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Cardiomyogenic differentiation of human multipotent stromal cells: a new approach fore regenerative medicine in cardiovascular disease.
Get PDFCardiovascular disease (CVD), which include hypertension, coronary heart disease (CHD), stroke, and congestive heart failure (CHF), are the leading and most common causes of mortality in both developing and industrialized countries, accounting for one third of all deaths globally.
Cardiomyocyte regeneration is limited in adult life and therefore the identification of a putative source of progenitors is of great interest to provide usable in vitro models and new perspectives for regenerative therapies. This appears to be a challenge due to the peculiar functional and anatomical heart properties. The goal of this project was to differentiate adult human Adipose tissue‐derived multipotent Stem Cells (ASCs) into cardiomyocytes or their close progenitors. Thus, the ultimate aim is to develop stem cell-based therapies for the cure of cardiovascular disease. More specifically, we investigated the application of two innovative approaches: the use of human growth factors in a well defined serum-free culture medium and the application of electrical and mechanical stimuli to mimic the physiological environment of the heart.
First of all, we developed a new protocol for the isolation of ASCs according with the standard GMP-procedure, which allows to extract a substantial number of cells for clinical application. The starting material was obtained from subcutaneous adipose tissue collected during an elective liposuction procedure. The isolated ASCs were characterized by flow cytometry and expanded until second passage. Several different cocktails of growth factors, cytokines and several culture conditions were tested for their ability to induce cardiomyogenesis. The goal was to identify the right combination of those factors and their time of application. One of the cardiogenic cocktails tested was indeed very effective in inducing the selected cardiac markers (e.g. NKX2-5 and MEF2C) and was chosen for the second part of this research work. The differentiation obtained was highly reproducible, as it was observed in 15 out of 15 samples from different patients. Interestingly, non induced ASCs expressed half of the selected cardiac markers (i.e. TBX5, GATA4 and BAF60C), suggesting the high Abstract V intrinsic potential and the inherent propensity of ASCs to differentiate into cardiac precursors. We further analysed the morphological changes that ASCs underwent during their differentiation by use of electron microscopy and immunofluorescence staining. By treating the cells with our selected induction cocktail, clear changes in their morphology were observed: the cells assumed a fusiform shape and became oriented.
The last milestone was to understand if the combination of the cardiac induction cocktail with a mechanical (and eventually electrical) stimulation on a scaffold would increase the efficiency of cardiogenic differentiation. For this, a customized device has been developed by the Swiss Stem Cell Foundation in collaboration with Scuola Universitaria Professionale della Svizzera Italiana (SUPSI) for the application of controlled mechanical and/or electrical stimuli to a scaffold over a desired lap of time. The scaffolds used were developed and characterized by Prof. Ciardelli’s Research Group (Materials in Bionanotechnology, Department of Mechanical and Aerospace Engineering, Politecnico di Torino). The preliminary results showed that the combination of growth factors, cytokines and mechanical stimulation for 10 days led to an at least 2 fold increase in mRNA expression of NKX2‐5, MEF2C, HAND2 and MHC when compared to the induction with cardiogenic cocktail alone on a simple 2D culture condition.
In conclusion, in this work we present a new protocol for the extraction of ASCs starting from adipose lipoaspirates, a new serum‐free based cardiogenic induction cocktail for ASCs and preliminary data about the differentiation in scaffold enhanced by mechanical stimulation opening a new perspective to cardiac regeneration
Effetti del trattamento cronico con nandrolone decanoato in ratti maschi adolescenti ed adulti sul comportamento emotivo ed interazione con il sistema endocannabinoide.
No full textIn this work we investigated the effects of chronic treatment with nandrolone decanoate (ND), a widely abused anabolic androgenic steroid, on the emotional profile of both adult and adolescent male rats. ND induced a complex depressive-like phenotype when it was administered during adolescence but not in adulthood, suggesting a more vulnerability of the adolescent brain to steroid abuse. In adolescent male rats this phenotype is characterized by alterations in emotional and cognitive behaviour, as well as in biochemical parameters such as pCREB levels and monoaminergic activity.
Since the endocannabinoid system (ECS) seems to be involved in the etiopathogenesis of depression and we observed alterations in the endocannabinoid receptor CB1 in rats exposed to ND during adolescence, we investigated the ability of the indirect CB1 receptor agonist URB597 to recover the depressive-like phenotype induced by ND. We observed that a 2-week URB597 treatment fully recovered the behavioural alterations but only partially rescued the biochemical changes and the monoaminergic activity alterations.
Finally, since cannabis is a drug widely abused among adolescents we evaluated a possible coabuse of THC, the main psychoactive component of cannabis, and ND in adolescent male rats. The co-administration of THC/ND prevented the occurrence of alterations in emotional behaviour. Overall, these data suggest that adolescent exposure to ND triggers the development of a complex depressive-like phenotype and that manipulation of the ECS could modulate the adverse effects induced by steroids on emotional behaviour
Corpo, potere e rappresentazione. L'immagine della sovranità tra teologia politica e antropologia filosofica.
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Vasospasmo cerebrale post-emorragico. Studio farmacologico dei meccanismi di vasodilatazione a livello del poligono di Willis. Studio morfologico della microcicolazione corticale e sue relazioni con eventi ischemici tardivi.
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The dual face of the endocannabinoid system in schizofrenia: experimental evidence.
Get PDFInvestigating the association between Cannabis, the endogenous cannabinoid system and schizophrenia must take into account two aspects of major relevance.
On the one hand, Cannabis is the most widely used illegal drug and there is substantial evidence that its consumption has to be classified as an independent risk factor for psychosis that may lead to a worse outcome of the disease. On the other hand, there are several lines of evidence that clearly indicate the presence of a dysregulation in the endocannabinoid system in animal models of psychosis and at least in a subgroup of schizophrenic patients.
Concerning the first point, our recent studies in female rats demonstrated that chronic THC treatment during adolescence induced a complex phenotype in adulthood characterized by the presence of anhedonia, behavioral despair in the forced swim test, reduced sociability as well as significant deficits in spatial working and object recognition memory. Moreover, adolescent THC administration also sensitizes to the locomotor activating effect induced by acute psychostimulant administration in adulthood. This response is consistent with the presence of a psychotic-like phenotype. Thus, the simultaneous presence of pronounced depressive-like behaviors, cognitive deficits as well as psychotic-like signs suggests that adolescent THC exposure had led to a behavioral phenotype in adulthood that reflects the presence of complex schizoaffective-like disorder. Interestingly, when the same protocol of THC exposure was performed in adult animals, no behavioral alterations were observed, thus highlighting the specific vulnerability of the adolescent brain to the long-lasting adverse effects of THC.
The neurobiology of cannabis-induced schizophrenia is still unknown. However, we recently demonstrated that adolescent exposure to THC produces-long lasting alterations in the endocannabinoid system, that ultimately could result in imbalances of excitatory and inhibitory neurotransmission within specific brain regions. Accordingly, our data demonstrate that the schizoaffective-like disorder induced by adolescent THC exposure in female rats is strictly associated with alterations in the inhibitory neurotransmission which are particularly prominent within the prefrontal cortex, a brain region that has been found to be severely affected in schizophrenia.
With regard to the second aspect, there are several lines of evidence that clearly indicate the presence of a dysregulation in the endocannabinoid system in different animal models of psychosis and based on these observations, the pharmacological modulation of the endocannabinoid system has been taken into account as a new therapeutic possibility for psychotic disorders. In line with this, we demonstrated that chronic treatment with the cannabinoid CB1 receptor antagonist, AM251, counteracts the psychotic-like phenotype in a neurodevelopmental model of schizophrenia in rats. This recovery at behavioral level was paralleled by the normalization of the endocannabinoid system functionality both in terms of endocannabinoid levels and CB1 receptor/G protein coupling in all the brain areas analyzed, possibly suggesting that the ability of AM251 to restore normal endocannabinoid system may account for its antipsychotic action.
As a whole, data reported in this thesis, strongly suggest an association between an altered endocannabinoid tone and the development of psychotic symptoms, thus supporting the exploitation of compounds acting on the endocannabinoid system as new therapeutic agents in the treatment of schizophrenia and related disorders
La variabilità nella risposta clinica alla Clozapina in pazienti schizofrenici. Ruolo dei polimorfismi genetici del CYP450 1 A2 e degli enzimi responsabili del trasporto della clozapina.
Get PDFNegli ultimi anni l’attenzione dei ricercatori si è focalizzata sulla farmacogenetica che permette di spiegare la variabilità interindividuale nella risposta alla clozapina (CLZ), nell’efficacia del trattamento e nella manifestazione di ADR’s. La farmacogenetica studia il ruolo dei polimorfismi del citocromo P450 (enzima che è responsabile del 70% del metabolismo della CLZ), del gene ABCB1 (che codifica il trasportatore plasmatico, la glicoproteina P) e del gene ABCG2 (che codifica un altro trasportatore, il Breast Cancer Resistence Protein) sul metabolismo, sulla concentrazione plasmatica e sulla risposta al trattamento con CLZ. Scopo del presente studio è la valutazione delle differenze genetiche dei pazienti che influenzano la risposta clinica al trattamento con CLZ, per stilare della linee guida al fine di ottimizzare l’uso di questo farmaco nella pratica clinica.
METODI
Pazienti. Nello studio sono stati arruolati in modo consecutivo tutti i pazienti con diagnosi di Schizofrenia che iniziano la terapia con CLZ afferenti al Dipartimento di Salute Mentale di Varese a partire dal 2011 e per un periodo di tre anni. I pazienti sono valutati all’inizio della terapia (I visita) e dopo 3 mesi dall’arruolamento (II visita). Durante la I visita è effettuata una raccolta anamnestica e una valutazione clinica e psicopatologica supportata da rating scale (PANSS, GAF e LUNSERS) ed è eseguito il prelievo per le analisi genetiche. Durante la II visita è ripetuta la valutazione psicopatologica al fine da valutare la risposta alla terapia con CLZ.
Genotipizzazione. I polimorfismi di Singolo Nucleotide (SNPs) nei geni CYP1A2 (C163A; G3860A), ABCB1 (C1236Te C3435T) e ABCG2 (C421A) sono stati identificati mediante Real Time-PCR utilizzando sonde TaqMan. Utilizzando il software SNPStats sono stati inoltre identificati i diversi aplotipi formati dagli SNPs considerati nello studio. Utilizzando un modello di regressione logistica sia i singoli SNPs che gli aplotipi sono stati correlati con la risposta alla terapia misurata attraverso la somministrazione della scala PANSS per gli aspetti psicopatologici e della scala GAF per il funzionamento psicosociale del soggetto.
RISULTATI
Sono stati arruolati complessivamente 46 pazienti, per 27 di questi è stato finora possibile effettuare le analisi genetiche e ottenere i dati clinici. I dati ottenuti dimostrano una correlazione statisticamente significativa tra la presenza degli SNPs ABCB1 1236T, ABCB1 3435 T e ABCG2 421A e una maggiore risposta clinica valutata mediante PANSS. Inoltre l’aplotipo TTA è significativamente più frequente nei soggetti che rispondono alla terapia con CLZ. Al contrario non è stato, finora, possibile determinare una correlazione tra la presenza di SNPs nel gene CYP1A2, così come non è stata identificata alcuna correlazione tra la presenza degli SNPs studiati e la risposta alla CLZ misurata mediante la scala GAF
Parkinson's disease biomarkers in peripheral blood T lymphocytes: identification through a proteomic approach
No full textAll the spots of the models or showing correlation with one of the mentioned parameters were excised from the gels and identified by LC-MS/MS fragmentation: some of the most important and recurring identification were beta fibrinogen and transaldolase; in particular, 4 spots correspond to beta fibrinogen and 2 spots correspond to transaldolase; all together, these 6 spots contribute to the prediction model in a very remarkable way.
For this reason, we validated by Western blot total beta fibrinogen and transaldolase changes to verify their proper identification and confirm the differences observed by 2-DE with another technique (Western blotting).
Worth of note, the level of some proteins was modified by levodopa and dopamine agonists therapy in T lymphocytes: this is a demonstration that these cells are sensitive to long term dopaminergic stimulation and that they are a valid tool to investigate dopaminergic imbalance at a peripheral level.
Additional experiments were conducted in order to deepen the role of fibrinogen in T cells, and the reason why its volume is reduced in PD patients: this study is currently going on, but preliminary results indicate that it is internalized by T cells from plasma.
In conclusion, in this thesis T lymphocyte proteome changes have been shown to be a valid tool to recognize and classify PD patients. It has been proposed a model using a rationale of similarity between affected cells (dopaminergic neurons) and probed system (T lymphocytes): we expect that our system will reasonably provide information on the effectiveness of future (or under development) drugs designed to interfere with specific cell death mechanisms targeting nigral neurons. Moreover, our system possess most features required for candidate PD biomarkers (specificity, indication of progression, accessibility).
Next step of this research is leading the current finding to the clinical practice. This objective will be reached only through large collaborative networks, that will allow us to select large cohorts of subjects with high heterogeneity in terms of gender, age, ethnicity, clinical phenotype; the use of a different and less time consuming technique in the clinical phase is advisable
Analysis and modeling of geological and environmental factors in site selection: the case of the Po Plain
No full textIl progetto di ricerca sviluppato nel corso del Dottorato ha l’obiettivo di illustrare come una rigorosa analisi geologica consenta di identificare, valutare e mitigare gli effetti di eventi naturali estremi, che possono costituire un serio fattore di rischio per gli impianti industriali e le aree metropolitane; a tale scopo, sono state selezionate due aree campione localizzate all’interno del bacino Padano, dove si sono condotte analisi geositologiche ed ambientali per caratterizzare le interazioni tra contesto naturale e impianto industriale: una discarica di rifiuti non pericolosi nel Comune di Cairo Montenotte (Sv) e l’area urbana di Como.
Nel caso di Cairo Montenotte, sono state condotte indagini geognostiche e geofisiche, prove in sito e di laboratorio e monitoraggio strumentale; altre indagini sperimentali sono state mirate alla caratterizzazione delle proprietà fisiche e meccaniche dei rifiuti, con particolare riferimento alla stabilità e all’evoluzione dei cedimenti del cumulo dei rifiuti.
La città di Como sorge su sedimenti di origine palustre molto recenti e soggetti a subsidenza; l’analisi di oltre 300 stratigrafie di sondaggio e delle prove geotecniche disponibili ha consentito di ricostruire l’assetto stratigrafico del sottosuolo mediante profili geologico-tecnici; il continuo monitoraggio della rete piezometrica comunale e dei parametri idrologici del bacino lacustre ha permesso di caratterizzare la circolazione idrica sotterranea. Sono stati poi approfonditi alcuni aspetti relativi alla presenza di lineamenti strutturali di importanza regionale, in particolare il Retroscorrimento di Monte Olimpino e infine sono state condotte alcune elaborazioni per ricostruire l’evoluzione paleo-ambientale dell’area a partire dalla deglaciazione.
The main goal of the project is to illustrate how the rigorous geological analysis allows to identify, assess, and mitigate extreme geological hazards, which might be a serious factor of environmental risk for the local industrial plants and large metropolitan areas. We selected 2 study areas in the Po Plain, a non-hazardous waste landfill located in Cairo Montenotte (Savona province), and the Como urban area. At these sites we perform several analysis in order to characterize the interactions between natural environment and industrial plants or critical facilities.
In the Cairo Montenotte case, core drillings, geophysical analysis and laboratory tests were conducted; moreover, the physical and mechanical properties of the waste were analyzed, specifically concerning waste stability and landfill subsidence. The mutual impacts between the industrial waste treatment plant and the natural environment have been analyzed and assessed.
The Como urban area lies on very recent palustrine sediments, an environment showing remarkable Holocene subsidence, and high potential for liquefaction and coastal flooding; the stratigraphic setting was reconstructed analyzing more than 300 borehole logs and geotechnical tests, and building new geological sections; wells monitoring allowed to characterize groundwater circulation. Some aspects related to the structural setting (e.g., Monte Olimpino Backthrust) and the Late Quaternary environmental evolution were also closely examined. The results of this comprehensive geological analysis have been used in order to assess the environmental impact of the new integrated defense system against floods, presently under construction along the Como lakeshore. This critical facility consists of rows of mobile gates which are intended to hydrologically isolate the town during events of very high level of the Lake Como.
In both cases it is possible to show how a proper approach for the geological siting is a critical tool for A) ensuring the environmental safety of the local industrial plant or critical facility, and B) reducing the operational costs by preventing from repeated readjustment of the project due to poorly defined design parameters
All medicines are drugs but not all drugs are medicines: pharmacoepidemiological approaches to assess the use of prescription/OTC medicines, herbs, dietary supplements and illicit drugs.
Get PDFMy PhD study programme has been focused on pharmacoepidemiologic topics.
Pharmacoepidemiology applies epidemiologic reasoning, methods and knowledge to the study of the uses and effects, beneficial and harmful, of medications in a defined population.
Pharmacoutilization studies, in particular, aim at collecting the prescriptive profile of the sample investigated.
Beside risk/effectiveness evaluations or the compliance assessment to a treatment, drug intake may be a indirect clue of the healthiness of the studied population since it is the marker of a disease severe enough to require a medication.
Data concerning the use of prescribed medications and over the counter (OTC) may also allow to evaluate potential risks of drug-drug interactions (DDI) which represents a particular concern especially in the elderly, where polypharmacy is extremely common.
DDI may be represented by a pharmacokinetic or pharmacodynamic interference of drugs on each other, which can determine an impaired effectiveness or an increased toxicity and is one of the main causes of adverse drug reaction, being responsible for up to 23% of hospital admissions (Secoli 2010).
In this context I have performed a pharmacoutilization study investigating the risk of potential DDI between treatment potentially used in dentistry and the prescribed medications/OTC used by outpatients attending a dental clinic.
Moreover, pharmacoepidemiology surveys offer the possibility to investigate use and
attitudes towards other agents such as dietary supplements (DS), herbal remedies (HR) and illicit substances such as doping.
Indeed, the use of the these substances appears to be raising, thought studies in literature are quite scarce.
Therefore, I decided to investigate the use, knowledge (benefits and risks) and attitudes towards these agents within sports.
I have also deepened the herbal remedies topic HR performing an extensive review of the existing literature and followinf s muticenter study in the setting of multiple sclerosis