Tesi sul tema "Settore BIO/16 - Anatomia Umana"

Magnetic resonance diffusion-based tractography techniques have offered a real breakthrough in brain studies. These methods allow, for the first time, to explore the anatomical organization of white matter pathways in humans, in-vivo and non-invasively. As any other method, diffusion-based tractography has limitations. The inherent limits related to the indirect measurement of the diffusion signal, and the strong dependence of this technique on acquisition, models and algorithm parameters, prevents the reliable reconstruction of some major white matter bundles. This dissertation targets the methods, limitations, improvements, and validation of tracking methods, with applications in neurobiological and clinical research. In particular, the main work focuses on a white matter bundle that represents a notable omission in tractography studies: the acoustic radiation (AR), a major projection sensory pathway conveying auditory information from the thalamus to the auditory cortex. Topographical knowledge of this bundle is scarce, and its in-vivo tractography reconstruction remains challenging, preventing the investigation of auditory and language functional mechanisms in humans. This dissertation investigates, for the first time, the topography of the AR using post-mortem blunt dissections and provides a detailed description of the trajectory of these fibres and their relationship with major neighbouring white matter bundles. The topographical information is then applied to conduct an investigation on the effects of different MRI acquisition and tractography parameters on the in-vivo tractography reconstruction of the AR. An optimal set of parameters is obtained for AR reconstructions and used to build the first tractography atlas of the acoustic radiation. The AR atlas is then applied to study congenital deaf patients. The optimized reconstruction parameters and the atlas generated in this dissertation may be used in future studies interested in identifying and characterizing the AR. The reliable 3D reconstruction of this bundle will improve our understanding of the functional mechanisms underlying hearing and language in healthy subjects and patients, as well as in neurosurgical applications.

Telemedicine consists in the use of telecommunication technologies to provide healthcare services, overcoming geographic, temporal, social, and cultural barriers. Today telemedicine is a developed field which includes about 50 different subspecialties: neuropsychology is one of them. Allowing the objective evaluation of the cognitive state of individuals, neuropsychology is a discipline of wide application; it also contributes significantly to an early diagnosis of subjects suspected to develop cognitive impairments due to Alzheimer disease or other degenerative dementias. Subjects at risk, and subjects who have already develop the illness, would particularly benefit of a telehealth intervention, which allows to overcome the barriers of space and time, and to provide an evaluation, as well as the therapy monitoring. These aspects would be particularly important for subjects who live far from health institutions , as those of rural areas. Obviously, we need to be sure that the results of the Tele-Neuropsychological assessment are comparable to those obtained via the classic '' face to face '' administration. This is the purpose of this research. We aimed in fact to compare the performances obtained in the two conditions at the MMSE test and the ADAS Cog test. To this purpose, we submitted a group of subjects affected by mild to moderate Alzheimer disease with associated vascular damages, to MMSE and the ADAS COG test, and performed a statistical analysis of data through a two sided Student ''t'' test. We found that the administration modality had no significant impact on the results. In fact, no significant difference was found neither in the MMSE, or in the ADAS_Cog scores administered by telehealth versus ''face to face''. While the results obtained at the MMSE confirm some previous data, this is, at our knowledge, the first study done on the ADAS_Cog, a test. Even if the conditions we employed in this research are not entirely superposable to those of patients staying at home (we evaluated the feasibility of teleheath by locating the patient and his caregiver in another room of the memory clinic ), we are confident that telehealth methodology, by video-conferencing, is as much reliable as the face to face modality. The small number of the subjects evaluated represents an obvious limitation of this study and suggests further studies involving larger number of subjects. However, our preliminary results give support to the idea that by tehealth the screening and the follow-up of the cognitive impairments age associated is feasible and valid.

BACKGROUND: Telemedicine consists in the use of telecommunication technologies to provide healthcare services overcoming geographic, temporal, social, and cultural barriers. Today telemedicine is a developed field, which includes about 50 different subspecialties: neuropsychology is one of them. Its main application consists in the early diagnosis of cognitive disorders of adult onset ,and particularly in the assessment of individuals potentially affected by degenerative conditions, such as Alzheimer disease (AD). These individuals would benefit significantly by an assessment by telemedicine. This study was aimed to assess the feasibility by telemedicine of two widely used neuropsychological test: the MMSE and the ADAS_COG test. While for the first some data are available in the literature, no study has investigated, to our knowledge, the ADAS_COG administration by videoconference in subjects with AD. We aimed thus to compare the results obtained by the ''face to face'' condition to those obtained by videoconference. MATERIALS AND METHODS: The ADAS_Cog and the MMSE test were administered to 28 subjects (8M) in two conditions: the face-to-face condition and the video-conference modality. Subjects were all participating to the randomized, double blind, placebo controlled ASCOMALVA study. The administration was done at the enrollment of the patients in the study, baseline, and after 6, 12, 18 and 24 months. Subjects were randomized in two groups. Group A subjects were submitted to the ADAS_Cog and the MMSE test by the face-to-face modality first; two weeks later the same tests were administered via telemedicine by videoconferencing. Group B were submitted vice-versa to the videoconference modality first, and then to the face to face. A two-tailed Student "t" test was done to evaluate the differences between the two modality. RESULTS: There was no significant difference in MMSE scores and ADAS_Cog scores (p minus .05) administered by telehealth versus face to face modality. In patient with moderate AD the administration via telemedicine gives worse results on average than the face to face mode CONCLUSIONS: The most employed tool to assess and measure the cognitive functions in subjects potentially affected by degenerative dementia are the MMSE and the ADAS_Cog. Both are required in the clinical trials to assess the severity of the damage and the effectiveness of new drugs. Their employ by videoconference should be very useful. We found that both the MMSE and the ADAS Cog test may be applied in subjects with mild to moderate Alzheimer disease, and that the scores obtained by videoconference are related to those obtained by the face to face condition. Videoconference may be very useful to reach patients with cognitive damages, to assess their stability or progression, to measure the efficacy of therapies.

Nei mammiferi si possono distinguere due tipi di tessuto adiposo che collaborando per un fine strategico e funzionale costituiscono l’ Organo Adiposo. Questi due tessuti sono il bianco (WAT) ed il bruno (BAT). Il BAT e’ specializzato nella produzione di calore, mentre il WAT e’ necessario all’ organismo per conservare energia in eccesso sottoforma di trigliceridi . L’ acclimatazione a freddo induce un incremento della componente bruna, senza modificare il numero totale di cellule. Questa forma di plasticita’ cellulare fino ad ora e’ stata associata a modelli animali che possiedono resistenza all’obesita’ ed al diabete. In questo lavoro e’ stato effettuato uno studio anatomico di topi adulti C57BL/6J (B6), linea molto sensibile allo sviluppo della sindrome metabolica con obesita’, iperglicemia e insulinoresistenza se alimentati con HFD . Lo scopo e’ stato quello di verificare la percentuale di adipociti bianchi e bruni di tutti i depositi che compongono l’Organo Adiposo di questo ceppo a 28°C (gruppo di controllo) ed a 6°C. Inoltre e’ stato verificato lo stato termogenico degli adipociti ML tramite l’espressione della proteina UCP1 ed effettuato uno studio sistematico sul numero di fibre noradrenergiche parenchimali TH (Tirosina idrossilasi) positive. La composizione cellulare di tutti i depositi era mista, con entrambi i citotipi rappresentati, non ne sono stati trovati di completamente puri. Questo dato conferma l’ esistenza di un Organo Adiposo . Nei controlli il 77% degli adipociti era UL, mentre negli animali acclimatati a freddo sono piu’ numerose le ML, che rappresentano il 60% di tutti gli adipociti, di questi il 79% era immunoreattivo per l’ anticorpo UCP1. Dopo l’esposizione a 6°C il numero totale di adipociti nell’ organo adiposo non era cambiato, il numero di ML era incrementato in modo significativo (+ 37% p =0.011) e il numero di UL era diminuito approssimativamente della stessa proporzione (- 41% p= 0.05). Non e’ stata trovata nessuna evidenza di apoptosi tra gli adipociti bianchi. Questi dati documentano una plasticita’ dell’ organo adiposo dei B6 che e’ stata osservata maggiormente nel deposito sottocutaneo anteriore (ASC) e nell’ addomino pelvico (AP), mentre in uno studio effettuato in passato su un ceppo piu’ resistente all’ obesita’ (SV129), che presentava una maggior quantita’ di ML sia a caldo che a freddo i depositi piu’ trasformati erano il sottocutaneo posteriore (PSC) ed il mesenterico (MES). La proteina UCP1 che riflette l’attivazione della termogenesi (non shivering) a freddo e’ espressa nel 34% degli adipociti bruni dei controlli, il loro numero incrementa significativamente a 6°C (79% p=0,02). L’ aumento dell’ espressione di UCP1 correla positivamente con quella della Tirosina-idrossilasi (TH), soprattutto negli animali esposti a freddo, suggerendo un ruolo fondamentale del sistema nervoso nella trasformazione dell’ organo adiposo. In molti depositi dei C57BL sia a caldo, ma soprattutto dopo acclimatazione a 6°C sono state osservate molte cellule “Pauciloculari”. In questa linea, come nei 129SV il volume totale dell’ organo si riduce e questo e’giustificato da una diminuzione di volume anche degli adipociti UL ed ML. Complessivamente tutte queste osservazioni suggeriscono un processo di Transdifferenziazione degli adipociti bianchi in adipociti bruni nei topi acclimatati a freddo, confermando quanto gia’ osservato nella linea SV129. concludendo possiamo affermare che la plasticita’ dell’ organo adiposo e’ indipendente dal background genetico, che invece potrebbe influire nel cambiamento di alcuni depositi piuttosto che altri.
In mammals, two functionally different type of adipose tissues are contained in a multi-depot organ: the adipose organ. It consists of several subcutaneous and visceral depots. Some areas of these depots are brown and correspond to brown adipose tissue, wich is specialized in energy expenditure, while many are white and correspond to white adipose tissue, the primary site of triglyceride storage. Cold acclimatisation induces an increase in the brown component without affecting the overall number of adipocytes; so far this form of plasticity was associated to obesity and diabetes resistance in experimental models. In this work we performed an anatomical study of adult C57BL/6J mice, which on HFD develop severe obesity, hyperglycemia and isulin resistance. The aim of this work was to check the percentage of white and brown adipocytes of all fat depots that make up the adipose organ of this strain at 28 ° C (control group) and 6 ° C. Was also monitored the thermogenic status of ML adipocytes through the expression of UCP1 protein and carried out a systematic study on the number of tyrosine hydroxylase parenchymal (noradrenergic)-positive nerve fibers (no. of fibers per 100 adipocytes). The cellular composition of all depots was mixed, with both cellular populations represented, there are not completely pure. This shows the existence of an adipose organ also in C57BL/6J. In the control animals the prevalent cell type were withe adipocytes (77% of all adipocytes), conversely in the cold-acclimated mice there was a prevalence of brown adipocytes (60% of all adipocytes); of these the 79% were immunoreactive for UCP1 antibody. After cold acclimation the total number of adipocytes in the adipose organ was unchanged, the number of brown adipocytes increased significantly (+37% p = 0.011) and the number of white adipocytes decreased by approximately the same amount (-41% p= 0.05). No evidence of apoptosis of white adipocytes was detected. The most prominent differences in cell composition (adipocyte plasticity) were found in the anterior subcutaneous (ASC) and in the abdomino- pelvic depots (AP). In a study conducted in 2005 on strain considered resistant to dietary induced obesity (SV129), was observed that the adipose organ contained a greater amount of ML in both conditions (28 ° C and 6 ° C) and the most changed depots were posterior subcutaneous (PSC) and the mesenteric (MES). In the control group the thermogenic protein UCP1 was expressed only in 34% of ML adipocyes, but their number increase significantly after cold exposure (79% p=0,02). The proportion of brown adipocytes UCP1+ is positively correlated with noradrenergic fiber density especially in the cold-acclimated mice, suggesting a crucial role of nervous system on the changes of the adipose organ after cold exposure. In many depots of C57BL (both 28°C and 6°C),especially after cold acclimation were observed Paucilocular cells (the “transdifferentiation marker”). In In this strain, like in SV129 the adipose organ shrank in volume; this reduction corresponded to the volume reduction of each brown and white adipocyte. Overall, these modifications of the adipose organ suggest a process of transdifferentiation of white into brown adipocytes in cold-acclimated mice confirming what was observed in the SV129 mice. In conclusion we can affirm that the phenotypic plasticity of the whole adipose organ is independent of genetic background, that could affect a greater change in a depot rather than in another .

E’ ben noto come i disturbi metabolici si associno all’obesità quasi esclusivamente quando l’obesità è di tipo viscerale. Ovvero quando i depositi adiposi che si espandono sono principalmente quelli viscerali. Il nostro gruppo ha dimostrato che il 90% dei macrofagi nel WAT dei soggetti obesi si dispone in maniera circolare attorno agli adipociti morti a formare strutture denominare CLS (crown-like structures). Inoltre, abbiamo recentemente osservato che, pur essendo valida la correlazione positiva tra dimensione degli adipociti e densità delle CLS (indice dell’ infiammazione) sia nel grasso viscerale che nel grasso sottocutaneo, negli animali geneticamente obesi, il grasso viscerale presenta una maggiore densità di CLS pur avendo adipociti più piccoli. Questa osservazione suggerisce che gli adipociti viscerali e i sottocutanei presentano una diversa predisposizione alla morte. L’infiltrazione macrofagica che caratterizza l’organo adiposo di soggetti obesi determina uno stato di infiammazione cronica di basso grado che genera insulino resistenza e diabete di tipo 2. In questo studio abbiamo valutato l’espressione di fattori associati allo stato d’infiammazione del tessuto adiposo e la presenza di alterazioni ultrastrutturali nelle cellule adipose dei depositi sottocutanei e viscerali di due modelli di obesità genetica (topi ob/ob e db/db). Nei due ceppi di topi, l’analisi immunoistochimica per PJNK e NF-kB (molecole associate ad infiammazione cronica) ha evidenziato l'espressione di entrambe le proteine da parte dei macrofagi che formano le CLS suggerendo che proprio queste strutture potrebbero essere la principale fonte di P-JNK e NF-kB. Lo studio degli aspetti ultrastrutturali ha evidenziato una riduzione dello spessore citoplasmatico, dell’area media dei mitocondri e della percentuale di superficie citoplasmatica libera da gocce lipidiche occupata dai mitocondri; queste alterazioni sono significative solo nei depositi viscerali, sito prevalente della morte adipocitaria e della conseguente formazione delle CLS. Questi dati confermano l’esistenza di una correlazione fra accumulo di grasso viscerale e incidenza dei disturbi associati all’obesità e suggeriscono che interventi mirati a ridurre lo stato di infiammazione del tessuto adiposo, impedendo il processo di ipertrofia adipocitaria e la conseguente morte degli adipociti, potrebbero rappresentare un promettente approccio per la prevenzione della Sindrome Metabolica.
It is well known that metabolic disorders are associated almost exclusively with obesity in the case of is visceral obesity. That is when the fat depots wich expand are mainly visceral. Our group has shown that 90% of macrophages in WAT of obese individuals surround the dead adipocytes forming structures called CLS (crown-like structures). In addition, we recently observed that, although an significant positive correlation exists between adipocyte size and CLS density (index of inflammation) in visceral and in subcutaneous depots, in genetically obese animals, visceral depot has a greater density of CLS although adipocytes are smaller, suggesting that visceral and subcutaneous adipocytes have a different susceptibility to death. The macrophage infiltration characterizing the adipose organ of obese subjects results in a state of chronic low-grade inflammation that produces insulin resistance and type 2 diabetes. In this study we evaluated the expression of factors associated with the adipose tissue inflammation state and the presence of ultrastructural alterations in adipocytes of subcutaneous and visceral depots of two genetic models of obesity (ob/ob mice and db/db). In both strains, immunohistochemistry analysis for NF-kB and PJNK (molecules associated with chronic inflammation) showed the expression of both proteins by macrophages that form the CLS suggesting that these structures could be the main source of P-JNK and NF-kB. The analysis of ultrastructural aspects showed a reduction of the cytoplasm thickness, mitochondria area, and of the percentage of free lipid droplets cytoplasmic area occupied by mitochondria; these alterations are significant only in the visceral depots, the main site of adipocyte death and the consequent formation of CLS. These data confirm the well known association between the accumulation of visceral fat and incidence of disorders associated with obesity and suggest that interventions aimed at reducing the state of inflammation of adipose tissue, by preventing the adipocyte hypertrophy process and the subsequent death of adipocytes, may represent a promising approach for the prevention of the metabolic syndrome.

Una delle conseguenze più comuni dell’ obesità, soprattutto dell’ obesità viscerale, è l’insorgenza nell’adulto del diabete mellito di tipo 2. Recenti studi suggeriscono che esso sia dovuto ad uno stato di lieve infiammazione cronica dell’ organo adiposo. Si è visto che l’organo adiposo degli obesi è infiltrato da macrofagi che producono citochine (soprattutto TNF-e IL-6) che sembrano essere responsabili dell’ insorgenza dell’ insulino resistenza che precede il diabete mellito di tipo 2. Queste citochine infatti, interferiscono con il substrato 1 del recettore insulinico rendendolo meno efficiente. E’ stato recentemente dimostrato che la grande maggioranza dei macrofagi che infiltrano il grasso si dispongono attorno agli adipociti morti formando strutture caratteristiche denominate “crown-like structures”. Questo fenomeno potrebbe essere causato da un’ eccessiva espansione degli adipociti obesi. Gli adipociti viscerali sembrano più suscettibili a questo tipo di morte offrendo una possibile spiegazione agli effetti più morbigeni dell’ accumulo di grasso viscerale. L’ ipotesi generalmente accettata per spiegare l’ insorgenza del diabete mellito di tipo 2 è che la resistenza insulinica comporti la necessità di una iperproduzione di insulina pancreatica che a lungo andare esaurisce la capacità compensativa delle isole di Langerhans sfociando quindi nel diabete franco. A causa dell’ espansione epidemica dell’ obesità, negli ultimi anni si è diffusa notevolmente la pratica del trattamento chirurgico di questa condizione morbosa. La chirurgia bariatrica negli Stati Uniti costituisce la prima causa di intervento chirurgico. L’ operazione di by-pass intestinale ha evidenziato un risultato inatteso sia nell’ uomo che in condizioni sperimentali: il miglioramento del diabete nei soggetti operati prima della perdita di peso. Questo suggerisce nuovi meccanismi che portano dalla resistenza insulinica alla franca condizione diabetica in quanto la modifica anatomica indotta dalla chirurgia ripristina la secrezione insulinica, suggerendo che vi sia una fase di inibizione della secrezione che precede la perdita di materiale delle cellule  nel pancreas. In questa tesi abbiamo indagato le isole di Langerhans in topi geneticamente obesi (ob/ob - privi di leptina; db/db – privi del recettore leptinico) e in topi indotti all’obesità madiante dieta grassa (HFD) a 15 settimane di età, per verificare se vi siano condizioni strutturali in grado di spiegare il meccanismo attraverso il quale la chirurgia bariatrica è in grado di migliorare in modo acuto e prima del calo ponderale, il metabolismo glucidico compromesso dalla condizione di obesità. I nostri risultati indicano che parallelamente all’ incremento di peso che insorge negli animali obesi, si assiste ad una progressiva ipertrofia e iperplasia delle isole di Langerhans (fase di compenso alla resistenza insulinica) soprattutto nei topi geneticamente modificati. A questo si accompagna un incremento dell’ insulino-resistenza che sfocia nel diabete franco come avviene nei topi db/db. Il diabete insorge prima di una evidente perdita di sostanza a livello delle isole di Langerhans suggerendo una fase di inibizione della secrezione insulinica. Il fenomeno assume maggiore gravità negli animali db/db rispetto agli ob/ob e agli HFD. L’ analisi delle isole rivela due aspetti che possono spiegare una progressiva inibizione della secrezione insulinica: il significativo e progressivo aumento dell’ innervazione parenchimale delle isole di Langerhans da parte di fibre adrenergiche (tirosina-idrossilasi immunoreattive) e la redistribuzione di elementi cellulari intrainsulari immunoreattivi per il neuopeptide Y (NPY). Entrambi questi aspetti possono indicare una progressiva inibizione della secrezione insulinica prima che intervengano fenomeni apoptotici in grado di determinare un esaurimento anatomico della secrezione insulinica. Questi dati suggeriscono quindi l’ipotesi che la chirurgia bariatrica possa promuovere meccanismi citofisiologici a partenza intestinale in grado di rimuovere tali aspetti inibitori. Inoltre, la presenza di cellule chemo-sensitive del primo tratto della parete intestinale suggerisce che tali elementi potrebbero essere implicati nel determinare il fenomeno inibitorio.
Among the most common consequences of the obesity, especially of visceral obesity, there is the onset of type 2 diabetes mellitus in adults. Recent studies suggest that this condition is due to a state of mild chronic inflammation of adipose organ. It has been shown that the body fat of obese subjects is infiltrated by macrophages that appear toproduce cytokines (especially TNF- and IL-6) responsible for the insurgence of insulin resistance that precedes type 2 diabetes mellitus. Indeed, they interfere with the insulin receptor substrate 1, making it less efficient. It has been recently shown that the vast majority of macrophages that infiltrate adipose tissue localize around dead adipocytes, forming characteristic structures known as "crown-like structures". This phenomenon could be caused by an over-expansion of obese adipocytes. Visceral adipocytes seem to be more susceptible to this kind of death by providing a possible explanation of dangerous effects of visceral fat accumulation. The widely accepted hypothesis to explain the onset of type 2 diabetes, is that insulin resistance results from the need of a pancreatic overproduction of insulin that eventually exhausts the compensatory capacity of the islets of Langerhans finally leading to frank diabetes. Because of the growing epidemic of obesity, in the last years the surgical practice for the treatment of this disease has significantly spread. Bariatric surgery in the United States is the main cause of surgery. Unexpectedly the intestinal bypass operation in humans and in experimental models results in the improvement of diabetes in patients that underwent surgery before weight loss. The fact that the anatomical changes induced by surgery are able to restore insulin secretion provides further explanations for the mechanisms responsible for the shift from the condition of insulin resistance to that of frank diabetes by suggesting the occurrence of a phase of insulin secretion inhibition preceding the loss of material in the cells of Langerhans. In this study we investigated the islets of Langerhans both in genetically modified obese mice (ob/ob : no leptin; db/db: leptin receptor-free) and mice with obesity induced by high - fat diet (HFD) at 15 weeks of age, to assess the existence of structural conditions that could explain the mechanism by which bariatric surgery can improve acutely the impaired glucose metabolism of obese subjects and before the weight loss. Our results indicate that in parallel to 'weight gain that occurs in obese animals, a progressive hypertrophy and hyperplasia of the islets of Langerhans (phase compensation to insulin resistance) occurs, especially in genetically modified mice. This is accompanied by an increase of insulin resistance that in mice leads to frank diabetes as we observed in db/db mice. The occurrence of diabetes before an evident loss of substance in the islets of Langerhans suggests the evidence of a phase of inhibition of insulin secretion. The phenomenon is more severe in db / db mice compared with ob / ob and the HFD mice. The analysis of the islets revealed two aspects that could explain a progressive inhibition of insulin secretion: the significant and progressive increase of' parenchymal innervation of the islets of Langerhans by adrenergic fibers (tyrosine hydroxylase, TH - immunoreactive) and the redistribution of cellular intrainsular elements that are immunoreactive for neuopeptide Y (NPY). Both these aspects indicate that a progressive inhibition of insulin secretion possibly occurs before apoptosis leading to a depletion of anatomical insulin secretion. These data provide the hypothesis that bariatric surgery could promote cytophysiological intestinal mechanisms through the removal of those inhibitory elements. In addition, the presence of chemo-sensitive cells in the first section of the bowel wall suggests that these factors may be involved in determining the inhibitory phenomenon.

Neuropsychiatric Systemic Lupus Erythematosus (NPSLE) is an autoimmune disease characterized by the production of brain-reactive autoantibodies. These autoantibodies are thought to be one of the main mediators of this condition, however little is known about their cellular and molecular target. To get inside this matter, we enrolled a cohort of 209 subjects including patients affected by Systemic Lupus Erythematosus (SLE, n=69), NPSLE (drug-naïve, n=36), multiple sclerosis (MS, n=22) as well as 82 age and gender matched healthy subjects. Sera from these subjects were used for immunostaining on rat brain through fluorescence microscopy and TEM, in parallel with immunofluorescence on human brain sections and cells (SH-SY5Y) to eventually confirm the results obtained in rat. As to the brain autoantibody target, western blot (WB) using wild type and transfected HEK293 cells, which do or do not express the RBFOXP3 gene (encoding the NeuN protein). Furthermore, ELISA was programmed to reveal the possible presence of the known circulating autoantibodies in SLE (anti: -Ro/SSA, -La/SSB, -RNP, -Sm, and -Rib-P) and autoantibodies to the aminoacidic sequence DWEYS. On rat brain sections, we observed autoantibody positive reaction in several areas (cortex, hippocampus, cerebellum, and others) exclusively using sera from patients with NPSLE and SLE but with prevalence and higher titer among the NPSLE patients (77,1% vs. 56,5%, respectively and 1:2,000-8,000 vs. 1:120-1,000, respectively). In order to consolidate the results obtained in rats, negative and positive sera from SLE and NPLSE patients as well as healthy and MS controls were selected (n=6) to be tested in human hippocampus and cerebellum sections. Autoantibody reactivity was found only using those sera positively reacting on rat tissues. Through ELISA, anti: -Ro/SSA, -La/SSB, -RNP, -Sm, -Rib-P and -DWEYS were found using rat-immunostained-positive sera from both NPSLE and SLE patients. In both patients’ populations, the presence of one or more types of these autoantibodies was not higher than 31,6% while a percentage was negative for all the above autoantibodies (17,9% and 31,6%, in NPSLE and SLE respectively). Hence, the presence of these autoantibodies could confirm only partially the reactivity according to the immunostaining results. In the rat cerebellum, the autoantibodies stained a cytoplasmic area also in proximity of the nucleus (namely perinuclear/cytoplasmatic pattern) or inside the nucleus (namely intranuclear pattern), both revealed using either fluorescence microscopy or TEM. In order to identify the cell type recognized by the autoantibodies in the cerebellum, we performed double staining, mixing the sera from patients with the antibody to NeuN (known to mark the granule cells), as well as antibodies to VGlut1, VAChT or GAD-65, marking glutamatergic axon terminals, cholinergic and GABAergic neurons, respectively. Autoantibody staining was localized exclusively within NeuN containing cells. Autoantibodies labelled NeuN containing neurons also in many other areas of the brain. We performed WB experiments to reveal NeuN as possible autoepitope using either wild type and transfected HEK293 cells, which do not or do express the RBFOXP3 gene, respectively. The patient’s sera never recognized the bands corresponding to the NeuN protein, despite these bands were labeled by the anti-NeuN antibody using transfected cells, proving that NeuN was not the autoantibody target. In conclusion, all together the da-ta obtained from this study demonstrate that (1) SLE and NPSLE could possess a mix of different types of autoantibodies reacting to neuronal cells (2) the entire brain could be the target of these autoantibodies (3) a high prevalence of brain reactive autoantibodies is found among NPLSE patients and (4) autoantibodies could react to the nucleus and/or cytoplasm of brain neuron cells.

Crucial for chronic lymphocytic leukaemia (CLL) development and progression are the iterative cycles of cell re-activation and proliferation that take place in lymphoid tissues. These iterative cycles are fundamental for the development and the progression of the disease. Since cellular fatty acid (FA) import and oxidation (FAO) were recently reported to be upregulated in CLL, compared to normal B lymphocytes, we explored the in vitro effects of ST1326, a reversible inhibitor of carnitine-palmitoyl transferase 1A (CPT1A), on leukaemic cells subject to activating microenvironment-mimicking stimuli. ST1326 induced dose-dependent mitochondrial dysfunction and cell death, which were remarkably higher in activated/proliferating than quiescent CLL cells. Drug sensitivity was observed irrespective of the presence of TP53 alterations or chromosomal abnormalities, which are known to cause chemoresistance in CLL patients. The treatment of normal B lymphocytes with ST1326, at doses lethal to CLL cells, causes only a slight inhibition of cell activation/proliferation, indicating a modest cytostatic, not cytotoxic, effect. ST1326 cytotoxicity in CLL was associated with decreased levels of intracellular Acetyl-CoA and down-regulation of signalling pathways that are crucial for leukaemic cell survival, activation and proliferation. In particular, environment-induced activation of STAT3 and STAT6 transcription factors, known to upregulate anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL, was impaired by ST1326. As a consequence, drug combination experiments with the BH3-mimetic ABT-199/Venetoclax, whose effects are counteracted by Mcl-1/Bcl-xL and cell proliferation, showed strong ST1326-mediated potentiation of ABT-199 cytotoxicity in activated/proliferating CLL cells. We also observed that ST1326 showed a synergic effect with Fludarabine in activated/proliferating CLL cells. The data indicate that CLL cells turning to an activated/proliferating state become more dependent on FAO and more sensitive to FAO-antagonists, and pave the way for ST1326 as an adjuvant tool in anti-CLL drug-combination regimens with drugs that lose efficacy on proliferating leukaemic cells.

Introduzione: Gli agonisti del recettore Toll-like 9 (TLR9) sono noti per la loro capacità di attivare le cellule del sistema immunitario innato e sono stati ampiamente studiati per il trattamento di diversi tipi di tumore. Tuttavia, ad oggi non sono stati raggiunti risultati soddisfacenti. Infatti, per prevenire reazioni immunitarie incontrollate e potenzialmente dannose, l’attivazione del TLR9 induce diversi meccanismi immunoregolatori che, in ultima istanza, possono inibire la risposta immunitaria. A questo proposito, è stato osservato che la stimolazione del TLR9 provoca un aumento dell’espressione del recettore PD-1 sulle cellule immuni. PD-1 è espresso dalle cellule del sistema immunitario sia innato sia adattativo e, in seguito al legame con i suoi due ligandi (PD-L1 e –L2), provoca lo spegnimento dell’attività del sistema immunitario. Per sfuggire alla risposta immunitaria, le cellule tumorali possono sfruttare l’asse PD-1/PD-Ls grazie all’over-espressione dei ligandi di PD-1. Il blocco di PD-1 mediante specifici anticorpi monoclonali, che impediscono l’interazione tra PD-1 e i suoi ligandi, può ripristinare l’attività delle cellule immunitarie. Tuttavia, solamente il 30% dei pazienti con tumore in stadio avanzato trae beneficio da questo tipo di terapia. Di conseguenza, una coterapia basata sull’attivazione del sistema immunitario mediante agonisti del TLR9, e sul blocco di un meccanismo immunosoppressivo, utilizzando un anticorpo anti-PD-1, rappresenta una promettente strategia immunoterapeutica per il trattamento del cancro. Scopo: L’efficacia della co-somministrazione di questi due farmaci è stata largamente studiata in molteplici studi preclinici e clinici, che si sono focalizzati principalmente sul ruolo del sistema immunitario adattativo. Poichè le cellule immunitarie innate, che possono esprimere PD-1, rappresentano i principali target degli agonisti del TLR9, lo scopo di questo studio è stato la valutazione del ruolo delle cellule del sistema immunitario innato nel determinare gli effetti della co-terapia. Metodi: Un agonista del TLR9 (CpG-ODN) e un anticorpo anti-PD-1 sono stati somministrati, singolarmente o in combinazione, a topi atimici xenotrapiantati intraperitonealmente con cellule IGROV-1 del carcinoma ovarico umano. I macrofagi sono stati depletati in vivo mediante clodronato incapsulato nei liposomi. Il microambiente tumorale è stato valutato con analisi di immunofluorescenza. Sono stati effettuati studi in vitro trattando la linea cellulare di macrofagi murini RAW264.7 con CpG-ODN e/o anticorpo anti-PD-1. L’effetto della combinazione sui macrofagi è stato valutato con analisi del profilo di espressione genica seguite da analisi bioinformatiche, Real-time PCR, multiplex ELISA e saggi di rilascio del 51Cr. Per investigare il ruolo specifico del sito di legame con l’antigene e del dominio del frammento cristallizzabile (Fc) dell’anticorpo anti-PD-1, sono stati utilizzati anticorpi anti-PD-1 ingegnerizzati. Il coinvolgimento del TRIM21 è stato valutato attraverso l’inibizione farmacologica ed esperimenti di silenziamento. Risultati: I topi che hanno ricevuto la combinazione CpG-ODN/anticorpo anti-PD-1 hanno mostrato un aumento della crescita tumorale e una marcata infiltrazione di macrofagi CD206+ e IL-10+ rispetto agli animali trattati con solo CpG-ODN. La deplezione dei macrofagi ha completamente annullato l’aumentata progressione tumorale osservata nel gruppo della combinazione. Gli esperimenti in vitro hanno evidenziato come le cellule RAW264.7 incubate sia con CpG-ODN sia con anticorpo anti-PD-1 esibiscano un profilo di espressione genica completamente differente da quelle trattate con il singolo farmaco, sviluppando un fenotipo caratterizzato dall’up-regolazione di markers associati sia a M1 sia a M2. I saggi multiplex ELISA hanno evidenziato un’aumentata secrezione di citochine relative sia a M1 sia a M2, validando i dati di espressione genica. I saggi funzionali hanno mostrato che i macrofagi stimolati con la combinazione sono in grado di ridurre l’attività citotossica delle cellule NK rispetto ai macrofagi incubati con CpG-ODN o anticorpo anti-PD-1. L’utilizzo di anticorpi anti-PD-1 ingegnerizzati ha rivelato che sia il riconoscimento del dominio Fc sia il sito di legame con l’antigene dell’anticorpo anti-PD-1 sono necessari per determinare I cambiamenti del profilo di espressione genica dei macrofagi osservati nella combinazione. Infine, si è dimostrato che il TRIM21 è coinvolto nelle modificazioni del fenotipo dei macrofagi mediante l’interazione con il dominio Fc dell’anticorpo anti-PD-1. Conclusioni: I risultati di questo studio mostrano che, quando nei macrofagi viene attivato il signaling del TLR9, la co-somministrazione con anticorpo anti-PD-1 induce in queste cellule immunitarie l’acquisizione di un fenotipo misto M1/M2 caratterizzato da funzionalità immunosoppressiva, in grado di compromettere la risposta immunitaria e promuovere la crescita tumorale. Poichè l’associazione di agonisti del TLR9 con anticorpi anti-PD-1 è tuttora investigata in diversi studi clinici, l’impatto di questi agenti faramacologici sui macrofagi dovrebbe essere preso in considerazione per evitare reazioni avverse potenzialmente pericolose, soprattutto nei tumori ad elevata infiltrazione di macrofagi.
Background: Toll-like receptor 9 (TLR9) agonists are known for their ability to activate innate immune cells and have been extensively tested for the treatment of different types of tumors. However, no satisfied results have been achieved so far. Indeed, to prevent uncontrolled and potentially harmful immune reactions, TLR9 activation induces several immunoregulatory mechanisms that, eventually, restrain the immune response. Among such processes, it has been observed that TLR9 triggering determines PD-1 receptor up-regulation on immune cells. PD-1 is reported to be expressed by adaptive and innate immune cells and to dampen immune system function upon binding to its two ligands (PD-L1 and -L2). By over-expressing such ligands, tumor cells exploit PD-1/PD-Ls axis to evade immune system attack. The blockade of PD-1 using specific monoclonal antibodies, disrupting PD-1/PD-Ls interaction, is able to restore the activity of the immune cells. However, only 30% of patients with advanced cancer benefit of this kind of therapy. Therefore, a combinatorial immunotherapeutic regimen based on the activation of the immune system, by TLR9 agonists administration, and on the abrogation of an immunosuppressive mechanism, utilizing anti-PD-1 antibody, represent a promising immunotherapeutic strategy in the treatment of cancer. Aim: The efficacy of the co-administration of these two immunotherapeutic drugs have been deeply investigated in several clinical and preclinical studies that mainly focused on the role played by the adaptive immune system. Considering that TLR9 agonists primarily targets innate immune cells and PD-1 can be also expressed by the innate immune system, the aim of the present study was to evaluate the contribution of the innate immune cells to the final outcome of this combinatorial therapy. Methods: Athymic mice, intraperitoneally xenografted with IGROV-1 human ovarian cancer cell line, received TLR9 agonist (CpG-ODN), anti-PD-1 antibody or their combination. In vivo macrophages depletion was achieved by liposomal-containing clodronate administration. Tumor immune contexture was evaluated by immunofluorescence analysis. In vitro studies were carried out by exposing RAW264.7 mouse macrophage cell line to CpG-ODN and/or anti-PD-1 antibody. The effect of the combinatorial treatment on macrophages was assessed by microarray profile and subsequent bioinformatic analysis, Real-time PCR, multiplex ELISA and 51Cr-release assays. To unravel the specific contribution of the antigen-binding site and the fragment crystallizable (Fc) domain of anti-PD-1 antibody, modified anti-PD-1 antibodies were utilized. The involvement of TRIM21 was investigated by pharmacological inhibition and silencing experiments. Results: Mice receiving CpG-ODN/anti-PD-1 antibody combination experienced an acceleration of tumor growth paralleled by a marked infiltration of CD206+ and IL-10+ macrophages compared to CpG-ODN-treated animals. The increased tumor progression observed in the combination group was completely abrogated by macrophage depletion. In vitro experiments showed that RAW264.7 cells stimulated with CpG-ODN and anti-PD-1 antibody exhibited a gene expression profile completely different to those exposed to each drug alone, acquiring a phenotype characterized by the up-regulation of both M1- and M2-related markers. Multiplex ELISA assays confirmed an augmented secretion of M1 and M2 cytokines, validating gene profile data. Functional assay showed that combination-primed macrophages were able to dampen NK cell cytotoxic activity compared to CpG-ODN- or anti-PD-1- antibody-treated cells. The use of engineered anti-PD-1 antibodies revealed that either the Fc domain and the antigen-binding site of anti-PD-1 antibody are necessary for determining the changes in gene expression profile on macrophage when combined with TLR9 agonists. Finally, TRIM21 was found to be involved in shaping macrophage phenotype by interacting with anti-PD-1 antibody Fc domain. Conclusions: Our results indicate that, when TLR9 signaling is activated, the co-administration of anti-PD-1 antibody induces in macrophages the acquisition of a “mixed” M1/M2 phenotype with enhanced immunosuppressive features, eventually negatively impacting on the immune response and promoting tumor growth. Since TLR9 stimulation and PD-1 blockade combinatorial immunotherapy is under investigation in different clinical trials, the impact of both agents on macrophages should be taken into consideration to avoid potentially harmful adverse effects, especially in tumors where the infiltration of macrophages is particularly abundant.

Scopo del lavoro Lo scopo principale del presente elaborato è stato quello di contribuire ad approfondire, attraverso l’analisi della cinematica mandibolare, la conoscenza riguardo al recupero funzionale post-operatorio del sistema stomatognatico, dopo trattamento combinato ortodontico-chirurgico e terapia per la riduzione della frattura di condilo mandibolare. Materiali e metodi Sono stati presi in considerazione, in 4 diversi esperimenti, pazienti sottoposti a chirurgia ortognatica e pazienti con esiti di frattura monolaterale di condilo, sottoposti ad intervento chirurgico di riduzione della frattura a cielo aperto o trattati non chirurgicamente mediante blocco intermascellare. I soggetti sono stati analizzati con metodiche non invasive, eseguendo una valutazione tridimensionale dei movimenti mandibolari mediante un sistema optoelettronico. I dati acquisiti nei diversi gruppi di studio sono stati confrontati con un gruppi di controllo formati da soggetti stomatologicamente sani, al fine di evidenziarne le eventuali discrepanze rispetto alla normale cinematica mandibolare. Sono stati acquisiti i movimenti limite della mandibola: massima apertura e chiusura buccale, laterotrusioni a destra e a sinistra, protrusione e retrusione. Inoltre sono stati analizzati i tracciati tridimensionali del punto interincisale e dei due condili, mentre per l’apertura è stata effettuata anche un’ulteriore scomposizione del movimento nelle due componenti rotatoria e traslatoria. Risultati I pazienti, dopo trattamento ortodontico combinato con chirurgia ortognatica o dopo riduzione di frattura di condilo, presentavano dal punto di vista clinico, un cinematica mandibolare comparabile con i soggetti normali. Ciononostante, nei diversi esperimenti, alcuni parametri sono risultati significativamente modificati: in particolare la percentuale di rotazione della mandibola durante il movimento di apertura e chiusura buccale, la quantità di protrusione mandibolare e i tragitti dei due condili. Conclusioni Grazie ai parametri utilizzati abbiamo dimostrato come, a breve e a medio termine, il recupero clinico dell’articolazione temporomandibolare sia totale e la funzione sia paragonabile a quella dei soggetti sani. Un’analisi dettagliata ha però evidenziato precisi deficit funzionali nella cinematica articolare. Non esistono ad oggi dati che mettono in relazione tali cambiamenti della cinematica mandibolare e condilare con l’insorgenza a lungo termine di patologie articolari. Le ricerche future saranno volte ad estendere ed approfondire tali analisi anche in pazienti che soffrono di patologie degenerative dell’ATM

Skin is a target organ for crucial side-effects of radiotherapy and the early effects on epidermis remain to be fully elucidated. Organotypic cultures of human skin can help in defining the early effects difficult to study in humans. To characterize our model, we performed a keratinocyte proliferation analysis in a time course study and then in irradiated samples. We evaluated the effects on terminal differentiation, intercellular adhesion and apoptosis by immunofluorescence and ultrastructural analysis. Bioptic fragments (n=8) were obtained from cosmetic surgery of young healthy women, cultured in Transwell, and harvested 24 or 48 hours after exposure to a single 2 Gy dose of gamma-rays. Epidermal proliferation progressively decreased in the time course study and significantly inhibited 24 hrs after gamma-rays. Patterns of CK10, INV, Dsg1 and Dsc1 proteins distribution were comparable in all samples. P53 was similarly detected in scattered positive keratinocytes in the spinous layer of 24 hrs samples, whilst in 48 hrs irradiated samples was expressed starting from basal layer. TEM analysis indicated that no desmosomal ultrastructural modifications occurred. In 24hrs irradiated skin the basal layer was undamaged but a condensation of intermediate filaments and apoptosis were evident in the upper spinous layers. In 48hrs irradiated skin apoptotic nuclei started from the basal layer. The epidermal homeostasis is rapidly altered by a single clinical dose of gamma-rays.

Age estimation is one of the most difficult and sensitive procedure in forensic pathology, and is characterized by a long tradition and a number of fields of application: the most common cases of age estimation in the forensic scenario deal with dead people, and in detail with personal identification; however, in the last years, age estimation has begun to include also other forensic cases, especially within the so-called Clinical Forensic Medicine, which is the branch of forensic pathology dealing with the living, and consequent evaluation of clinical data for judicial purposes. The procedure of age estimation therefore is now applied to the living for the ascertainment of imputability before trial, or for the correct assignation of scholar classes of adopted children. In recent times, also the age estimation of the living adult, which in the most challenging and difficult field of application, has begun to be performed in order to verify the age of retirement, usually in old immigrants. One of the most interesting phenomena recorded during this long evolution is the constant and progressive modification of methods of age estimation according to the specific field of application: some methods cannot be applied in specific cases, and this lack of tools has led to the evolution of new procedures, or the amelioration of the existing ones. However, the more specific the field of application, the fewer are the methods which can be applied. The extreme evolution of this phenomenon concerns the most recent field of application of age estimation in the living which deals with 2D images: the ascertainment of age in case of photos may be judicially important, since most Countries state the crime of juvenile pronography, which concerns all the images reproducing minors in a pornographic context. In these cases the forensic anthropologist may be requested to provide an age estimation of the persons in the photo: this is the final evolution of any procedure of age estimation, which has passed in the last year from the dead subject (where any investigative method is substantially allowed), to the living person (where only few methods can be applied) up to the image of the living person itself. One can clearly consider the pitfalls included in such as operation, since the forensic anthropologist or pathologist is usually requested to give an indication concerning the biological profile of a real person on his photos. If in case of the living age estimation has tried to change its methods, now with photos the forensic anthropologist faces a new challenge which consists not only in the development of new tools, but also in verifying if such analyses can be performed also in photos. The tasks are therefore two, and both of them difficult to achieve: the first one consists in finding biological information related with age, and the second in ascertaining if such information is also verifiable in photo. As one can imagine, the procedure of age estimation in cases of images faces new challenges, with new questions which are still waiting for an answer. This study aims at exposing the results of different investigations performed during the PhD course, aimed at verifying the relation between facial measurements and age, in vivo and in photo: the line of research followed a project aiming first at ascertaining the reliability of linear measurements in photos, both for subjects aged under and above 18 year threshold, and then at verifying the chance of extrapolating new biological parameters from the face useful for age estimation. The first steps will consist in finding a standardization of facial metrical assessment by an analysis of reliability of collocation of facial landmarks. After this phase, the study will attempt at verifying the reliability of linear measurements for age estimation in photos in subjects aged under 18 years. A similar experiment will be applied as well to young adults aged over 18 years, after a preliminary study of in vivo measurements in order to verify the correlation of such parameters with age. In conclusion, the study will attempt at finding new biological geometrical parameters (in detail, facial surfaces) for age estimation, in vivo and in photo. This path led to interesting results which may be sumed up in the following points: 1) the study performed on facial landmarks showed that not all the points are reliable for a standardized procedure of facial assessment: in detail, the anatomical landmarks are the most trustable, probably because are defined by anatomical structures which can be easily detected, and are consequently less subjective. More interestingly, such landmarks show the same reliability in photos taken from subjects with different ages, and this means that anatomical points are crucial also for the issue of age estimation from 2D images; 2) the second study pointed out that ratios between linear measurements show a correlation with age also in photos, and therefore may be used for age estimation, although with a high error range, and only taking into consideration the main age thresholds; from this point of view, such parameters may provide a method for verifying if a subject is close to one of the chosen age limits, but data are too limited to provide adequate regression formulae useful to put in relation age and measurements. However, these results represent the first step for the development of an age estimation method useful for reconstructing age with higher precision; 3) the third study confirmed that some linear measurements show a correlation with age which may be promising for the development of regression formulae useful for age estimation in photos; however, the error range is estimated in 3-4 years, which means that age may be reconstructed in an interval of several years: however, this error range matches with difficulties to the actual forensic purposes, where an age estimation with a more limited error is often requested; the need for the search for new facial parameters is therefore even more evident; 4) the fourth study attempted at verifying the modifications of facial parameters in juvenile adults and subjects in transition phase, and pointed out that ear and mouth show a constant increase also after 18 years; from a general point of view, the parameters which are related with age in vivo should show the same relation also in photos, and therefore may be useful for age estimation; 5) the fifth study focused on verifying the applicability of ear characteristics to age estimation in photos: results show that neither the ratios between linear measurements nor the areas show a correlation with age; these results therefore confirm that photograph is an independent manner of acquisition of reality, with its own rules and relations between facial parameters and age, which not necessarily correspond to those observed in vivo. These results mean that the relation of facial parameters should be verified directly in photos, and underline the need for determining new facial measurements useful for forensic purposes; 6) the last study aimed at verifying the relation of a novel facial parameter (in detail, facial surfaces) with age; results show that the correlation are lower than those showed by linear measurements: the analysis performed in photos confirmed the independence of measurements taken in photos and in vivo (actually, on the digital 3D models of the face), since some parameters are related with age in vivo, not in photos, and vice versa. Anyway, facial surfaces are too variable and are influenced by too many environmental and individual factors to be usable for the development of an age estimation method. In conclusion, facial metrical assessment actually cannot be considered a scientifically valid method for forensic purposes: in detail, linear measurements have been widely explored by past literature which showed their constant modification with age. However, this information finds a limited importance in forensic practice, and is affected by a relevant limit in 2D images, which deals with the distorsion of measurements in two dimensions. The introduction of the new 3D acquisition systems now allows the operators to perform a more detailed analysis of face by the measurements of surfaces and volumes; however, literature concerning this topic is still at the beginning, and mainly deals with geometrical measurements which are more standardized, but also more influenced by other variability factors than age, such as the weight, etc. From this point of view, the analysis of surfaces and volumes adds precious information to the morphology of face, and it will be one of the main field of research of the modern anatomy. From the anatomical point of view, the experimental project pointed out relevant suggestions for different issues: in detail, the chromatic analysis of facial modifications may provide a new useful tool for standardizing and quantifiying variables such as increasing or decreasing of weight, the influence of facial expressions, the resemblance of children with their parents. The application of modern 3D image acquisition system may therefore radically improve the anatomical study of faces, adding new information potentially useful also for clinical purposes.

Psoriasis has recently been defined as “a T-cell mediated inflammatory skin disease with T helper cell type 1 (Th1), type 17 (Th17) and IL-22-producing CD4+ T cells as principal mediators”. The interplay between pro-inflammatory cytokines such as Tumour Necrosis Factor (TNF)-alpha, interleukin (IL)-17 and IL-22 and the two main epidermal cytotypes, i.e. keratinocytes and Langerhans cells, is a central issue in the pathogenesis of psoriasis. The aim of our study was to evaluate the early, direct, and specific effect of TNF-alpha, IL-17 and IL-22 alone or in combination by immunofluorescence on keratinocyte proliferation, keratin (K) 10, 14 and 17 expression, the molecular composition of intercellular junctions (desmocollin 1, E-cadherin, occludin and filaggrin), number of epidermal Langerhans cells. Transmission electron microscopy (TEM) was used to analyse the fine structure of the skin. An innovative model of human skin culture standardized in our laboratory, in which a psoriatic microenvironment was reproduced, was used. Skin explants were obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent. Bioptic fragments were divided before adding TNF-alpha or IL-17 or IL-22 or a combination of the three cytokines (Triple), and harvested 24, 48, and 72 hours after cytokine incubation. Interestingly, keratinocyte proliferation was inhibited after exposure to TNF-alpha, IL-17 and the combination of cytokines while this parameter was not affected by IL-22 incubation. In all experimental groups, starting from 24 hours, occludin immunostaining was not homogeneously distributed. K10 immunostaining gradually decreased in scattered clusters in the spinous layer only after exposure to IL-22 or to a combination of the three cytokines. K14 staining became widely discontinuous with both IL-22 and the triple, starting from 48 hours. K17 expression was induced and progressively increased with time in the suprabasal layers of epidermis in all experimental groups, except TNF-alpha group. No differences were found in the expression of desmocollin 1 and the distribution of E-cadherin, at any time point. By immunofluorescence analysis with anti-human Langerin antibody we calculated the percentage of Langerhans cells/mm2 of living epidermis after 24 and 48 hours of incubation (considering control as 100%). At 24 hours Langerhans cells number was significantly higher in samples treated with a combination of IL-17 and TNF-alpha (216.71 + 15.10%; p < 0.001) and in TNF-alpha (125.74 + 26.24%; p < 0.05). No differences were observed in IL-17- treated samples (100.14 + 38.42%). After 48 hours, the number of epidermal Langerin-positive cells in IL-17- and TNF-alpha treated samples slightly decreased (94.99 + 36.79 % and 101.37 + 23% vs. their controls, respectively). With the combination of IL-17 and TNF-alpha epidermal Langerhans cells strongly decreased (120 +13.36%). At ultrastructural level, after IL-22 incubation we observed keratin aggregates in the perinuclear cytoplasm of cells, while the combination of the three cytokines induced an enlargement of intercellular spaces. By TEM, upon TNF-alpha stimulus, Langerhans cells appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical Birbeck granules. Upon IL-17 stimulus, Langerhans cells showed a cytoplasm with many mitochondria and numerous Birbeck granules close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of a combination of IL-17 and TNF-alpha did not affect Langerhans cells ultrastructure. Altogether, our results support the hypothesis that a synergistic effect exists among the pro-inflammatory cytokines of psoriasis and that they interplay with each other in the microenvironment of the psoriatic plaque, with consequent amplification of the effect of each cytokine.

Brain disorders are the first leading cause of disability and the second leading cause of death worldwide. Decoding the mechanisms of brain diseases is mandatory to improve diagnosis, neuroprotective strategies, and treatments. Although there are many different pathological and clinical manifestations, as well as causes and molecular pathways underlying neurological disorders, increasing evidence suggest that synapses, having a crucial role in neuronal communication and brain functions, are the first station that degenerates and loses functionality in neurological diseases. This concept has led to the theory of brain disease, from neurodevelopmental to neurodegenerative, as synaptopathies, in which the synaptic impairment is a shared pathogenic feature. The main aim of this thesis was to dissect this concept, focusing on a specific pathway particularly involved in brain functions and dysfunctions: the c-Jun N-terminal Kinase (JNK) signalling pathway. More in details, we studied the role of JNK in two neurodevelopmental syndromes, Rett and Angelman, and in a chronic brain illness, Alzheimer disease, focusing on the synaptopathy as the first initial mechanism of many different brain diseases. In Rett Syndrome, a rare severe developmental disease, we studied the synaptic dysfunction of two different murine models identifying JNK as an important actor downstream MECP2, the gene mutated in the pathology. We demonstrated that the specific inhibition of JNK, by D-JNKI1 treatment, strongly improved the symptoms and the molecular disorganization of the PSD region in both mice models. Then, we proved JNK activation in human neurons, differentiated from human MECP2-mutated iPSCs (MECP2mut), compared to the isogenic control expressing wild-type MECP2 allele (MECP2wt). The JNK signal was activated in the MECP2-mutated iPSCs and not in control iPSCs, and D-JNKI1 blocked the MECP2mut -induced neuronal death. In Angelman Syndrome, we analysed the synaptic dysfunction in the UBE3A+/- mouse model. JNK was strongly activated in the brain of these mice, suggesting its important role also in this disorder. The D- JNKI1 treatment improved the behavioural defects, and this correlated with the stabilization of the synaptic biomarkers. Finally, we focused on the Alzheimer’s synaptic dysfunction, the most characterize synaptopathy, to study JNK role in a chronic illness. Alzheimer mice model 5XFAD presented a powerful JNK activation together with synaptic dysfunction and cognitive impairment. We then centre our attention on the selective brain JNK isoform, JNK3, the most responsive to stress stimuli, finding that 5XFAD mice presented a powerful increase of JNK3 levels. Therefore, we decided to test the potential neuroprotective effect of the specific inhibition of JNK3. In fact, thanks to the collaboration with Prof. Falconi, we have in the laboratory the specific JNK3 inhibitor, dSIMBA2. Before testing this in-vivo, we studied the effect of JNK3 inhibition in an in-vitro model of synaptic dysfunction induced by Ab oligomers (ABO) to define the right dose and the neuroprotection against ABO-induced synaptic dysfunction, finding that dSIMBA2 prevents ABO toxicity and the induced-synaptopathy. 5 To summarize, the data obtained in this thesis participate in the reinforcement of JNK as a central actor in the degeneration mechanisms of the synapses that characterized both neurodevelopmental and neurodegenerative diseases. In addition, this work adds new elements to define JNK3, the most responsive JNK isoform to stress, as a key player in the AD, and an important target for the treatment of synaptic injury, potentially also in other brain illnesses. Developing new compounds able to inhibit one of the most responsive kinases to stress stimuli may be an intriguing field to explore to prevent synaptic dysfunction.

In the mouse nervous system, Sox2 is expressed in stem cells and early precursors, and in few mature neurons (Zappone et al., 2000; Ferri et al., 2004). Adult Sox2-deficient mice, in which Sox2 expression is decreased by about 70%, exhibit neural stem/precursor cell proliferative defects in the hippocampus and periventricular zone (Ferri et al., 2004). I performed in vitro differentiation studies on neurosphere derived neural cells. Neural stem cells from Sox2-deficient mice produce reduced numbers of neural progenitors. Moreover, I found the SOX2 is important for the neural progenitors cell cycle progression. I demonstrated that SOX2 promoted the proliferation of neural stem cells through facilitating the G1/S transition with the levels of cyclin D1 and cyclin E expression in neural progenitors samples. Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production. Mash1 is required for the generation of neocortical neurons with characteristics of GABAergic interneurons. Moreover, in vivo Sox2 deficiency causes a reduction of GABAergic neurons (Cavallaro M. et al., 2008). These observations point to a possible role for SOX2 togheter with Mash1 in the genesis of neural progenitors of GABAergic neurons. The early neural progenitors, derive from Sox2-deficient mice produce reduced numbers of MASH1 positive cells. In vivo Chromatin immunoprecipitation assays reveal interactions of SOX2 and Mash1 in early neural progenitor cells. My data also suggest that the specific recruitment of these protein to the Mash1 in the ventral telencephalon defines the spatiotemporal activity of MASH1 in the developing nervous system.

Glial cells in both central and peripheral nervous systems are connected by gap junctions, which allow electrical and metabolic coupling between them. This study investigated coupling between satellite glial cells that envelope the spinal ganglion neurons in mice aging from 90 to 730 days using the dye coupling technique and electron microscopy. Experiments were done on spinal ganglia L4 and L5. Both the dye coupling technique and the electron microscopy showed that clusters of two-six neurons sharing a common satellite cell sheath were present in mice of all ages. Dye coupling incidence between satellite cells associated with a single neuron increased from 24.2% at 90 days to 50.5% at 730 days. Dye coupling incidence between satellite cells that are in contact with two or more neurons increased from 2.7% at 90 days to 18.6% at 730 days. Examination of the ganglia by the electron microscope showed that the density of gap junctions (number of gap junctions per 100 µm2 of surface area of satellite cells) was about seven fold greater in 730 days than in 90 days both in satellite cells associated with a single neuron and in satellite cells that are in contact with two or more neurons. The mean length of individual gap junction did not change with age. These results provide strong evidence for an increase of functional coupling between satellite cells during life. This increase is apparently due to an increase in the total area of the system of gap junctions connecting these cells.

The use of platelet-enriched plasma has been proposed as a source of growth factors in tissue regeneration. The molecular mechanism at the basis of PRP action is not yet understood. The recent finding that PRP is a potent chemoattractive agent for osteoblasts, prompted us to investigate whether the increased cell migration might be due to cytoskeleton rearrangements. PRP pretreatment of SaOS-2 cells (osteoblastic cell line) increases the capacity of migration in a dose and time dependent manner. Interestingly, PRP pre-treatment induces cytoskeleton reorganization of the actin microfilaments, promoting the acquisition of a migratory phenotype in a time dose dependent manner. We also investigated the effects of a PRP pretreatment on microtubules and also in this case we observe an acquisition of a migratory phenotype. Adding an antibody against PDGF in PRP pretreatment the effect on migration capacity and actin remodelling was completely abolished. We observe also that a PRP pretreatment induces the induction of the mRNA espression of alpha chain of PDGF receptor in a short time, while for a long time the pretreatment inhibits the mRNA espression of the same chain. In conclusion we can say that the pretreatment with PRP is able to increase the migratory ability of osteoblastic cells by cytoskeleton reorganization. The molecular pathway linking PRP to microfilaments and microtubules rearrangement is probably mediated by PDGF signalling.

Accumulating evidence indicate that immune deregulation or inflammation are linked to several types of psychiatric diseases (Patterson, 2009). Accordingly, perinatal infections have been associated with increased risk for neurodevelopmental disorders (Boksa, 2010; Meyer et al., 2006), including schizophrenia (Miller, Culpepper, Rapaport, & Buckley, 2013), autism (Shi, Fatemi, Sidwell, & Patterson, 2003) and epilepsy (Pineda et al., 2014). By using the POLY I:C (polyinosinic-polycytidylic acid) model of inflammation, we addressed the possibility that a single inflammatory event during pregnancy may alter neurodevelopmental processes, thus leading to neurologic disorders in the offspring. Our results demonstrate that a single injection of POLY I:C at gestation day 9 causes higher susceptibility to kainate-induced seizures in the offspring and other behavioral aberrations. This is associated with increased permeability of the Blood-Brain Barrier (BBB), due to alterations in tight junctions, as demonstrated by a reduction of Claudin-5 expression levels. The inflammatory status of adult animals doesn’t represent a susceptibility factor; only early alterations are visible. No changes in microbiota was observed in the offspring of POLY I:C-treated mothers. Wnt/β-catenin pathway and pericytes markers alterations are instead had found both in developmental and adult stages of POLY I:C-treated mothers offspring. Our results thus open to the possibility that inflammation during early pregnancy may increase susceptibility to psychiatric diseases and epilepsy by interfering with normal brain vascularization and BBB formation.

OPTOELECTRONIC MOTION ANALYSIS OF SPINE AND THORAX IN SCOLIOSIS Protocol design and application in a pilot-study group Vincenzo Giovanni FERRARA Matr. R09064 The forward bending test according to Adams (AT) and rib hump quantification by scoliometer are common clinical examination techniques in idiopathic scoliosis, although precise data about the change of angular axial rotation in forward bending posture are not available (1). The aims of the present research is to create a motion analysis protocol to investigate the movement of spine and thorax in a sample of adolescent idiopathic scoliosis (AIS) patients during Adams test. Ten patients (5 men, 5 women; age 16,2+2,3) with idiopathic scoliosis with mean Cobb-angles (19°+2,11°) were recruited. We recorded the axial spine and thorax surface rotation during forward bending movement using an optoelectronic three-dimensional motion analyzer (BTS Smart System) with 60 Hz sampling rate. Twenty-six passive markers (diameter 0,5 and 1 cm) were used to define chest wall, spine and pelvis. Spine was reconstructed as follows: we placed six markers on the spinous processes (SP) of the second (D2), fifth (D5), ninth (D9) thoracic vertebra, above the SP of the third (L3), the fourth (L4) lumbar vertebra and above SP of the first sacral vertebra (S1). Thorax was reconstructed by placing fourteen markers: six markers above the rear corners of the second (2ANGdx, 2ANGsx), fifth (5ANGdx, 5ANGsx), ninth (9ANGdx, 9ANGsx) couple of ribs, one marker above the xiphoid process (PX), one marker above incisura jugularis(FG), six markers over the joints between the ribs and the cartilage of the second (2SCdx, 2SCsx) fifth (5SCdx, 5SCsx), ninth (9SCdx, 9SCsx ) pair of ribs. Pelvis was reconstructed by placing four markers upon right and left anterior superior iliac spine (SIASdx, SIASsx), and upon right and left posterior superior iliac spine (SIPSdx, SIPSsx). Joint coordinate system according to International Society of Biomechanics recommendation was adopted (axes X for inclination, axes Z for flexion/extension, axes Y for rotation) (2). To study the movement of the spine and thorax we have designed four virtual planes. The first (p1) between D2-2SCdx-2SCsx, the second (p2) between D5-5CCdx-5CCsx), the third (p3) between D9-9CCdx-9CCsx, the fourth (R) between S1-SIASdx-SIASsx. Each patient was examined during the forward flexion of the trunk starting from a standing position (F1) and from a seated position (F2) to exclude the influence of leg length inequalities. All the angular values of D5-D9-L3-L5 and of p1, p2, p3, R were calculated. We used Student t-Test for paired samples to test by means the difference pattern of forward bending between F1 and F2 for the whole sample. The significance level was set at 5% for all statistical analyses (p<0,05). We observed no differences between the amplitudes of the movements in rotation and tilt during F1 an F2 of D5-D9-L3-L5 (p>0,05). In addition, no significant correlation was found for all comparison between angular values of p1 versus p2 versus p3 versus R during F1 and F2 around X and Y. Instead a significant correlation between F1 and F2 were found in p1 versus p3 around Z (p<0,001) and p3 versus R around Z (p<0,001). During AT we observe in all subjects a rigid spine with the exception of the dorsal spine with fulcrum in D9/p3. The present study confirm the possibility to use this non-invasive protocol for deformity assessment in AIS patients. Further investigations into this matter should be extended to a larger sample of participants to investigate the movement of the spine and thorax in AIS. REFERENCES 1) Wu Ge, Van der Helm F, Veeger H, et al. Isb recommendation on definition of joint coordinate system of various joint for reporting of human joint motion – Part I: ankle, hip and spine. Journal of Biomechanics 2002; 35: 543-48. 2) Stokes IA, Moreland MS. Measurement of the shape of the surface of the back in patients with scoliosis. The standing and forward-bending positions. Journal of Bone Joint Surgery America 1987; 69(2):203-11.

Nell'uomo e negli animali da esperimento, la somministrazione del fattore neurotrofico ciliare (CNTF) riduce l'assunzione di cibo e il peso corporeo. Studi su modelli animali hanno costantemente dimostrato che il CNTF esogeno non solo riduce l'assunzione di cibo, agendo sui centri di alimentazione ipotalamica e del tronco encefalico, ma migliora anche l'iperglicemia, l'iperinsulinemia e l'iperlipidemia associate all'obesità esercitando effetti metabolici attraverso azioni sugli organi periferici, incluso il tessuto adiposo bianco, muscolo scheletrico e fegato. Pochissimi dati sono ancora disponibili su un possibile ruolo del CNTF endogeno come modulatore del bilancio energetico. In questo progetto abbiamo testato se il CNTF endogeno potrebbe essere coinvolto nella regolazione del bilancio energetico valutando il fenotipo metabolico dei topi in cui è stato deleto il CNTF (CNTF-/-) alimentati con la dieta normale (NCD) o una dieta ricca di grassi (HFD). I principali risultati ottenuti sono stati: 1) i topi CNTF-/- maschi adulti alimentati con HFD per 13 settimane hanno guadagnato un peso corporeo significativamente maggiore rispetto ai littermates alimentati con HFD (WT); 2) quando comparati con gli animali WT, i topi CNTF-/- nutriti con NCD hanno mostrato un'ipertrofia adipocitaria più elevata nei depositi di tessuto adiposo bianco viscerale mesenterico ed epididimale (WAT) mentre i topi CNTF-/- alimentati con HFD hanno mostrato una maggiore infiltrazione di macrofagi e una maggiore predisposizione alla morte degli adipociti in questi depositi WAT viscerali; 3) squilibri cellulari nel tessuto adiposo bianco viscerale nei topi CNTF-/- correlano con la sovraespressione della proteina MCP1 e alla citochina infiammatoria TNFα, mentre l'espressione dell’adiponectina si è ridotta nel deposito epididimale dei topi nutriti con HFD; 4) la mancanza di CNTF nei topi nutriti con NCD e HFD comportava una compromissione della segnalazione dell’insulina nel muscolo scheletrico e nel fegato dove, inoltre, una steatosi massiccia era particolarmente evidente nei topi CNTF-/- alimentati con HFD rispetto ai topi WT mantenuti nelle stesse condizioni nutrizionali; 5) I topi CNTF-/- alimentati con HFD hanno mostrato livelli di insulina circolanti e un indice HOMA più elevati rispetto ai loro controlli alimentati con HFD. Collettivamente, questi dati suggeriscono che il CNTF endogeno, possibilmente prodotto e secreto nel sangue da una/qualche fonte/i cellulare non ancora identificata, può raggiungere organi periferici rilevanti dal punto di vista metabolico che portano il suo recettore specifico ed esercitare effetti metabolici in condizioni fisiologiche o patofisiologiche distintive.
In humans and in experimental animals, administration of ciliary neurotrophic factor (CNTF) reduces food intake and body weight. Studies of animal models have consistently demonstrated that exogenous CNTF not only reduces food intake, by acting on hypothalamic and brainstem feeding centres, but also improves obesity-associated hyperglycaemia, hyperinsulinemia and hyperlipidaemia by exerting metabolic effects through actions on peripheral organs, including white adipose tissue, skeletal muscle and the liver. Very few data are yet available on a possible role of endogenous CNTF as a modulator of energy balance. In this project we tested whether endogenous CNTF might be involved in energy balance regulation by assessing the metabolic phenotype of CNTF ablated mice (CNTF-/-) fed normal chow diet (NCD) or an high-fat diet (HFD). The main results obtained were: 1) adult male CNTF-/- mice challenged with an HFD for 13 weeks gained significant more body weight than the littermate controls fed the HFD (WT); 2) when compared with the respective WT animals, NCD fed CNTF-/- mice exhibited a higher adipocyte hypertrophy in mesenteric and epididymal visceral white adipose tissue (WAT) depots whereas the HFD fed CNTF-/- mice showed increased macrophage infiltration and an higher proneness of adipocyte to death at these visceral WAT depots; 3) visceral WAT cellular derangements in the CNTF-/- mice matched with the overexpression of the monocyte chemoattractant protein 1 and the proinflammatory cytokine tumour necrosis α, whereas adiponectin expression was found to be reduced in the epididymal depot of HFD fed mice; 4) lack of CNTF in both NCD and HFD fed mice involved an impairment of insulin signalling in the skeletal muscle and in the liver where, in addiction, a massive steatosis was especially evident in the HFD fed CNTF-/- mice in comparison with the WT mice kept under the same nutritional conditions; 5) HFD fed CNTF-/- mice exhibited higher circulating insulin and HOMA index in comparison with their HFD fed controls. Collectively, these data suggest that endogenous CNTF, possibly produced and secreted in the blood by a/some yet unidentified cellular source/s may reach peripheral metabolically relevant organs that bear its specific receptor and exert metabolic effects under distinctive physiological or pathophysiological conditions.

ABSTRACT BACKGROUND The availability of organs and tissues, obtained in laboratory from biological material, is one of the main demands in the event of loss of substance, due to congenital or post-traumatic defects. For this purpose, bioengineered tissues have been developed through decellularization and recellularization processes. AIM OF THE STUDY The objectives of this study were: • Development of a new type of biological scaffold derived from omentum that preserves the structural integrity of the extracellular matrix and the vascular network of the native tissue even after a phase of decellularization. • Development of a further biological scaffold from skeletal muscle, derived from the decellularization of the rectus muscle of the rabbit, for the reconstruction of a defect in the abdominal wall. MATERIAL AND METHODS As regards the first part of the experiment, samples of omentum, from rat and human, were decellularized through physical reactions (freezing/thawing and mechanical agitation), chemical (EDTA and isopropanol) and enzymatic (trypsin, lipases and endonucleases) methods involving the removal of cells and their lipid content. Samples were analyzed at various stages by histological staining (hematoxylin and eosin, azan Mallory, van Gieson, PAS, Sudan, Oil Red), immunohistochemical reactions (anti- CD31, -CD34, α-smooth muscle actin) and stained with DAPI. Furthermore, evaluations about the quantitative concentration of genomic DNA at the end of decellularization were carried out with a spectrophotometer. Attempts have been made to recellularize scaffold by cells of the stromal vascular fraction (SVF), isolated from samples of human lipoaspirate. It has, also, developed an additional scaffolds by decellularization of samples of the rectus muscle of the rabbit. Samples were subjected to physical (freezing/thawing), chemical (EDTA and Triton X -100) and enzymatic (DNase) methods and results were analyzed by histological staining (hematoxylin and eosin, azan Mallory, van Gieson). Thereafter, the scaffold was implanted in a rabbit receiver in correspondence with a surgical defect in the abdominal wall with loss of substance. After three weeks, the implant was studied by ultrasound examination in vivo and, after sacrifice of the animal, it was analyzed with the histological stainings mentioned above. RESULTS Histological stainings confirmed the effectiveness of the decellularization process with the total loss of its lipid component of the omentum. This led to the creation of an acellular scaffold in which the three-dimensional organization of the collagen, elastic, and reticular fibers was preserved. Also, the preservation of the vascular network was highlighted. Preliminary studies have shown, moreover, the possibility of recellularization of the scaffold through the introduction of SVF cells. Regarding the second part of the project, it was shown that the muscle-derived scaffold has maintained the integrity of the extracellular matrix and the structure of the vascular channels, in the removal of cellular elements. Following implantation in the receiving rabbit, a solution of continuity with the adjacent tissues has been highlighted, by ultrasound in vivo, with the absence of herniation . After the sacrifice of the animal, histological analysis of the scaffold showed the presence of reparative tissue and new vascular channels in the seat of the abdominal defect. Future perspectives will be to recellularize the scaffold with progenitor cells to promote muscle regeneration. CONCLUSIONS We obtained a new type of acellular biological structure resulting from the omentum, through the application of the process of decellularization, which allows to minimize possible alterations of the extracellular matrix during the removal of the cells. The scaffold was preliminarily recellularized with cells of the stromal vascular fraction, but further experiments will focus on the further evolution of in vitro recellularization and in vivo implantation. The decellularization of the muscle tissue and its implantation in vivo has allowed to identify a biological structure capable of offering a valid alternative to the materials currently used for the defects repair in the abdominal wall. Also in this direction we will evaluate in the future the possibility of recellularization of the scaffold with progenitor cells.
RIASSUNTO PRESUPPOSTI DELLO STUDIO La disponibilità di organi e tessuti ottenuti in laboratorio da materiale biologico è, ad oggi, una delle maggiori richieste nei casi di perdita di sostanza a causa di difetti congeniti o post-traumatici. A fronte di ciò, si è cercato di realizzare dei tessuti bioingegnerizzati mediante l’applicazione di processi di decellularizzazione e successiva ricellularizzazione. SCOPO DELLO STUDIO Gli obiettivi del presente lavoro sono stati i seguenti: • Sviluppare un nuovo tipo di scaffold biologico derivato dall'omento che conservi l’integrità strutturale della matrice extracellulare e della rete vascolare del tessuto nativo anche dopo una fase di decellularizzazione. • Sviluppare un ulteriore scaffold biologico da muscolo scheletrico, derivato dalla decellularizzazione del muscolo retto del coniglio, per la ricostruzione di un difetto della parete addominale. MATERIALI E METODI Per quanto riguarda la prima parte sperimentale, campioni di omento, di ratto e di uomo, sono stati decellularizzati mediante l'impiego di una serie di reazioni fisiche (congelamento/scongelamento ed agitazione meccanica), chimiche (EDTA e isopropanolo) ed enzimatiche (tripsina, endonucleasi e lipasi) che comportano la rimozione delle cellule e della loro componente lipidica. I campioni sono stati analizzati nelle varie fasi mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson, PAS, Sudan, Oil Red), reazioni immunoistochimiche (anti-CD31, -CD34, -α actina del muscolo liscio) e colorazione con DAPI. Inoltre, sono state effettuate valutazioni quantitative allo spettrofotometro della concentrazione di DNA genomico al termine della decellularizzazione. Sono stati effettuati tentativi di ricellularizzazione dello scaffold mediante cellule della frazione stromale vascolare (SVF), isolate da campioni di lipoaspirato umano. È stato altresì sviluppato un ulteriore scaffold mediante decellularizzazione di campioni del muscolo retto di coniglio. Questi sono stati sottoposti a reazioni fisiche (congelamento/scongelamento), chimiche (EDTA e Triton X-100) ed enzimatiche (DNasi) ed i risultati sono stati analizzati mediante colorazioni istologiche (ematossilina ed eosina, azan Mallory, van Gieson). In seguito, lo scaffold è stato impiantato a tutto spessore in un coniglio ricevente in corrispondenza di un difetto della parete addominale con perdita di sostanza prodotto chirurgicamente. Dopo tre settimane, l’impianto è stato studiato mediante esame ecografico in vivo e, dopo sacrificio dell’animale, è stato analizzato mediante le colorazioni istologiche di cui sopra. RISULTATI Le colorazioni istologiche hanno confermato l'efficacia del procedimento di decellularizzazione con la perdita totale della componente lipidica propria dell'omento. Ciò ha portato alla realizzazione di uno scaffold acellulare nel quale è stata mantenuta l’organizzazione tridimensionale delle fibre collagene, elastiche e reticolari. Inoltre è stata evidenziata la preservazione della rete di canali vascolari. Studi preliminari hanno dimostrato, inoltre, la possibilità di ricellularizzare lo scaffold mediante introduzione di cellule appartenente alla SVF. Riguardo la seconda parte del progetto, è stato dimostrato che lo scaffold di derivazione muscolare ha mantenuto l'integrità della matrice extracellulare e della struttura dei canali vascolari, di cui rimangono apprezzabili i diversi strati che li compongono. A seguito dell’impianto nel coniglio ricevente è stata evidenziata, mediante ecografia in vivo, l’integrazione dello scaffold con i tessuti adiacenti con buone proprietà meccaniche. Dopo il sacrificio dell’animale l’analisi istologica dello scaffold ha delineato la presenza di tessuto riparativo e di nuovi canali vascolari nella sede del difetto addominale. Obiettivo futuro sarà quello di ricellularizzare lo scaffold con cellule progenitrici muscolari per favorire la rigenerazione in senso muscolare dello scaffold stesso. CONCLUSIONI Abbiamo ottenuto un nuovo tipo di struttura acellulare biologica derivante dall'omento, mediante l'applicazione del processo di decellularizzazione, il quale permette di minimizzare il più possibile le alterazioni della matrice extracellulare durante la rimozione delle cellule. In via preliminare lo scaffold è stato ricellularizzato con cellule appartenenti alla frazione stromale vascolare ma ulteriori esperimenti si focalizzeranno sul potenziamento della ricellularizzazione in vitro e su impianto in vivo. La decellularizzazione del tessuto muscolare ed il suo impianto in vivo ha permesso di identificare una struttura biologica in grado di offrire una valida alternativa ai materiali attualmente impiegati per la riparazione dei difetti di parete addominale. Anche in questa direzione verrà valutata in futuro la possibilità di ricellularizzare lo scaffold con cellule progenitrici.

Il gene “VGF” fu scoperto perché è uno dei prodotti più potentemente indotti da NGF (NERVE GROWTH FACTOR) in cellule di feocromocitoma di ratto, in coincidenza con il loro passaggio ad un fenotipo neuronale indotto dal NGF stesso. Un primo trascritto è tradotto nel precursore proVGF, che è accumulato in granuli secretori e poi liberato in seguito a vari stimoli. La localizzazione di VGF e dei suoi peptidi derivati è altamente specifica e ristretta a sottopopolazioni neuronali del sistema nervoso centrale e periferico e gruppi di cellule endocrine. Noi abbiamo studiato presenza e modulazione di VGF e dei suoi frammenti nel surrene di diverse specie animali di interesse zootecnico e sperimentale. Parallelamente abbiamo sviluppato un metodo immuno-chimico che per la prima volta ha consentito la misurazione di VGF e dei suoi frammenti.

The synchronizing effects of the physical exercise: a research path. Three studies, concerning influences of the time of training on the circadian structure in humans, are presented. Goals of the researches are: to define how the time of training modifies the circadian structure; to evaluate if is possible face diseases related with biological rhythms disorders thanks to the training; to define guide lines for athletes, trainers and specialists. The works: STUDY 1) Influences of the time of training on the HR circadian rhythm; STUDY 2) reduction of the Jet Lag symptoms pre-adjusting the circadian structure by a training plan before an eastward trans-meridian flight; STUDY 3) synchronization state and effects of the training on the sleep and the circadian structure in lack of daylight conditions above the Artic Circle in north of Norway. The studies indicate it is possible to asses that the time of training influences the circadian structure, in particular an evening training induces a delay (3 hours in average) of the Acrophases of several Marker Rhythms (like HR and Activity Levels). This phenomenon can be used to induce a pre-adjustment of the circadian structure before an east-ward flight to prevent or reduce the symptoms of the Jet Lag syndrome, thanks to a reduction of the phase shift. It also seems that a regular morning training can reduce the psycho-physical symptoms that the people who live into the Circumpolar Circle commonly experience during the winter months, increasing the activity levels and improving the sleep quality.

Il fattore neurotrofico ciliare (ciliary neurotrophic factor, CNTF) è un fattore neurotrofico attivo sia su neuroni che su cellule gliali. Poiché in epoca postnatale il CNTF mantiene il trofismo dei circuiti motori, eventuali effetti terapeutici di Axokine, analogo ricombinante del CNTF umano, sono stati valutati su pazienti affetti da patologie motorie. Mentre gli effetti sulle abilità motorie si sono rivelati scarsi, i pazienti trattati con Axokine hanno presentato un significativo calo ponderale legato ad una riduzione dell’appetito. Numerosi gruppi di ricerca hanno quindi valutato l’azione del CNTF nell’ipotalamo, sede dei circuiti neuronali che nei mammiferi regolano il bilancio energetico. Nel complesso, il CNTF esogeno determina sazietà, riduzione dell’introito di cibo e aumento della spesa energetica, modulando funzione e struttura dei circuiti neuronali ipotalamici sensibili alla leptina, insulina ed altri fattori circolanti. Nulla è noto circa la presenza e la distribuzione del CNTF nell’ipotalamo, in condizioni normali o patologiche, e in questo studio condotto su topi, ci siamo proposti di valutare 1) se il CNTF è espresso nell’ipotalamo, 2) quali tipi cellulari lo producono e 3) se l’espressione ipotalamica del CNTF è modulata da differenti condizioni nutritive. I dati di RT-PCR e Western blotting mostrano che il CNTF è costitutivamente espresso nell’ipotalamo di topi tenuti in condizioni standard. L’analisi immunoistochimica mostra che il CNTF è prodotto da alcune cellule gliali in sede subependimale e dall’ependima del terzo ventricolo, dove sono intensamente positivi i taniciti dell’ipotalamo tuberale e mammillare, regioni ipotalamiche che contengono le popolazioni neuronali che regolano il comportamento alimentare. Al fine di verificare un possibile ruolo del CNTF endogeno nella regolazione del bilancio energetico, l’espressione del CNTF è stata studiata in topi resi obesi da dieta ad alto contenuto lipidico e, all’opposto, in topi magri, tenuti in condizione di restrizione calorica (60% del normale introito calorico giornaliero). I risultati hanno evidenziato che l’obesità da dieta iperlipidica determina un significativo incremento dell’espressione di CNTF nei taniciti delle regioni tuberale e mammillare dell’ipotalamo, mentre la restrizione calorica ha un effetto opposto. Per confermare il coinvolgimento del CNTF nella regolazione del bilancio energetico a livello ipotalamico, abbiamo quindi studiato l’espressione del recettore specifico. I dati di RT-PCR e Western blotting hanno mostrato che, parallelamente a quanto osservato per il ligando, l’obesità da dieta iperlipidica determina un aumento significativo dell’espressione ipotalamica del recettore per il CNTF, mentre la restrizione calorica si associa ad una sua significativa riduzione. Non essendo disponibili anticorpi specifici per il recettore del CNTF, i target cellulari del CNTF sono stati individuati mediante valutazione immunoistochimica dell’attivazione della specifica via di trasduzione (Jak1/2-STAT3 pathway) in topi trattati in acuto con bolo di CNTF. Le principali cellule target del CNTF sono le cellule ependimali, tra cui i taniciti. A conferma dei dati di biologia molecolare, nei topi obesi la responsività di queste cellule al CNTF aumenta, all’opposto nei topi tenuti in condizioni di restrizione calorica si riduce. In conclusione, il CNTF è prodotto dall’ependima del terzo ventricolo, ed in particolare dai taniciti della regione tuberale e mammillare. Esso rappresenta un nuovo fattore di sazietà coinvolto nella regolazione fisiopatologica dei circuiti ipotalamici del bilancio energetico. La modulazione farmacologica della sua espressione e/o del recettore specifico potrebbe rappresentare in futuro un approccio al trattamento farmacologico dell’obesità umana.
The ciliary neurotrophic factor (CNTF) is a neurotrophic factor acting on both neurons and glial cells. As it is involved in the maintenance of motor circuits in the postnatal period, possible therapeutic effects of Axokine, an analog of human recombinant CNTF, have been evaluated in patients affected by motor disorders. Despite effects on motor skills were not encouraging, the patients treated with Axokine showed a significant weight loss related to a reduction in appetite. Starting from these observations, many research groups evaluated the effects of CNTF administration in the hypothalamus, the pivotal center of the energy balance regulation in mammals. Collectively, these studies confirmed that CNTF induces satiety, reduction of food intake and increase of energy expenditure through an action on the hypothalamic neuronal circuits that are responsive to leptin, insulin and other circulating factors. As little is known about the presence and distribution of CNTF in the hypothalamus, in normal or pathological conditions, here we aimed to assess in mice 1) whether CNTF is expressed in the hypothalamus, 2) which cell types produce it, and 3) whether the hypothalamic expression of CNTF is modulated by different nutritional conditions. The results from RT-PCR and Western blotting analyses suggest that CNTF is constitutively expressed in the hypothalamus of mice kept in standard conditions. By immunohistochemistry, CNTF is produced by some subependymal astrocytes and ependymal cells. The tanycytes of the tuberal and mammillary hypothalamus, where the neuronal populations involved in the regulation of feeding behavior are located, were strongly positive for CNTF. To assess a possible role of endogenous CNTF in the regulation of energy balance, its expression was evaluated in obese mice fed with an high-fat diet (HFD) and, conversely, in lean mice, kept in conditions of calorie restriction (60% of normal daily caloric intake). The results showed that obesity induced by a HFD is characterized by a significant increase of the expression of CNTF in the tanycytes of the tuberal region of the hypothalamus, whereas the caloric restriction determines the opposite effect. To confirm the involvement of CNTF in the regulation of energy balance in the hypothalamus, we then analyzed the expression of its specific receptor. RT-PCR and Western blotting revealed that, similar to the ligand, obesity induced by a HFD caused a significant increase in the hypothalamic expression of CNTF receptor; the opposite was true for the calorie restriction condition. Given the unavailability of specific antibodies against the CNTF receptor, CNTF-responsive cells were detected by P-STAT3 immunohistochemistry in mice acutely treated with CNTF. Ependymal cells, importantly tanycytes, were the main hypothalamic cells responding to CNTF. In agreement with molecular data, in obese HFD mice the responsiveness of the ependyma to CNTF increased, whereas it decreased in calorie restricted mice. In conclusion, the CNTF is produced by the ependymal cells of the third ventricle, in particular by the tanycytes of the tuberal and mammillary regions of the hypothalamus. It represents a new satiety factor involved in the pathophysiological regulation of the energy balance. The pharmacological modulation of its expression and/or of its specific receptor could be an innovative approach to the treatment of human obesity.

La Butirrilcolinesterasi (BChE) è un enzima idrolitico appartenente alla famiglia delle carbossilesterasi prodotto e secreto dal fegato. È presente in tutti i mammiferi e ricopre un ruolo molto importante nella neurotrasmissione grazie alla sua azione nei confronti del neurotrasmettitore acetilcolina (ACh). Diversamente dal ruolo ben definito dell’acetilcolinesterasi (AChE) nella regolazione del signalling colinergico, per molti anni non è stata attribuita una vera funzione fisiologica alla BChE. Nonostante alcune informazioni relative alla sua attività enzimatica siano già state scoperte, un ruolo fisiologico chiaro deve ancora essere attribuito a questo enzima. Studi recenti hanno accresciuto l’interesse nei suoi confronti delineando un coinvolgimento della BChE nella regolazione dell’appetito e nell’insorgenza dell’obesità, individuando tale enzima come possibile responsabile dell’idrolisi dell’ormone grelina. Dato l’interesse crescente per la BChE e il suo rapporto con aspetti metabolici nell’ambito della regolazione dell’appetito, in questo lavoro di tesi è stata realizzata una mappatura morfologica dettagliata della distribuzione di questo enzima nel tratto gastrointestinale di topo. Attraverso tecniche di immunoistochimica e studi di immunofluorescenza con doppie marcature abbiamo realizzato una mappatura dell’enzima dalla porzione più rostrale (ghiandole salivari ed esofago), fino alla più caudale (intestino tenue e colon). Per ogni organo del tratto gastrointestinale analizzato le reazioni di immunoperossidasi qui svolte mostrano la distribuzione della BChE, mentre le doppie marcature identificano attraverso microscopia a fluorescenza confocale gli esatti citotipi che esprimono l’enzima. Cellule positive per la BChE sono state identificate nel fegato, nella porzione duttale delle ghiandole salivari, nell’epitelio cheratinizzato dell’esofago, nella porzione ghiandolare della mucosa gastrica (cellule parietali), nelle cripte intestinali (cellule di Paneth), e nelle cellule a secrezione mucosa del colon e duodenali (ghiandole di Brunner). È interessante notare come la BChE sia presente vicino alle nicchie proliferative gastrointestinali. È stata riportata qui per la prima volta la presenza di BChE nei colangiociti epatici, nelle cellule di Paneth e nella porzione ghiandolare dello stomaco: risultati che possono suggerire un ruolo di controllo per la BChE nei confronti dell’omeostasi e della rigenerazione tissutale. Un altro possibile ruolo fisiologico per la BChE derivante dall’ interpretazione dei nostri dati potrebbe essere la sua azione come agente detossificante nei confronti dei composti introdotti con la dieta. Inoltre, la correlazione spaziale tra le cellule che producono grelina e le cellule parietali positive alla BChE nella mucosa ossintica gastrica, potrebbe suggerire un ruolo idrolitico verso la grelina tramite un’azione paracrina, fin’ora mai proposto. Complessivamente, i dati morfologici proposti in questo lavoro di tesi possono essere visti come un importante punto di partenza per progetti sperimentali e studi futuri volti a chiarire l’esatto ruolo della BChE nel tratto gastrointestinale.
The Butyrylcholinesterase (BChE) is an hydrolytic enzyme produced and released by liver belonging to the carboxylesterases superfamily. It is present in all mammals and plays an important role in neurotransmission due to its action towards the neurotransmitter Acethylcholine (Ach). In contrast to the long-established and well-defined role of Acethylcholinesterase (AChE) in regulating cholinergic signaling, a true physiological function for BChE remained elusive over many decades. Despite BChE enzymatic activity have already been elucidated, a proper physiological role for that enzyme has yet to be defined. Recent studies rise the interest towards BChE proposing a new challenging and interesting role for that enzyme linked to obesity and appetite regulation: the hydrolysis of the neuropeptide gut hormone, ghrelin. Due to the growing interest for BChE and its role in the field of feeding and appetite regulation, in this thesis a detailed morphological study of the distribution of BChE along the mouse gastrointestinal tract has been performed. Through immunohistochemistry and double labelling immunofluorescence studies, a map of the distribution of the enzyme has been proposed here from the more rostral portion (salivary glands and esophagus) to the more caudal one (large intestine and colon). For each organ of the gastrointestinal tract analyzed, the immunoperoxidase stainings described the distribution of BChE, while the double labelling studies identified the exact cytotype expressing this enzyme. BChE-positive cells were found in the liver, in the ductal portion of salivary glands, in the keratinized layer of the squamous epithelium of the esophagus, in the gastric mucosa (parietal cells), in the intestinal crypts (Paneth cells) and in mucus secreting cells of the duodenal Brunner glands and of the large intestine. Interestingly, the presence of BChE was frequently associated withto the distribution of gastro-intestinal proliferative niches. The presence of BChE in hepatic cholangiocytes, in intestinal Paneth cells and in the glandular portion of the stomach has been reported here for the first time: these results may suggest a role for BChE in the homeostatic control of tissues regeneration. Another possible physiological function for BChE coming from the interpretation of our results could be its action as a detoxifying agent towards ingested compounds. Moreover, the spatial correlation between ghrelin producing cells and BChE-positive parietal cells in the gastric oxyntic mucosa could suggest a BChE hydrolytic function towards ghrelin through a paracrine action. Collectively, the morphological results coming from this thesis work can be proposed as the onset for further experimental projects and studies aiming to clarify the role of BChE in the gastrointestinal tract.

HEPATOCELLULAR CARCINOMA IN THE CIRRHOTIC LIVER. IMMUNOHISTOCHEMICAL AND MORPHOMETRIC ANALYSIS. Objective: To explore how morphometry can minimize subjectivity in the assessment of liver nodules in cirrhosis using a novel classification tool. Study design: 8 hepatocellular carcinomas (HCC), 5 high-grade dysplastic nodules (HGDN), 8 large regenerative nodules (LRN), and 18 regenerative (cirrhotic) nodules (CN) obtained from cirrhotic explant livers, were analyzed using a Kontron-Zeiss KS400 image analyzer. We generated a morphometric model based on the analysis of volume fractions occupied by hepatocyte nuclei/cytoplasm, sinusoidal endothelium and lumen, neoplastic acini, fibrosis, centrilobular veins, portal arteries, veins and bile ducts, individual lesional arteries (smooth muscle actin), capillarized sinusoids (CD34), new vessels (CD31) and on surface fraction occupied by reticulin, and number in unit volume and size distribution of hepatocyte nuclei, and mean hepatocyte nucleus diameter and volume. Results: Volume fraction of capillarized sinusoids, new vessels and individual lesional arteries were more prominent in HCC and HGDN, when compared with LRN and CN, whereas surface fraction of reticulin was markedly decreased in HCC. The morphometric values of these three features were integrated into our classification tool to construct a hybrid system, which reclassified the nodules in the same categories. Conclusions: Our novel hybrid classification tool may minimize subjectivity in the histological assessment of nodular lesions in cirrhosis.

Obesity is nowadays a growing global health pandemic disease requiring an urgent intervention. High dietary fat intake promotes the development of obesity in humans and animal models as a result of an imbalance between energy intake and energy expenditure that is determinant for the excessive energy storage in the form of fat and so redundant accumulation of adipose tissue. When the energy balance is positive, the adipose tissue prevalently undergoes an increment of white adipocytes, that become at first hypertrophic and then, due to a close relationship between one adipocyte and its neighbor, hyperplastic. Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, defined adipokines, via which adipose tissue is strictly linked with other organs. An excessive accumulation of fat tissue during obesity alters adipokines secretion: this imbalance in adipose tissue homeostasis lead to the development of both cardiovascular and renal dysfunctions linking obesity with hypertension, atherosclerosis and nephropathy. Numerous studies have investigated the prevention and treatment of obesity and its associated dysfunctions using naturally-occurring antioxidants. We focused our attention on the inducible isoform of heme oxygenase (HO-1). HO-1 catalyzes the breakdown of heme, a potentially harmful pro-oxidant, into its products biliverdin and carbon monoxide, with a concomitant release of iron. HO-1 acts as a protective system against oxidative stress and inflammation. Moreover, it is considered to have a role in the regulation of vascular tone and it may provide renal cytoprotection. The epoxyeicosatrienoic acid (EET), derived from the metabolism of arachidonic acid by cytochrome P450 epoxygenase enzyme, induces HO-1 expression and activity and so it plays various biological activities. It is also considered a cytoprotective mediator because its function in activating AMPK, LKB1 and Akt, kinases that play a pivotal role in the regulation of energy metabolism. Moreover, EET could upregulate endothelial nitric oxide synthase (eNOS) and so increase the release of nitric oxide (NO), modulating vascular tone and homeostasis. In this study we at first analyzed the consequences of high fat diet on adipose tissue, aorta and kidney and then we hypothesized that the induction of HO-1, through EET administration would ameliorate fat accumulation and the resulting dysfunctions in a mice model of obesity. C57BL/6 mice were divided into four groups: lean, lean fed with a high fat diet (HF), lean fed with a high fat diet and treated with EET (HF EET), lean fed with a high fat diet and treated with EET and MSPPOH, an inhibitor of EET (HF EET MSPPOH). Food intake, body weight, blood levels of glucose, adiponectin and pro-inflammatory cytokines, along with HO-1, AMPKα1/pAMPKα1, LKB1/pLKB1, Akt/pAkt and eNOS expression were analyzed. We demonstrated that adipose tissue plays a focal role in the pathogenesis of obesity: we observed in fact white hypertrophic and hyperplastic adipocytes and an imbalance of adipokine secretion in the HF group. The HF mice showed low level of HO-1 and adiponectin and high systemic levels of the pro-inflammatory TNF-α and IL-6. The intraperitoneally EET administration prevented weight gain, decreased subcutaneous fat content, increased adiponectin and decreased TNF-α and IL-6. The HO-1 expression in adipose tissue, aorta and kidney consequently increased pAMPKα1, pLKB1, pAkt, and eNOS. The co-treatment with EET and MSPPOH countered the beneficial effects of EET, underling its capability of inhibitor of EET synthesis. In conclusion, this study provides strong evidence confirming the fundamental endocrinal function of the adipose tissue and its pivotal role in the pathogenesis of obesity. Moreover, HO-1 has important anti-obesity effects, manifested by a decrease of fat content, an increased number of low size adipocytes and with reciprocal increases in adiponectin, pAMPKα1, pLKB1, pAkt and eNOS, all effects that ameliorate both energy metabolism and vascular tone and so the deleterious effect of a high dietary fat intake.

L'imaging a risonanza magnetica (MRI) è sempre più utilizzato in ambiente medico per la sua abilità di produrre in modo non invasivo immagini di altà qualità dell'interno del corpo umano. Sin dalla sua introduzione nei primi anni 70, techiche di acquisizione via via più complesse sono state proposte, portando l'MRI ad essere utilizzata su uno spettro di applicazioni sempre più ampio. Le tecniche più innovative, tra cui la risonanza magnetica funzionale e di diffusione, richiedono tecniche di analisi ed algoritmi di elaborazione molto complessi per estrarre informazioni utili dai dati acquisiti. Lo scopo di questa tesi è stato quello di sviluppare e ottimizzare tecniche avanzate di elaborazione per applicarle all'analisi di dati di risonanza magnetica sia in ambiente preclinico che clinico. Durante il corso di dottorato sono stato coinvolto attivamente in diversi progetti di ricerca, ed ogni volta mi sono trovato ad affrontare problematiche diverse. In questa tesi, tuttavia, saranno riportati i risultati ottenuti nei tre progetti più interessanti a cui ho preso parte. Tali progetti avevano come obiettivo (i) l'implementazione di un protocollo sperimentale innovativo per imaging funzionale in animali da laboratorio, (ii) lo sviluppo di nuovi metodi per l'analisi di dati di Dynamic Contrast Enhanced MRI in modelli sperimentali di tumore e (iii) l'analisi di dati di diffusione in pazienti affetti da ischemia cerebrale. Particolare enfasi sarà posta sugli aspetti tecnici che riguardano gli algoritmi ed i metodi di elaborazione utilizzati nel processo di analisi.
Magnetic resonance imaging (MRI) is increasingly being used in medical settings because of its ability to produce, non-invasively, high quality images of the inside of the human body. Since its introduction in early 70’s, more and more complex acquisition techniques have been proposed, raising MRI to be exploited in a wide spectrum of applications. Innovative MRI modalities, such as diffusion and functional imaging, require complex analysis techniques and advanced algorithms in order to extract useful information from the acquired data. The aim of the present work has been to develop and optimize state-of-the-art techniques to be applied in the analysis of MRI data both in experimental and clinical settings. During my doctoral program I have been actively involved in several research projects, each time facing many different issues. In this dissertation, however, I will report the results obtained in three most appealing projects I partecipated to. These projects were devoted (i) to the implementation of an innovative experimental protocol for functional MRI in laboratory animals, (ii) to the development of new methods for the analysis of Dynamic Contrast Enhanced MRI data in experimental tumour models and (iii) to the analysis of diffusion MRI data in stroke patients. Particular emphasis will be given to the technical aspects regarding the algorithms and processing methods used in the analysis of data.

Il carcinoma della vescica è uno dei tumori di più frequente riscontro da parte dell’urologo, rappresentando la seconda principale causa di decesso fra tutte le neoplasie del tratto genitourinario. Più del 90% delle neoplasie maligne della vescica è rappresentato dai carcinomi delle cellule uroteliali. La scarsità di procedure non invasive attendibili per la diagnosi precoce, cui va aggiunta la complessa eterogeneità biologica che questo tumore riscontra nella pratica clinica, sono alla base dei decennali tentativi della comunità scientifica nell’individuazione di biomarcatori tumorali, configurabili come indicatori precoci dell’esistenza del processo neoplastico, da utilizzare anche per la sua sorveglianza a lungo termine. Alla luce di tali considerazioni, la necessità di identificare nel tumore vescicale alcuni indicatori biochimici e genetici altamente specifici, sensibili e valutabili, oltre che su frammenti di tessuto, anche su liquidi biologici (urine e plasma), è tutt’oggi al centro di numerosi studi scientifici. Lo scopo di questo lavoro è stato quello di analizzare l’espressione della serina proteasi HtrA1, che è nota fungere da soppressore tumorale in diversi tumori solidi, nel tessuto vescicale uroteliale umano in condizioni fisiologiche e tumorali, al fine di valutarne una eventuale alterazione dei livelli in presenza di neoplasia. Inoltre, abbiamo voluto estendere lo studio all’analisi dei fluidi biologici e alla valutazione di un possibile coinvolgimento dell’HtrA1 nella progressione della malattia. Infatti, studi più o meno recenti hanno dimostrato come l’HtrA1 sia una molecola in grado di esercitare una azione di controllo sulla crescita e proliferazione cellulare e di indurre la morte cellulare stimolando l’apoptosi. Sono stati reclutati per lo studio pazienti affetti da neoplasia vescicale uroteliale a diverso grado e stadio, soggetti sani e con cistite. Di ciascun individuo, sono stati raccolti campioni bioptici tissutali assieme ad urine e plasma. Gli studi di immunoistochimica da noi condotti hanno dimostrato che l’HtrA1 è una molecola espressa dall’urotelio della vescica in condizioni fisiologiche ed in patologie infiammatorie come la cistite batterica. Al contrario, la proteina è risultata assente in tutti i casi esaminati di carcinoma uroteliale con diverso grado di malignità e a differenti stadi di infiltrazione, fin dalle fasi più precoci di comparsa visibile della neoplasia. Una diversa espressione dell’HtrA1 tra i tessuti patologici ed i tessuti sani, nonostante simili livelli del trascritto, è stata evidenziata dall’analisi western-blotting, dalla quale è emersa la presenza di due forme dell’HtrA1, una nativa di peso molecolare di ~50 kDa ed una, che si origina per autoproteolisi della prima, di ~38 kDa. Solo la forma a più basso peso molecolare ha mostrato una diminuzione significativa in tutti i tessuti patologici analizzati rispetto ai sani, dimostrandosi idonea ad essere considerata un buon biomarcatore tumorale. Poiché questa proteina fu originariamente descritta come una proteasi di secrezione, abbiamo ipotizzato che essa potesse essere secreta da parte dell’urotelio nella cavità vescicale o dal tessuto nel sangue. Abbiamo quindi esaminato la presenza dell’HtrA1 anche nelle urine e nel plasma di tutti i soggetti reclutati, dimostrando un incremento significativo della proteina nell’urina e nel plasma dei pazienti con carcinoma rispetto ai soggetti sani. Il presente lavoro ha quindi evidenziato l’HtrA1 quale possibile marker tissutale e urinario/plasmatico utile nella diagnosi del carcinoma uroteliale della vescica. Inoltre, dati di biologia molecolare supportati dai risultati ottenuti in vivo ci hanno suggerito che, anche nella vescica umana, l’HtrA1 può assumere il ruolo di soppressore tumorale e che probabilmente sia l’urotelio normale adiacente a quello tumorale a determinare un aumento dell’HtrA1 nelle urine dei soggetti con carcinoma piuttosto che l’urotelio interessato dalla neoplasia, forse come risposta di protezione alla progressione della malattia.
Bladder cancer is one of the cancers most commonly encountered by the urologist, making it the second leading cause of death among all cancers of the genito-urinary tract. More than 90% of malignant neoplasms of the bladder is represented by the carcinomas of the urothelial cells. The lack of reliable non-invasive procedures for early diagnosis, together with the complex biological heterogeneity that this tumor has in clinical practice, are the basis of decades of efforts of the scientific community in identifying cancer biomarkers, configurable as early indicators of the existence of the neoplastic process, to be used also for its long-term surveillance. In the light of these considerations, the need to identify some highly specific and sensitive biochemical and genetic markers in bladder cancer, to be valued, as well as in tissue fragments, even in biological fluids (urine and plasma), is still the focus of numerous scientific studies. The purpose of this work was to analyze the expression of serine protease HtrA1, which is known to act as a tumor suppressor in various solid tumors, in human urothelial bladder tissue under physiological and neoplastic conditions, in order to assess a possible alteration of its levels in presence of cancer. In addition, we wanted to extend the study to the analysis of biological fluids and evaluation of possible involvement of HtrA1 in the progression of the disease. In fact, more or less recent studies, showed how the HtrA1 is a molecule capable of exerting a control action on cell growth and proliferation and to induce cell death by stimulating apoptosis. We recruited for the study patients with urothelial bladder cancer at different grade and stage, healthy subjects and with cystitis. Of each individual, tissue biopsy samples were collected along with urine and plasma. The immunohistochemical studies carried out showed that HtrA1 is a molecule expressed in bladder urothelium under physiological conditions and in inflammatory diseases, such as bacterial cystitis. On the contrary, the protein was absent in urothelial carcinoma with different degree of malignancy and at different stages of infiltration, right from the earliest stages of visible appearance of the neoplasm. A different expression of HtrA1 between the pathological and normal tissues, despite similar levels of the transcript, was detected by Western blotting, which revealed the presence of two forms of HtrA1, a native form with the molecular weight of ~ 50 kDa and another, which originates by autoproteolysis from the native one, of ~ 38 kDa. Only the HtrA1 form with lower molecular weight showed a significant decrease in all analyzed pathological tissues compared to the healthy counterparts, proving to be suitable to be considered a good cancer biomarker. Since this protein was originally described as a secreted protease, we hypothesized that it might be secreted by the urothelium in the bladder cavity or by tissue into blood. Thus, we examined the presence of HtrA1 also in the urine and plasma of all patients enrolled, demonstrating a significant increase of the protein in the urine and plasma of cancer patients compared to healthy subjects. The present work has therefore shown that HtrA1 may be considered a possible tissue and urinary/plasma biomarker, useful in the diagnosis of urothelial carcinoma of the bladder. In addition, data of molecular biology supported by the results obtained in vivo have suggested that, even in the human bladder, the HtrA1 can assume the role of tumor suppressor and that, probably, the normal urothelium adjacent to the tumor is responsible of the increase of HtrA1 in the urine of patients with carcinoma rather than the urothelium affected by cancer, perhaps as a protective response to disease progression.

I leiomiomi (fibromi) uterini sono neoplasie benigne estremamente comuni. L’incidenza è di oltre il 60% in donne in età fertile e pertanto, rappresentano uno dei maggiori problemi di sanità pubblica. I leiomiomi sono spesso multipli, originano nello strato muscolare dell’utero, e si ritiene che ogni fibroma origina da una singola cellula trasformata. Sebbene si sta iniziando a comprende la patofisiologia dei leiomioma, la loro origine resta sconosciuta. Negli ultimi anni, i ricercatori hanno considerato i leiomiomi come una patologia fibrotica. La fibrosi è un processo continuo ed esagerato di guarigione innescato da danno tissutale ed è caratterizzata da eccessiva produzione di proteine della matrice extracellulare (ECM), in particolare collagene. La fibrosi è un meccanismo che include due eventi principali: reclutamento di cellule infiammatorie nel sito di lesione e l'attivazione di cellule – miofibroblasti - che producono collagene. La prima parte del nostro studio è stato condotta con un approccio morfologico. I nostri obiettivi erano di capire: 1. Se lo stimolo infiammatorio cronico che agisce sul miometrio può innescare, in cellule riparative ancora non identificate, la proliferare e la sintesi di quantità esagerate di ECM; 2. Se i miofibroblasti del leiomioma possono avere origine dai fibroblasti stromali CD34-positivi. I risultati ottenuti hanno mostrato la presenza di un gran numero di cellule infiammatorie all'interno del leiomioma e nel tessuto circostante, rispetto al miometrio autologo lontano dal leiomioma. Un forte aumento è stato osservato nella quantità di macrofagi, individuati da un anticorpo specifico per il CD68. I macrofagi tissutali sono cellule chiave nel processo riparativo e di cicatrizzazione, e nel processo di reclutamento e attivazione dei miofibroblasti. Pertanto, un aumento del numero di cellule infiammatorie nei leiomiomi è a favore e supporta l'ipotesi di una loro patogenesi di tipo infiammatoria. Una seconda serie di risultati morfologici suggeriscono la presenza, nei leiomiomi, di miofibroblasti che producono ECM. Il ruolo dell’activina A nel pathway infiammatorio è stato ampiamente documentato in diversi sistemi biologici. I livelli di activina A sono elevati durante l'infiammazione e l’activina A aumenta la produzione di ECM in diverse condizioni patologiche. Pertanto uno dei nostri scopi è stato anche quello di testare se l’activina-A può avere un ruolo nel processo di fibrosi uterina e nello sviluppo e crescita dei leiomioma, effettuando degli studi in vitro. Abbiamo visto che l’activina-A aumenta significativamente i livelli di mRNA della fibronectina, del collagene 1A1 e del versican nelle colture primarie di cellule di leiomioma, e ha aumentato in maniera significativa i livelli di mRNA della fibronectina ma non del collagene 1A1 e del versican in colture cellule primarie di cellule del miometrio. L'aumento della fibronectina indotto dall’activina-A nelle cellule di leiomioma è stato visto anche a livello proteico, mediante western blot e immunocitochimica. Considerato che l’activina A ha ruolo pro-fibrotico nei leiomioma uterini, abbiamo anche valutato se e quali molecole infiammatorie sono in grado di stimolare l'espressione di questa citochina profibrotica in vitro nelle cellule di leiomioma e di miometrio. Abbiamo trovato sia nelle cellule primarie che nelle linee cellulari che il TNF-α aumenta sostanzialmente i livelli di espressione di mRNA dell’activina. A tutt’oggi, non esiste ancora una terapia medica efficace contro i leiomiomi uterini. L’isterectomia, soluzione definitiva contro i leiomiomi, è uno dei principali interventi chirurgici addominali che determina un aumento del rischio di morbilità postoperatoria e che portano all’infertilità. Tranilast è una farmaco sintetico che presenta molteplici effetti terapeutici per diverse condizioni patologiche. Siccome non ci sono studi sui fibromi uterini, ci siamo chiesti se il tranilast avesse effetti sulle proteine della ECM nei leiomiomi. Nel presente studio abbiamo dimostrato che il tranilast ha un effetto anti-fibrotico sulle cellule primarie di miometrio e di leiomioma. Nel complesso, la nostra ricerca presenta un nuovo approccio per lo studio della patogenesi del leiomioma. I dati presentati supportano la presenza di infiammazione all’interno del fibroma uterino, l'attivazione di cellule produttrici di collagene-miofibroblasti e l'eccessiva produzione di proteine della ECM, che conducono al rimodellamento tissutale e alla crescita del leiomioma.
Uterine leiomyomas (fibroids) are extremely common benign neoplasms. The incidence is over 60% in women of reproductive age and therefore represents one of the major public health problems. Leiomyomas are often multiple, originate in the smooth muscle layer of the uterus, and it has been proposed that each fibroid originates from a single transformed cell. Although the pathophysiology of leiomyomas is beginning to be understood their etiology still remains unknown. Over the last years, researchers have considered leiomyoma as a fibrotic disorder. Fibrosis is an exaggerated and continuous wound healing process triggered by tissue injury and characterized by excessive production of extracellular matrix (ECM) proteins, in particular collagens. Fibrosis is a mechanism that includes two main happenings: recruitment of inflammatory cells to the site of injury and activation of collagen producing cells- myofibroblasts. Our first study was morphological. We aimed to research: 1. If chronic inflammatory stimulus acting on the myometrium may trigger yet unidentified reparative cells to proliferate and to synthesize exaggerated amounts of ECM; 2. If leiomyoma myofibroblasts may derive from CD34-positive stromal fibroblasts. Our results showed the presence of a large number of inflammatory cells inside leiomyomas and in the surrounding tissue, when compared to autologous myometrium far from the leiomyoma. A large increase was observed in the amount of macrophages, identified by an antibody against CD68. Tissue macrophages are key cells in the reparative and scarring process, and in myofibroblasts recruitment and activation. Therefore, an increased number of inflammatory cells inside the leiomyoma support and agree with the hypothesis of an inflammatory pathogenesis of uterine leiomyoma. A second set of morphological results suggest the presence of myofibroblasts producing ECM in leiomyomas. The role of activin A in inflammatory pathways of different biological systems has been well documented. Since the level of Activin A is elevated during inflammation and is responsible for increase production of ECM in different pathological conditions. Therefore our aim was also to investigate if activin-A may have role in fibrosis process in uterus and leiomyoma development and growth, using an in vitro approach. We found that activin-A significantly increased fibronectin, collagen1A1 and versican mRNA expressions in primary leiomyoma cells, and it significantly increased fibronectin mRNA expressions but not collagen1A1 and versican expression in primary myometrial cells. The increased fibronectin expression by activin-A in leiomyoma cells was seen also at the protein level, by western blot and immunocytochemistry. Based on the findings that activin A has a pro-fibrotic role in uterine leiomyoma, we also evaluated whether and which inflammatory molecules are able to stimulate the expression of this profibrotic cytokine in leiomyoma and myometrial cells in vitro. We found in both, primary and immortalized cells, thatTNF-α was able to substantially increase activin A mRNA expression. To date, there is still no effective medical therapy against uterine leiomyomas. Hysterectomy, a permanent solution against leiomyomas, is a major abdominal surgical procedures caring on an increased risk of postoperative morbidity and leading to the loss of female reproductive potential. Tranilast is a synthetic drug that exhibits multiple therapeutic effects in diverse pathologic conditions. Since limited work has been reported in uterine fibroid biology, we aimed to research tranilast effect on ECM in leiomyoma. In the present study we demonstrated that tranilast has anti-fibrotic effect on human primary myometrial and leiomyoma cells. Overall, our research presents novel approach to leiomyoma pathogenesis. The presented data support the presence of inflammation inside the human uterine leiomyoma tissue, activation of collagen producing cells-myofibroblasts and the excessive production of ECM proteins, leading to the tissue remodeling and leiomyoma growth.

Genetic chaperonopathies manifest themselves from very early in life. Chaperonopathies related to neurodegenerative disorders discussed in “Introduction” section are a heterogeneous group of disorders which affect one or more of the various physiological systems, for example, the nervous system. This heterogeneity is due, in particular, to the not fully known molecular activity, which every single molecular chaperone has within a specific tissue. My general questions about them were 1) why a mutation on a molecular chaperone that is expressed by most, if not all cytotypes, seems to affect the functioning of a single physiological system? 2) why do different mutations on the same molecular chaperone cause apparently different pathologies especially in terms of clinical manifestations? This heterogeneity limits the research approach on diseases, which now is conducted towards every single mutation without being able to generalize a unique molecular process. I spent the first 18 months of my Ph.D. project at the SBARRO Department of Temple University in Philadelphia to study the V98I mutation on chaperone Hsp60 causing hereditary spastic paraplegia (SPG13). For a better understanding of the associated diseases, it would be highly beneficial to examine the impact of mutant chaperone genes during development, starting with fertilization and proceeding throughout the entire ontogenetic process. Zebrafish is amenable to such embryonal analysis as well as studies during adulthood. In addition, the zebrafish genome contains a wide range of genes encoding proteins similar to those that form the chaperoning system in humans. Due to the very complex roles played by Hsp60 in cell and tissue homeostasis, the gene is highly conserved during evolution. Nucleotide and amino acid sequences of Hsp60 in zebrafish have 88% of identity with its human orthologous. The first aim of my research was the establishment of a zebrafish model as an innovative approach for the study of the molecular basis of SPG13 and define the role of missense mutations V98I. In the last 18 months of my Ph.D. project, I was at the BIND Department of the University of Palermo and I had the possibility to study a new mutation that occurred in subunit number 5 of CCT complex. This mutation was found in a pediatric patient who is now being treated by the Department Of Sciences For The Promotion Of Health And Childhood "G. D'Alessandro" at the University of Palermo. Thus, I focused my attention on this novel variant. The main aims were 1) understanding, with the help of bioinformatics software, the type of mutation and if it causes some alteration of chaperonin molecular anatomy; 2) define the morphological changes caused by the mutation in skeletal muscle tissue.

Glioblastomas multiforme (GBM) are the most common malignant primary brain tumors in adults. They are highly aggressive and have an overall survival of <15 months despite maximal surgical resection and chemoradiation (Ostrom, 2019). GBMs are typically heterogeneous with a wide range of genetic and epigenetic variations among tumor cells. Extracellular vesicles (EVs) represent one of the plausible ways through which can be obtained a better understanding of the heterogeneous subpopulations of GBM / molecular signatures. EVs hold promise for the discovery of potential tumours biomarkers useful in clinical managment for GBM patient diagnosis and follow-up. Isolating EVs from body fluids and screening their protein content may serve as a complementary approach to assess the heterogeneous molecular landscape of GBM as tumors evolve. Many reports support the idea that Hsps are implicates in the pathogenesis and in the progression of different human neoplasms, by uncertain metabolic mechanism. Athough Hsps perform their canonical “chaperoning” functions in both prokariotic and eukaryotic cells, they have also acquired, probly during evolution, “extra-chaperoning” roles. Among these roles, there are some involved in the mechanism of cancerogenesis. In this study, we evaluate the exspression of some Hsps (in particular Hsp10, Hsp27, Hsp60, Hsp70, and Hsp90) through experiments of immnohistochemistry in samples of GBM and healthy controls, and also by immunofluorescence analysis on priamry and secondary cell lines of GBM. We also focused to research these proteins in EVs isolated from plasma obatained from patients with GBM, before and after surgery. The isolation was followed by a morphological and biochemical characterization of the EVs, in order to better study the characteristics of these potential natural carriers for the tool development for diagnostic and, possibly, also follow-up biopsy.

The introduction of new technologies has provided, in the last years, a significant contribution to anthropometry. In this context, facial anthropometry has greatly benefited from optical instruments such as laser scanners and stereophotogrammetry. The latter technique has proven to be accurate, repeatable and fast; therefore, taking into consideration its non-invasive nature, it has been increasingly applied to medicine, due to the relevant support that anthropometry can provide to this field. A facial anthropometric assessment can provide reliable morphometric details about the presence of deformities and peculiar features connected to underlying pathological conditions, not always easily recognizable. In the case of certain neurologic diseases, it can also provide new insights about the genotype/phenotype correlation taking the close relationship between facial and cerebral development into consideration. Furthermore, the three-dimensional morphometric evaluation of the face can reveal objective parameters useful for the planning and assessment of maxillo-facial and dental treatments, thus facilitating the clinical decisions and increasing the patients’ compliance. The facial morphometric evaluations presented in the current thesis were performed through the VECTRA M3 3D stereophotogrammetric system (Canfield Scientific, Fairfield, NJ, USA). All the patients and control subjects involved were marked with a set of facial landmarks (adapted according to the different study purposes), before the acquisitions. Once the three-dimensional models were obtained, they were elaborated through the software of the stereophotogrammetric system. Data were analysed through different statistical techniques, according to the type of study executed. The morphometric evaluations were divided in two groups: facial morphometric analyses performed through a landmark-based approach and through a surface- based approach. The first group included the studies: 1) “The face of adult patients affected by Dravet Syndrome: a 3D stereophotogrammetric preliminary assessment”, 2) “3D Craniofacial morphometric analysis of GLUT-1 DS patients” and 3) “Stereophotogrammetric analysis of a case of holoprosencephaly”. The second group included the studies: 4) “3D stereophotogrammetric assessment of labial symmetry in a girl treated for a lymphatic malformation” and 5) “Facial reanimation assessment performed through 3D-3D superimposition: a new method”. 7 For both assessed syndromes, study 1 and 2 allowed the individuation of facial features common among the patients, whose recognition can have a role in the diagnosis of the disease, both in children (study 2) and in adult cases (study 1). Study 3 allowed the identification of the presence of dysmorphic facial features in a girl affected by holoprosencephaly with an apparently normal aspect, thus sustaining the potential of the 3D stereophotogrammetric facial analysis in the morphometric characterisation of the face. Study 4 and 5 showed the usefulness of this technique for performing an objective surgical follow-up and final evaluation of maxillo-facial treatments, helping clinicians in their decisions and motivating the patients. In conclusion, all the studies sustained the usefulness, for medical purposes, of an anthropometric assessment of the human face, performed through a three-dimensional stereophotogrammetric analysis. Moreover, they highlighted its applicability to different categories of patients, including children and people with intellectual disability; thus again justifying the increasing diffusion of stereophotogrammetry in clinical and research centres.

Lo scopo di questa tesi e` quello di studiare l’espressione di alcuni enzimi del metabolismo lipidico in cellule epatiche di un mammifero ibernante, il moscardino (Muscardinus avellanarius), nelle diverse fasi del ciclo annuale. Infatti il metabolismo lipidico subisce profonde modificazioni nel corso del ciclo annuale dell’animale ibernante (fase di attivita`, fase di ibernazione e fase di risveglio primaverile) e questo potrebbe essere associato a variazioni nella espressione o nell’attivita` di enzimi. In particolare per questa ricerca abbiamo focalizzato la nostra attenzione su due enzimi chiave della via di sintesi e del catabolismo degli acidi grassi: Acido grasso sintetasi (FAS) Acil CoA sintetasi (ACSL-1) La loro espressione è stata analizzata nel fegato per il ruolo critico giocato da questo organo nel metabolismo dei lipidi. L’espressione di questi enzimi e` stata studiata tramite immunolocalizzazione ultrastrutturale, tecnica che permette una visualizzazione ad alta risoluzione dei comparimenti cellulari associata con la localizzazione di molecole funzionali nei compartimenti stessi. In particolare il lavoro si e` svolto nei seguenti step sperimentali: Valutazione della qualita` morfologica dei campioni di fegato prelevati da moscardino ed inclusi in resina per la microscopia elettronica Messa a punto del protocollo immunocitochimico per la rivelazione dei due enzimi scelti Marcatura immunocitochimica dei due enzimi in moscardini nelle tre fasi del ciclo annuale con valutazione della distribuzione della marcatura nei diversi compartimenti cellulari Analisi semi-quantitativa della densita` di marcatura dei due enzimi nelle tre diverse fasi del ciclo annuale e analisi statistica della densita` di marcatura osservata
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La valutazione combinata ed integrata della fisiologia e del comportamento permette una completa e coerente valutazione pre-clinica delle funzioni del sistema nervoso centrale. E’ stato sviluppato un sistema integrato di elettroencefalografia per via telemetrica e di video (Video-tEEG), che permette la registrazione simultanea e continua per lunghi periodi delle tracce EEG e delle immagini video. La presente ricerca e’ stata focalizzata sul set-up strumentale e sulla messa a punto della tecnica chirurgia per la registrazione combinata dei segnali EEG corticale, EEG ippocampale e elettromiogramma (EMG) mediante telemetria nel ratto. Il recupero post-operatorio degli animali e’ stato monitorato per periodi di 24 ore circa in occasione del Giorno 1, 6 e 15 dopo la chirurgia, mediante la registrazione delle trace EEG e dell’attivita’ motoria generale utilizzando i video. I risultati ottenuti suggeriscono che la tecnica chirurgica applicata per l’impianto del trasmettitoro telemetrico consente un recupero graduale degli animali entro 15 giorni dall’intervento chirurgico. Durante tutto il periodo di recupero post-chirurgico, i paramateri comportamentali e locomotori non hanno messo in evidenza cambiamenti del ritmo circadiano-giornaliero di buoi-luce e ritornano ai loro valori basali entro un periodo di 15 giorni. Utilizzando una connessine meccanica tra l’elletrodo penetrante in area profonda del cervello e l’elettrodo telemetrico, il sistema e’ in grado di acquisire segnali di EEG ippocampale di buona qualita’ a partire da 15 giorni dopo l’impianto. La ricerca ha dimostrato il possibile uso di questa tecnica di impianto chirurgico e l’applicazione del sistema integrato di Video-tEEG in diverse aree di ricerca pre-clinica compresa la Safety Pharmacology, con vantaggi per un uso etico degli animali nella ricerca.
The combined evaluation of physiology and behaviour allows a complete and comprehensive pre-clinical assessment of central nervous system (CNS) functions. An integrated video-telemetric electroencephalography (Video-tEEG) system, which allows the simultaneous and continuous recording of EEG and video images for long periods, was developed. This research work focuses on the equipment set-up and surgical methodology for combined recording of cortical, hippocampal EEG and electromyogram (EMG) waveforms by telemetry in freely moving rats. The post-operative recovery of animals was monitored by recording of EEGs and the general activity by videos for approximately 24 hours on Day 1, 6 and 15 after surgery. The results suggest that the applied surgical technique for the implantation of telemetric transmitter allows a gradual recovery of animals within 15 days. During all the recovery period the behavioural and locomotor parameters showed that there were no changes of the light-dark circadian cycle and they return to background values within a 15-day period. Using a mechanical connection between the deep and the telemetric electrodes, the recording system is able to acquire hippocampal EEG of good quality starting from 15 days after surgical implantation. The present research work demonstrated the possible use of this surgical technique and the application of the integrated Video-tEEG system in different pre-clinical research areas and also in Safety Pharmacology, with advantages for an ethical use of animals.

Introduzione: Il disturbo da Deficit di Attenzione/Iperattività (ADHD) è uno dei piu’ comuni disturbi psichiatrici infantili, con una prevalenza di circa il 5% nei bambini di età scolare. Secondo il manuale diagnostico statistico dei disturbi mentali dell’ associazione americana di psichiatria, 4° edizione rivista (DSM-IV-TR), l’ ADHD è caratterizzato da un pattern persistente e inappropriato per l’ età di in attenzione, iperattività-impulsività o entrambe. L’ esatta eziopatogenesi alla base dell’ ADHD non è completamente nota. E’ probabile che l’ ADHD sia un’ entità sindromica con una eziopatogenesi multifattoriale, che comprende fattori genetici e ambientali. Diverse linee di evidenza, presentate in dettaglio nella prima parte della tesi, suggeriscono che la carenza di ferro potrebbe essere coinvolta nell’ eziopatogenesi dell’ ADHD. Primo, il ferro è un co-fattore di enzimi necessari per la sintesi e il catabolismo dei neurotrasmettitori aminergici (dopamina, serotonina e noradrenalina), che sono coinvolti nella fisiopatologia dell’ ADHD. Secondo, la carenza di ferro è associata a un decremento dei recettori dopaminergici D2 e del trasportatore della dopamina nei gangli della base (in particolare a livello dello striato), che sono coinvolti nella patogenesi dell’ ADHD. Terzo, numerosi studi mostrano che la carenza di ferro (con o senza anemia associata) si associa a deficit cognitivi e comportamentali, inclusi deficit dell’ attenzione e iperattività. Quarto, la carenza d ferro nei gangli della base è anche considerata un fattore importante nella patogenesi della Sindrome delle Gambe senza Riposo, che puo’ essere in co-morbidità con l’ ADHD, suggerendo cosi’ una possibile eziopatogenesi comune in cui la carenza di ferro potrebbe avere un ruolo importante. Infine, il soprappeso, che è piu’ comune nei soggetti con ADHD, puo’ associarsi a carenza di ferro. Razionale: Finora, gli studi che hanno esaminato la carenza di ferro nell’ ADHD sono basati sulla misura della ferritina sierica, un marker periferico di carenza di ferro. Tuttavia, attualmente non è chiaro quanto i markers periferici di carenza marziale correlino con il contenuto di ferro cerebrale. Poiché è il ferro cerebrale che puo’ influenzare le funzioni cerebrali, una valutazione del ferro cerebrale risulta indispensabile. Finora, nessuno studio ha valutato i livelli di ferro cerebrale mediante Risonanza magnetica nucleare (RMN). Scopi: Gli scopi dello studio, presentato nella seconda parte della tesi, sono i seguenti: Scopo principale: confrontare i livelli di ferro cerebrale (stimati mediante RMN) in un campione di bambini con ADHD, in un gruppo di bambini con altri disturbi psichiatrici (diversi dall’ ADHD) e in un gruppo di controlli sani. I livelli di ferro sono stati valutati in quattro regioni cerebrali ricche di ferro: talamo, putamen, pallidum, caudato. Obiettivi secondari: valutare la relazione fra livelli di ferritina sierica e valori stimati di ferro cerebrale nei tre gruppi. Metodi: Soggetti: I pazienti (età: 6-14 anni) con ADHD e quelli con altri disturbi psichiatrici sono stati reclutati nel Servizio di psicopatologia del bambino e dell’ adolescente dell’ ospedale Robert Debrè a Parigi nel periodo 2006-2008. I controlli sani sono stati reclutati fra i figli dei dipendenti dello stesso ospedale. Criteri di non inclusione: presenza di uno o piu’ disturbi neurologici, supplementazione di ferro attuale o pregressa, anemia e uso attuale di ogni farmaco con effetti sulle funzioni cognitive. Procedura: Valutazione psichiatrica: la diagnosi di ADHD e di altri disturbi psichiatrici è stata effettuata secondo i criteri del DSM-IV-TR e confermata dall’intervista semistrutturata Kiddie-SADS-PL. Valutazione dei livelli periferici di ferro: Misura della ferritina (metodo Tinanquant), ferro sierico (metodo Ferrozine), ed emoglobina. Valutazione del ferro cerebrale: la valutazione dei livelli di ferro cerebrale è stata effettuata mediante la misura del parametro T2* alla risonanza magnetica nucleare (RMN). L’inverso di T2* (R2*) è inversamente correlato ai livelli di ferro cerebrale. Le analisi di RMN sono state eseguite su una macchina Philips 1.5 T. Analisi statistica: I valori di T2* sono stati confrontati nei tre gruppi usando una analisi oneway ANOVA con la procedura di confronto multiplo di Bonferroni. La correlazione fra i livelli di ferritina sierica e i livelli stimati di ferro cerebrale è stata effettuata mediante analisi di Spearman. Tutte le analisi statistiche sono state effettuate mediante software SPSS v. 15.0 (SPSS, Inc., Chicago, IL, USA). Risultati: Per l’ analisi statistica sono stati usati i dati relativi a 18 bambini con ADHD, 9 pazienti con altre psicopatologie (disturbi d’ ansia, disturbi dell’ umore, disturbo ossessivo compulsivo, schizofrenia precoce) e 9 controlli sani. In base all’ analisi della potenza statistica, un campione di tali dimensioni consente di rilevare una differenza di due punti nei valori di T2* con un potere statistico di circa l’ 85%. L’ analisi dei dati ha rilevato che i valori di T2* erano significativamente piu’ alti (e quindi i valori di ferro cerebrale erano significativamente inferiori) nel talamo, sia a destra (p=0.015) che a sinistra (p=0.010) nei bambini con ADHD confrontati con i controlli sani. Non sono state riscontrate altre differenze significative nelle altre regioni di interesse. I bambini con ADHD presentavano valori di ferritina sierica significativamente inferiori rispetto ai bambini con altri disturbi psichiatrici (p=0.015) e inferiori, seppure non significativamente, rispetto ai controlli sani (p=0.10). I valori di ferritina sierica sono risultati inversamente correlati con valori di T2*, ma la correlazione non è risultata significativa in alcuna regione di interesse (p> 0.005). Discussione: Questo è il primo studio che ha valutato i livelli di ferro cerebrale nei bambini con ADHD. I risultati dello studio indicano che nei bambini con ADHD si riscontrano valori inferiori di ferro rispetto ai controlli sani a livello del talamo, suggerendo che cio’ potrebbero compromettere il funzionamento di tale struttura. Anche se il talamo è stato scarsamente investigato nell’ ADHD, si presume che esso sia una struttura molto critica nei processi attentivi nel soggetto normale. La carenza di ferro potrebbe avere una ripercussione negativa sul funzionamento talamico anche in altri disturbi psichiatrici. Tuttavia, ulteriori studi sono necessari per comprendere se la carenza di ferro è specifica dell’ ADHD o si puo’ riscontrare anche in altri disturbi psichiatrici. I valori di ferritina sierica sono risultati inversamente correlati ai valori di T2*, ma la correlazione non è risultata significativa. Anche se valori bassi di ferro periferico potrebbero influenzare negativamente il contenuto cerebrale di ferro, i nostri risultati suggeriscono che i livelli di ferritina sierica possono rappresentare solo una stima approssimativa del ferro cerebrale. Conclusioni e prospettive future: Il presente studio fornisce un contributo significativo alla comprensione della fisiopatologia dell’ADHD, suggerendo che la carenza di ferro cerebrale potrebbe contribuire alla fisiopatologia di tale disturbo ( e potenzialmente di altri disturbi) mediante la commissione delle funzioni del talamo, che è coinvolto nei processi attentivi e di vigilanza. Ulteriori studi, mediante nuove metodiche di RMN che possono fornire una stima migliore dei livelli cerebrali di ferro, sono necessari per confermare i nostri dati. Questo insieme di studi potrebbe permettere una migliore comprensione della fisiopatologia alla base del cluster di sintomi comportamentali dell’ ADHD. L’ approccio di tali studi è innovativo nel campo dell’ADHD e, in generale, della psichiatria infantile, favorendo la transizione da un approccio basato sulla descrizione di sindromi comportamentali a un basato sulla fisiopatologia.
Background: Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most common childhood psychiatric disorders, estimated to affect about 5% of school-aged children worldwide. According to the Diagnostic and Statistical Manual of Mental Disorders-4th edition-Text Revision (DSM-IV-TR), ADHD is defined by a persistent and age-inappropriate pattern of inattention, hyperactivity-impulsivity or both. The exact etiopathogenesis underlying ADHD is not completely understood. It is likely that ADHD is an heterogeneous syndromic entity with a multifactorial etiopathogenesis, including genetic and environmental factors. Several lines of evidence, reviewed in detail in the first part of the thesis, suggest that iron deficiency (ID) might be involved in the etiopathogenesis of ADHD. First, iron is a co-factor of enzymes necessary for the synthesis and catabolism of the aminergic neurotransmitters (dopamine, serotonin, and noradrenaline), which have been shown to be involved in the pathophysiology of ADHD. Second, iron deficiency is associated with a decrease in dopamine D2 receptors, as well as of dopamine transporter in basal ganglia (in particular in the striatum), which have been implicated in ADHD pathogenesis. Third, there is a large body of research showing that ID with or without anemia in childhood is associated with cognitive and behavioral impairments, including poor attention and hyperactivity. Fourth, ID in basal ganglia is also increasingly recognized as a central factor in the pathophysiology of Restless Legs Syndrome, which may be co-morbid with ADHD, thus suggesting possible common pathophysiologic pathways in which iron deficiency may play a role. Finally, overweight, which is more common in children with ADHD than controls, has been associated with iron deficiency. Rationale: To date, available studies on ID in ADHD are based on the measure of serum ferritin, a peripheral marker of ID. However, how well peripheral iron indices correlate with central (i.e. brain) iron content is still unclear. Since it is central iron that may impact on brain function, there is a need to assess brain iron levels in children with ADHD. No published study has assessed brain iron levels in children with ADHD by means of Magnetic Resonance Imaging (MRI). Aims: The aims of the study, presented in the second part of the thesis, are the following: Primary: To compare brain iron levels, estimated by means of MRI, in a sample of children with ADHD, in a group of children with other psychiatric disorders (different from ADHD), and in a group of healthy controls. Iron levels were estimated in four regions which have been shown to contain iron: thalamus, putamen, pallidum, and caudate. Secondary: To assess the relationship between serum ferritin levels and estimated brain iron levels in the three study groups. Methods: Subjects: Patients (6-14 years) with ADHD, as well as those with other psychiatric disorders, were recruited from the Child and Adolescent Psychopathology Unit of the Hospital Robert Debré in Paris (2006-2008). Healthy controls were recruited from relatives of hospital employees. Non-inclusion criteria were the presence of one or more neurologic disorders, a previous or ongoing iron supplementation, anemia, and the current use of any drug that could significantly affect cognitive function. Procedures: Psychiatric evaluation: The diagnosis of ADHD, as well as of other psychiatric disorders, was made according to DSM-IV-TR criteria and was confirmed by the semi-structured interview Kiddie-SADS-PL. The Kiddie-SADS-PL confirmed the absence of any relevant psychiatric disorder in the control group. Assessment of peripheral iron status: A complete blood count and measurement of serum ferritin levels, as well as of serum iron and hemoglobin (Tinaquant and Ferrozine method) were obtained. MRI measurements: Estimation of brain iron was obtained on the basis of T2*. The inverse of T2* (R2*) are directly correlated with iron stores. MRI examinations were performed on a 1.5 T Philips Unit. Statistical analysis: T2* were compared in the three study groups using one-way ANOVA analysis with Bonferroni multiple comparison procedure. The correlation between serum ferritin levels and estimated brain iron levels in the four regions was assessed by means of the Spearman correlation. All statistical analyses were performed using SPSS v. 15.0 (SPSS, Inc., Chicago, IL, USA). Results: Data from eighteen children with ADHD, nine patients with other psychopathologies (Anxiety Disorders, Mood Disorders, Obsessive Compulsive Disorder, and Early Schizophrenia), and nine healthy controls were used for the statistical analysis. According to the power analysis, this sample size allowed for a detection of a difference of 2 points in T2* with a power of about 85%. It was found that T2* were significantly higher (meaning that iron levels were significantly lower) in thalamus, both in right (p= 0.015) and in left thalamus (p= 0.010) in children with ADHD compared to healthy controls. No other significant differences were found for the other regions of interest. Children with ADHD had serum ferritin levels significantly lower than children with other psychiatric disorders (p =0.006) and healthy controls (p=0.001). Serum ferritin levels were inversely correlated with T2*, but the correlations were not significant in any regions of interest (p> 0.005). Discussion: This is the first study to assess brain iron levels in children with ADHD. MRI data suggest that low iron levels in thalamus might impair its functioning in children with ADHD. Although the thalamus has been scarcely investigated in ADHD, it is presumed to be a very critical brain region subserving normal attention processes. Iron deficiency might negatively impact thalamic functioning also in other psychiatric disorders, but further studies are needed to assess to what extent iron deficiency is specific of ADHD or can be found in other psychiatric disorders. Serum ferritin levels inversely increased with T2*, but the correlation was not significant. Although low peripheral iron levels may negatively impact on brain iron, our results suggest that serum ferritin levels might be only a proxy for brain iron but can not estimate it accurately. Conclusions and future perspectives: This study provides a significant contribution to our understanding of the pathophysiology of ADHD, suggesting that brain iron deficiency might contribute to the pathophysiology of ADHD (and perhaps of other childhood psychiatric disorders) via its impact on thalamic functioning, which is part of neuronal circuits serving attention and alertness. Further studies, with novel MRI approaches to better estimate brain iron, are needed to confirm our results. This body of research might contribute to advance our knowledge on the etiopathogenesis and pathophysiologic pathways underlying the cluster of ADHD symptoms. The approach which underlies the rationale of these studies is an innovative one in the field of ADHD, and, in general, in child psychiatry, moving from the description of syndromes to pathophysiologybased disorders.

AIMS: We sought to determine the changes in content and organization of non-collagenic constituents of the extracellular matrix (EC), focusing in particular on highly sulphated proteoglycans (HSPGs) throughout the normal human. METHODS. Histochemical staining with Alcian Blue pH 0.2 allowed for tinctorial distinction of HSPGs on complete 1-cm transverse rings removed from the ascending and descending thoracic aorta and abdominal supraceliac, suprarenal, and infrarenal aorta of young subjects at autopsy. The same protocol has been applied to Sprague-Dawley rats’ aorta to confirm the human findings. Counterstaining with Orcein and Sirius Red allowed comparing with the total content of elastin and collagen. RESULTS. Collagen/elastin ratio increases from the proximal to distal aorta. There is a significant decrease in elastin between the supradiaphragmatic and the abdominal aorta. The proportion of HSPGs does not change significantly throughout the thoracic and thoraco-abdominal aorta, while a significant increase in total HSPGs content is observed at the infrarenal site. The same findings were confirmed on rat’s tissues. CONCLUSION. The infrarenal aorta differs histologically and biochemically from the remainder of the aorta. Highly sulphated proteoglycans in the distal aorta bear an increased load as compared to the proximal aorta. A decrease in infrarenal elastin without a corresponding decrease in collagen may effect the compliance and integrity of the distal aorta. These anatomic differences may be important in predisposing the infrarenal aorta to atherosclerosis and aneurysm formation. Rats represent a feasible model to investigate the normal asset of EC constituents of the aorta.

Abstract Epithelial-to-Mesenchymal Transition (EMT) is a stepwise and complex biological mechanism which plays an important role in tumor progression. During EMT, epithelial cells undergo a phenotypical switch to mesenchymal cells, acquiring motility and invasion capability. We aimed at clarifying the role of this complex mechanism in the Pancreatic Ductal Adenocarcinoma (PDAC) focusing on the expression of EMT markers in different cell culture conditions, such as monolayer and spheroids. For this study we used immunofluorescence and confocal microscopy, zymography, differential proteomics, real-time pcr and western blot to analyse and characterize three commercial PDAC cell lines ( HPAF-II, HPAC and PL45) cultured as monolayer or 3D- spheroids. Our analyses revealed that PDAC cells cultured in monolayer show different epithelial characteristics, such as retention of the E-cadherin/β-catenin complex – and, consequently, keep zonula adherens junctions – as well as some mesenchymal and EMT markers, such as high levels of matrix metalloproteinases (MMPs). The analysis of 3D-spheroids revealed that the E-cadherin/ β-catenin complex was similarly expressed at the cell boundaries on the plasma membrane in 2D-monolayers as well as in the 3D-spheroids, but cleavage fragments were detectable in lysates obtained from monolayers. By contrast, N-cadherin expression was observed in few HPAC cells grown in monolayers but increased in 3D-spheroids, and some cells expressing collagen type I were observed in 3D-spheroids. Podoplanin and α-smooth muscle actin were similarly expressed in both experimental conditions, as well as Snail, Slug and Zeb1. Our results confirm that EMT plays a role in PDAC. In particular, the concomitant retention of an epithelial phenotype based on the retention of zonula adherens junctions together with the expression of mesenchymal markers and proteins involved in actin cytoskeleton reorganization, strongly suggest a collective migration mechanism for PDAC cells. Moreover, the overall information provided by this study support the use of 3D cultures in biomedical research to bridge the gap between traditional cell cultures and in vivo settings. 3D cultures seem provide powerful information on PDAC cells phenotype possibly better reflecting the in vivo organization than monolayer cell culture systems, and could therefore represent a pre-clinical model to identify and validate tumor markers and to study new molecular tools to inhibit signalling pathways, or to target EMT transcription factors.

The present study focuses on active cervical movements in healthy women of different ages. The aim of the study is to perform a three-dimensional qualitative and quantitative analysis of active head-cervical RoM and of the overall movement execution in healthy women to assess the relationship with age. Age-related variations in active cervical RoM are still partially unknown: some investigations demonstrated that age has no effect whatsoever on the primary movements40 while other studies found an inverse proportionality between age and cervical RoM6,13,17. A few investigations17,47 tried to compare the entirety of the movement, not limiting themselves to the RoM but the results are varied and not conclusive. Three groups of women were compared: 22 aged 15 to 18 years (adolescents), 25 aged 20 to 30 years (young adults) and 16 aged 35 to 45 years (mid-aged women). Active flexion and extension, lateral bending and axial rotation were recorded via an optoelectronic system19,48. After the mathematical exclusion of thoracic movements, cervical RoM was referred to head local reference system and calculated using the tilt/twist method. Data were compared using ANOVA. Subsequently all the data where reconstructed through an high order polynomial interpolation, the result curves averaged per age group and movement type and compared through correlation analyses. Flexion and extension were larger in the adolescents (137°) than in the young adults (128°) and mid-aged women (127°). Lateral bending had similar ranges in the three groups: 103° for adolescents, 101° for young adults and 100° for mid-aged women. Axial rotation had similar ranges in the adolescents and in the mid-aged women (143°) and a slightly larger range in the young adults (151°). Primary movements were always associated with out-of-plane components and correlation was found between primary and coupled movements.

L'organo adiposo dei mammiferi è caratterizzato da grande plasticità. Dopo acclimatazione al freddo, ad esempio, gli adipociti bianchi possono trasformarsi in adipociti bruni che producono calore per sostenere le esigenze termogenetiche del corpo. Al contrario, sotto sovraccarico lipidico, gli adipociti bruni possono transdifferenziare in adipociti bianchi che immagazzinano lipidi per tamponare l'eccesso di nutrienti introdotti con gli alimenti. Recentemente abbiamo raccolto dati nella ghiandola mammaria di topi femmine gravide, gli adipociti bianchi non delipidano e acquisiscono una posizione pericitica, come si pensa, ma invece transdifferenziano in cellule epiteliali alveolari che producono latte. In particolare, tale transdifferrenziazione è reversibile, perché alla fine della lattazione le cellule epiteliali alveolari rapidamente riconvertono in adipociti bianchi che accumulano lipidi. Nel tentativo di comprendere i segnali molecolari coinvolti nel processo di transdiffernziazione adipo-epiteliale, abbiamo ricreato un sistema di cocultura dove siamo stati in grado di riprodurre in qualche misura la transdifferenziazione di adipo-epiteliale. L'analisi molecolare dei fattori che intervengono nel nostro sistema sperimentale ha evidenziato il possibile ruolo del Fibroblast Growth Factor come un possibile candidato in grado di indurre la transdifferenziazione adipo-epiteliale anche in vivo.
The mammalian adipose organ is characterized by great plasticity. After cold acclimation, for example, white adipocytes can convert into heat-producing brown adipocytes to sustain the thermogenetic needs of the body. Conversely, under lipid overload brown adipocytes transdifferentiate into lipid-storing white adipocytes to buffer the excess of nutrients introduced with the aliments. We recently collected evidences that in the mammary gland of pregnant female mice, white adipocytes do not slim, dedifferentiate and acquire a pericytic position, as generally thought, but instead do transdifferentiate into milk-producing epithelial alveolar cells. Notably, such a transdifferentiation is reversible, because at the end of lactation alveolar epithelial cells quickly re-convert to lipid-storing white adipocytes. In the attempt to detect the molecular cues involved in the adipoepithelial transdifferentiation process we established a coculture system where we were able to reproduce to a some extent the adipoepithelial transdifferentiation. The analysis of the molecular players intervenying in our experimental setting stressed the possible role of the basic Fibroblast Growth Factor as a possible candidate directing adipoepithelial transdiffferentiation also in vivo.

I leiomiomi uterini (detti anche fibromi, miomi) sono tumori benigni che originano dallo strato muscolare dell’utero (miometrio) e rappresentano la principale indicazione dell’isterectomia nel mondo. I leiomiomi uterini colpiscono circa il 77% delle donne in eta fertile e circa il 25% di esse presenta tumori con sintomatologia clinica evidente, tra cui la presenza di forte o anomalo sanguinamento uterino, dolore o pressione pelvica, infertilità e aborti ricorrenti. È comunemente noto che questi tumori sono caratterizzati da una elevata proliferazione cellulare ed una eccessiva deposizione di matrice extracellulare (ECM). Si ritiene che la crescita dei leiomiomi dipenda dall’azione degli ormoni ovarici mediante elementi intermedi come citochine e fattori di crescita. La Proteina Inibitore della Raf Chinasi (RKIP) ha un ruolo emergente come regolatore in diversi pathway molecolari ed è associato a un numero crescente di malattie, essendo coinvolto indiverse vie di trasduzione del segnale. Lo scopo della presente tesi è stato quello di indagare la presenza e il ruolo dell’RKIP nel leiomioma. Abbiamo dimostrato che l’RKIP è espresso nel miometrio e nel leiomioma. Per individuare il ruolo dell’RKIP, abbiamo eseguito esperimenti in vitro con un composto chimico quale la locostatina, capace di legarsi all’RKIP bloccandolo. Abbiamo dimostrato che il trattamento con la locostatina porta all’attivazione della via di segnale MAPK (fosforilazione di ERK), fornendo una opportuna validazione dell’efficacia nel bloccare l’RKIP. Inoltre, abbiamo dimostrato che l'inibizione dell’RKIP con la locostatina riduce le componenti della ECM, tra cui il collagene 1A1, la fibronectina, e il versican. In aggiunta, l'inibizione dell’RKIP con la locostatina riduce la proliferazione cellulare e la migrazione sia nelle cellule miometriali che di leiomioma. Infine, abbiamo dimostrato che il trattamento con la locostatina riduce l’espressione del GSK3β. Pertanto, anche se l'attivazione delle MAPK dovrebbe far aumentare la proliferazione e la migrazione, la destabilizzazione e l’inattivazione del GSK3β porta alla riduzione della proliferazione e della migrazione delle cellule miometriali e di leiomioma.
Uterine leiomyomas (fibroids, myomas) are benign (non-cancerous) tumors that origin from the smooth muscle layer of the uterus (myometrium), and are the most common indication for hysterectomy in the world. Uterine leiomyomas affect about 77% of women of reproductive-age, and approximately 25% of them bear clinically apparent tumors with symptoms like heavy or abnormal uterine bleeding, pelvic pain or pressure, infertility, and recurrent pregnancy loss. It is commonly known that these tumors are characterized by increased cell proliferation and excessive deposition of extracellular matrix (ECM). Growth of leiomyoma is thought to be dependent on ovarian hormones activity through intermediate elements such as cytokines and growth factors. Raf Kinase Protein Inhibitor (RKIP) has emerging roles as regulator of multiple signaling networks and is associated with an increasing number of diseases through its involvement with signal transduction pathways. The aim of the present thesis was to investigate the presence and the role of RKIP in leiomyoma. We demonstrated that RKIP is expressed in human myometrial and leiomyoma tissue. In order to define the RKIP role, we performed in vitro experiments with the chemical compound locostatin, known to bind and block RKIP. We showed that locostatin treatment results in the activation of the MAPK signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, we showed that RKIP inhibition by locostatin reduces ECM components, including collagen1A1, fibronectin, and versican. Moreover, the inhibition of RKIP by locostatin impairs cell proliferation and migration in both leiomyoma and myometrial cells. Finally, we demonstrated that locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization and inactivation of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells.