Дисертації з теми "Modello animale di iperossia"

the serpins are a family of serine protease inhibitors involved in several biological functions and in the maintenance of cell homeostasis. SERPINB3 (known as SCCA1), a member of the ovalbumin family, is normally expressed in squamous epithelium, and it is over-expressed in tumours of epithelial origin and in hepatocellular carcinoma. The SERPINB3 involvement in the control of proteolytic processes has important implications in neoplastic transformation, whereas the balance between proteases and their inhibitors can affect cell motility, invasiveness, proliferation and apoptosis. Considering the potential role of SERPINB3 and the lack of human biological tissues for clinical research, the possibility to use a transgenic animal model to explore in vivo the role of this serpin in several cell processes could be of great value. The aim of the present study was to assess the SERPINB3 involvement in several pathologies by a transgenic mouse model for human SERPINB3, expressed in different organs as brain, lung and liver.
Le serpine sono una famiglia di inibitori delle proteasi seriniche implicata in molte funzioni biologiche e nei processi di controllo dell’omeostasi cellulare. SERPINB3 (chiamata anche SCCA1), appartenente alle ov-serpine, è espressa normalmente negli epiteli squamosi, ma si trova iper-espressa nelle cellule neoplastiche di origine epiteliale e nell’epatocarcinoma. Il coinvolgimento di SERPINB3 nella regolazione dei processi proteolitici ha importanti implicazioni a livello dei processi neoplastici, dal momento che l’equilibrio tra le proteasi ed i loro inibitori, può influenzare la mobilità, l’invasività, la proliferazione e la morte cellulare stessa. Dato il potenziale ruolo di SERPINB3 ed il limite determinato dalla disponibilità di materiale biologico per la ricerca clinica sull'uomo, risulta importante poter utilizzare un modello animale che permetta di sperimentare in vivo il ruolo che la proteina può rivestire in molteplici processi cellulari. Lo scopo dello studio è quello di sperimentare il coinvolgimento della serpina in diverse patologie utilizzando un modello animale, costituito da un topo transgenico per SERPINB3 umana, caratterizzato dall’espressione della serpina in diversi organi quali il cervello, i polmoni ed il fegato.

Bone, a specialized connective tissue, consists of cells and mineralized extracellular matrix. The main cell types of bone tissue are: the osteoblasts, the osteocytes and the osteoclasts. Osteoblasts produce extracellular matrix, osteoclasts are responsible of its resorption, hence bone physiology is a delicate balance between synthesis of new bone and resorption of the old one. Osteoporosis is a disease in which catabolic activity of osteoclasts overtakes anabolic activity of osteoblasts leading to increased bone resorption and progressive bone fragility. Primary osteoporosis is a common disease among post-menopausal female population. Pathologies as diabetes mellitus, hyperparathyroidism and long-term treatment with glucocorticoids cause secondary osteoporosis. Glucocorticoid-induced osteoporosis is the most common type of secondary osteoporosis. Glucocorticoid treatment is a well known method to induce osteoporosis in animal models, hence it could be an example of “translational model” in which injured bone could be repopulated by stem cells or progenitors in clinical trials. The aim of this thesis is to investigate whether preosteoblasts could repopulate injured bone in an animal model treated with glucocorticoids. Preosteoblasts have been isolated from newborn calvariae of GFP mice. In these cells, expression of the osteogenic marker Runx2 has been assessed by Real time PCR, while osteogenic potential has been analysed by cytochemistry assays to detect alkaline phosphatase and mineralized bone nodules (Alizarin Red and Von Kossa staining). To realize the in vivo model, C57BL/6 three months aged mice have been divided into three groups [group I (n=4): mice not treated with drug and not infused with cells, group II (n=4): mice treated with drug and not infused with cells, group III (n=4): mice treated with drug and infused with cells]. Drug (methylprednisolone) has been administered for one month with a dose of 75 mg/Kg/week. In mice of group III, 5 x 105 GFP preosteoblasts, previously expanded in vitro, have been infused with injection into the tail vein. Mice have been sacrificed, tibial and femoral bones have been harvested, processed and analysed by istomorphometry and immunoistochemistry. Expression of Runx2, osteonectin (SPARC) and alkaline phosphatase (ALP) in these tissues has been detected with Real time PCR. In vitro preosteoblasts produce alkaline phosphatase during early time in culture with normal medium, while the level decreases in differentiating conditions with medium containing ascorbic acid and β-glycerophosphate. Preosteoblasts maintained in differentiation medium for 30 days are positive to Alizarin and Von Kossa staining, hence they are able to produce mineralized extracellular matrix that is a feature of functional mature osteoblasts. Runx2 expression increases during differentiating conditions; in cells maintained in differentiation medium for 30 days there is an increase of 50% compared to cells maintained in normal medium (p<0.05). In mice of group III an increased level of parameters concerning osteoid has been detected (O.Th, OS/BS, OV/BV) and an increased number of active osteoblasts (during synthetic activity) has been observed compared to group II. Between these two groups, significant variations of bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) have not been detected. Microarchitecture parameters (Nd.N/TV, Nd/Tm) have not been affected. Similar results have been obtained from inderect microarchitecture parameters as Marrow Star Volume and Fractal Dimension. Real time PCR analysis revealed a reduction in osteogenic gene expression in group II compared to group I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05). In group III there is a recovery of expression of osteogenic markers (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%; p<0.01) compared to group II. Immunoistochemistry is under investigation. In our glucocorticoid-induced osteoporosis model we sacrificed mice only one week after infusion of cells, this preparatory investigation shows that our model induces the engraftment of preosteoblasts in injured bone. However, a longer time, at least of 1-2 months, is needed to investigate if preosteoblasts are able not only to graft onto host tissue, but also to proliferate in vivo and to differentiate in full mature and functional osteoblasts.
L’osso è un tessuto connettivo specializzato costituito da cellule e matrice extracellulare mineralizzata. Le cellule principali sono gli osteoblasti, gli osteociti e gli osteoclasti. Gli osteoblasti depongono la matrice ossea, mentre gli osteoclasti sono i responsabili del suo riassorbimento. La fisiologia dell’osso è quindi il risultato di un delicato equilibrio tra deposizione di matrice ossea e suo riassorbimento. Quando l’azione catabolica degli osteoclasti è maggiore rispetto a quella anabolica degli osteoblasti si produce una progressiva fragilità ossea che porta ad un quadro clinico osteoporotico. L’osteoporosi primaria colpisce soprattutto la popolazione femminile dopo la menopausa. Molte patologie come il diabete mellito, l’iperparatiroidismo ed il trattamento a lungo termine con glucocorticoidi causano l’osteoporosi secondaria. L’osteoporosi indotta da glucocorticoidi è la più comune causa di osteoporosi secondaria. Il trattamento con glucocorticoidi è un noto procedimento di induzione dell’osteoporosi in modelli animali e può dunque rappresentare un primo esempio di modello “traslazionale” potenzialmente applicabile in clinica per indurre un ripopolamento dell’osso con cellule staminali mesenchimali o precursori osteogenici. Lo scopo di questo lavoro è stato pertanto valutare nel modello animale se sia possibile ripopolare l’osso danneggiato con preosteoblasti. I crani di topi neonati transgenici (GFP) sono stati prelevati e messi in coltura per ottenere preosteoblasti. Nelle colture in vitro è stata valutata l’espressione del gene Runx2 con la tecnica di Real time PCR, mentre la capacità osteogenica è stata analizzata con colorazioni citochimiche per la fosfatasi alcalina e per la deposizione di matrice ossea mineralizzata (Alizarin Red e Von Kossa). Per la realizzazione del modello in vivo topi C57BL/6 maschi di 3 mesi sono stati divisi in 3 gruppi [gruppo I (n=4): topi non trattati con farmaco e non infusi con cellule; gruppo II (n=4): topi trattati con farmaco non infusi con cellule; gruppo III (n=4): topi trattati con farmaco ed infusi con cellule]. Il farmaco (metilprednisolone) è stato somministrato per un mese alla dose di 75 mg/Kg/settimana. Negli animali appartenenti al gruppo III sono state infuse, attraverso iniezione nella vena della coda, 5 x 105 preosteoblasti GFP precedentemente espansi in vitro. I topi sono stati sacrificati, le tibie ed i femori sono stati prelevati e processati per l’analisi istomorfometrica e della microarchitettura ossea e per l’ immunoistochimica. In questi tessuti, l’espressione genica di Runx2, osteonectina (SPARC) e fosfatasi alcalina (ALP) è stata valutata tramite Real time PCR. In vitro i preosteoblasti producono fosfatasi alcalina durate i primi giorni di coltura in medium non differenziante, mentre il livello decresce in condizioni differenzianti, cioè in medium contenente acido ascorbico e β-glicerofosfato. I preosteoblasti mantenuti in medium di differenziamento per 30 giorni sono positivi alle colorazioni Alizarin Red e Von Kossa, quindi sono in grado di produrre matrice ossea mineralizzata, caratteristica degli osteoblasti funzionali e maturi. L’espressione del gene Runx2 aumenta durante il differenziamento, si ha un aumento del 50% nelle cellule differenziate per 30 giorni rispetto alle cellule non differenziate (p<0.05). L’inoculazione dei preosteoblasti nei topi del gruppo III ha evidenziato un aumento dei parametri statici di neoformazione ossea relativi all’osteoide (O.Th, OS/BS, OV/BV) ed un aumento del numero di osteoblasti attivi, cioè in corso di deposizione di osteoide, rispetto al gruppo II. Tra questi due gruppi non si sono osservate, invece, variazioni significative in termini di volume osseo (BV/TV), spessore trabecolare (Tb.Th) numero delle trabecole (Tb.N) e separazione fra esse (Tb.Sp). Non sono state rilevate, inoltre, differenze dei parametri di microarchitettura (Nd.N/TV, Nd/Tm). Risultati simili sono emersi dalla valutazione dei parametri indiretti di microarchitettura (Marrow Star Volume e Fractal Dimension). L’espressione genica ha dimostrato che nel gruppo II si ha una riduzione dell’espressione dei geni osteogenici rispetto al gruppo I (ALP: -50%, p<0.01; Runx2: -56.75%, p<0.01; SPARC: -44.5%, p<0.05). Nel gruppo III si è avuto un recupero dell’espressione dei geni osteogenici (ALP: +40%, p<0.05; Runx2: +66.28%, p<0.001; SPARC: +55%, p<0.01) rispetto al gruppo II. I campioni di tessuto per l’ immunoistochimica devono essere processati. Nel nostro modello sperimentale di osteoporosi indotta da glucocorticoidi nel topo, abbiamo sacrificato gli animali solo una settimana dopo l’infusione delle cellule; questi dati preliminari dimostrano che il nostro modello induce l’engraftment dei preosteoblasti nell’osso danneggiato. Tuttavia è richiesto un tempo di osservazione più lungo, di almeno 1-2 mesi per valutare se le cellule trapiantate siano in grado, non solo di integrarsi nel tessuto dell’ospite, ma anche di proliferare in vivo e di differenziare in osteoblasti maturi e funzionali.

The establishment of a correlation between behavior and structure changes is essential to understand disorders diagnosed on the bases of “behavioral” disturbances. Among these Parkinson’s Disease (PD) has been traditionally considered as a "pure" movement disorder secondary to focal degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra pars compacta (SNc). However, in recent years the clinical phenotype has been revised, showing that PD is a multisystem neurodegenerative disorder with motor and non-motor features showing different time evolutions. Several animal models have been employed in experimental studies concerning the pathogenetic mechanism of PD and therapeutic strategies. A commonly used animal model of PD is the unilateral injection of 6-hydroxydopamine (6-OHDA) into the SNc of rodents capable of inducing a stable nigrostriatal lesion within 3 weeks. The time-course of the anatomical, metabolic and behavioral changes that occur at the late-phase has been extensively studied. However, despite a large body of evidence has been provided about the 6-OHDA PD model, some crucial questions concerning the early-phase PD lesioned-rat behavior remain unanswered. Indeed, it is very interesting to study the early molecular and functional changes time-course observed in the emergence of the Parkinsonian lesion in the standard 6-OHDA-induced by high-DAergic depletion model and whether they might be predictive of the severity of the lesion. In this PhD thesis we investigated the early-phase time course of behavioral and structural changes in the nigrostriatal pathway during the degeneration of SNc neurons induced by intranigral injection of 6-OHDA (8 μg/4 μL) in the right hemisphere. In particular, we investigated if early (1 to 7 days post-lesion) motor behavioral deficits can be fully characterized and whether they may be predictive of the severity of the DAergic lesion at later time points (14, 21 days post lesion). To determine the time-course of the emergence of potential spontaneous motor deficits, we modified the Tail Suspension Test, here indicated as Tail Suspension Swing Test (TSST).We compared the TSST results to those obtained with the standard rotation test in the Open Field Test (OFT).We recorded spontaneous and apomorphine-induced behavioral asymmetries using a modified implementation of the TSST and the standard OFT, before and following 6-OHDA injection. The accuracy of the 6-OHDA lesion was evaluated by means of ex vivo whole-brain high-resolution Magnetic Resonance Imaging (MRI). The DAergic lesion was then evaluated by immunohistochemical staining for tyrosine hydroxylase (TH) to detect TH-immunoreactive (TH-ir) neurons and fibers in the SNc and the striatum(ST), respectively. Moreover, we studied the behavioral features of the animals after 21 days of 6-OHDA-induced lesion of the DAergic nigrostriatal system, providing a T-Pattern Analysis (TPA) of the sequential organization of the behavior, in terms of sequences detected and features. Our main experimental result is that TSST is more sensitive to reveal the spontaneous motor asymmetries. On the other hand, the results of the TSST and OFT tests after acute and repetitive apomorphine treatment, well correlated with immunohistochemical striatal DA staining and nigral DAergic neuronal loss seen at the different time-points. The TSST can therefore be useful for the selection of those animals to study early pathophysiological mechanisms, that occur from the first day after the 6-OHDA lesion and new neuroprotective strategies without the confounding apomorphine administration. Moreover, our TPA results at 21 days post-lesion show for the first time that the temporal structure and fine characteristics of behavioral sequencing are altered in the late phase of the PD rodent model obtained with a unilateral SNc injection of 6-OHDA.

Levodopa-induced dyskinesia (LID) is a disabling motor complication occurring in Parkinson's disease patients (PD) after long-term l-DOPA treatment. Although its etiology remains unclear, there is accumulating evidence that LID relies on an excessive dopamine receptor transmission, particularly at the downstream signaling of D1 receptors. We previously reported that the inhibition of 5-alpha reductase (5AR), the rate limiting enzyme in neurosteroids synthesis, rescued a number of behavioral aberrations induced by D1 receptor-selective and non-selective agonists, without inducing extrapyramidal symptoms. Thus, this study was aimed at investigating whether the pharmacological blockade of 5AR by the two prototypical irreversible inhibitors finasteride (FIN) and dutasteride (DUTA), might elicit antidyskinetic properties in a rodent model of Parkinson's disease. Specifically, we tested the effects of FIN and DUTA on dyskinesias induced by dopaminergic agonists in 6-hydroxydopamine (6-OHDA)-lesioned rats. Acute and chronic effect of different doses of FIN (30-60mg/kg) and DUTA (15-30 mg/Kg), was assessed on LID in male 6-OHDA-lesioned dyskinetic rats. Thereafter, to fully characterize the therapeutic potential of these inhibitors on LID and their impact on l-DOPA efficacy, we tested abnormal involuntary movements and forelimb use in hemiparkinsonian male rats chronically injected with FIN (30-60mg/kg/24days) and DUTA (30-60mg/kg/24days) either prior to- or concomitant with l-DOPA administration. In addition, to investigate whether the antidyskinetic properties on LID may be ascribed to a modulation of the D1- or D2/D3-receptor function, dyskinesias were assessed in l-DOPA-primed 6-OHDA-lesioned rats that received FIN in combination with selective direct dopaminergic agonists. Finally, we set to investigate whether FIN may produce similar effect in female hemiparkinsonian rats, as seen in males. The results indicated that both FIN and DUTA administrations significantly dampened LID in all tested treatment regimens, without interfering with the ability of l-DOPA to ameliorate forelimb use in the stepping test. The antidyskinetic effect appears to be due to modulation of both D1- and D2/D3-receptor function, as FIN also reduced abnormal involuntary movements induced by the selective D1 receptor agonist SKF-82958 and the D2/D3 receptor agonist ropinirole. Significant dampening of LID was also observed in female rats, although only at the higher tested dose. To our knowledge, these findings for the first time highlight 5AR enzyme as a new therapeutic target for the treatment of dyskinesia in PD. Clinical investigations are warranted to assess whether similar protection from dyskinesia might be reproduced also in PD patients.

La sindrome dell’ovaio policistico (PCOS) è un comune disordine endocrinologico e riproduttivo che si riscontra nel 6-10% della popolazione femminile. In generale, è considerata una malattia metabolica multifattoriale caratterizzata da varie manifestazioni cliniche quali iperandrogenismo, ovaie policistiche e disfunzioni ovulatorie, che la rendono la causa più comune di infertilità da anovulazione nelle donne, ma anche da problemi metabolici come obesità, insulino-resistenza, iperinsulinemia e diabete di tipo II, che la associano a complicanze cardiovascolari, neurologiche e psicologiche, come ansia e depressione. Come è emerso recentemente, un aumento di AGE ha un ruolo chiave nelle disfunzioni ovariche e nella riduzione della fertilità associata alla PCOS. I risultati presentati in questa tesi dimostrano, per la prima volta, che una condizione di stress glicativo MG-dipendente si instaura nell’ambiente ovarico di topi PCOS. Questa condizione risulta associata a cambiamenti della funzione di SIRT1 nella regolazione della fisiologia mitocondriale e della sopravvivenza cellulare. Nel nostro studio, ci siamo basati su un modello ben studiato di topo PCOS indotto da DHEA e abbiamo approfondito la caratterizzazione del micro-ambiente ovarico nei topi DHEA. Abbiamo studiato, inoltre, l’efficacia di diverse formulazioni di carnitine (L-Carnitina, Acetil-L-Carnitina e Propionil-L-Carnitina) su un modello in vitro murino, sottoposto a danno ossidativo, noto essere una delle possibili cause della ridotta qualità ovocitaria associata alla PCOS. Infine è stata valutata l’efficacia di queste formulazioni di carnitine sui topi DHEA. Le carnitine sono essenziali nel metabolismo degli acidi grassi e possono agire proteggendo dal danno mitocondriale e da condizioni di bilancio energetico alterato come quelle presenti nella sindrome dell'ovaio policistico (PCOS), evidenziate anche dai ridotti livelli di L-carnitina nel siero delle pazienti affette da questa patologia. Ripristinare l'equilibrio energetico e fornire adeguate riserve di energia all’ovocita durante la follicologenesi e la maturazione può rappresentare un’importante strategia per migliorare l’ambiente intraovarico e aumentare la probabilità di gravidanza. In questo contesto, composti metabolici, come le carnitine, con effetti positivi sull’attività mitocondriale e sullo scavenging dei radicali liberi, possono contribuire a mitigare gli effetti di questa sindrome.

Objectives: The isthmic coarctation of the aorta accounts for 5-7% of all congenital heart diseases. Despite a full anatomic correction, almost 20-40% of the patients develop systemic hypertension later in their lives. Long-term follow-up studies demonstrate a reduced life expectancy for the patients born with an aortic coarctation because of increased risk for coronary artery disease and cerebral vascular incidents. The pathogenesis of systemic hypertension following a coarctation repair seems to be related to the inappropriate response of the aortic baroceptors to the increased stiffness of the ascending aorta, which is present in every patient affected by an aortic coarctation. The systemic hypertension, increasing the afterload, determines hypertrophy of the myocytes of the left ventricle. The reduced contractility of the hypertrophied myocytes is responsible for driving new hypertophy which eventually is responsible for heart failure. Besides surgical therapies for relieving of the isthmic coarctation, nowadays the employment of endovascular stents have been successful in treating adolescents and adults affected by an aortic coarctation. Despite the extremely encouraging acute results of these endovascular techniques, no data are available regarding the long term results. Few data are available regarding the hemodynamic effect secondary to the presence of an stent and no data are available regarding the possible endotelial dysfunction secondary to the flow disturbance caused by the stent. Aim of this animal study is to understand the hemodynamic impact of a bare metal stent in the isthmic region and how the disturbance of the normal laminar flow at the level of the stent might by responsible for dysfunction of the endothelium. Materials and methods: Eight female sheep of the same breed have been employed in this animal study. Between three to five months of age all the animals have been catheterized and in four of them a bare metal stent have been implanted in the aortic isthmus. At the time of the first catheterization all the animals underwent a full echocardiographic examination collecting data regarding the systolic and diastolic functionality of the left ventrcle as well as data of its dimension and thickness. All the animals have been followed every three months for a total of twelve months after the first catheterization. Every three months the blood pressure of each animals has been checked as well a new echocardiographic evaluation has been performed. At the time of the follow-up windows blood has been drawn and stored for subsequent analysis of the renin-angiotensin-aldosterone levels and kidney function. At the end of the twelve months a new catheterization was performed collecting two different subsets of hemodynamic data: baseline and under simulated stress condition by continous infusion of Isopenaline titrated up to 10 mcgs/kg/min. After this catheterization each animals were sacrificed in order to collect tissue samples of the ascending and descending aorta and the heart for microscopic and molecular biology evaluations. Results: Since the beginning of the study in June 2008, a total of twelve animals have been catheterized and in eight of them a bare metal stent was successfully implanted. Three of the eight sheep in which a stent was implanted didn't survived: one animal died during the procedure because of aortic dissection, and two animals died in the first week of follow-up because of sepsis. Another animal was excluded form the study because it developed severe aortic insufficiency duirng the follow-up. Eight animals completed the twelve months of follow-up and were sacrificed. At the end of the follow-up there were no statistical differences in the weigh and growth between the sham and the stented animals as no differences were recorded in terms of blood pressure. From an echocardiographic point of view the two groups didn't show any statistical difference in terms of LV hypertrophy and function. The hystological analysis failed to show any difference in terms of hypertrophy and fibrosis of the left ventricle and in the architecture of the ascending and descending aorta between the sham and operated animals. Since the two groups of animals didn't differ in any of the variables analyzed, we decided to not analyze the renin-angiotensin-aldosterone levels since we didn't expect any difference between the two groups. The analysis of the expression of genes involved in the oxydative stress (MMP-9 and Caspase-3) failed to demonstrate any statistical significance between the two groups of animals. Conclusions: In this animal model the presence of a rigid metallic stent doesn't seem to impact the homeostatic regulation of the cardiovascular system and it isn't responsible for the development of systemic arterial hypertension in a growing animal. Despite a trend of significance in the different expression of the MMP-9 in the aorta of the aninals with the stent compared to the sham ones, we cannot conlude the stent is responsible for the development of endothelial dysfunction. The main limitations of this study are the small size of the sample, the limited time of follow-up and the different structure of the aorta of the sheep compared to the aorta of humans affected by coarctation of the aorta. Our results might open new insights into the understanding of the development of the systemic hypertension after a coarctation repair. The increased stiffness of the aorta of these subjects might be secondary to the underline presence of endothelial dysfunction especially in those subjects corrected with implantation of a stent. Further studies are needed to confirm our hypothesis
Obiettivi: La coartazione istmica dell'aorta rappresenta il 5-7% di tutte le cardiopatie congenite. Nonostante un'efficace correzione anatomica, circa il 20-40% dei pazienti sviluppa un'ipertensione tardiva durante il follow-up. Studi di follow-up a lungo termine hanno dimostrato in questi malati una ridotta aspettanza di vita per un'aumentata incidenza di malattia coronarica e incidenti cerebro-vascolari. L'eziopatogenesi dell'ipertensione tardiva sembra essere l'anomala risposta dei brocettori aortici conseguente all'aumentata rigidità dell'aorta ascendente presente sin dalla nascita in tutti i soggetti con coartazione aortica. L'ipertensione arteriosa determinando un incremento dell'afterload del ventricolo sinistro causa ipertrofia dei miociti ventricolari che riducendo la loro capacità contrattile innesca un circolo vizioso che porta a scompenso cardiaco. Al giorno d'oggi l'impianto di stent endovascolari ha sostituito le tradizionali tecniche chirurgiche nella correzione della coartazione aortica degli adolescenti e adulti. Nonostante gli eccellenti risultati immediati, non sono ancora disponibili dati certi del follow-up di questi pazienti a lungo termine. Sebbene sia stato dimostrato che la presenza di uno stent in regione istmica non sia in grado di alterare la compliance aortica, nulla si sa riguardo la possibile induzione di disfunzione endoteliale da alterazione del flusso ematico laminare a livello dell'interfaccia tra stent e aorta nativa. Scopo del presente progetto di ricerca è studiare gli effetti emodinamici conseguenti all'impianto di uno stent sia in termini pressori che in termini di disfunzione endoteliale. Materiali e Metodi: Nel progetto sperimentale sono state impiegate otto pecore femmina di raazza Alpagota. A un'età compresa tra tre e cinque mesi tutti gli animali sono stati sottoposti a un cateterismo cardiaco e in quattro è stato impiantato uno stent in regione istmica. Tutti gli animali sono stati sottoposti a prima dello studio emodinamico a un'ecocardiografia completa per la determinazione della funzionalità sistolica e diastolica del ventricolo di sinistra così come per la determinazione dei volumi e spessori ventricolari. A intervalli di tre mesi e per un totale di dodici mesi ciascun animale è stato sottoposto a regolari controlli ecocardiografici. A ciascun follow-up è stato poi prelevato un campione ematico per una successiva analisi dei livelli plasmatici della renina-angiotensina-aldosterone. Al completamento dei dodici mesi di follow-up è stato ripetuto un nuovo studio emodinamico per la determinazione invasiva della pressione arteriosa di base e dopo stress test mediante infusione di dobutamina sino al dosagggio massimo di 10 mcg/kg/min. Terminato il cateterismo cardiaco ciascun animale è stato sacrificato e sottoposto a studio autoptico per il prelevamento di campioni tissutali dell'aorta ascendente e discendente e del miocardio del ventricolo di sinistra e dei reni per studi istologici e di biologia molecolare. Risultati: Dall'inizio dello studio nel giugno del 2008, un totale di dodici animali sono stati sottoposti a studio emodinamico e in otto è stato impiantato un stent metallic in regione istmica. Delle otto pecore in cui uno stent è stato impiantato, una è deceduta in sede operatoria per dissezione aortica, due sono deceduti nella prima settimane di follow-up per sepsi, una è stata esclusa dallo studio per lo sviluppo di un sevra insufficienza aortica durante il follow-up. Otto animali hanno completato I dodici mesi di follow-up e sono stati sacrificati. I due gruppi di animali non hanno mostrato differenza statisticamente signnificative né in termini del comportamento pressorio né in termini di funzionalità e volumetrica del ventricolo di sinistra. A livello istologico non abbiamo riscontrato differenze in termini di fibrosi e ipertrofia del ventricolo di sinistra e nell'architettura della parete aortica. I livelli plasmatici degli ormoni renina-angiotensina-aldosterone non sono stati determinati vista la mancanza di differenza statisticamente significative delle variabili esplorate. L'analisi dell'espressione genica dei geni attivati in condizioni di stress ossidativo (MMP-9 e Caspasi-3) non ha evidenziato differenze nei due gruppi. Conclusioni: I risultati di questo progetto sperimentale sembrerebbero suggerire l'ininfluenza della presenza di uno stent metallico sull'omeostasi pressoria. Nonostante vi siano delle differenze nei livelli di espressività delle MMP-9 a livello dell'aorta degli animali portatori di stent, queste differenze, verosimilmente a causa della bassa numerosità del campione oggetto di studio, non si sono dimostrate statisticamente significatieve. I limiti maggiori del nostro progetto sperimentale risiedono nella bassa numerosità del campione di studio, nel periodo limitato di follow-up e nelle differenze strutturali esistenti tra l'aorta degli ovini e l'aorta dei soggetti affetti da coartazione aortica. L'irrigidimento aortico caratteristico dei soggetti nati con coartazione aortica potrebbe essere secondario alla presenza di una disfunzione endoteliale e la presenza di uno stent in aorta potrebbe essere motivo di aggravamento di tale alterazione. Ulteriori studi saranno necessari al fine di valutare l'appropriatezza delle nostre ipotesi

Introduction: Cardiac fibrosis is a pathological response that causes abnormalities in cardiac conduction and mechanical function, thereby contributing to the pathophysiology of a variety of cardiac conditions, including hypertrophy, failure, and arrhythmias. Atrial fibrillation (AF) is the most common sustained clinical arrhythmia and is a major cause of population morbidity and mortality. Recent studies have demonstrated that structural remodeling, involving prominent fibrotic changes, is a fundamental determinant of the perpetuation of AF, and contributes synergistically with electrical remodeling to the AF substrate. Atrial fibrosis is a hallmark feature of arrhythmogenic structural remodeling in clinical AF. Increased amounts of fibrous tissue occur not only in AF patients with identifiable cardiac diseases, but also in those with lone AF.There is a positive correlation between the amount of fibrosis and the persistence of AF,7 suggesting that AF may itself cause structural remodeling that in turn promotes AF. Evidence supporting this idea comes from animal studies showing that even when the ventricular rate is well-controlled, rapid atrial activation causes atrial fibrosis,9 and from work indicating that rapidly-activated atrial-derived cardiomyocytes secrete substances that enhance collagen synthesis by atrial fibroblasts. Methods We studied 60 male adult inbred Fisher rats. We divided the animals in 3 groups of 20 rats each. The first group received vagal stimulation and induce atrial fibrillation protocol (AF + VNS) and follow for 60 days, after 24 h 10 rats were treated with angiotensin II drugs for 60 days (AF + VNS + Ang II). The second group received vagal stimulation (VNS) and follow for 60 days, after after 24 h 10 rats were treated with angiotensin II drugs for 60 days (VNS + Ang II). The third group received induce atrial fibrillation protocol (AF) and follow for 60 days, after 24 h 10 rats were treated with angiotensin II drugs for 60 days (AF + Ang II). Results α1(I) and α1(III) procollagen mRNA levels increased statistically significant in the groups with atrial fibrillation associated or not with VNS in contrast with the group in sinus rhythm. The treatment with Angiotensin II receptor antagonist decrease proportionally in all groups the expression of mRNA levels for ventricular and atrial α1(I) and α1(III) procollagen. The VNS increase the expression of mRNA levels for ventricular and atrial α1(I) and α1(III) procollagen statistically significant between the two group in atrial fibrillation but not statistically significant when the group were treated with Angiotensin II receptor antagonist. The level of right ventricle was almost two time compare with both atria analysis or left ventricle analysis, the VNS increase the level of of mRNA levels for ventricular and atrial α1 (I) and α1(III) procollagen in the right ventricle also in the sinus rhythm group (p=0.5). Conclusion The study demonstrates that long-term VNS in the rats with permanent atria fibrillation induce an increase in cardiac procollagen mRNAs, cardiac fibrosis histologically evident with the presence of inflammatory cells and myocyte necrosis in both groups with atrial fibrillation with statistically significant of the increases in case of VNS; the highest increases of the cardiac procollagen mRNAs was observed in the right ventricle more that in the both atria, this increase was prevented from angiotensine II antagonist receptor therapy. We showed that VNS increased the inducibility of AF. We observed moreover the VNS associates with atrial fibrillation decrease significant the rate ventricular response.

Le cellule mesenchimali stromali (MSC) sono cellule multipotenti e numerosi studi hanno mostrato i loro effetti benefici nel danno renale acuto ma non sono ancora stati dimostrati potenziali effetti nella malattia renale cronica. L'ostruzione ureterale unilaterale (UUO) è un modello di fibrosi interstiziale nel quale l'attivazione di molecole vasoattive, citochine profibrotiche e infiammatorie gioca un ruolo patogenetico nello sviluppo dell'apoptosi e atrofia tubulare. Il sistema renina-angiotensina (RAS) gioca un ruolo chiave nello sviluppo della fibrosi renale e i farmaci che hanno come target l'angiotensina II, principale mediatore del RAS, sono attualmente la terapia più efficace nel ridurre la progressione della malattia renale cronica. E' noto che gli ACE-inibitori (ACEi) inducono un aumento compensatorio della renina plasmatica per la mancaza del feedback negativo sulla sua produzione. Tuttavia, la renina (R) promuove il danno renale non solo stimolando la produzione di ANGII, ma anche up-regolando geni profibrotici attraverso l'attivazione del recettore renina/prorenina. Lo scopo dello studio è stato indagare se l'infusione di MSC riduceva il danno renalein un modello animale di UUO e comparare gli eventuali effetti protettivi di ACEi e MSC in UUO. Abbiamo studiato 5 gruppi di ratti. A: sham operati. B: ratti sottoposti a UUO che ricevevano soluzione salina. C: ratti sottoposti a UUO che ricevavano MSC 3X106 nella vena della coda al giorno 0. D:ratti sottoposti a UUO che ricevevano lisinopril dal g 1 al g 21. E: ratti sottoposti a UUO che ricevevano MSC 3X106 nella vena della coda al giorno 0 e lisinopril dal g 1 al g 21. I ratti sono stati sacrificati al giorno 7 e 21. I risultati dello studio mostrano che MSC in UUO prevengono l'aumento della renina, riducono la generazione di ANGII e che in terapia combinata con ACEi riducono ulteriormente l'ANGII, determinando una sinergia nel miglioramento della fibrosi renale.
Mesenchymal stromal cells (MSC) are multipotent cells with immunomodulant and anti-inflammatory effect. Several studies have shown MSC beneficial effect in acute kidney injury but are not yet known MSC effects in chronic kidney disease. Unilateral ureteral obstruction (UUO) is a model of renal interstitial fibrosis, the final common pathway for progressive renal diseases where the activation of vasoactive molecules, inflammatory and profibrotic cytokines plays a pathogenetic role in the development of cell apoptosis and tubular atrophy. Renin-angiotensin system (RAS) plays a pivotal role in renal fibrosis and agents that target angiotensin II, principal mediator of RAS, are the most effective therapy to reduce progression of chronic kidney disease. Its known that angiotensin-converting enzyme inhibitors (ACEi) induce a compensatory increase in plasma renin levels because of the disruption of the negative feedback of its production. However renin (R) promotes renal injury not only by stimulating angiotensin II (ANG II) generation, but also up-regulating pro-fibrotic genes through renin/prorenin receptor activation. The aim of the study was to investigate whether MSC infusion attenuate renal injury in a rat model of UUO and to compare the protective effects of ACEi and MSC in UUO. We studied 5 groups of rats. A: rats sham operated. B: rats UUO received saline solution on day 0 (vehicle control). C: rats UUO received MSC 3X106 on day 0 via tail vein. D: rats UUO received lisinopril from d 1 to 21. E: rats UUO received MSC on d 0, and lisinopril from d 1 to 21. Rats were sacrified on d 7 and 21. Our results show that MSC in UUO rat model prevent renin increase, reduce angiotensin II generation and in combination therapy with ACEi suppress further angiotensin II block, thereby lead to a synergy in ameliorating renal fibrosis.

Le cellule mesenchimali stromali (MSC) sono cellule multipotenti e numerosi studi hanno mostrato i loro effetti benefici nel danno renale acuto ma non sono ancora stati dimostrati potenziali effetti nella malattia renale cronica. L'ostruzione ureterale unilaterale (UUO) è un modello di fibrosi interstiziale nel quale l'attivazione di molecole vasoattive, citochine profibrotiche e infiammatorie gioca un ruolo patogenetico nello sviluppo dell'apoptosi e atrofia tubulare. Il sistema renina-angiotensina (RAS) gioca un ruolo chiave nello sviluppo della fibrosi renale e i farmaci che hanno come target l'angiotensina II, principale mediatore del RAS, sono attualmente la terapia più efficace nel ridurre la progressione della malattia renale cronica. E' noto che gli ACE-inibitori (ACEi) inducono un aumento compensatorio della renina plasmatica per la mancaza del feedback negativo sulla sua produzione. Tuttavia, la renina (R) promuove il danno renale non solo stimolando la produzione di ANGII, ma anche up-regolando geni profibrotici attraverso l'attivazione del recettore renina/prorenina. Lo scopo dello studio è stato indagare se l'infusione di MSC riduceva il danno renalein un modello animale di UUO e comparare gli eventuali effetti protettivi di ACEi e MSC in UUO. Abbiamo studiato 5 gruppi di ratti. A: sham operati. B: ratti sottoposti a UUO che ricevevano soluzione salina. C: ratti sottoposti a UUO che ricevavano MSC 3X106 nella vena della coda al giorno 0. D:ratti sottoposti a UUO che ricevevano lisinopril dal g 1 al g 21. E: ratti sottoposti a UUO che ricevevano MSC 3X106 nella vena della coda al giorno 0 e lisinopril dal g 1 al g 21. I ratti sono stati sacrificati al giorno 7 e 21. I risultati dello studio mostrano che MSC in UUO prevengono l'aumento della renina, riducono la generazione di ANGII e che in terapia combinata con ACEi riducono ulteriormente l'ANGII, determinando una sinergia nel miglioramento della fibrosi renale.
Mesenchymal stromal cells (MSC) are multipotent cells with immunomodulant and anti-inflammatory effect. Several studies have shown MSC beneficial effect in acute kidney injury but are not yet known MSC effects in chronic kidney disease. Unilateral ureteral obstruction (UUO) is a model of renal interstitial fibrosis, the final common pathway for progressive renal diseases where the activation of vasoactive molecules, inflammatory and profibrotic cytokines plays a pathogenetic role in the development of cell apoptosis and tubular atrophy. Renin-angiotensin system (RAS) plays a pivotal role in renal fibrosis and agents that target angiotensin II, principal mediator of RAS, are the most effective therapy to reduce progression of chronic kidney disease. Its known that angiotensin-converting enzyme inhibitors (ACEi) induce a compensatory increase in plasma renin levels because of the disruption of the negative feedback of its production. However renin (R) promotes renal injury not only by stimulating angiotensin II (ANG II) generation, but also up-regulating pro-fibrotic genes through renin/prorenin receptor activation. The aim of the study was to investigate whether MSC infusion attenuate renal injury in a rat model of UUO and to compare the protective effects of ACEi and MSC in UUO. We studied 5 groups of rats. A: rats sham operated. B: rats UUO received saline solution on day 0 (vehicle control). C: rats UUO received MSC 3X106 on day 0 via tail vein. D: rats UUO received lisinopril from d 1 to 21. E: rats UUO received MSC on d 0, and lisinopril from d 1 to 21. Rats were sacrified on d 7 and 21. Our results show that MSC in UUO rat model prevent renin increase, reduce angiotensin II generation and in combination therapy with ACEi suppress further angiotensin II block, thereby lead to a synergy in ameliorating renal fibrosis.

Bortezomib (BTZ) is a proteasome inhibitor that shows potent antineoplastic effects mainly in the treatment of the multiple myeloma. However, BTZ induces a painful neuropathy that causes a significant impairment of patient’s quality of life. CR4056 is a novel imidazoline-2 (I2) ligand that acts modulating the levels of monoamino oxidase (MAO), and it is characterized by a relevant efficacy in several animal models of pain. This study was designed to test the efficacy of CR4056 in a chronic model of BTZ-induced neurotoxicity, compared with two well known analgesic, buprenorphine and gabapentin. Besides, the effect of CR4056 in preventive and therapeutic schedule was evaluated. After 8 weeks of treatment, BTZ reduced nerve conduction velocity (NCV) and induced allodynia. CR4056, gabapentin or buprenorphine in neuropathic animals did not reverse the impaired NCV. By contrast, the optimal CR4056 dose reversed BTZ-induced allodynia. This effect was persistent along the treatment period without rebound after suspension. These results promote the use of CR4056 as a potential treatment in the chronic neuropathic pain therapy.

Hunter Syndrome (mucopolysaccharidosis type II, MPS II) is an inherited metabolic disease belonging to the wide group of lysosomal storage disorders (LSDs), including nearly 50 different pathologies. Although individually rare, these pathologies have a collective incidence from 1:4000 to 1:7000 live births, depending on the analyzed population. Most LSDs are due to single, or more rarely multiple, deficit of lysosomal enzymes, responsible of macromolecules degradation. Unmetabolized substrates result in multiorgan and multisystem diseases, which in the vast majority of the patients seriously affect also the central nervous system. Very little is known on LSDs pathophysiology and even less on the determinants of their neurological impairment. Since ERT (enzymatic replacement therapy) is getting promising results only in treatment of the LSDs without neurological involvement, because the enzyme cannot efficiently cross the blood brain barrier, more attention should be put in handling the cognitive and behavioral components of these pathologies. In this scenario, the comprehension of the neurological pathogenesis of these diseases, and in this specific case, of Hunter syndrome, becomes mandatory. Such understanding, although quite complex, might allow, among others, the development of new specific therapeutic strategies targeted to the brain. In this pre-clinic investigation, the murine MPS II model was used for a complex molecular analysis through high-throughput technology. RNAs from two different brain areas, cortex and midbrain, have been analyzed with next generation sequencing, by using SOliD® (Sequencing by oligo ligation and detection) technology. Although this technology is considered the most specific for RNA sequencing, it has not been extensively used so far due to its very high costs and to the complexity of the data analysis requiring advanced bioinformatics competence and a good capacity of software handling. RNA sequencing is a very powerful technology that, in contrast to microarray, can point out every single cellular transcript indistinctly. This work is a comparative study between brain areas derived from the IDS-knock-out and the healthy control mice. Data obtained, after alignment and filtration, have been classified according to Gene Ontology Domains, and analyzed by functional categories. The analysis has clearly pointed out the involvement of a wide group of genes and pathways implicated in neurological processes. Altered structures and cellular functions have been highlighted both through the analysis of terms that have the same alteration in the two cerebral areas and, in a more specific way, through the analysis of terms related to each area. This approach can underline genes whose altered expression is directly related to the cell pathological condition and, at the same time, genes with differential expression, representing instead specific pathways for the function of the brain area considered. Also the detection of alterations in genes involved in some common neurodegenerative diseases (as Parkinson’s and Alzheimer’s) could be very interesting; common pathways could be hypothesized for the disease development or as a consequence of the pathological state. The last part of this work has focused on some selected pathways that have been chosen as the most interesting candidates for the pathogenesis of the disease: heparan sulphate binding protein, calcium homeostasis, oxidative stress, autophagy, axon guidance, neuroinflammation, correlation with other neurodegenerative diseases and the growth hormone. A significant endocellular alteration, due to progressive increase of glycosaminoglican deposits and also of secondary deposits as gangliosides, could justify the involvement of proteins related to calcium metabolism, detected by the present analysis; since calcium plays an important ubiquitous messenger function in different biological processes, its role of leitmotiv in many pathways emerging from this analysis is not surprising. In conclusion, although very complex, the analysis here presented has highlighted the great power of this technology, due to its ability to detect, not only pathways obviously related to this disease, but also non-suspected pathways, whose role in the determination of the pathological condition has not been yet clarified.
La Sindrome di Hunter (o Mucopolisaccaridosi di tipo II, MPS II) è una patologia ereditaria appartenente al più vasto gruppo delle malattie da accumulo lisosomiale (Lysosomal Storage Disorders, LSDs), comprendente quasi 50 diverse patologie. Tali patologie, sebbene individualmente molto rare, presentano un’incidenza complessiva che va da 1:4000 a 1:7000, a seconda della popolazione considerata. Le LSDs, che risultano per lo più da deficit singoli, e più raramente multipli, di enzimi lisosomiali deputati alla degradazione di molecole complesse, sono devastanti malattie multiorgano e multisistemiche che, in buona parte dei casi, comprendono un coinvolgimento neurologico grave. Poco è noto tuttora sulla patofisiologia di queste sindromi e, ancor meno, sulle cause del loro deficit neurologico. Nel momento in cui alcune di queste patologie trovano finalmente beneficio dall’applicazione della terapia enzimatica sostitutiva, risalta maggiormente la problematica del trattamento della componente cognitiva e comportamentale. Essa infatti non trova beneficio da questi nuovi approcci terapeutici, poiché gli enzimi impiegati non sono in grado di attraversare la barriera emato-encefalica. Nasce da qui la necessità di comprendere la patogenesi neurologica di queste sindromi e, nel caso specifico di questo lavoro, della sindrome di Hunter. Tale comprensione, seppure molto complessa, potrebbe consentire, tra le altre cose, lo sviluppo di specifiche strategie terapeutiche mirate al cervello. In questo lavoro di ricerca preclinica, il modello murino per la MPS II è stato impiegato per effettuare una valutazione molecolare complessa attraverso l’impiego di tecnologie high throughput. RNA derivati da 2 aree cerebrali, la corteccia e il mesencefalo, sono stati analizzati mediante next generation sequencing, impiegando la procedura SOLiD® (Sequencing by Oligo Ligation and Detection). Questa tecnologia, seppure estremamente indicata proprio per il sequenziamento dell’RNA, è stata finora poco utilizzata a questo scopo, a causa dei costi ancora piuttosto elevati, ma soprattutto, a causa della complessità dell’analisi che richiede notevoli competenze bioinformatiche e capacità di gestione dei software. Il sequenziamento dell’RNA è una tecnologia estremamente potente in grado di evidenziare, a differenza della tecnica del microarray, tutti i trascritti cellulari in maniera indistinta. Il lavoro qui presentato è un’analisi di tipo comparativo tra le aree cerebrali dell’animale knock-out per l’IDS e le corrispondenti aree dell’animale sano di controllo. I dati, dopo la fase di allineamento e di filtrazione, sono stati classificati secondo i domini della Gene Ontology e analizzati in base alle principali categorie funzionali. L’analisi ha chiaramente evidenziato il coinvolgimento di molti geni e di parecchie vie specificamente implicati in processi neurologici. L’alterata struttura e funzione cellulare sono state evidenziate sia in modo generico, attraverso analisi di termini ugualmente alterati nelle due diverse aree cerebrali, sia in modo specifico, all’interno di ciascuna area cerebrale. Ciò consente di mettere in risalto i geni la cui alterata espressione è direttamente correlata allo stato di accumulo patologico della cellula e i geni con espressione differenziale che, invece, rappresentano pathways specifici per le funzioni svolte da quell’area cerebrale. Molto interessante potrebbe risultare anche l’alterazione di geni implicati in alcune comuni patologie neurodegenerative croniche quali il morbo di Alzheimer e di Parkinson. Vie comuni potrebbero essere ipotizzate per l’instaurarsi delle malattie o come conseguenza dello stato patologico. La parte finale dell’analisi ha preso in considerazione le vie di segnale e le correlazioni più interessanti, alcune delle quali già precedentemente considerate come potenziali candidati nella patogenesi delle malattie da accumulo lisosomiale: l'eparan solfato binding protein, l'omeostasi del calcio, lo stress ossidativo, il processo dell’autofagia, l'axon guidance, la neuroinfiammazione, la correlazione con altre malattie neurodegenerative e l'ormone della crescita. Una forte alterazione del comparto endocellulare dovuta al progressivo, patologico aumento dei depositi primari di glicosaminoglicani, ma anche di quelli secondari quali i gangliosidi, potrebbe giustificare il coinvolgimento delle proteine implicate nel metabolismo del calcio cellulare, rilevato da questo lavoro. Essendo poi il calcio un messaggero ubiquitario coinvolto in differenti processi biologici non stupisce il ruolo di filo conduttore in molti pathways, evidenziato da questa analisi. In conclusione, seppure fortemente complessa, l’analisi intrapresa in questo studio ha evidenziato le enormi potenzialità della procedura, dovute alla sua caratteristica capacità di mettere in luce, oltre a processi correlati in modo sospetto alla patologia, anche pathways non sospetti o la cui implicazione nella determinazione dello stato patologico non sia ancora stata definita.

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

Il Sorafenib è l’unica terapia sistemica approvata per l’epatocarcinoma (HCC) avanzato. Tuttavia, molti tumori sviluppano resistenze. La chemioterapia metronomica sembrerebbe avere un effetto antiangiogenetico. La Capecitabina metronomica è potenzialmente efficace nell’HCC avanzato. Lo scopo dello studio è stato valutare il comportamento di un modello murino di HCC sottoposto a Sorafenib, Capecitabina e terapia combinata, per dimostrarne un eventuale effetto sinergico. Il modello è stato creato in topi scid mediante inoculazione sottocutanea di 5 milioni di cellule HuH7. I topi sono stati suddivisi in 4 gruppi: gruppo 1 sottoposto a terapia con placebo (9 topi), gruppo 2 a Sorafenib (7 topi), gruppo 3 a Capecitabina (7 topi) e gruppo 4 a terapia combinata Sorafenib+Capecitabina (10 topi). I topi sono stati studiati al giorno 0 e 14 con ecografia B-mode e con mezzo di contrasto (CEUS). Al giorno 14 sono stati sacrificati e i pezzi tumorali sono stati conservati per l’analisi Western Blot. Un topo del gruppo 1 e 4 topi del gruppo 4 sono morti precocemente e quindi sono stati esclusi. Il delta di crescita tumorale al giorno 14 rispetto al giorno 0 è risultato di +503 %, +158 %, +462 % e +176 % rispettivamente nei 4 gruppi (p<0.05 tra i 4 gruppi, tra il gruppo 1 e 2, tra il gruppo 1 e 4, tra il gruppo 2 e 3, tra il gruppo 3 e 4). Alla CEUS non si sono evidenziate differenze statisticamente significative nei cambiamenti di perfusione tumorale al giorno 14 nei 4 gruppi. L’analisi Western Blot ha mostrato livelli di VEGFR-2 inferiori nel gruppo dei topi trattati con Sorafenib. La terapia di associazione di Sorafenib e Capecitabina non comporta un beneficio, in termini di riduzione della crescita tumorale, in un modello murino di HCC rispetto al solo Sorafenib. Inoltre, può essere sospettato un incremento di tossicità.
Introduction and aim: Sorafenib, an anti-angiogenic agent, is the only approved systemic therapy for the treatment of advanced hepatocellular carcinoma (HCC), but the rate of response is suboptimal. Metronomic chemotherapy is well tolerated and has been shown to act on dividing endothelial cells, resulting in an anti-angiogenic effect. Metronomic capecitabine is potentially active in human HCC. Aim of the study was to evaluate the combination of sorafenib and capecitabine to demonstrate a possible synergistic effect in a HCC xenograft mice model. Methods: heterotopic mice model was created in SCID mice. Once mass diameter of 5-10 mm was reached, mice were randomized to receive placebo (n=9), sorafenib (n=7), capecitabine(n=7) or combination of them (n=10). Animals were studied at day 0 and +14 with B-mode ultrasound (US) and with contrast-enhanced (CE) US, then they were sacrificed. Western-Blot analysis was performed. Results: four mice belonging to the combination group and 1 belonging to placebo group were found dead in cages. After exclusion of these 5 mice, increase in median tumor volume at day +14 versus day 0 was +503 mm3 (range 109-1112) in the placebo group, +158 mm3 (36-331) in the sorafenib group, +462 mm3 (254-653) in the capecitabine group and +176 mm3 (29-338) in the combination group (p=0.002 across categories; p<0.05 between placebo and sorafenib or combination and between capecitabine and sorafenib or combination). Percentage of perfused areas at CEUS did not differed across categories neither at day 0 nor at day +14. VEGF levels at Western-Blot analysis were lower in the sorafenib group. Conclusions: the combination of sorafenib and capecitabine is not superior to mono-therapy with sorafenib in the treatment of HCC and an increased toxicity appears suggested.

Il Sorafenib è l’unica terapia sistemica approvata per l’epatocarcinoma (HCC) avanzato. Tuttavia, molti tumori sviluppano resistenze. La chemioterapia metronomica sembrerebbe avere un effetto antiangiogenetico. La Capecitabina metronomica è potenzialmente efficace nell’HCC avanzato. Lo scopo dello studio è stato valutare il comportamento di un modello murino di HCC sottoposto a Sorafenib, Capecitabina e terapia combinata, per dimostrarne un eventuale effetto sinergico. Il modello è stato creato in topi scid mediante inoculazione sottocutanea di 5 milioni di cellule HuH7. I topi sono stati suddivisi in 4 gruppi: gruppo 1 sottoposto a terapia con placebo (9 topi), gruppo 2 a Sorafenib (7 topi), gruppo 3 a Capecitabina (7 topi) e gruppo 4 a terapia combinata Sorafenib+Capecitabina (10 topi). I topi sono stati studiati al giorno 0 e 14 con ecografia B-mode e con mezzo di contrasto (CEUS). Al giorno 14 sono stati sacrificati e i pezzi tumorali sono stati conservati per l’analisi Western Blot. Un topo del gruppo 1 e 4 topi del gruppo 4 sono morti precocemente e quindi sono stati esclusi. Il delta di crescita tumorale al giorno 14 rispetto al giorno 0 è risultato di +503 %, +158 %, +462 % e +176 % rispettivamente nei 4 gruppi (p<0.05 tra i 4 gruppi, tra il gruppo 1 e 2, tra il gruppo 1 e 4, tra il gruppo 2 e 3, tra il gruppo 3 e 4). Alla CEUS non si sono evidenziate differenze statisticamente significative nei cambiamenti di perfusione tumorale al giorno 14 nei 4 gruppi. L’analisi Western Blot ha mostrato livelli di VEGFR-2 inferiori nel gruppo dei topi trattati con Sorafenib. La terapia di associazione di Sorafenib e Capecitabina non comporta un beneficio, in termini di riduzione della crescita tumorale, in un modello murino di HCC rispetto al solo Sorafenib. Inoltre, può essere sospettato un incremento di tossicità.
Introduction and aim: Sorafenib, an anti-angiogenic agent, is the only approved systemic therapy for the treatment of advanced hepatocellular carcinoma (HCC), but the rate of response is suboptimal. Metronomic chemotherapy is well tolerated and has been shown to act on dividing endothelial cells, resulting in an anti-angiogenic effect. Metronomic capecitabine is potentially active in human HCC. Aim of the study was to evaluate the combination of sorafenib and capecitabine to demonstrate a possible synergistic effect in a HCC xenograft mice model. Methods: heterotopic mice model was created in SCID mice. Once mass diameter of 5-10 mm was reached, mice were randomized to receive placebo (n=9), sorafenib (n=7), capecitabine(n=7) or combination of them (n=10). Animals were studied at day 0 and +14 with B-mode ultrasound (US) and with contrast-enhanced (CE) US, then they were sacrificed. Western-Blot analysis was performed. Results: four mice belonging to the combination group and 1 belonging to placebo group were found dead in cages. After exclusion of these 5 mice, increase in median tumor volume at day +14 versus day 0 was +503 mm3 (range 109-1112) in the placebo group, +158 mm3 (36-331) in the sorafenib group, +462 mm3 (254-653) in the capecitabine group and +176 mm3 (29-338) in the combination group (p=0.002 across categories; p<0.05 between placebo and sorafenib or combination and between capecitabine and sorafenib or combination). Percentage of perfused areas at CEUS did not differed across categories neither at day 0 nor at day +14. VEGF levels at Western-Blot analysis were lower in the sorafenib group. Conclusions: the combination of sorafenib and capecitabine is not superior to mono-therapy with sorafenib in the treatment of HCC and an increased toxicity appears suggested.

L’obiettivo di questo studio è valutare gli effetti sull’emodinamica splancnica dopo la somministrazione di terlipressina in associazione all’infusione endovenosa di octeotride. La terlipressina trova impiego clinico nella terapia selettiva delle emorragie da varici esofagee, principale complicanza dell’ipertensione portale, senza provocare alterazioni della dinamica emocoagulativa. L’uso dell’octreotide è ben conosciuto come valida terapia per emorragia delle varici in pazienti cirrotici attraverso la riduzione della pressione portale e il flusso sanguigno portocollaterale. Lo scopo del presente studio è valutare gli effetti dell’associazione farmacologica di terlipressina e octreotide nell’emodinamica epatica, attenzionando l’interazione tra il flusso portale e quello arterioso epatico nel maiale.
The objective of this study was to evaluate the effects of splanchnic hemodynamics after administration of terlipressin in combination intravenous infusion of octreotide. Terlipressin is used clinically in the treatment of selective variceal bleeding, the main complication of portal hypertension, without causing any of the dynamics emocoagulativa. The use of octreotide is well known as an effective therapy for variceal bleeding in cirrhotic patients by reducing portal pressure and blood flow portocollaterale. The purpose of this study was to evaluate the pharmacological effects of the association of terlipressin and octreotide in hemodynamics of the liver, with particular attention to the interaction between the hepatic arterial and portal flow in the pig.

Hearing impairment is an increasingly common disease. In Italy deaf people are about seven million, including half a million adults with disabling hearing loss and over one thousand births per year with congenital deafness. This causes difficulty in integration in society for adults and prevents the language acquisition for children (Fekete, 1999). As hearing loss has high social costs, the expectation for the development of new treatments is extensive and diseases leading to hearing damage are increasingly studied from clinic and base research. Hearing loss (HL) can have genetic causes, can be associated with aging or exposure to noise or ototoxic substances, and the aetiology can be attributed to vascular injury, trauma, tumours, infections or autoimmune response. All of these factors could be ascribed to alterations in cochlear microcirculation resulting in hypoxia. This condition can damage cochlear hair cells and neurons possibly leading to HL. Hypoxia and ischemia can then be identified as possible factors contributing to the aetiology of deafness, but they have not been experimentally studied yet. The purpose of this work is to develop animal models of ischemia and infarction suitable for the study of cochlear vascular damage, and to characterize them with electrophysiology and gene/protein expression analyses. For this reason it was decided to monitor the effects of ischemia in thrombosis mimicked by complete temporary carotid occlusion, and in stroke mimicked by incomplete permanent left coronary artery. In particular this study focused on the analysis of: organ of Corti and spiral ganglion structures, coagulation, oxidative stress and apoptosis. A further aim was to compare these models with other models of ototoxic damage, such as noise and cisplatin. These models are both characterized for electrophysiology, oxidative stress and apoptosis, but the possible involvement of vascular damage has not been investigated yet. This comparison helped us to characterize the new models of vascular injury in the oxidation and apoptosis expression patterns. In our models, both infarction and ischemia cause a small but significant hearing loss, localized at the cochlear apex. Furthermore, there is a slight induction of the coagulation cascade, both in procoagulant and anticoagulant part, and an activation of JNK, that may lead to cell survival. In addition, only in the carotid ischemia the cuticular plate of outer hair cells is damaged. Even noise and cisplatin cause vascular damage, but while in noise-treated animals the coagulation genes show only an mRNA expression increase, after cisplatin administration an mRNA and protein increase of the tissue factor is detected, which leads to the coagulation cascade activation. In the ischemic models we did not detect any apoptosis activation, while in the other models we noticed opposite reactions: in noise there is an increased transcription of the anti-apoptotic genes that leads to cell survival, while cisplatin activates pro-apoptotic factors. The activation of apoptosis is documented in literature and is confirmed in both conditions by OHC loss detected in histological sections, which leads to a more severe deafness than in the ischemia models. In conclusion, the two models of ischemia developed are suitable for the study of cochlear vascular damage, as they produce a slight hearing loss and give modifications in coagulative, oxidative and apoptotic factors gene expression. Furthermore, the comparison with two other widely used models allowed us to specify the pathways involved. We can therefore say that all types of damage taken into consideration act on the inner ear with vascular damage and oxidative mechanisms.

The aim of our study is to evaluate a new surgical technique for the in utero correction of the diaphrgmatic hernia (CDH) obcluding the left main bronchus in an animal model (sheep). The first part of the project will be based on the examiantion of the anatomy of the trachea and main bronchi. On the basis of the anatomical records will be developed some devices for its endoluminal obstruction. After that, will be performed the study of the endoscopical anatomy of the lower respiratory tract of the lamb to identify landmarks useful to allow the obstruction of the left main bronchus. The introductions of the devices in the trachea will be performed in three ways: without their direct vision of the catheter (“blind mode”) or under ultrasound or endoscopical guidance. Being the first time that we are working on this disease, a part of the study will be spent to evaluate the effectiveness of techniques for its creation and correction. The rationale of this experiment is based on the fact that the lung ipsilateral to the diaphragmatic lesion is usually the most severely affected by hypoplasia. Obstructing the left main bronchus we suppose to obtain an hyperplastc left lung allowing the right to develop physiologically decreasing the respiratory distress of the newborn. All lambs will be euthanized and their lungs will be used to obtain histological and imunohistological sections to value the number of type II pneumocytes and arterial thickness. The results will be compared between animals with CDH, animals with the correction of the disease achieved by tracheal ligation and healthy animals.

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination and dysarthria. The mutational mechanism in spinocerebellar ataxia type1 (SCA1), a dominantly inherited form of SCA, consists of an expanded trinucleotide CAG repeat that encodes a polyglutamine tract in ataxin1. Mutant SCA1 transgenic mice present pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss, at least in the early stage of life; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. In the present study we correlated the earliest histopathological changes in both homozygous and heterozygous transgenic SCA1 mice, 2 and 6 months old, to the mitochondrial oxidative metabolism in cerebellar cells. Our results showed selective Cytochrome c Oxidase (COX) deficiency in Purkinje cells (PC). Analysis on COX-competent and -deficient PC, isolated by laser-microdissector, demonstrated that the observed oxidative dysfunction is related to mitochondrial DNA (mtDNA) depletion. In conclusion, we provide evidence of a selective oxidative metabolism defect in neuronal PC expressing mutant ataxin. This defect could represent one of the earliest pathogenetic step of the Purkinje cells’ suffering in SCA1 disease.

Depression frequently coexists with substances abuse, including heavy or problematic cannabis use. To mimic this comorbidity, we combined the olfactory bulbectomy model of depression with intravenous drug self-administration procedure to verify whether depressive-like rats display higher voluntary intake of the cannabinoid CB1 receptor agonist WIN 55,212-2 (WIN, 12.5 μg/kg/infusion). The olfactory-bulbectomized rat is a well-established model that exhibits a high degree of behavioural and neurochemical similarity to depression. Male Lister Hooded rats were either olfactory-bulbectomized (OBX) or sham-operated (SHAM), and following 21 days they were implanted with intravenous catheters. A 3-week period is necessary for the development of a depressive-like phenotype following OBX surgery, which was verified by means of the motility and sucrose preference test. Indeed, the presence of anhedonia and hypermotility in response to a novel aversive environment are two hallmarks of OBX rats. Only OBX rats displaying a depressive-like phenotype were allowed to self-administer WIN by lever-pressing under a continuous (FR-1) schedule of reinforcement in 2h daily sessions, as previously described (Fattore et al. 2001). Our results show that both OBX and SHAM rats developed stable WIN intake. Yet, rates of responding for WIN was constantly higher in OBX than in SHAM rats starting soon after the first week of training. This finding is in line with previously reported higher intake of methamphetamine (Kucerova et al. 2011) and oral nicotine (Vieyra-Reyes et al. 2008) in OBX than in SHAM animals, which confirms the hypothesis of higher drug intake in depressive-like conditions. Finally, response latency (defined as time elapsed from commencement of the experimental session until the first active lever press) was significantly different between the two groups. More specifically, response latency was shorter in OBX than SHAM rats from day 9 onward, which suggests that after initial exposure to the CB1 receptor agonist the bulbectomized rats may be more motivated than controls to obtain it. Moreover, analysis of temporal patterns of responses revealed quantitative but not qualitative differences between OBX and SHAM rats during self-administration training, since the response rate was typically slow and evenly distributed throughout the 2h test session in both groups. These findings showed that olfactory bulbectomy markedly affects cannabinoid self-administration, likely through a reduction of the cannabinergic rewarding effects to which animals compensate by increasing WIN intake. A number of human and animal studies demonstrated a causal link between altered serotonin 5-HT1B receptor activity and development of neuropsychiatric conditions, including depression and drug addiction Therefore, we evaluated the effect of acute pre-treatment with the serotonin 5-HT1B receptor agonist CGS-12066B (CGS, 2.5-10 mg/kg) on the WIN self-administration. Acute intraperitoneal (IP) pre-treatment with CGS (2.5, 5 and 10 mg/kg) did not significantly modify WIN intake in OBX and SHAM rats. Moreover, since WIN self-administration was shown to be associated to an increased dopamine transmission in the shell of the nucleus accumbens (NAc) (Fadda et al., 2006), we used the in vivo microdialysis technique to test whether OBX and SHAM rats displayed similar increase in dopamine levels within the NAc shell in response to a challenge of WIN at a dose (0.3 mg/kg, IP) that mimics the daily mean amount of drug typically self-administered by trained rats. We found that, contrary to SHAM rats, dopamine levels in the nucleus accumbens of OBX rats did not increase in response to a WIN challenge, suggesting a dopaminergic dysfunction in bulbectomized rats. Altogether our results suggest that WIN is not affected by acute stimulation of 5-HT1B receptors, and that a dopaminergic rather than a 5-HT1B mechanism is likely to underlie enhanced WIN self-administration in OBX rats. To verify whether prolonged stimulation of CB1 receptors could affect the depressive-like phenotype of OBX rats, we also investigated the effect of chronic (2-weeks) treatment with WIN 55,212-2 (0.5 mg/kg, IP) in OBX and SHAM. Although not statistically significant, a trend was found as concerns a potential antidepressive effect of chronic WIN administration in OBX rats. Finally, a [3H]CP-55,940 binding autoradiography study was performed to investigate CB1 receptor-mediated signaling in the brain of olfactory bulbectomized rats. A reduced functionality of CB1 receptors was detected in the caudate-putamen and in the nucleus accumbens core, which confirmed an altered CB1 receptor-mediated limbic signaling in OBX rats. Fattore et al. (2001) Psychopharmacology 156: 410-416. Kucerova et al. (2011) Int J Neuropsychopharmacol. 15:1503-1511. Vieyra-Reyes et al. (2008) Brain Res. Bull. 77: 13-28. Fadda et al. (2006) Neuroreport 17: 1629-1632.

La sostituzione Gly (G)→Arg (R) a livello del codone 972 (G972R) è il più diffuso tra tutti i polimorfismi noti a carico di IRS-1, il principale substrato del recettore insulinico; i risultati degli studi volti a confermare l’associazione tra questo polimorfismo e la patogenesi del Diabete di tipo 2 sono discordanti; in vitro è però evidente come questa variante determini la riduzione dell’azione dell’insulina in modo specifico per il tipo cellulare e, a livello molecolare, una più debole associazione tra IRS-1 e la PI3-K. In questo studio, abbiamo voluto valutare in vivo l’incidenza del polimorfismo G972R sulla patogenesi del Diabete di tipo 2: abbiamo quindi generato un modello murino sovraesprimente tale variante (i topi Tg972), studiandone il fenotipo anche in relazione all’età o ad un fattore ambientale come l’obesità, valutando l’efficienza dell’azione dell’insulina nel fegato, nel muscolo e nel tessuto adiposo e l’omeostasi del glucosio nei topi sottoposti ad una dieta normale o ricca di grassi. I topi transgenici sottoposti a dieta normale diventano intolleranti al glucosio ed insulino-resistenti in modo dipendente dall’età, la trasduzione del segnale insulinico risulta più debole rispetto ai WT; inoltre, nonostante non vi siano differenze nella morfologia del pancreas rispetto ai topi di controllo, la secrezione insulinica dei transgenici è gravemente ridotta ad ogni concentrazione di glucosio utilizzata. Al termine della dieta grassa, il peso corporeo e del tessuto adiposo dei topi Tg972 aumenta sensibilmente rispetto ai WT; la dieta grassa, inoltre, accelera l’insorgenza di una grave intolleranza al glucosio e dell’insulino-resistenza nei transgenici. Alla luce di questi risultati, riteniamo che i topi Tg972 siano un ottimo modello per studiare l’interazione tra fattori genetici ed ambientali nella patogenesi del Diabete di tipo 2 e per la sperimentazione di nuove terapie.
Molecular scanning of human insulin receptor substrate (Irs) genes revealed a single Irs1 prevalent variant, a glycine to arginine change at codon 972 (G972R); previous in vitro studies had demonstrated that the presence of this variant results in an impaired activation of the insulin signalling pathway, while human studies gave controversial results regarding its role in the pathogenesis of insulin resistance and related diseases. To address in vivo the impact of this IRS-1 variant on whole body glucose homeostasis and insulin signalling, we have generated transgenic mice overexpressing it (Tg972) and evaluated insulin action in the liver, skeletal muscle and adipose tissue and assessed glucose homeostasis both under a normal diet and a high-fat diet. We found that Tg972 mice developed age-related glucose and insulin intolerance and hyperglycemia, with insulin levels comparatively low. Glucose utilization and insulin signalling were impaired in all key insulin target tissues in Tg972 mice. There were no differences in pancreatic morphology between Tg972 and wild-type mice, however when insulin secretion was evaluated in isolated islets, it was significantly reduced in Tg972 mice islets at any glucose concentration tested. Under a high-fat diet, Tg972 mice had increased body and adipose tissue weight, and were more prone to develop diet-induced glucose and insulin intolerance. So, we believe that Tg972 mice may represent a useful model to elucidate the interaction between genetic and environmental factors in insulin resistance pathogenesis. Furthermore, they may become an important tool to test novel tailored therapies.

Introduction: The progression from adolescence strong use of Cannabis to heroin abuse in adulthood, theorized by Kandel as Gateway Hypothesis (GH) doesn't adequately account the importance of phenotype vulnerability as predisposing factor involved in the development of a drug abuse-related behavioral disorder. Addiction prone Lewis (LEW) and addiction resistant Fischer (F344) inbred rat strains represents a useful animal model of different drug vulnerability in light of the different susceptibility to opiates and psychostimulants reinforcing properties. Aim: Thus, the purpose of this study was to investigate the influence of Δ9-THC adolescence exposure on the addictive properties of heroin in LEW and F344 strain, during heroin self-administration (SA) in adulthood. Methods: On the 6th postnatal (PN) week rats were administered twice a day with increasing doses of Δ9-THC (2, 4, 8 mg/kg, i.p.) for three consecutive days. In adulthood (12th PN), LEW and F344 rats were trained to acquire heroin SA behavior (0.025 mg/kg/48 μl, 1-h daily session), under Fixed Ratio-1 (FR-1) (1NP = 1 infusion) schedule of responding. When criterion of acquisition was met, LEW and F344 rats were subjected to self administer heroin: i) under Fixed Ratio (FR-3 and FR-5) schedule of responding (Exp I), to better understand how Δ9-THC could influence heroin reinforcing properties; ii) under Progressive Ratio (PR3-4) schedule of reinforcement (Exp II) in order to evaluate if Δ9-THC adolescent exposure affect the motivational value of heroin; iii) during daily 4-h SA (long access, LA), with increasing doses of heroin (0.025, 0.050 and 0.100 mg/kg), under FR-1 schedule (Exp. III) to determine the role of Δ9-THC as predisposing factor in the vulnerability of opiate abuse by escalation of heroin intake. Results: In each experiments, adolescent pre-exposure to Δ9-THC induced higher operant responding activity and greater adaptation to all experimental conditions (Exp I, II, III) of opiate-reinforced SA behavior in adult LEW rats as well as progressive escalation of heroin intake when exposed to higher doses of heroin, compared to LEW vehicle as well as to F344 strain (Exp III). Furthermore, LEW Δ9-THC pretreated rats readily acquire PR schedule, showing greater nose poking behavior and higher breaking point values compared to their controls as well as to the counterpart F344 groups. No such differences were observed in the F344 rats strain. Conclusion: Genetic vulnerability plays a critical role as a predisposing factor in the progression to opiate abuse and related behavioral disease. The results of my research strongly demonstrate that Δ9-THC pre-treatment in adolescence differentially affects both heroin rewarding properties and motivational value, in adulthood, in a strain-related way, and for the first time clearly indicate that heavy Cannabis use during adolescence could have a gateway effect in the adulthood only on individuals provided by an addiction prone genetic and/or phenotype background.

“Naturally occurring cancers in pet dogs and humans share many features, including histological appearance, tumour genetics, molecular targets, biological behaviour and response to conventional therapies. Studying dogs with cancer is likely to provide a valuable perspective that is distinct from that generated by the study of human or rodent cancers alone. The value of this opportunity has been increasingly recognized in the field of cancer research for the identification of cancer-associated genes, the study of environmental risk factors, understanding tumour biology and progression, and, perhaps most importantly, the evaluation and development of novel cancer therapeutics”.(Paoloni and Khanna, 2008) In last years, the author has investigated some molecular features of cancer in dogs. The Thesis is articulated in two main sections. In section 1, the preliminary results of a research project aimed at investigating the role of somatic mutations of Ataxia-Telangiectasia mutated (ATM) gene in predisposing to cancer in boxer dogs, are presented. The canine boxer breed may be considered an unique opportunity to disclose the role of ATM somatic mutation since boxer dogs are known to be dramatically susceptible to cancer and since they may be considered a closed gene pool. Furthermore, dogs share with human the some environment. Overall, the abovementioned features could be considered extremely useful for our purposes. In the section 2, the results of our studies aimed at setting up accurate and sensitive molecular assays for diagnosing and assessing minimal residual disease in lymphoproliferative disorders of dogs, are presented. The results of those molecular assay may be directly translated in the field of Veterinary practice as well as the may be used to improve our objective evaluation of new investigational drugs effectiveness in canine cancer trials.

“Naturally occurring cancers in pet dogs and humans share many features, including histological appearance, tumour genetics, molecular targets, biological behaviour and response to conventional therapies. Studying dogs with cancer is likely to provide a valuable perspective that is distinct from that generated by the study of human or rodent cancers alone. The value of this opportunity has been increasingly recognized in the field of cancer research for the identification of cancer-associated genes, the study of environmental risk factors, understanding tumour biology and progression, and, perhaps most importantly, the evaluation and development of novel cancer therapeutics”.(Paoloni and Khanna, 2008) In last years, the author has investigated some molecular features of cancer in dogs. The Thesis is articulated in two main sections. In section 1, the preliminary results of a research project aimed at investigating the role of somatic mutations of Ataxia-Telangiectasia mutated (ATM) gene in predisposing to cancer in boxer dogs, are presented. The canine boxer breed may be considered an unique opportunity to disclose the role of ATM somatic mutation since boxer dogs are known to be dramatically susceptible to cancer and since they may be considered a closed gene pool. Furthermore, dogs share with human the some environment. Overall, the abovementioned features could be considered extremely useful for our purposes. In the section 2, the results of our studies aimed at setting up accurate and sensitive molecular assays for diagnosing and assessing minimal residual disease in lymphoproliferative disorders of dogs, are presented. The results of those molecular assay may be directly translated in the field of Veterinary practice as well as the may be used to improve our objective evaluation of new investigational drugs effectiveness in canine cancer trials.

Lo scopo della tesi è stato quello di ottenere un valido modello sperimentale di sclerosi sistemica (SSc), patologia caratterizzata da fibrosi della cute e degli organi interni e da microvasculopatia periferica. A tal fine è stata allestita e caratterizzata una colonia di topi knockout per il gene codificante il recettore dell'attivatore di tipo urochinasico del plasminogeno (uPAR o CD87). uPAR è infatti un componente chiave del sistema fibrinolitico ed è coinvolto in vari processi fisiopatologici, quali la trombolisi, il rimodellamento tissutale e l'angiogenesi. Il sistema fibrinolitico è costituito da un pro-enzima inattivo, il plasminogeno, che una volta convertito in plasmina degrada la fibrina e trasforma le pro-metalloproteasi di matrice (pro-MMP) in MMP attive, che a loro volta degradano i componenti della matrice extracellulare (ECM). Sono stati identificati due attivatori fisiologici del plasminogeno, uno tissutale (tPA) ed uno di tipo urochinasico (uPA o urochinasi), che si lega al proprio recettore uPAR. uPAR è un recettore di superficie, è costituto da tre domini extracellulari (D1-D2-D3), ed è espresso in diversi tipi cellulari, quali monociti, neutrofili, cellule T attivate, fibroblasti, cellule endoteliali e cellule muscolari lisce dei vasi. Studi recenti hanno dimostrato che le cellule endoteliali microvascolari ed i fibroblasti ottenuti dal derma di pazienti SSc presentano un recettore uPAR troncato (D2-D3) ed ipofunzionale che determina un severo deficit angiogenetico. E' stato inoltre recentemente provato che l’assenza di uPAR causa un’attivazione dei fibroblasti e che il suo clivaggio è una tappa cruciale nel differenziamento dei fibroblasti in miofibroblasti, i principali responsabili dell'eccessiva deposizione di collagene in corso di fibrosi. Un’alterata espressione di uPAR nelle cellule endoteliali e nei fibroblasti potrebbe pertanto essere coinvolta nei due principali aspetti della SSc: la microvasculopatia periferica, caratterizzata da un deficit angiogenetico e dalla perdita di capillari, ed il processo fibrotico. Per l'allestimento della colonia di topi da utilizzare negli esperimenti, topi maschi uPAR-/- sono stati incrociati, inizialmente, con femmine wild type, in modo tale da ottenere animali eterozigoti che, ulteriormente incrociati tra loro, hanno permesso di generare topi uPAR-/- ed uPAR+/+ con lo stesso background genetico ed appartenenti alla stessa colonia di allevamento. La presenza o l'assenza del gene uPAR attivo è stata determinata mediante PCR, eseguita sul DNA genomico estratto da biopsie prelevate dall'estremità della coda di ciascun animale. I topi sono stati allevati e al momento del sacrificio, a 12 e a 24 settimane di età, da essi sono stati prelevati i polmoni e biopsie di cute del dorso. Una parte dei campioni è stata processata per l'inclusione in paraffina e le successive analisi istopatologiche ed immunoistochimiche, mentre un’altra parte è stata processata per l’analisi del contenuto di collagene mediante il dosaggio dell’idrossiprolina. Le sezioni incluse in paraffina, tagliate al microtomo, sono state colorate con Ematossilina-Eosina, Tricromica di Masson e Rosso Sirio Picrato e sottoposte ad analisi immunoistochimica, sia in perossidasi che in fluorescenza. I risultati ottenuti permettono di proporre il topo uPAR-/- come un possibile nuovo modello murino di SSc. Nella cute degli animali uPAR-/- sono state infatti riscontrate gran parte delle caratteristiche patologiche della SSc umana: 1) lo spessore del derma, il contenuto di collagene ed il numero dei miofibroblasti sono risultati significativamente aumentati nei topi uPAR-/- rispetto ai controlli. Il derma dei topi uPAR-/- presenta inoltre fasci di collagene ispessiti, abbondante fibrosi perivascolare ed una parziale sostituzione del tessuto adiposo sottocutaneo da parte del tessuto connettivo fibroso; 2) nel derma dei topi uPAR-/- è risultata evidente una marcata sovraespressione delle citochine profibrotiche TGF-b, CTGF/CCN2 ed ET-1, notoriamente associate alla SSc umana; 3) nei topi uPAR-/- i cambiamenti fibrotici a carico della cute sono accompagnati da apoptosi delle cellule endoteliali e da una severa perdita del microcircolo; Il modello del topo uPAR-/- presenta caratteristiche patologiche simili a quelle riscontrate nella SSc umana anche a livello polmonare. Il pattern istopatologico osservato è infatti paragonabile a quello di una pneumopatia interstiziale aspecifica, con vaste aree di parenchima polmonare caratterizzate da infiammazione cellulare diffusa ed accumulo di collagene.

In Sicily, the ovine livestock production has strong traditional connotations, with an almost exclusively extensively character. Moreover, the dairy branch is an important part of the ovine sector and shows several critical issues. Unfortunately, we have little informations about the hygienic sanitary quality of ovine dairy products. The aim of the study was to perform a microbiological characterization of ovine milk, ricotta and tuma from different Sicilian districts, using rapid and high reliability technologies, in order to collect data on the microbiological quality of local products. For this purpose, the prevalence of isolated strains belonging to the Family of Enterobacteriaceae has been evaluated, along with their identification with mass spectrometry techniques. Finally, all identified strains were tested for the phenotypic expression of ESBL (Extended spectrum β-lactamase) production. Then, the ESBL positive strains were selected for the elaboration of an experimental model. This work lead to the implementation of the mass spectra database with Superspectra highly specific for the phenotypic expression of ESBL. Data collected during the study revealed poor hygienic-sanitary conditions of the dairyproducts. Milk samples showed a mean value of 6.4±1.4 Log CFU/ml for Aerobic mesophilic count and 3.8±1.8 CFU/ml for Enterobacteriaceae count. Same considerations can be made for ricotta and tuma samples, in which, in addition to those two parameters, also Staphylococcal charges resulted unsatisfactory, with a mean value of 3.9±1.3 Log CFU/g and 3.8±1.5 Log CFU/g respectively. In one sample of ricotta was also detected Listeria monocytogenes with a charge of 3.3 Log CFU/g. Among the isolated strains belonging to the family of Enterobacteriaceae (n.355), Hafnia alvei reported the highest prevalence in all the samples (40.3%), followed by E. coli (13.2%), Citrobacter spp (10.1%) and other enterobacteria in lower percentage. Considering the phenotypical exhibition of antibiotic resistance, the 70% of strains resulted ESBL+, with different expression rates depending on the species. Finally, 5 samples of ESBL + and ESBL - strains for species (Hafnia alvei, E. coli, E. cloacae, K. oxytoca) were selected in order to generate mass spectra that were analyzed for peaks similarities. The results of this comparing algorithm have conducted to a new hierarchical clustering of the samples and to the implementation of the data set with new Superspectra ESBL+ and ESBL-, that allowed both the rapid identification at the genus level and the ESBL expression of each sample. The reliability of our model ranged from 50% to 80% of correct identifications; so mass spectrometry could be routinely employed in microbiological diagnostics as novel, time-saving and cost-reducing tool.