Hypocretin 1 and 2 (HCRT, also called Orexin A and B) are neuropeptides released by neurons in the lateral hypothalamus. HCRT neurons widely project to the entire neuroaxis. HCRT neurons have been reported to participate in various hypothalamic physiological processes including cardiovascular functions, wake-sleep cycle, and they may also influence metabolic rate and the regulation of body temperature. HCRT neurons are lost in narcolepsy, a rare neurological disorder, characterized by excessive daytime sleepiness, cataplexy, sleep fragmentation and occurrence of sleep-onset rapid-eye-movement episodes. We investigated whether HCRT neurons mediate the sleep-dependent cardiovascular adaptations to changes in ambient temperature (Ta). HCRT-ataxin3 transgenic mice with genetic ablation of HCRT neurons (n = 11) and wild-type controls (n = 12) were instrumented with electrodes for sleep scoring and a telemetric blood pressure (BP) transducer (DSI, Inc.). Simultaneous sleep and BP recordings were performed on mice undisturbed and freely-behaving at 20 °C, 25 °C, and 30 °C for 48 hours at each Ta. Analysis of variance of BP indicated a significance of the main effects of wake-sleep state and Ta, their interaction effect, and the wake-sleep state x mouse strain interaction effect. BP increased with decreasing Ta. This effect of Ta on BP was significantly lower in rapid-eye-movement sleep (REMS) than either in non-rapid-eye-movement sleep (NREMS) or wakefulness regardless of the mouse strain. BP was higher in wakefulness than either in NREMS or REMS. This effect of sleep on BP was significantly reduced in mice lacking HCRT neurons at each Ta, particularly during REMS. These data suggest that HCRT neurons play a critical role in mediating the effects of sleep but not those of Ta on BP in mice. HCRT neurons may thus be part of the central neural pathways which mediate the phenomenon of blood pressure dipping on passing from wakefulness to sleep.

Mental retardation in Down syndrome (DS) has been imputed to the decreased brain volume, which is evident starting from the early phases of development. Recent studies in a widely used mouse model of DS, the Ts65Dn mouse, have shown that neurogenesis is severely impaired during the early phases of brain development, suggesting that this defect may be a major determinant of brain hypotrophy and mental retardation in individuals with DS. Recently, it has been found that in the cerebellum of Ts65Dn mice there is a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting that failure of Shh signaling may underlie the reduced proliferation potency in DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. We found that the expression levels of the Shh receptor Patched1 (Ptch1) were increased compared to controls both at the RNA and protein level. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. We further found that the overexpression of Ptch1 in trisomic NPCs is related to increased levels of AICD, a transcription-promoting fragment of amyloid precursor protein (APP). Increased AICD binding to the Ptch1 promoter favored its acetylated status, thus enhancing Ptch1 expression. Taken together, these data provide novel evidence that Ptch1 over expression underlies derangement of the Shh pathway in trisomic NPCs, with consequent proliferation impairment. The demonstration that Ptch1 over expression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

La possibilità di indurre stati ipotermici ed ipometabolici come il torpore o l’ibernazione in animali non ibernanti può avere dei risvolti utili nella pratica medica, in quanto permetterebbe di trarre vantaggio dagli effetti benefici dell’ipotermia senza gli effetti compensatori negativi causati dalla risposta omeostatica dell’organismo. Con questo lavoro vogliamo proporre un nuovo approccio, che coinvolge il blocco farmacologico dell’attività dei neuroni nel bulbo rostroventromediale (RVMM), un nucleo troncoencefalico che si è rivelato essere uno snodo chiave nella regolazione della termogenesi attraverso il controllo dell’attività del tessuto adiposo bruno, della vasomozione cutanea e del cuore. Nel nostro esperimento, sei iniezioni consecutive del agonista GABAA muscimolo nel RVMM, inducono uno stato reversibile di profonda ipotermia (21°C al Nadir) in ratti esposti ad una temperatura ambientale di 15°C. Lo stato ipotermico/ipomentabolico prodotto dall’inibizione dei neuroni del RVMM mostra forti similitudini col torpore naturale, anche per quanto concerne le modificazioni elettroencefalografiche osservate durante e dopo la procedura. Come negli ibernati naturali, nei ratti cui viene inibito il controllo della termogenesi si osserva uno spostamento verso le regioni lente delle spettro di tutte le frequenze dello spettro EEG durante l’ipotermia, ed un forte incremento dello spettro EEG dopo il ritorno alla normotermia, in particolare della banda Delta (0,5-4Hz) durante il sonno NREM. Per concludere, questi risultati dimostrano che l’inibizione farmacologica selettiva di un nucleo troncoencefalico chiave nel controllo della termogenesi è sufficiente per indurre uno stato di psuedo-torpore nel ratto, una specie che non presenta stati di torpore spontaneo. Un approccio di questo tipo può aprire nuove prospettive per l’utilizzo in ambito medico dell’ipotermia. / The possibility to mimic hypomethabolic and hypothermic states like torpor or hibernation in non-hibernating animals may be useful in medical practice since the beneficial effects of deep hypothermia could be obtained without the concomitant elicitation of adverse autonomic compensatory responses. Until now, tentatives of induction of such states have been focused on the inhibition of cellular metabolism at systemic level but worked only in animals already able to enter torpor. Here we show a new approach, that involves the pharmacological blockade of the activity of the neurons within the Rostral Ventromedia Medulla (RVMM), a brainstem area that has been shown to be a key thermogenetic relay that controls the brown adipose tissue, the cutaneous blood vessels and the heart. In our experiment, six consecutive injections, aimed to block the thermogenetic drive by the activation of the GABAA receptors in RVMM neurons, induced a reversible deep hypothermia (21°C at nadir) state in free behaving rats exposed to 15°C ambient temperature. The hypothermic/hypometabolic state produced by prolonged inhibition of RVMM neurons strongly resembles the naturally occurring torpor, even in the EEG changes observed during and after the procedure. Like animals that spontaneously show hibernation or torpor, rats with the thermogentic drive blocked show a left-shift of all the EEG frequencies during the hypothermia, and a strong increase in EEG power spectrum after the return to thermoneutrality, in particular in the EEG Delta band (0.5-4.0 Hz) during NREM sleep. In conclusion, these results show that the selective pharmacological inhibition of a key brainstem area of the cold-defence pathway is sufficient in inducing a topor-like state in the rat, a species that does not enter torpor spontaneously. Such an approach may open new perspectives in the use of deep hypothermia in several medical conditions.

This thesis presents SEELF (Sustainable EEL fishery) Index, a methodology for evaluation of European eel (Anguilla anguilla) for the implementation of an effective Eel Management Plan, as defined by EU Regulation No.1100/2007. SEELF uses internal and external indices, age and blood parameters, and selects suitable specimen for restocking; it is also a reliable tool for eel stock management. In fact, SEELF Index, was developed in two versions: SEELF A, to be used in field operations (catch&release, eel status monitoring) and SEELF B to be used for quality control (food production) and research (eel status monitoring). Health status was evaluated also by biomarker analysis (ChE), and data were compared with age of eel. Age determination was performed with otolith reading and fish scale reading and a calibration between the two methods was possible. The study area was the Comacchio lagoon, a brackish coastal lagoon in Italy, well known as an example of suitable environment for eel fishery, where the capability to use the local natural resources has long been a key factor for a successful fishery management. Comacchio lagoon is proposed as an area where an effective EMP can be performed, in agreement with the main features (management of basins, reduction of mortality due to predators,etc.) highlighted for designation of European Restocking Area (ERA). The ERA is a new concept, proposed as a pillar of a new strategy on eel management and conservation. Furthermore, the features of ERAs can be useful in the framework of European Scale Eel Management Plan (ESEMP), proposed as a European scale implementation of EMP, providing a more effectiveness of conservation measures for eel management.

La malattia da reflusso gastroesofageo (GERD) si divide in due categorie: malattia non erosiva (NERD) ed erosiva (ERD). Questi due fenotipi di GERD mostrano caratteristiche patofisiologiche e cliniche differenti. NERD è la forma più comune. Anche se ERD e NERD sono difficili da distinguere a livello clinico, la forma NERD possiede caratteristiche fisiologiche, patofisiologiche, anatomiche, e istologiche uniche. La replicazione cellulare dello strato basale si pensa sia una delle cause implicate nella resistenza della mucosa e nella difesa strutturale dell’epitelio. Diversi studi hanno dimostrato che la proliferazione cellulare è ridotta nella mucosa esofagea esposta ad insulti acidi e peptici cronici, in pazienti GERD, in più uno studio recente ha dimostrato che il recettore per i cannabinoidi CB1 era implicato nella riparazione delle ferite nella mucosa del colon. Sulla base di questi dati abbiamo valutato la presenza del recettore CB1 in biopsie della mucosa esofagea, di pazienti ERD, NERD e di controlli sani, tramite analisi Western Blot, Immunoistochimica e Real-Time PCR, dimostrando per la prima volta la presenza di questo recettore nell’epitelio dell’esofago e una riduzione dei suoi livelli di espressione nei pazienti ERD, camparati con i NERD e con i controlli sani. Successivamente, per chiarire meglio i meccanismi molecolari che caratterizzano ERD e NERD, abbiamo effettuato un analisi proteomica con la tecnica shotgun, la quale ha evidenziato un patter proteico di 33 proteine differenzialmente espresse in pazienti NERD vs ERD, sette delle quali confermate in wester Blot, e quattro in immunoistochimica. Concludendo i nostri risultati hanno confermato che ERD e NERD sono due entità distinte a livello proteico, e hanno proposto dei candidati biomarker per la diagnosi differenziale di ERD e NERD. / Gastroesophageal reflux disease (GERD) falls into one of two categories: non-erosive reflux disease (NERD) or erosive reflux disease (ERD). The two main phenotypes of GERD show different pathophysiological and clinical characteristics. NERD is the most common phenotypic presentation among GERD. Although separation of ERD and NERD on a clinical level is difficult, there are physiological, pathophysiological, anatomical, and even histological characteristics that are unique to NERD. Cell replication of basal layers is hypothesized to be one of the causes implicated in the resistance of the mucosa and structural epithelial defense. Several studies demonstrated that cell proliferation was reduced in esophageal mucosa exposed to chronic acid-peptic insult, in patients with GERD. In addition, a recent study demonstrated that cannabinoid receptor 1 was implicated in colon mucosa wound healing. Based on these findings, we investigated the presence of CB1 receptors in biopsies of esophageal mucosa, collected from GERD patients and healthy subjects. The biopsies were analyzed by Western Blot, Immunoistochemistry and Real-Time PCR. Our results showed, for the first time, the presence of CB1 receptors in human esophageal epithelium. In addition, we demonstrated that the levels of CB1 mRNA and protein in ERD subjects showed a reduction in comparison with NERD patients. In order to elucidate molecular features that characterize NERD an ERD, a shotgun proteomics approach was assessed. The comparison of the protein patterns between NERD and ERD subjects demonstrated a differentially expression of 33 proteins, seven of them were confirmed by Western Blot analysis, and four of them by immunoistochemistry. In conclusion our results confirmed that NERD and ERD disease could be considered two distinct entities of the same pathology, at protein level. This study proposes an array of candidate biomarkers to discriminate between non-erosive and erosive reflux disease.

I microRNA sono una classe di piccole molecole di RNA non codificante che controllano la stabilità di numerosi RNA messaggeri, perciò sono considerati come “master regulator” dell’espressione genica. Ogni tumore è caratterizzato da un profilo di espressione alterato dei microRNA. Il miR-101 è un oncosoppressore represso nei tessuti tumorali ed è candidato come biomarcatore del cancro colon-rettale. È regolato da numerosi eventi fisiologici e patologici, come angiogenesi e carcinogenesi. Gli eventi molecolari coinvolti nella regolazione dell’espressione del miR-101 sono scarsamente conosciuti, poiché è trascritto da due loci genici non caratterizzati. L’obiettivo di questo lavoro è di caratterizzare i geni del miR-101 ed individuarne i regolatori molecolari coinvolti nella cancerogenesi colon-rettale. / MicroRNAs are a class of small, non-coding RNAs that control the stability of many messenger RNAs and serve as “master regulators” of gene expression. The alterations in the expression of microRNAs is a common feature of cancers. The oncosuppressor miR-101 is hardly expressed in tumor samples and is a candidate biomarker in colorectal cancer. It is regulated under a number of physiological and pathological conditions, including angiogenesis and tumors. Little is known about the molecular factor involved in the regulation of miR-101 expression, because it has 2 unknown genetic loci. The aim of this work is the characterization of miR-101 genes and shed light on its molecular regulators involved in colorectal carcinogenesis.

We usually perform actions in a dynamic environment and changes in the location of a target for an upcoming action require both covert shifts of attention and motor planning update. In this study we tested whether, similarly to oculomotor areas that provide signals for overt and covert attention shifts, covert attention shifts modulate activity in cortical area V6A, which provides a bridge between visual signals and arm-motor control. We performed single cell recordings in monkeys trained to fixate straight-ahead while shifting attention outward to a peripheral cue and inward again to the fixation point. We found that neurons in V6A are influenced by spatial attention demonstrating that visual, motor, and attentional responses can occur in combination in single neurons of V6A. This modulation in an area primarily involved in visuo-motor transformation for reaching suggests that also reach-related regions could directly contribute in the shifts of spatial attention necessary to plan and control goal-directed arm movements. Moreover, to test whether V6A is causally involved in these processes, we have performed a human study using on-line repetitive transcranial magnetic stimulation over the putative human V6A (pV6A) during an attention and a reaching task requiring covert shifts of attention and reaching movements towards cued targets in space. We demonstrate that the pV6A is causally involved in attention reorienting to target detection and that this process interferes with the execution of reaching movements towards unattended targets. The current findings suggest the direct involvement of the action-related dorso-medial visual stream in attentional processes, and a more specific role of V6A in attention reorienting. Therefore, we propose that attention signals are used by the V6A to rapidly update the current motor plan or the ongoing action when a behaviorally relevant object unexpectedly appears at an unattended location.

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Speeding the VO2 kinetics results in a reduction of the O2 deficit. Two factors might determine VO2 kinetics: oxygen delivery to muscle (Tschakovsky and Hughson 1999) and a muscle 'metabolic inertia' (Grassi et al. 1996). Therefore, in study 1 we investigated VO2 kinetics and cardiovascular system adaptations during step exercise transitions in different regions of the moderate domain. In study 2 we investigated muscle oxygenation and cardio-pulmonary adaptations during step exercise tests before, after and over a period of training. Study 1 methods: Seven subjects (26 ± 8 yr; 176 ± 5 cm; 69 ± 6 kg) performed 4 types of step transition from rest (0-50W; 0-100W) or elevate baseline (25-75W; 25-125W). GET and VO2max were assessed before testing. O2 uptake and were measured during testing. Study 2 methods: 10 subjects (25 ± 4 yr; 175 ± 9 cm; 71 ± 12 kg) performed a step transition test (0 to 100 W) before, after and during 4 weeks of endurance training (ET). VO2max and GET were assessed before and after of ET (40 minutes, 3 times a week, 60% O2max). VO2 uptake, Q and deoxyheamoglobin were measured during testing. Study 1 results: VO2 τ and the functional gain were slower in the upper regions of the moderate domain. Q increased more abruptly during rest to work condition. Q τ was faster than VO2 τ for each exercise step. Study 2 results: VO2 τ became faster after ET (25%) and particularly after 1 training session (4%). Q kinetics changed after 4 training sessions nevertheless it was always faster than VO2 τ. An attenuation in ∆[HHb] /∆VO2 was detectible. Conclusion: these investigations suggest that muscle fibres recruitment exerts a influence on the VO2 response within the moderate domain either during different forms of step transition or following ET.

Bone remodelling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton to mechanical loads. Skeletal unloading leads to severe hypoxia (1%O2) in the bone microenvironment resulting in imbalanced bone remodelling that favours bone resorption. Hypoxia, in vivo, is a physiological condition for osteocytes, 5% O2 is more likely physiological for osteocytes than 20% O2, as osteocytes are embedded deep inside the mineralized bone matrix. Osteocytes are thought to be the mechanosensors of bone and have been shown to orchestrate bone formation and resorption. Oxygen-deprived osteocytes seem undergo apoptosis and actively stimulate osteoclasts. Hypoxia and oxidative stress increase 150-kDa oxygen-regulated protein (ORP 150) expression in different cell types. It is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. It well known that ORP 150 plays an important role in the cellular adaptation to hypoxia, as anti-apoptotic factor, and seems to be involved in osteocytes differentiations. The aims of the present study are 1) to determine the cellular and molecular response of the osteocytes at two different conditions of oxygen deprivation, 1% and 5% of O2 compared to the atmospheric oxygen concentration at several time points. 2) To clarify the role of hypoxic osteocytes in bone homeostasis through the detection of releasing of soluble factors (RANKL, OPG, PGE2 and Sclerostin). 3) To detect the activation of osteoclast and osteoblast induced by condition media collected from hypoxic and normoxic osteocytes. The data obtained in this study shows that hypoxia compromises the viability of osteocytes and induces apoptosis. Unlike in other cells types, ORP 150 in MLO-Y4 does not seem to be regulated early during hypoxia. The release of soluble factors and the evaluation of osteoclast and osteoblast activation shows that osteocytes, grown under severe oxygen deprivation, play a role in the regulation of both bone resorption and bone formation.