 y) are absolutely far from the theoretical expectations. The experiments investigating the 2ϵ and ϵβ+ processes in 174Hf are also reported; the obtained half-life limits are set at the level of 1016–1018 y. Moreover, we estimate the T1/2 of 2ν2ϵ of 174Hf decay at the level of (0.3–6) × 1021 y (at now the related measured lower limit is 7.1×1016 y)

Ricerca di processi rari con rivelatore a Xenon liquido

L'argomento di questo lavoro di tesi è la ricerca di vari processi rari con l'apparato a Xenon liquido dell'esperimento DAMA nei Laboratori Nazionali del Gran Sasso dell’INFN. Tale apparato è uno scintillatore puro, ha ~2 l di volume sensibile (~6.5 kg di liquido) e può utilizzare alternativamente due gas di Xenon: uno arricchito in 129Xe al 99.5%, l'altro in 136Xe al 68.8% e in 134Xe al 17.1% (questo ha permesso anche di effettuare in questa tesi uno studio preliminare del VPIE [vapour pressure isotope effects] in Xenon). In particolare, le ricerche oggetto di questo lavoro sono state realizzate utilizzando i dati raccolti dall’apparato in 8823.54 ore di misura utilizzando il gas arricchito in 136Xe. L’apparato e le sue caratteristiche sono descritti in dettaglio in questa tesi. Le analisi dati hanno riguardato i seguenti processi rari: (i) processi di decadimento 2b- dello 134Xe e dello 136Xe; (ii) possibili decadimenti del nucleone, di-nucleone e tri-nucleone in canali invisibili nello 136Xe; (iii) possibile decadimento 136Xe®136Cs con violazione della conservazione della carica elettrica. Per ognuno degli argomenti trattati, oltre alla descrizione delle procedure di analisi utilizzate e ai risultati ottenuti, vengono fornite anche una introduzione teorica e una descrizione dello stato attuale della ricerca; è, inoltre, sottolineata la competitività dei risultati ottenuti. Le caratteristiche dell'apparato hanno permesso di raggiungere, nelle varie ricerche effettuate, risultati o unici o molto competitivi. In particolare, per quanto riguarda i decadimenti 2b- dello 134Xe e dello 136Xe, tutti i limiti (90%C.L.) posti sui tempi di dimezzamento dei canali studiati migliorano di fattori tra 3 e 700 i risultati precedenti oppure sono stati ottenuti per la prima volta: 134Xe 2b- 0n(0+ ® 0+) T1/2 >5.8·1022 anni 136Xe 2b- 0n(0+ ® 0+) T1/2 >1.2·1024 anni 2b- 0n(0+ ® 2+) T1/2 >2.6·1022 anni 2b- 0n(0+ ® 2+) T1/2 >4.2·1023 anni 2b- 0nM(0+ ® 0+) T1/2 >5.0·1023 anni 2b- 2n(0+ ® 0+) T1/2 >1.0·1022 anni 2b- 2n(0+ ® 2+) T1/2 >9.4·1021 anni Inoltre il limite posto sul modo di decadimento 2b- 0n(0+ ® 0+) dello 136Xe è attualmente il secondo miglior limite sperimentale ottenuto con esperimenti di rivelazione diretta, se si considerano tutti gli isotopi candidati al decadimento 2b- e tutti i modi di decadimento (piu’ stringente di questo è solo il limite sul 2b- 0n(0+ ® 0+) del 76Ge). Tali risultati hanno permesso anche di porre limiti superiori sulla massa efficace del neutrino di Majorana, ámnñ, e sulla costante di accoppiamento efficace Majorone-neutrino, ágMñ. Per quanto riguarda lo studio di possibili decadimenti del nucleone, di-nucleone e tri-nucleone in canali invisibili nello 136Xe, il nuovo approccio utilizzato (già introdotto per la prima volta dal gruppo DAMA in collaborazione con il gruppo dell’INR di Kiev e che consente di avere un branching ratio e un'efficienza di rivelazione ~1) ha permesso di ottenere limiti competitivi, pur disponendo di un rivelatore di massa modesta rispetto ad altri apparati di grande massa che hanno studiato processi analoghi con altri approcci: tn > 3.3·1023 anni tnn - tnnn - tp > 4.5·1023 anni tnp > 3.2·1023 anni tnnp > 1.4·1022 anni tpp > 1.9·1024 anni tnpp > 2.7·1022 anni tppp > 3.6·1022 anni I limiti posti (90% C.L.) sono validi per tutti i canali di decadimento invisibili e, in particolare, per il decadimento in particelle che interagiscono debolmente con la materia e per la possibile “scomparsa” nelle extra-dimensioni. Inoltre, i limiti sui decadimenti del tri-nucleone sono stati ottenuti sperimentalmente per la prima volta. Infine, per quello che riguarda la ricerca del possibile decadimento che viola la conservazione della carica elettrica: 136Xe ® 136Cs, il limite ottenuto sulla vita media: t >1.3·1023 anni (90% C.L.) è ben competitivo e rappresenta la prima misura sperimentale per tale possibile canale di decadimento.The subject of this thesis is the search for several rare processes with the liquid Xenon set-up of the DAMA experiment, in the Gran Sasso National Laboratory of the INFN. The detector is used as a pure scintillator and has ~2 l of sensitive volume (~6.5 kg of liquid) that can be filled alternatively with Xenon gas enriched in 129Xe at 99.5% or with Xenon gas enriched in 136Xe at 68.8% and 134Xe at 17.1% (this feature has also allowed to realise a preliminary study of the VPIE [vapour pressure isotope effects] in Xenon). In this thesis, the set-up and its characteristics are discussed in detail. In addition the data collected in 8823.54 hours of measure, by using the gas enriched in 136Xe, to search for: (i) 2b- decay of 134Xe and 136Xe; (ii) nucleon, di-nucleon and tri-nucleon decay into invisible channels in 136Xe; (iii) charge-non-conserving decay 136Xe®136Cs are considered. For each one of the considered processes, not only a description of the used analysis procedures and the obtained results are provided but also a theoretical introduction and a description of the present state of the search are presented; moreover, the competitiveness of the obtained results is pointed out. The unique features of the set-up have allowed to achieve, for the several searches carried out, results that are unique or very competitive. In particular, for the studied 2b- decay modes of 134Xe and 136Xe, the obtained half live limits (90%C.L.) are improved of factors from 3 to 700 or they have been obtained for the first time: 134Xe 2b- 0n(0+ ® 0+) T1/2 >5.8·1022 years 136Xe 2b- 0n(0+ ® 0+) T1/2 >1.2·1024 years 2b- 0n(0+ ® 2+) T1/2 >2.6·1022 years 2b- 0n(0+ ® 2+) T1/2 >4.2·1023 years 2b- 0nM(0+ ® 0+) T1/2 >5.0·1023 years 2b- 2n(0+ ® 0+) T1/2 >1.0·1022 years 2b- 2n(0+ ® 2+) T1/2 >9.4·1021 years Moreover, the obtained limit for the 2b- 0n(0+ ® 0+) decay mode of 136Xe is at the present the second best experimental limit achieved by direct detection experiments, considering all the nuclei and all the decay modes searched for (the best limit has been obtained for the 2b- 0n(0+ ® 0+) of 76Ge). These limits have also allowed to derive upper limits on the effective Majorana neutrino mass, ámnñ, and on the effective coupling constant Majoron-neutrino, ágMñ. The search for nucleon, di-nucleon and tri-nucleon decay into invisible channels in 136Xe, has been performed following a new approach (already introduced for the first time by the DAMA group in collaboration with the INR Kiev group). This approach assures a high detection efficiency and a branching ratio close to 1 while other different approaches are necessarily pursued with very large mass apparata to compensate the lower values of these quantities. In this way competitive limits have been set (90% C.L.): tn > 3.3·1023 years tnn - tnnn - tp > 4.5·1023 years tnp > 3.2·1023 years tnnp > 1.4·1022 years tpp > 1.9·1024 years tnpp > 2.7·1022 years tppp > 3.6·1022 years The obtained limits hold for each invisible decay channel, including “disappearance” in extra-dimensions or decay in particles which weakly interact with the matter. Moreover the life time limits for the tri-nucleon decays into invisible channel have been set here for the first time. Finally, the lower limit on the life time of the charge-non-conserving decay 136Xe®136Cs has been obtained by searching for the subsequent b- decay of 136Cs. The achieved limit: t >1.3·1023 years (90% C.L.) is well competitive and represents the first experimental measurement for this possible decay channel

Techniques for Background Identification in the Search for Rare Processes with Crystal Scintillators

In astroparticle, nuclear and subnuclear physics, low-counting experiments play an increasingly important role in the investigation of rare processes such as dark matter, double beta decay, some neutrino processes and low-background spectrometry. Extremely low-background features are more and more required to produce detectors and apparata of suitable sensitivity. Over time, a great deal of interest and attention in developing experimental techniques suitable to improve, verify and maintain the radiopurity of these detectors has arisen. In this paper, the characterization of inorganic crystal scintillators (such as, e.g., NaI(Tl), ZnWO4 and CdWO4) using α, β and γ radioactive sources and the main experimental techniques applied in the field to quantitatively identify the radioactive contaminants are highlighted; in particular, we focus on inorganic crystal scintillators, widely used in rare processes investigation, considering their applications at noncryogenic temperatures in the framework of the DAMA experiment activities at the Gran Sasso National Laboratory of the INFN (National Institute for Nuclear Physics, INFN)

Possible implications of the channeling effect in NaI(Tl) crystals

The channeling effect of low energy ions along the crystallographic axes and planes of NaI(Tl) crystals is discussed in the framework of corollary investigations on WIMP dark matter candidates. In fact, the modeling of this existing effect implies a more complex evaluation of the luminosity yield for low energy recoiling Na and I ions. In the present paper related phenomenological arguments are developed and possible implications are discussed at some extent