NEWLY IDENTIFIED PHOX2B-REGULATED GENES AS POSSIBLE DRUG TARGETS FOR THE PHARMACOLOGICAL INTERVENTION IN CONGENITAL CENTRAL HYPOVENTILATION SYNDROME (CCHS)

Congenital Central Hypoventilation Syndrome (CCHS, OMIM #209880) is a very rare neonatal neurological disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing (Weese-Mayer et al., 2017). It is often associated with Hirschsprung\u2019s disease (HSCR) and neural crest-derived tumours (i.e. neuroblastoma). Frameshift mutations (5%) and polyalanine triplet expansions (from 4 to 13 residues) (95%) have been detected in the coding region of PHOX2B, a transcription factor required for the development of neurons that regulate the cardiovascular, respiratory and digestive organs, forming the sensory and motor arms of the visceral reflex circuits. Consistent with its role as transcriptional regulator, transcriptional dysregulation might be an important mechanism of CCHS pathogenesis. CCHS is a life-long disorder for which the only treatment option is ventilatory support provided by tracheotomy, nasal mask or diaphragm pacing by phrenic nerve stimulation, as pharmacological respiratory stimulants have proved to be ineffective. A strong limitation to the comprehension of the pathogenesis of CCHS, and the development of new and effective treatment for this disease, is the missing knowledge of target genes regulated by PHOX2B, whose expression may be eventually dysregulated. Very little is known about the genes regulated by PHOX2B. Most of the genes identified so far are regulatory genes that encode for transcription factors and enzymes that control downstream processes involved in the survival and differentiation of specific neural structures, such as TH, DBH (Lo et al., 1999; Adachi et al., 2000), PHOX2A (Flora et al., 2001), TLX2 (Borghini et al.,2006), RET (Bachetti et al., 2005) MSX-1 (Revet et al., 2008), SOX10 (Nagashimada et al., 2012), ALK (Bachetti et al., 2010) and PHOX2B itself (Cargin et al., 2005). The aim of this thesis was to identify new potential pharmacological targets for the development of drugs in order to improve the respiratory symptoms and the quality of life of CCHS patients. In the first part of my project we investigated the molecular mechanisms underlying the recovery of chemosensitivity, observed in two CCHS patients, following the administration of the progestinic desogestrel (Straus et al., 2010). In SK-N-BE(2)C cell clones, stably expressing nuclear progesterone receptor isoforms PR-B and PR-A, we demonstrated that 3-KDG treatment, active metabolite of the desogestrel, reduces the expression of PHOX2B, its target genes as well as PHOX2B +7 alanine expanded protein, by means of a post-transcriptional mechanism. This finding provided the evidence of a direct molecular link between PHOX2B and desogestrel and suggested the possibility that reduction of PHOX2B mutant protein may contribute to the positive effects observed in the two CCHS patients. In the second part of my project we proposed to identify new PHOX2B target genes that can be deregulated in the pathology, in the future perspective that might be potential pharmacological targets, alternative to PHOX2B. In particular, we demonstrated that transcriptional dysregulation and dysfunctions of K+ and Na+ channels activity may contribute to the onset of respiratory problems associated with CCHS. Target directly the de-regulated PHOX2B target genes is an alternative pharmacological strategy to by-pass the effect on PHOX2B in the perspective of rescuing their activity. Since several drugs targeting these proteins are already used in clinics, the potential progress toward a therapeutic intervention to treat CCHS is today more than concrete

Measurements and optimization of the light yield of a TeO2_2 crystal

Bolometers have proven to be good instruments to search for rare processes because of their excellent energy resolution and their extremely low intrinsic background. In this kind of detectors, the capability of discriminating alpha particles from electrons represents an important aspect for the background reduction. One possibility for obtaining such a discrimination is provided by the detection of the Cherenkov light which, at the low energies of the natural radioactivity, is only emitted by electrons. This paper describes the method developed to evaluate the amount of light produced by a crystal of TeO$_2$ when hit by a 511 keV photon. The experimental measurements and the results of a detailed simulation of the crystal and the readout system are shown and compared. A light yield of about 52 Cherenkov photons per deposited MeV was measured. The effect of wrapping the crystal with a PTFE layer, with the aim of maximizing the light collection, is also presented

New experimental limits on the alpha decays of lead isotopes

For the first time a PbWO4 crystal was grown using ancient Roman lead and it was run as a cryogenic detector. Thanks to the simultaneous and independent read-out of heat and scintillation light, the detector was able to discriminate beta/gamma interactions with respect to alpha particles down to low energies. New more stringent limits on the alpha decays of the lead isotopes are presented. In particular a limit of T_{1/2} > 1.4*10^20 y at a 90% C.L. was evaluated for the alpha decay of 204Pb to 200Hg

New application of superconductors: high sensitivity cryogenic light detectors

In this paper we describe the current status of the CALDER project, which is developing ultra-sensitive light detectors based on superconductors for cryogenic applications. When we apply an AC current to a superconductor, the Cooper pairs oscillate and acquire kinetic inductance, that can be measured by inserting the superconductor in a LC circuit with high merit factor. Interactions in the superconductor can break the Cooper pairs, causing sizable variations in the kinetic inductance and, thus, in the response of the LC circuit. The continuous monitoring of the amplitude and frequency modulation allows to reconstruct the incident energy with excellent sensitivity. This concept is at the basis of Kinetic Inductance Detectors (KIDs), that are characterized by natural aptitude to multiplexed read-out (several sensors can be tuned to different resonant frequencies and coupled to the same line), resolution of few eV, stable behavior over a wide temperature range, and ease in fabrication. We present the results obtained by the CALDER collaboration with 2x2 cm2 substrates sampled by 1 or 4 Aluminum KIDs. We show that the performances of the first prototypes are already competitive with those of other commonly used light detectors, and we discuss the strategies for a further improvement

Characterization of the KID-Based Light Detectors of CALDER

The aim of the Cryogenic wide-Area Light Detectors with Excellent Resolution (CALDER) project is the development of light detectors with active area of $5\times5$ cm$^2$ and noise energy resolution smaller than 20 eV RMS, implementing phonon-mediated kinetic inductance detectors. The detectors are developed to improve the background suppression in large-mass bolometric experiments such as CUORE, via the double read-out of the light and the heat released by particles interacting in the bolometers. In this work, we present the characterization of the first light detectors developed by CALDER. We describe the analysis tools to evaluate the resonator parameters (resonant frequency and quality factors) taking into account simultaneously all the resonance distortions introduced by the read-out chain (as the feed-line impedance and its mismatch) and by the power stored in the resonator itself. We detail the method for the selection of the optimal point for the detector operation (maximizing the signal-to-noise ratio). Finally, we present the response of the detector to optical pulses in the energy range of 0-30 keV

Energy resolution and efficiency of phonon-mediated Kinetic Inductance Detectors for light detection

The development of sensitive cryogenic light detectors is of primary interest for bolometric experiments searching for rare events like dark matter interactions or neutrino-less double beta decay. Thanks to their good energy resolution and the natural multiplexed read-out, Kinetic Inductance Detectors (KIDs) are particularly suitable for this purpose. To efficiently couple KIDs-based light detectors to the large crystals used by the most advanced bolometric detectors, active surfaces of several cm$^2$ are needed. For this reason, we are developing phonon-mediated detectors. In this paper we present the results obtained with a prototype consisting of four 40 nm thick aluminum resonators patterned on a 2$\times$2 cm$^2$ silicon chip, and calibrated with optical pulses and X-rays. The detector features a noise resolution $\sigma_E=154\pm7$ eV and an (18$\pm$2)$\%$ efficiency.Comment: 5 pages, 5 figure

TeO2_2 bolometers with Cherenkov signal tagging: towards next-generation neutrinoless double beta decay experiments

CUORE, an array of 988 TeO$_2$ bolometers, is about to be one of the most sensitive experiments searching for neutrinoless double-beta decay. Its sensitivity could be further improved by removing the background from $\alpha$ radioactivity. A few years ago it has been pointed out that the signal from $\beta$s can be tagged by detecting the emitted Cherenkov light, which is not produced by $\alpha$s. In this paper we confirm this possibility. For the first time we measured the Cherenkov light emitted by a CUORE crystal, and found it to be 100 eV at the $Q$-value of the decay. To completely reject the $\alpha$ background, we compute that one needs light detectors with baseline noise below 20 eV RMS, a value which is 3-4 times smaller than the average noise of the bolometric light detectors we are using. We point out that an improved light detector technology must be developed to obtain TeO$_2$ bolometric experiments able to probe the inverted hierarchy of neutrino masses.Comment: 5 pages, 4 figures. Added referee correction

Desogestrel down-regulates PHOX2B and its target genes in progesterone responsive neuroblastoma cells

The paired-like homeobox 2B gene (PHOX2B) encodes a key transcription factor that plays a role in the development of the autonomic nervous system and the neural structures involved in controlling breathing. In humans, PHOX2B over-expression plays a role in the pathogenesis of tumours arising from the sympathetic nervous system such as neuroblastomas, and heterozygous PHOX2B mutations cause Congenital Central Hypoventilation Syndrome (CCHS), a life-threatening neurocristopathy characterised by the defective autonomic control of breathing and involving altered CO2/H+ chemosensitivity. The recovery of CO2/H+ chemosensitivity and increased ventilation have been observed in two CCHS patients using the potent contraceptive progestin desogestrel. Given the central role of PHOX2B in the pathogenesis of CCHS, and the progesterone-mediated effects observed in the disease, we generated progesterone-responsive neuroblastoma cells, and evaluated the effects of 3-Ketodesogestrel (3-KDG), the biologically active metabolite of desogestrel, on the expression of PHOX2B and its target genes. Our findings demonstrate that, through progesterone nuclear receptor PR-B, 3-KDG down-regulates PHOX2B gene expression, by a post-transcriptional mechanism, and its target genes and open up the possibility that this mechanism may contribute to the positive effects observed in some CCHS patients

Development of a Li2MoO4 scintillating bolometer for low background physics

We present the performance of a 33 g Li2MoO4 crystal working as a scintillating bolometer. The crystal was tested for more than 400 h in a dilution refrigerator installed in the underground laboratory of Laboratori Nazionali del Gran Sasso (Italy). This compound shows promising features in the frame of neutron detection, dark matter search (solar axions) and neutrinoless double-beta decay physics. Low temperature scintillating properties were investigated by means of different alpha, beta/gamma and neutron sources, and for the first time the Light Yield for different types of interacting particle is estimated. The detector shows great ability of tagging fast neutron interactions and high intrinsic radiopurity levels (< 90 \muBq/kg for 238-U and < 110 \muBq/kg for 232-Th).Comment: revised versio