Littérature scientifique sur le sujet « Doppio Beta »

Abstract Introduction: Approved angiogenesis inhibitors generally target certain growth factors or their receptors, which exist in both normal and cancerous cells; this results in suppression of cell-signaling pathways throughout the body and causes adverse effects. For this reason, there is a need for a new angiogenesis inhibitor that can specifically target the tumor vasculature. In our previous study, we revealed that doppel, a prion-like protein, was overexpressed specifically in the tumor vasculatures, but not in normal endothelium, and its expression enhanced blood vessel formation. To develop our new anti-angiogenic agent, we produced monoclonal antibodies which target doppel as the antigen. Methods: We evaluated whether the doppel antibody inhibits angiogenesis by a spheroid assay. We cultured human colonic tumor-associated endothelial cells (HCTEC) and collected the cells by a hanging drop method. After producing compact aggregates, the spheroids were collected and embedded into collagen-based 3D cultures. The culture media/growth factor/doppel antibody mixture was added to each well that contained 3D spheroid cultures. After 24 hours, the number of sprouts was counted and analyzed. Further, HCTECs were incubated with culture media, growth factor, and doppel antibodies for investigating the underlying mechanisms. Each well was lysed, and we conducted western blot and microarray experiments using the lysates. In addition, we injected fluorescent dye-conjugated doppel antibody into tumor xenograft mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS). Results: In spheroid assays, we found that doppel antibody SNU-H01 and SNU-H02 inhibit angiogenesis (by 32% and 29%, respectively) compared to control. In anti-phosphorylation assays using western blot, the amount of phosphorylated VEGFR2 and phosphorylated FGFR1 was decreased in doppel antibody SNU-H01 and SNU-H02-treated groups. Also, when SNU-H02-treated lysate was added to the microarray kit, STAT5A and beta-catenin were inhibited. Inhibition of STAT5A may promote apoptosis and increase sensitivity to anticancer drugs, and inhibition of beta-catenin may further increase suppression of angiogenesis. In addition, doppel antibody SNU-H04 accumulates in the tumor significantly higher compared to IgG injected mouse group. Conclusion: Doppel antibodies in development, SNU-H01, -H02, -H03, -H04, will be screened and selected for their anti-angiogenic efficacy and favorable pharmacodynamic features. We hope that doppel antibody is expected to be a new and superior tumor vasculature-specific angiogenesis inhibitor that can overcome the limitations of existing anti-angiogenic agents. Citation Format: Ha Kyeong Lee, Ruby Maharjan, Yeojin Jeon, Jeong Uk Choi, Youngro Byun. Anti-doppel monoclonal antibody as a tumor endothelial cell-specific angiogenesis inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 339.

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide.

In this study, the effects of a series of phorbol esters with different spectra of biological activities and different patterns of activation of the isoenzymes of protein kinase C (PKC) have been studied in human neutrophils. The aim was to gain more information on which isoenzymes of PKC are involved in neutrophil activation, specifically inhibition of fMet-Leu-Phe (fMLP)-stimulated bivalent cation influx and stimulation of O2-. release (either alone or potentiation of the response to fMLP). Prior addition of both phorbol 12-myristate 13-acetate (PMA) and sapintoxin A (SAPA) inhibited fMLP-stimulated Mn2+ influx. Higher concentrations of resiniferatoxin (RX) were also inhibitory, inhibition being more apparent at longer preincubation times. However, 12-deoxyphorbol 13-O-phenylacetate (DOPPA) showed only a slight inhibitory effect and required a prolonged preincubation. PMA, SAPA and RX, but not DOPPA, stimulated O2-. release by themselves. Lower concentrations of PMA, SAPA and RX, which were ineffective alone, considerably potentiated O2-. release stimulated by fMLP, whereas DOPPA had little or no effect. These results rule out a major role for PKC-delta (not activated by SAPA) and PKC-beta 1 (activated by DOPPA), but suggest the involvement of RX kinase in addition to PKC in the inhibition of fMLP-stimulated Mn2+ influx and potentiation of fMLP-stimulated O2-. release. However, when the cytosolic free Ca2+ concentration ([Ca2+]i) was elevated with the Ca2+ ionophore ionomycin, DOPPA was able to stimulate O2-. release, which probably reflects the known Ca2+ requirement for activation of PKC-beta 1 by DOPPA in vitro. The effects of the other phorbols were also enhanced when [Ca2+]i was elevated; all of the phorbols synergize, to variable extents, with Ca2+ to activate PKC in vitro. Enhancement of RX-stimulated O2- release by elevation of [Ca2+]i was unexpected, since RX kinase has been reported to be inhibited by high concentrations of Ca2+ in vitro. Finally, use of fura-2 and SK&F 96365 to manipulate the fMLP-stimulated rise in [Ca2+]i showed that when fMLP was able to evoke its normal rise in [Ca2+]i (to a peak of 700-900 nM), O2-. release was potentiated by PMA, SAPA and RX. However, when fMLP was only able to evoke a small increase in [Ca2+]i (to a peak of 400 nM), potentiation by PMA was unaffected but potentiation by SAPA and RX was considerably reduced. This observation agrees with published data demonstrating that activation of PKC in vitro by SAPA is more Ca(2+)-dependent than activation by PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Background:Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that regulates cholesterol metabolism through low-density lipoprotein receptor degradation and that has been linked with cardiovascular (CV) risk. Patients with spondyloarthritis (SpA) are prone to an increased and premature prevalence of atherosclerosis that has been linked to an atherogenic lipid profile among these individuals.Objectives:The purpose of the present study was to examine whether PCSK9 levels are related to both abnormalities in the lipid profile and the severe atherosclerosis that occur in patients with SpA.Methods:Cross-sectional study that encompassed 545 individuals; 299 patients with SpA and 246 statins intake-matched controls. PCSK9 and lipoproteins serum concentrations, standard lipid profile and carotid intima-media thickness and carotid plaques were assessed in patients and controls. A multivariable analysis, adjusted for standard CV risk factors, was performed to evaluate the influence of PCSK9 on SpA related dyslipidemia, disease related data, and subclinical carotid atherosclerosis.Results:Lipid profiles showed that most lipid panel parameters (total cholesterol, HDL- and LDL-cholesterol, lipoprotein A and apoliprotein A1) were lower in SpA patients compared to controls. Contrary, Apo B:Apo A1 and LDL:HDL cholesterol ratios were higher in SpA. The mean PCSK9 serum levels were significantly lower in SpA patients compared to controls (249 ± 105 vs. 199 ± 74, ng/ml, p=0.000) in the univariate analysis. An additional multivariable analysis adjusting for demographics and CV risk factors plus all the lipid-related molecules (that were found to be different between patients and controls) disclosed that PCSK9 (beta coef. -44 [95%CI -60- -27] % mg/dl, p=0.000) conserved its downregulation in SpA patients.Traditional CV risk factors were not related to PCSK9 in controls and patients. Concerning lipid profile, some correlations were found between lipid-related molecules and PCSK9. In this sense, triglycerides were positively associated with PCSK9 in both patients and controls; total cholesterol and apolipoprotein A1 serum levels were associated with PCSK9 in patients but not in controls and; Lipoprotein (a) was related to PCSK9 only in controls (Table 1).Regarding disease-related data, disease duration (log beta coef. 10 [0-20], p=0.043), and ASDAS-CRP (12 [95%CI 4-20], p=0.004) and BASFI (log beta coef. 12 [95%CI 0-25], p=0.049) scores,were positively related to PCSK9. Moreover, patients in the very high disease activity ASDAS-CRP category disclosed higher serum levels of PCSK9 compared to those in the remission category (32 [95%CI 2-63], p=0.038). Remarkably, while patients on current prednisone showed higher serum levels of PCSK9 (55 [95%CI 24-8]) ng/ml, p=0.001), patients under anti-TNF alpha therapies exhibited inferior levels (beta coef. -26 [95%CI -43- -9], p=0.003). PCSK9 in SPA patients with carotid plaque was higher compared to patients without plaque, however, this difference was lost after multivariable analysis.Conclusion:PCSK9 is downregulated in SpA patients independently of other inflammation-related lipid profile modifications that occur in the disease. Disease activity is positively associated with PCSK9 serum levels. PCSK9 is univariately related to the presence of carotid plaque.Disclosure of Interests:Juan Carlos Quevedo-Abeledo Speakers bureau: Abbvie, Laura de Armas-Rillo: None declared, Vanessa Hernández-Hernández Speakers bureau: Pfizer, Abbvie, MSD, delgado frias esmeralda Speakers bureau: Pfizer, Abbvie, MSD, Antonia de Vera-González: None declared, Alejandra Delgado-González: None declared, Jose Antonio García-Dopico: None declared, Iván Ferraz-Amaro Grant/research support from: Pfizer, Abbvie, Speakers bureau: Pfizer, Abbvie, MSD.

In this study, collagen hydrolysates (CHDs) were fabricated with honey-propolis wax (HPW), structurally modified as a sponge matrix, and experimentalized on wound healing in a mouse model. The scaffold was characterized by means of in vitro enzymatic degradation; in vitro HPW release; and in vivo wound-healing mouse model, wound-healing-specific RNA, transcripts, and protein markers. The functional activity of the HPW extracted from raw propolis was determined using total flavonoids, antioxidant scavenging assays, and anti-hemolytic principles. The results indicated that HPW had a high flavonoid content (20 μg/mL of wax) and antioxidant activities. The effective concentration (EC50) of HPW was estimated (28 mg/mL) and was then used in the subsequent in vivo experiments. Additionally, the dopped mixture of CHDs and HPW substantially enhanced the wound-healing process and regulated wound biochemical markers such as hexoseamine and melondialdehyde. CHDs- HPW upregulated the expression of growth factors including vascular endothelial growth factor (VEGF) (2.3-fold), fibroblast growth factor (FGF) and epidermal growth factor (EGF) (1.7-fold), and transforming growth factor-beta (TGF-β) (3.1-fold), indicating their potential capacity to perform wound re-epithelialization and the loading of ground tissue. Pro-inflammatory markers IL-1 β (51 pg/mL) and TNF-α (220 pg/mL) were significantly reduced in the CHD-HPW-treated wound. These interesting results were further confirmed using mRNA and protein growth factors from the wound, which enhanced the load of collagen-I in the wound site. In conclusion, CHDs-HPW exhibited a significant reduction in inflammation and inflammatory markers and helped to obtain a faster wound-healing process in a mouse model. The newly engineered biosponge could be developed as a promising therapeutic approach for the regeneration and repair of damaged human skin in the future.

Background: External counterpulsation (ECP) is a noninvasive method used to augment cerebral perfusion but the optimal use of ECP in ischemic stroke has not been well documented. We aim to investigate the effects of increasing ECP treatment pressure on cerebral blood flow and blood pressure (BP). Methods: We recruited 38 acute ischemic stroke patients with large artery occlusive disease and 20 healthy elderly. Mean cerebral blood flow velocities (CBFV) of bilateral middle cerebral artery were monitored using transcranial dopper. Continuous beat-to-beat BP was measured via finger cuffs. We started ECP treatment pressure from 150mmHg, then gradually increased to 187.5mmHg, 225mmHg and 262.5mmHg. CBFV and BP were recorded before ECP and during each pressure increment respectively for 3 minutes. CBFV data of patients was analyzed based on whether it was ipsilateral or contralateral to the infarct. Results: Median NIHSS of stroke patients was 5.5 and mean time after stroke onset was 5.24 days. Mean BP was significantly elevated from baseline in both groups after ECP started. BP increase percentages of two groups similarly kept augmented following raised ECP pressure and reach maximium at 262.5mmHg (patients 16.9% vs. controls 16.52% compared with baseline). Under different ECP pressures, ipsilateral mean CBFV of stroke patients increased 5.15% (150mmHg), 4.35% (187.5mmHg), 4.55% (225mmHg) and 3.52% (262.5mmHg) from baseline. All were significantly higher than baseline but did not differ among different pressures. Contralateral mean CBFV changed likewise (5.16%, 4.02%, 3.7% and 3.34% increase from baseline). Mean CBFV of controls under ECP pressures did not increase from baseline. Conclusion: The increasing treatment pressure of external counterpulsation continuously augments blood pressure but not cerebral blood flow velocity of ischemic stroke patients with large artery occlusive disease. Cerebral blood flow velocity of stroke patients significantly increases from baseline under ECP pressure of 150mmHg but it reaches a plateau as ECP pressure further raises. Among 4 ECP pressures above, 150mmHg is the optimal treatment pressure for ischemic stroke due to higher risks of hypertension-related complications in acute stroke with higher ECP pressure.

L’attività di ricerca da me svolta durante il dottorato di ricerca è stata legata all’applicazione e sviluppo di rivelatori a scintillazione criogenici, pensati per la ricerca del decadimento doppio beta senza emissione di neutrini (0νββ). Ho analizzato due possibili implementazioni di questa tecnica: rivelatori ad alte prestazioni basati sulla lettura della luce di scintillazione e rivelatori bolometrici a scintillazione con doppia lettura. Il rivelatore per 0νββ basato sulla scintillazione che ho studiato è costituito da cristalli scintillanti, contenenti l’isotopo candidato al 0νββ, accoppiati otticamente a rivelatori a deriva in silicio (Silicon Drift Detectors, SDD), utilizzati come lettori di luce e operati a 120K. Il mio lavoro è stato dedicato alla caratterizzazione degli SDD, per mezzo di misure dirette con raggi X e misure di scintillazione. Le misure con raggi X sono state utilizzate per caratterizzare gli SDD progettati appositamente per questa applicazione dalla Fondazione Bruno Kessler (FBK), che sono i dispositivi di questo tipo con singolo anodo più grandi mai prodotti. I risultati ottenuti mostrano il livello estremamente basso di rumore elettronico raggiunto da questi rivelatori, dimostrando che questa caratteristica non ne limita le prestazioni. Le misure di scintillazione, invece, sono state realizzate accoppiando un cristallo di CdWO4 al SDD, permettendo di valutare la risoluzione energetica del rivelatore complessivo. Con diverse misure, ho dimostrato che la risoluzione energetica è limitata dalla efficienza di raccolta di carica del SDD, non uniforme sulla superficie del rivelatore. Per risolvere questo problema, una nuova serie di SDD che correggono questa caratteristica è stata progettata e prodotta da FBK. Al fianco di questo sviluppo tecnico, ho lavorato come membro della collaborazione CUPID-0 all’analisi dei dati raccolti dall’esperimento. CUPID-0 è un rivelatore bolometrico a scintillazione composto da 26 cristalli di ZnSe, 24 dei quali arricchiti in 82Se, un candidato al 0νββ. Combinando la buona risoluzione energetica del canale di calore con l’identificazione delle particelle resa possibile dal segnale di luce, CUPID-0 è stato in grado di raggiungere il fondo più basso mai raggiunto da un esperimento bolometrico per la ricerca del 0νββ. Grazie a questo risultato, è stato possibile estrarre il miglior limite sulla vita media del 0νββ del 82Se, dimostrando le alte prestazioni di questo approccio sperimentale. Con il mio lavoro ho ottenuto un ulteriore miglioramento della risoluzione energetica, correggendo la correlazione tra luce e calore. Anche grazie a questo nuovo passaggio di analisi, un nuovo limite sulla vita media del 0νββ del 82Se è stato ottenuto, utilizzando tutti i dati raccolti nella prima fase dell’esperimento. Ho anche contribuito alla definizione del modello del fondo dell’esperimento, sviluppando un sistema di analisi per caratterizzare le contaminazioni superficiali dei cristalli. Sfruttando i risultati del modello di fondo, ho poi potuto mettere un limite alla violazione della simmetria di Lorentz nelle interazioni dei neutrini, ottenuto analizzando la forma dello spettro del decadimento doppio beta a due neutrini del 82Se. Il lavoro da me svolto mi ha consentito di acquisire esperienza nell’operazione e analisi dati di scintillatori operati a temperature criogeniche, spendibili sulle diverse tecniche sperimentali che ho approfondito e estendibili ad altre applicazioni basate su questo tipo di rivelatori.
The research activity during my PhD has been devoted to the application and development of cryogenic scintillation detectors for the search of neutrinoless double beta decay (0νββ). I investigated two implementations of this design: high performance detectors based on the light output readout and the double-readout scintillating bolometers. The scintillation based 0νββ detector that I investigated is based on scintillating crystals, containing a 0νββ candidate, optically coupled to Silicon Drift Detectors (SDDs) operated as light detectors at 120K. My work was dedicated to the characterization of the SDD detectors, performing both X-ray and scintillation measurements. The X-ray measurements served as a testing ground for the devices designed and produced for this application by Fondazione Bruno Kessler (FBK), which are the biggest single anode SDDs ever built. The obtained results showed that these devices have excellent noise performances, proving that the electronic noise is not a limiting factor in this application. The scintillation measurements performed with a CdWO4 crystal coupled with a SDD, instead, allowed to evaluate the attainable energy resolution. With different measurement, I showed that the energy resolution is limited by the variable charge collection efficiency of the SDD surface. To solve this problem, a new series of SDDs with uniform efficiency is going to be designed and produced at FBK. Alongside this technical development, I worked as a member of the CUPID-0 collaboration in the data analysis. CUPID-0 is a scintillating bolometer composed by 26 ZnSe crystals, 24 of which are enriched in 82Se, a 0νββ candidate. Combining the good energy resolution given by the heat channel readout and the particle identification given by the light signal, CUPID-0 was able to reach the lowest background ever reached by a bolometric experiment in the region of interest for the 0νββ. Consequently, the best limit on the 0νββ of 82Se could be extracted, showing the efficiency of this technological approach. I obtained an ulterior enhancement of the attainable energy resolution in the heat channel, by the means of the light/heat correlation correction. With this new analysis step, a new limit was obtained on the 0νββ half-life of 82Se, calculated with the total statistic of the first phase of the experiment. I also worked in the definition of the background model for the CUPID-0 experiment, developing an analysis routine to characterize the surface contamination of crystals. Exploiting the result of the background model I could put a limit on the Lorentz violation in the neutrino sector, analyzing the shape of the 2νββ spectrum. My research work allowed me to acquire a solid know-how on scintillators operated at cryogenic temperatures, expendable in between the two technological routes I have followed and extendable to other scintillation-based applications.

The GERmanium Detector Array (GERDA) experiment, located underground at the INFN Laboratori Nazionali del Gran Sasso (LNGS) in Italy, uses high-purity germanium detectors to search for neutrinoless double beta decay (0νββ) of Ge-76. The first phase of the experiment lasted from November 2011 to May 2013 and collected data with a total exposure of 21.6 kg · yr. In this thesis, a thorough analysis of these data was performed. A background model was developed to decompose the observed energy spectrum in its individual contributions. The region around the Q-value of 0νββ, Qββ , at 2039 keV was studied in great detail. As main contributions to the background in this region, alpha and beta decays of the U-238 chain, beta decays of the Th-232 chain, and beta decays of K-42 were identified. It was shown that the background around Qββ can be approximated with a flat distribution. Neutrino accompanied double beta decay (2νββ) is a lepton number conserving process allowed by the Standard Model. Due to the low background in the experiment, in the region dominated by 2νββ a signal-to-background ratio of 3 : 1 could be reached. This allowed to measure the half-life of the decay with a precision unprecedented by previous experiments, T1/2^2ν = (1.96 ± 0.13) · 10^21 yr. Several beyond-Standard Model theories predict neutrinoless double beta decay with majoron emission (0νββχ(χ)). Depending on the theory, this process can be lepton number violating or lepton number conserving. A search in the GERDA Phase I data gave no indication of contributions to the observed energy spectra for any of the majoron models. The lower limit on the half-life for the ordinary majoron model (spectral index n = 1) was determined to be T1/2^0νχ > 4.15 · 10^23 yr (90 % quantile). This limit and the limits derived for the other majoron modes constitute the most stringent limits on 0νββχ(χ) of Ge-76 measured to date. The primary scope of the GERDA experiment was the search for 0νββ of Ge-76. This lepton number violating decay is expected by extensions of the Standard Model. The observation of 0νββ would be the proof that the neutrino has a non-vanishing Majorona mass component. The analysis of the GERDA Phase I data did not reveal any hint for the presence of a signal from 0νββ. A lower limit on the half-life was derived, T1/2^0ν > 1.83 · 10^25 yr (90 % quantile).
L’esperimento GERmanium Detector Array (GERDA), situato nei Laboratori Nazionali del Gran Sasso (LNGS) dell’INFN, utilizza rivelatori al germanio ultra-puro per la ricerca del doppio decadimento beta senza neutrini (0νββ). Tali rivelatori sono arricchiti nell’isotopo Ge-76. La prima fase dell’esperimento è durata da novembre 2011 a maggio 2013 ed ha raccolto dati con un’esposizione totale di 21.6 kg · yr. In questa tesi è stato sviluppato dapprima un modello dei fondi per scomporre lo spettro energetico osservato nei suoi singoli componenti. La regione intorno al Q-valore della reazione 0νββ, Qββ , a 2039 keV è stata studiata in modo dettagliato. I contributi principali al fondo in questa regione sono: i decadimenti alfa e beta della catena del U-238, i decadimenti beta della catena del Th-232 ed i decadimenti beta del K-42. È stato dimostrato inoltre che il fondo intorno a Qββ può essere descritto con una costante. Il doppio decadimento beta con emissione di due neutrini (2νββ) è un processo che conserva il numero leptonico ed è previsto dal Modello Standard. Nella regione dominata dagli eventi 2νββ è stato raggiunto un rapporto fra segnale e fondo di 3 : 1. Questo risultato ha permesso di misurare il tempo di dimezzamento del decadimento con una precisione ineguagliata dagli esperimenti precedenti, T1/2^2ν = (1.96 ± 0.13) · 10^21 yr. Alcuni modelli di fisica oltre il Modello Standard prevedono il doppio decadimento beta senza neutrini con emissione di uno o due majoroni (0νββχ(χ)). In base alla teoria, questo processo può violare o conservare il numero leptonico. Un’analisi dei dati della prima fase di GERDA non ha fornito alcun riscontro di contributi di uno di questi modelli agli spettri energetici osservati. Il limite inferiore sul tempo di dimezzamento per il modello ordinario del majorone (indice spettrale n = 1) è stato stimato pari a T1/2^0νχ > 4.15 · 10^23 yr (quantile del 90 %). Questo valore e quelli ricavati per altri modelli del majorone costituiscono i limiti più stringenti su 0νββχ(χ) nel Ge-76 misurati fino ad ora. Lo scopo primario dell’esperimento GERDA è la ricerca del 0νββ nel Ge-76. Questo processo, che viola il numero leptonico, è previsto dalle estensioni del Modello Standard e la sua osservazione dimostrerebbe che la massa del neutrino ha una componente di tipo Majorana. L’analisi dei dati della prima fase di GERDA non ha rivelato nessun cenno della presenza di un segnale di 0νββ. È stato così determinato un limite inferiore sul tempo di dimezzamento, T1/2^0ν > 1.83 · 10^25 yr (quantile del 90 %).

Introduzione: La cirrosi viene frequentemente complicata dall’incremento della pressione portale e dal conseguente sviluppo di varici esofago-gastriche, ascite e sanguinamento da varici. L'azione dei β-bloccanti nel ridurre il valore dell’HVPG, dipende da un diminuito afflusso portale, secondario alla riduzione dell'indice e alla vasocostrizione splancnica Purtroppo, le resistenze vascolari intraepatiche sono scarsamente influenzate dall’azione dei beta-bloccanti. Il 5-metiltetraidrofolato (5-MTHF) può migliorare la funzione vascolare attraverso la sua azione sulla eNOS e sullo stress ossidativo vascolare Attualmente non è noto se il 5-MTHF possa avere lo stesso effetto sulla microcircolazione epatica. Obiettivi: Valutare l’efficacia del trattamento per 3 mesi con 5-MTHF in associazione con propranololo rispetto a propranololo + placebo, in termini di riduzione dell’HVPG nei pazienti cirrotici con ipertensione portale (PI). Obiettivi secondari sono: a) Valutare la tollerabilità del 5-MTHF nei pazienti cirrotici con PI; b) Valutare se vi sia un miglioramento del tono vascolare intraepatico mediato dalla diminuzione dello stress ossidativo e dalla maggiore biodisponibilità di NO nei pazienti con cirrosi e PI. Risultati: In entrambi i gruppi si evidenzia una riduzione del valore dell’HVPG rispetto al valore basale, ma la modificazione risultava significativamente maggiore nel gruppo trattato con 5-MTHF rispetto al gruppo placebo (20.8%, SD 15.4 nel gruppo 5-MTHF vs 9.2%, SD 21.3 nel gruppo placebo; p=0.033). Analoga riduzione si evidenzia per il valore dell’elastometria misurata con FibroScan® (21.8%, SD 34.7 nel gruppo 5-MTHF vs 4.4%, SD 32.5 nel gruppo placebo, p=0.064). Discussione: l'analisi di confronto tra il gruppo di trattamento e il gruppo placebo mostra una differenza statisticamente significativa in termini di entità di riduzione del gradiente pressorio porto-epatico. Ulteriori studi multicentrici, su coorti più ampie sono auspicabili per validare l’utilizzo del 5-MTHF+placebo nel ridurre il valore di pressione portale misurato con HVPG
Introduction: Cirrhosis is frequently complicated by the increase of portal pressure and development of esophageal varices, ascites and upper gastrointestinal bleeding. The mechanism of action of β-blockers in reducing the value of HVPG depends on a decreased portal inflow, secondary to the reduction of the index and to splanchnic vasoconstriction. Unfortunately, intrahepatic vascular resistance is poorly influenced by the action of beta-blockers. 5-methyltetrahydrofolate (5-MTHF) can improve vascular function through its action on eNOS and vascular oxidative stress Currently it is not known whether 5-MTHF may have the same effect on liver microcirculation. Objectives: to evaluate the efficacy of 3 months treatment with 5-MTHF in combination with propranolol compared to propranolol + placebo in terms of reduction of HVPG in cirrhotic patients with portal hypertension (PI). Secondary objectives are: a) to evaluate the tolerability of 5-MTHF in cirrhotic patients with PI; b) to evaluate if there is an improvement in the intrahepatic vascular tone mediated by the decrease of oxidative stress because of greater bioavailability of NO in patients with cirrhosis and PI. Results: in both groups there was a reduction in the HVPG value compared to baseline, but the change was significantly higher in the 5-MTHF group compared to the placebo group (20.8%, SD 15.4 in the 5-MTHF vs 9.2%, SD 21.3 in the placebo group, p=0.033). A similar reduction was noted for the value of elastometry measured with FibroScan® (21.8%, SD 34.7 in the 5-MTHF group vs. 4.4%, SD 32.5 in the placebo group, p = 0.064). Discussion: the comparison analysis between the treatment group and the placebo group shows a statistically significant difference in terms of reduction of porto-systemic pressure gradient. Further multi-center studies on larger cohorts are desirable to validate the use of 5-MTHF + placebo in reducing the portal pressure value measured with HVPG

Premessa I microRNA occupano una posizione gerarchicamente critica nella regolazione delle networks cellulari e sono quindi candidati di rilievo per un coinvolgimento patogenetico in patologie sistemiche, quali le neoplasie e le malattie degenerative. Di conseguenza, queste molecole sono concordemente ritenute i biomarcatori di nuova generazione piu' promettenti per la diagnosi, per la prognosi ed il disegno di interventi terapeutici innovativi: e' facile ipotizzare che la disponibilita ' di batterie di microRNA, ben caratterizzati dal punto di vista molecolare, strettamente associati alla patologia di interesse, e presenti nel siero, dovrebbe modificare in modo radicale le modalita' operative e le prospettive di successo nell' ambito della Medicina clinica. Obiettivo del nostro lavoro e' stato quello di contribuire alla caratterizzazione del ruolo dei microRNA nella patogenesi del Diabete Mellito (DM), ed identificare e caratterizzare un set di microRNA da utilizzare quali biomarcatori molecolari e potenziali bersagli terapeutici di tale patologia, la cui incidenza e' aumentata a livello mondiale a tal punto da giustificare l ' utilizzo del termine epidemia. Nonostante una estesa bibliografia, e' evidente che le basi molecolari del DM non sono state sufficientemente studiate a livello di sistema. Materiali e Metodi Abbiamo sfruttato un sistema modello che mima gli eventi infiammatori occorrenti in vivo in pazienti diabetici. In particolare abbiamo analizzato il trascrittoma di 518 microRNA in cellule alfa e beta pancreatiche murine (alfaTC1 e betaTC1), mediante analisi High Throughput (HT) effettuata con TaqMan Low Density Arrays per la PCR Real-Time. Le cellule sono state analizzate sia a steady state che dopo induzione di apoptosi con un cocktail di citochine (IFN-gamma, IL-1beta, TNF-alfa) per diversi tempi di esposizione (24h e 48h). Sono stati considerati differenzialmente espressi (DE) i microRNA che presentavano variazioni nell ' espressione (Fold change) di almeno 3 volte (verso l ' alto o verso il basso) nei trattati rispetto ai relativi controlli (FC maggiore o uguale a 3 o minore o uguale a 0.33) e comuni a tre controlli endogeni (snoRNA135, snoRNA202 e MammalU6). I microRNA differenzialmente espressi ottenuti sono stati ulteriormente filtrati effettuando un Paired T-Test. Una successiva analisi in silico ci ha permesso di individuare i targets predetti e validati dei miRNA DE. Risultati e Discussione Le nostre analisi hanno permesso di verificare che l ' esposizione prolungata e ad alte concentrazioni delle linee cellulari murine di glucagonoma (alfaTC1) e di insulinoma (betaTC1) al cocktail di citochine pro-infiammatorie induce marcati cambiamenti nell ' espressione dei microRNA rispetto a quanto avviene nelle stesse cellule in condizioni non perturbate ed in misura maggiore nelle betaTC1 piuttosto che nelle alfaTC1. Abbiamo identificato, nel complesso, il 6,18% (32/518) di microRNA con espressione significativamente alterata in entrambe le linee cellulari (tra questi il miR-146a, il miR-21 e il miR-34a risultano essere gia' noti in letteratura come coinvolti nel processo di secrezione insulinica nelle beta cellule e nella loro proliferazione e apoptosi). Nelle alfaTC1 sono risultati differenzialmente espressi 12 miRNA, di cui 4 risultano essere specifici del time point a 24h, 6 del time point a 48h, e 2 presentano una disregolazione costante durante l ' intero time course. Nelle betaTC1 mostrano espressione differenziale 25 miRNA, di cui 2 risultano essere specifici del time point a 24h, 13 del time point a 48h, e 10 mostrano una espressione alterata sia a 24h che a 48h. La costante disregolazione nei due time point suggerisce che tali miRNA possano avere un importante ruolo nella cascata di segnali scatenata dalle citochine. Considerando l ' intero time course si nota che 7 miRNA sono alterati in maniera specifica nelle alfaTC1, 20 nelle betaTC1 e 5 (miR-125b-5p, miR-146a, miR-155, miR-203 e miR-21) sono sovraespressi in entrambi i fenotipi cellulari. L ' induzione di miRNA sia nelle alfaTC1 che nelle betaTC1 potrebbe rappresentare un meccanismo fisiologico di risposta all ' azione delle citochine comune fra due fenotipi cellulari che condividono la stessa origine endodermica. La nostra attenzione si e' rivolta in modo particolare ai miRNA che presentano una importante alterazione dell ' espressione in una linea rispetto all ' altra, considerando anche il confronto tra i due fenotipi cellulari a steady state; maggiore importanza e' stata data ai microRNA che mostrano variazione di espressione di segno opposto nelle due linee cellulari dopo trattamento, e livelli di espressione opposti nella stessa linea cellulare prima e dopo l ' esposizione a citochine. Da questo punto di vista miR-216a, miR-216b e miR-217 rappresentano microRNA particolarmente interessanti sui quali svolgere esperimenti di genomica funzionale ai fini di verificare un possibile coinvolgimento nel Diabete Mellito (in letteratura non e' attualmente riportata alcuna correlazione tra questi miRNA e la malattia). Inoltre alcuni tra i targets predetti e validati dei DE miRNA ottenuti mediante analisi in silico sono risultati appartenere al set di geni di seconda classe candidati per il Diabete ottenuto in un lavoro recentemente completato dal nostro gruppo di ricerca [Barbagallo et al., in preparazione]: tra questi Stat3 (bersaglio dei miR-125b-5p e miR-21) e Bbc3, target del miR-148a. Conclusioni I nostri dati hanno descritto per la prima volta l ' assetto molecolare dei microRNA nelle alfa cellule pancreatiche dopo loro esposizione ad un cocktail di citochine pro-infiammatorie, oltre che gettare le basi per una migliore comprensione dei meccanismi molecolari responsabili della morte delle beta cellule. Prospettive future di questo lavoro sono: l ' identificazione delle alterazioni di pathways e networks coinvolte dopo trattamento delle cellule in vitro; la validazione dei piu' credibili tra i microRNA candidati mediante trasfezione di anti- o di pre-miRNA per determinarne il silenziamento o l ' attivazione funzionale. Mediante l ' integrazione dei dati ottenuti verra' prodotta una lista completa di microRNA, dei loro potenziali bersagli proteici e delle pathways coinvolte nell ' alterazione del fenotipo cellulare e molecolare delle cellule alfa e beta pancreatiche, in un sistema in vitro che riproduce in modo credibile le condizioni che si verificano in vivo nei pazienti durante l ' insorgenza del Diabete Mellito.
Background MicroRNAs are in a critically hierarchic position within cellular networks, whose functioning they regulate: therefore, they are important candidates for being involved in the pathogenesis of complex systemic diseases like cancer and degenerative diseases. Accordingly, these molecules are unanimously considered the most promising new generation ' s biomarkers for diagnosis, prognosis and designing novel therapeutic interventions: it is easy to expect that the availability of batteries of well characterized microRNAs, closely associated to the disease of interest and present in the serum, would radically change the operating modes and the prospects of success in Clinical Medicine. The main aims of this project was to contribute to the characterization of microRNAs' role in the pathogenesis of Diabetes Mellitus (DM) and to identify and characterize a set of microRNAs to be used as molecular biomarkers and potential therapeutic targets of DM. The incidence of this disease is increasing worldwide, so that many authors have defined this phenomenon an epidemy. Despite the large volume of literature data on DM, it is evident that our knowledge of its pathogenetic mechanisms should be greatly expanded. Methods We used a model system that mimics the inflammatory events occurring in vivo in diabetic patients. In particular, we analyzed the transcriptome of 518 microRNAs in mouse pancreatic alpha and beta cells (alphaTC1 and betaTC1), by performing a High Throughput (HT) analysis with TaqMan Low Density Arrays (TLDA) for PCR Real-Time. We analyzed cells both at steady state and after inducing apoptosis with a cocktail of cytokines (IFN-gamma, IL-1beta, TNF-alpha) for different times of exposure (24h and 48h). We considered as up- or downregulated those miRNA that have a natural logarithm of expression fold change of at least 1 or greater and -1 or less, respectively. Data normalization was performed by using snoRNA135, snoRNA202 and MammalU6 as endogenous controls. Differentially expressed microRNAs (DE miRNAs) obtained were further filtered by performing a Paired T-Test. A subsequent in silico analysis allowed us to identify the predicted and validated targets of DE miRNAs. Results and Discussion Our experimental data demonstrate that prolonged exposure of alphaTC1 and betaTC1 to high concentrations of pro-inflammatory cytokines induces marked changes in miRNAs expression compared to the same cells under physiological conditions, and to a greater extent in betaTC1 than in alphaTC1. We identified 6.18% (32/518) of microRNAs with significantly altered expression in both cell lines (miR-146a, miR-21 and miR-34a had been previously reported to be involved in beta cells insulin secretion, proliferation and apoptosis). In alphaTC1 post treatment (PT) we found 12 differentially expressed miRNAs, 4 of which are specifically altered at 24h and 6 specifically altered at 48h, whereas 2 miRNAs show a constant dysregulation throughout the time course. In betaTC1 we observed that 25 miRNAs show differential expression post cytokines treatment, 2 of which are specifics of 24h time point, 13 are specifics of 48h and 10 show altered expression in both time points. The altered expression of these miRNAs at 24h and 48h PT in the 2 cell lines could suggest their important role within the signaling cascade triggered by cytokines. Considering the entire time course, 7 miRNAs are specifically altered in alphaTC1, 20 are specifically altered in betaTC1, and 5 miRNAs (miR-125b-5p, miR-146a, miR-155, miR-203 and miR-21) are over expressed in both cell phenotypes. The induction of some miRNAs in both alphaTC1 and betaTC1 could be a physiological response to cytokines action common to both cell phenotypes sharing the same endodermal origin. We have focused our attention particularly to miRNAs with an important altered expression in a cell line compared to the other, considering also the comparison between the two cell phenotypes at steady state; we gave more importance to microRNAs showed expression variation of opposite sign in the two cell lines after treatment, and opposite expression levels in the same cell line before and after cytokines exposure. From this point of view, miR-216a, miR-216b and miR-217 are particularly interesting DE miRNAs; these miRNAs will be selected to perform functional genomics experiments in order to verify a possible involvement in Diabetes (in literature is not currently reported any correlation between these miRNAs and Diabetes). In addition, some of the DE miRNAs predicted and validated targets obtained by in silico analysis were found to belong to the set of Diabetes candidate genes obtained in a work recently completed by our research group [Barbagallo et al., manuscript in preparation]: these include Stat3 (target of miR-125b-5p and miR-21) and Bbc3, target of miR-148a. Conclusions and Perspectives It is important to emphasize that this is the first HT profiling study of microRNAs in pancreatic alpha cells, both at steady state and after treatment with cytokines; we have also contribute to a better understanding of the molecular mechanisms responsible for beta cells death. Future aims of this project are to reconstruct through computational analysis the pathways and networks involved and to identify their alterations after apoptosis induction, and validate the most credible among candidates microRNAs by transfection of anti- or pre-miRNA to determine silencing or functional activation. Through the integration of these data we will obtain a complete list of microRNAs, their targets proteins and corresponding pathways, involved in alteration of the cellular and molecular phenotype of pancreatic alpha and beta cells, in an in vitro system that credibly reproduces the conditions that occur in vivo in patients during the onset and progression of Diabetes.

BACKGROUND & AIMS: Diabetes occurring as a direct consequence of loss of liver function is usually characterized by non-diabetic fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) levels and should regress after orthotopic liver transplantation (OLT). This observational, longitudinal study investigated the relationship between the time-courses of changes in all 3 direct determinants of glucose regulation, i.e., β-cell function, insulin clearance and insulin sensitivity, and diabetes regression after OLT. METHODS: Eighty cirrhotic patients with non-diabetic FPG and HbA1c levels underwent an extended oral glucose tolerance test (OGTT) before and 3, 6, 12 and 24 months after OLT. The OGTT data were analysed with a mathematical model to estimate derivative control (DC) and proportional control (PC) of β-cell function and insulin clearance (which determine insulin bioavailability), and with the Oral Glucose Insulin Sensitivity (OGIS)-2 h index to estimate insulin sensitivity. RESULTS: At baseline, 36 patients were diabetic (45%) and 44 were non-diabetic (55%). Over the 2-year follow-up, 23 diabetic patients (63.9%) regressed to non-diabetic glucose regulation, whereas 13 did not (36.1%); moreover, 4 non-diabetic individuals progressed to diabetes (9.1%), whereas 40 did not (90.9%). Both DC and PC increased in regressors (from month 3 and 24, respectively) and decreased in progressors, whereas they remained stable in non-regressors and only PC decreased in non-progressors. Insulin clearance increased in all groups, apart from progressors. Likewise, OGIS-2 h improved at month 3 in all groups, but thereafter it continued to improve only in regressors, whereas it returned to baseline values in the other groups. CONCLUSIONS: Increased insulin bioavailability driven by improved β-cell function plays a central role in favouring diabetes regression after OLT, in the presence of a sustained improvement of insulin sensitivity.

La poli(ADP-ribosilazione) è una modifica post-traduzionale del proteoma, che svolge un ruolo importante nell ambito di diverse funzioni cellulari (e.g., la regolazione della struttura della cromatina a steady state e durante lo sviluppo ed il differenziamento, la modulazione della espressione genica, i meccanismi di riparazione del DNA, l attivazione e la regolazione del processo apoptotico, la necrosi). La reazione è catalizzata dalle proteine PARP [Poly(ADP-ribose)Polymerases], una famiglia di enzimi che catalizzano l aggiunta di polimeri di poli(ADP-ribosio) alle proteine bersaglio, le cui attività molecolari e funzioni biologiche vengono di conseguenza modulate. Nell ambito di questo progetto di ricerca, abbiamo determinato la struttura genomica dei geni della famiglia PARP in Homo sapiens e in Mus musculus ed abbiamo analizzato la loro espressione in cellule alfa e beta del pancreas di topo, sia a steady state che dopo trattamento con citochine con la conseguente induzione di apoptosi: secondo un consenso generale, questo modello sperimentale consente di riprodurre in vitro alcuni dei fenomeni che sono correlati all insorgenza del Diabete Mellito (DM). I nostri risultati dimostrano una notevole attivazione della espressione di diverse PARP, soprattutto nelle cellule beta, e suggeriscono quindi un loro possibile ruolo nella patogenesi del DM. Per converso, questi stessi dati dovrebbero consentire anche una ulteriore definizione delle funzioni biologiche e del ruolo fisiologico di queste proteine, Infine, questi dati suggeriscono il possibile utilizzo di inibitori delle PARP per applicazioni di Medicina Traslazionale al trattamento clinico del Diabete Mellito.

The Cadmium-Zinc-Telluride 0-Neutrino-Double-Beta Research Apparatus (COBRA-Experiment) investigates the theoretically predicted neutrinoless double beta decay (0νββ-decay) to indirectly determine the effective Ma- jorana mass of the electron-neutrino by a measurement of the half-life of the 0νββ-decay using room-temperature semiconducting Cadmium-Zinc- Telluride-detectors (CZT). The detectors are made of elements containing several isotopes that decay via double beta decay (ββ-decay). In such a con- figuration the detector itself becomes the source of the decay and, hence, the efficiency for the detection of such events rises. This work covers the investigations and characterizations made on the CZT detectors used in the COBRA-Experiment, currently running. Prior to in- stallation the physical properties of the detectors are analyzed and during operation the stability of the detectors is monitored. For the laboratory analysis three dedicated setups are developed that allow for detailed inves- tigations of different properties of the detectors. Beside the working point determination and the analysis of the temperature dependence of the de- tector performance, the spatial detector response to localized irradiation is analyzed and a setup to generate a library of specific pulse shapes is designed and operated. Furthermore, an investigation for a possible discrimination of α- and β-decay events based on pulse shape discrimination is performed as well as an analysis of the long term stability of underground operated CZT detectors.