Pharmacogenetic testing provides an outstanding opportunity to improve prescribing safety and efficacy. In Public health policy pharmacogenetics is relevant for personalized therapy and to maximize therapeutic benefit minimizing adverse events. CYP2D6 is known to be a key enzyme responsible for the biotransformation of about 25-30% of extensively used drugs and genetic variations in genes coding for drug-metabolizing enzymes might lead to adverse drug reactions, toxicity or therapeutic failure of pharmacotherapy. Significant interethnic differences in CYP2D6 allele distribution are well established, but immigration is reshaping the genetic background due to interethnic admixture which introduces variations in individual ancestry resulting in distinct level of population structure. The present thesis deals with the genetic determination of the CYP2D6 alleles actually present in the Emilia-Romagna resident population providing insights into the admixture process. A random sample of 122 natives and 175 immigrants from Africa, Asia and South America where characterized considering the present scenario of migration and back migration events. The results are consistent with the known interethnic genetic variation, but introduction of ethnic specific variants by immigrants predicts a heterogeneous admixed population scenario requiring, for drugs prescription and pharmacogenetics studies, an interdisciplinary approach applied in a properly biogeographical and anthropological frame. To translate pharmacogenetics knowledge into clinical practice requires appropriated public health policies, possibly guiding clinicians to evaluate prospectively which patients have the greatest probability of expressing a variant genotype.

No description available.

La farmacogenetica fornisce un importante strumento utile alla prescrizione farmacologica, migliorando l’efficacia terapeutica ed evitando le reazioni avverse. Il citocromo P450 gioca un ruolo centrale nel metabolismo di molti farmaci utilizzati nella pratica clinica e il suo polimorfismo genetico spiega in gran parte le differenze interindividuali nella risposta ai farmaci. Con riferimento alla terapia della narcolessia, occorre premettere che la narcolessia con cataplessia è una ipersonnia del Sistema Nervoso Centrale caratterizzata da eccessiva sonnolenza diurna, cataplessia, paralisi del sonno, allucinazioni e sonno notturno disturbato. Il trattamento d’elezione per la narcolessia include stimolanti dopaminergici per la sonnolenza diurna e antidepressivi per la cataplessia, metabolizzati dal sistema P450. Peraltro, poiché studi recenti hanno attestato un’alta prevalenza di disturbi alimentari nei pazienti affetti da narcolessia con cataplessia, è stata ipotizzata una associazione tra il metabolismo ultrarapido del CYP2D6 e i disturbi alimentari. Lo scopo di questa ricerca è di caratterizzare il polimorfismo dei geni CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP3A5 e ABCB1 coinvolti nel metabolismo e nel trasporto dei farmaci in un campione di 108 pazienti affetti da narcolessia con cataplessia, e valutare il fenotipo metabolizzatore in un sottogruppo di pazienti che mostrano un profilo psicopatologico concordante con la presenza di disturbi alimentari. I risultati hanno mostrato che il fenotipo ultrarapido del CYP2D6 non correla in maniera statisticamente significativa con i disturbi alimentari, di conseguenza il profilo psicopatologico rilevato per questo sottogruppo di pazienti potrebbe essere parte integrante del fenotipo sintomatologico della malattia. I risultati della tipizzazione di tutti i geni analizzati mostrano un’alta frequenza di pazienti con metabolismo intermedio, elemento potenzialmente in grado di influire sulla risposta terapeutica soprattutto in caso di regime politerapico, come nel trattamento della narcolessia. In conclusione, sarebbe auspicabile l’esecuzione del test farmacogenetico in pazienti affetti da narcolessia con cataplessia. / Pharmacogenetics represents an important tool to improve prescribing decisions, enhancing treatment outcomes and avoiding adverse drug reactions. Cytochrome P450 enzymes play a central role in drug metabolism and its genetic polymorphism explains a large portion of interindividual differences in drug response. Referring to the therapy of narcolepsy, it should be specified that narcolepsy with cataplexy is a serious central nervous system hypersomnia characterized by severe daytime sleepiness, cataplexy, sleep paralysis, hallucinations and awakening during sleep. First-line treatment of narcolepsy includes dopaminergic stimulants for excessive sleepiness and antidepressants for cataplexy, which are extensively metabolized by the cytochrome P450 system. Moreover, due to the high prevalence of eating disorders in narcoleptic patients showed by recent studies, an association between CYP2D6 UM phenotype and eating disorders has been hypothesized. The aim of this study was to characterize the polymorphism of CYP2D6, CYP2C9, CYP2C19, CYP3A4, CYP3A5 and ABCB1 genes, involved in drug metabolism and transport, in a total of 108 samples belonging to narcoleptic patients, and evaluate the metabolizer status in a subgroup of patients displaying a psychopathologic profile consistent with eating disorders. The results show no statistical association between CYP2D6 ultrarapid metabolism and eating disorders in narcoleptic patients, proving that these disorders seems to be an integral part of the narcolepsy phenotype. Results from genotyping of all genes analyzed in the study show a an high frequency of patients with intermediate metabolism, potentially affecting therapeutic response especially in case of polytherapy, as in narcolepsy treatment. In conclusion, pharmacogenetic testing it would be beneficial in patients with narcolepsy with cataplexy.

Ischemic heart disease is a major cause of death worldwide. Identifying the mechanisms mediating cardiac resistance to injury (‘cardioprotection’) can contribute to therapies for cardiac injury. These studies investigated the role of the membrane channel forming protein connexin43 (Cx43), a downstream effector of PKCε-mediated cardioprotection. In isolated cardiomyocytes FGF-2/PKCε –induced cytoprotection is mediated by Cx43 phosphorylation at PKCε-target site, serine(S)262. Hypothesis (1). PKCε-mediated cardioprotection increases Cx43 phosphorylation at PKCε-target sites and prevents ischemia and/or reperfusion-induced Cx43 remodeling. Rat hearts were subjected to protective treatments (ischemic preconditioning, FGF-2, diazoxide), followed by 30 min global ischemia and/or 60 min reperfusion. All treatments elicited above-physiological levels of phospho(p)S262-Cx43 and pS368-Cx43 (P*Cx43), which were sustained during global ischemia, and reperfusion, and accompanied by attenuation of ischemia-induced Cx43 dephosphorylation, and prevention of Cx43 lateralization. Cx43 is present in cardiac subsarcolemmal (SSM) but not interfibrillar (IFM) mitochondria. Hypothesis (2). FGF-2 exerts protective effects on both mitochondrial populations, but is associated with mitochondrial (mito) P*Cx43 state in SSM, and mediated by mitoCx43 function. FGF-2 treatment increased calcium tolerance in SSM and IFM by 2.9- and 1.7-fold, respectively, compared to controls. In the presence of Gap27, a Cx43 hemi-channel blocker, the salutary effect of FGF-2 were lost in SSM but not IFM, indicating a functional role for Cx43. FGF-2 increased levels of PKCε, pPKCε and Tom-20 translocase in SSM and IFM. In SSM, FGF-2 increased pS262-and pS368-Cx43 by 30-fold and 8-fold, compared to controls. Stimulation of untreated SSM with a PKC activator (phorbol 12-myristate 13-acetate; PMA) also increased pS262-and-S368-Cx43 and calcium tolerance, which was prevented by εV1-2, a PKCε-inhibiting peptide. The effect of FGF-2 on isolated cardiac mitochondria is unknown. Hypothesis (3). FGF-2 exerts a direct protective effects on SSM. Direct stimulation of SSM with FGF-2 increased pS262-and-S368-mitoCx43, and calcium resistance to mPTP, and was dependent on mitoPKCε. FGF receptor inhibitors, SU5402 and FGFR neutralizing antibodies, blocked the effect of FGF-2, suggesting a FGFR1-like protein is responsible for the direct protective effects of FGF-2. This new intracellular mechanism of cytoprotection implies that endogenous intracellular levels of FGF-2 may determine constitutive levels of cardiac mitochondrial resistance to mPTP.

L’abuso dell’anziano è una condizione estremamente diffusa e ha un grande costo umano e finanziario. La dimensione reale del problema del maltrattamento dell’anziano non è stata correttamente definita, in particolare i casi segnalati ufficialmente di abuso sono solo la "punta dell'iceberg". In questi anni il problema è stato riconosciuto a livello internazionale come un fenomeno comune, che viene sottostimato e degno di attenzione da parte della comunità scientifica. In Italia non ci sono molti dati, la prevalenza degli abusi e maltrattamenti può essere circa il 5% e il problema è ampiamente sottovalutato, perché sono scarse le segnalazioni di abusi. Le evidenze disponibili dimostrano che l'abuso sugli anziani in Italia costituisce una questione pubblica, tuttavia, la legislazione e le politiche per affrontare il fenomeno sono scarse, riflettendo la mancanza di una strategia nazionale coerente e globale. Il codice civile italiano non contiene alcun riferimento esplicito a persone anziane, e pochi sono anche gli articoli del codice penale che riguardano le persone anziane. Gli articoli più rilevanti interessate sono le seguenti: art. 572: "Maltrattamenti contro familiari e conviventi" e art. 643: "circonvenzione di persone incapaci". Il presente studio ha lo scopo di analizzare la situazione attuale del fenomeno nel nostro Paese, al fine di valutare la dimensione del problema e le relazioni di abuso e maltrattamenti tra vittime, caregiver e operatori sanitari alle autorità. / Elder abuse is an extremely widespread condition and has a big human and financial price. The real dimension of the problem of elder maltreatment has not been properly defined, notably the officially reported cases of abuse are only the “tip of the iceberg”. In these years the problem has been recognized on an international level as a common phenomenon, which is underestimated and worthy of attention by the scientific community. In Italy there are not a lot of data, the prevalence of elder abuse and mistreatment may be around 5% and the problem is widely underestimated because there are few reports of abuse. The evidence available shows that elder abuse in Italy constitutes a public issue; nevertheless, legislation and policies tackling the phenomenon are scattered, reflecting the lack of a coherent and comprehensive national strategy. Moreover the elderly are not specifically protected by law. The Italian Civil Code does not contain any explicit reference to elderly people, very few articles of the Penal Code consider elderly people. The most relevant articles concerned are: art. 572: “Mistreatment of family members or children” and art. 643: “Circumvention of incapacitated persons”. The present study aim to analyze the actual situation of the phenomena in our country in order to evaluate the dimension of the problem and the reports of elder abuse and mistreatment by the victims, caregivers or health operators to the authorities.

Con questo mio lavoro di tesi ho voluto esplorare il fenomeno del maltrattamento e dell’abuso sui minori delineandone, sulla base della letteratura scientifica nazionale ed internazionale, gli aspetti clinici, epidemiologici ed i fattori di rischio. Un breve excursus giuridico illustrerà, poi, gli articoli di legge relativi alla tutela dei minori; in particolare, ci si soffermerà ad illustrare quegli articoli che normano i doveri dei sanitari nei confronti dell’autorità giudiziaria nel caso si sospetti un abuso su minori. La conoscenza della semeiotica dell’abuso, il suo riconoscimento all’interno di una diagnostica differenziale clinico-forense, la corretta repertazione e la consapevolezza che, un approccio autenticamente tutelante verso la vittima, deve sempre realizzarsi all’interno di una cornice giuridica fanno si che, il ruolo della medicina legale, competente in tutti questi ambiti, diventi senza ombra di dubbio “primum inter pares” all’interno dei team multidisciplinari ed interistituzionali che si occupano di minori vitti. Il gruppo scientifico in cui sono stata inserita e con cui si è proceduto alla redazione del manuale è il Gruppo di lavoro per l’abuso e il maltrattamento dell’infanzia coordinato dalla dott.ssa Maria Rosa Giolito ed ha coinvolto professionisti italiani afferenti a tre differenti aree sanitarie: quella ginecologica, quella medico-legale e quella pediatrica. Il testo elaborato è stato immaginato come un aiuto ai professionisti medici che si trovano a valutare un soggetto prepubere con sospetto di abuso sessuale. Non costituisce una linea-guida per la diagnosi di abuso sessuale, ma definisce alcuni requisiti essenziali e diffonde alcune conoscenze per evitare errori che possano ripercuotersi negativamente sulla valutazione clinica e sull’eventuale conseguente iter giudiziario. / With my thesis I wanted to explore the phenomenon of child abuse and child abuse by sketching on the basis of national and international scientific literature, clinical, and epidemiological risk factors. A brief review will discuss legal, then the statutory provisions relating to the protection of minors, in particular, we will look to illustrate those articles that organize the duties of health in relation to the judicial authority in cases of suspected child abuse. The semiotics of knowledge of the abuse, its identification in a forensic clinical differential diagnosis, the correct repertazione and the realization that a truly tutelante approach towards the victim, must always take place within a legal framework makes it that the role of forensic medicine, competent in all these areas, without a doubt become "first among equals" within multidisciplinary teams and institutions that deal with child victims. The Panel in which they are inserted and which is being drafted in the manual is the Working Group for the abuse and mistreatment of children coordinated by Dr. Maria Rosa Giolito and involved professionals belonging to three different Italian areas of health: the gynecological, the forensic and the children. The text drafted was imagined as an aid to medical professionals who are evaluating a person suspected of prepubertal sexual abuse. This is not a guideline for the diagnosis of sexual abuse, but it defines the essential requirements and disseminates knowledge to avoid some errors that could adversely affect the clinical evaluation and possible subsequent judicial process.

Cette thèse avait pour but d’élargir le champ d’application des ultrasons en cardiologie, dans le domaine de l’imagerie et dans le domaine de la thérapie. Concernant l’imagerie, ce sont les capacités, les atouts et les limites des ultrasons à hautes cadences d’image (ultrafast echo) qui ont été explorés. Dans un premier temps, nous avons montré la possibilité et l’intérêt d’estimer la rigidité du myocarde par ultrason en pratique clinique, chez l’enfant et chez l’adulte. La technique de l’élastographie par onde de cisaillement, utilisant une nouvelle approche de l’imagerie par sommation cohérente harmonique ultrarapide (imagerie non-linéaire), a été appliquée pour la première fois chez l’humain en cardiologie. De plus, nous avons montré que la rigidité du foie, également estimée grâce à l’élastographie par onde de cisaillement, était directement corrélée aux pressions de remplissage du cœur droit, qui sont difficiles à évaluer de manière quantitative en pratique clinique. Dans un second temps, nous nous sommes intéressés à caractériser l’orientation des fibres myocardiques durant le cycle cardiaque en développant l’imagerie du tenseur de rétrodiffusion ultrasonore en trois dimensions. Le but était de réaliser la preuve de concept sur un cœur battant afin d’ouvrir sur les possibilités d’applications. Dans un troisième temps, le doppler de puissance en ultrafast echo nous a permis d’estimer les capacités des ultrasons à visualiser et analyser les flux (et donc les débits) intra coronariens. C’est à l’heure actuelle la seule technique d’imagerie clinique ayant une résolution spatiale (et temporelle) suffisante pour voir les flux dans des vaisseaux ayant un diamètre inférieur à 500 micromètres. Enfin, l’ultrafast echo nous a permis de visualiser les micro déplacements du myocarde, qui sont eux-mêmes initiés par l’activité électrique intra-myocardique, dans un intervalle de temps très réduit. Ceci est le couplage électromécanique, et y avoir accès par ultrason ouvrirait de multiples perspectives. Nous avons commencé à l’appliquer chez le fœtus humain et chez l’adulte en transthoracique. Concernant la thérapie, nous avons exclusivement utilisé les effets mécaniques des ultrasons focalisés à hautes intensités (phénomène de cavitation). Plusieurs équipes avaient déjà montré que cela était applicable en médecine cardiovasculaire, notamment pour perforer le septum inter-atrial (zone musculaire séparant les deux oreillettes cardiaques) ou pour détruire un thrombus intravasculaire. Nous avons décidé de montrer que le phénomène de cavitation peut avoir deux autres applications distinctes : 1) la section de cordage valvulaire mitral, qui est une technique utilisée actuellement en chirurgie (donc à cœur ouvert et en circulation extra-corporelle) visant à diminuer une fuite valvulaire à cause de cordage restrictif ; 2) l’assouplissement des feuillets valvulaires calcifiés, qui est une maladie représentant un enjeu de santé publique touchant des millions de personnes, et n’ayant à ce jour aucune stratégie non invasive disponible et applicable en pratique clinique. Pour ces deux applications, nous avons réalisé des preuves de concept in vitro puis in vivo à cœur battant, sur modèle animal (ovin). L’application humaine sera la prochaine étape. C’est donc la translation de nouvelles technologies ultrasonores vers des applications cliniques en cardiologie qui a motivé et guidé ce travail de thèse. / No abstract

Gap Junctions als wichtigste Elemente der Zelle zur Ermöglichung einer interzellulären Kommunikation erlauben eine koordinierte Antwort auf externe und interne Stimuli und somit ein Zusammenspiel von Zellgruppen und Organen im Gesamtorganismus. In der vorliegenden Arbeit wurde der Einfluss einer mittelfristigen und chronischen Stimulation neonataler Rattenkardiomyozyten durch Insulin und den Wachstumsfaktoren Insulin-like Growth Factor-1 (IGF-1), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-β (TGF-β) und Fibroblast Growth Factor-2 (FGF-2) auf die Expression der Connexine 40, 43 und 45 untersucht. Dabei zeigte sich unter der Insulin-Stimulation eine konzentrationsabhängige Regulation der Connexin 43 (Cx43) Expression. Die Exposition gegenüber IGF-1 hatte einen signifikanten Anstieg der Cx43 Proteinmenge zur Folge. Unter 24stündiger VEGF- oder FGF-2-Stimulation fand sich dagegen diesbezüglich kein relevanter Unterschied. Die Analysen nach langfristiger Exposition gegebenüber TGF-β zeigten eine signifikante Abnahme der Cx43 Proteinmenge bei unveränderter Cx43 mRNA. Zur Erfassung mittelfristiger Veränderungen wurden die Kardiomyozyten jeweils 3 Stunden mit den Wachstumsfaktoren VEGF und TGF-β inkubiert. Dabei zeigte sich jeweils eine signifikante Zunahme der Cx43 Proteinmenge und –mRNA. Die Connexine 40 und 45 waren in den ventrikulären Kardiomyozyten nur spärlich nachweisbar und durch keinen der untersuchten Faktoren signifikant induzierbar.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.

As junções GAP são canais intercelulares responsáveis pela comunicação de células vizinhas, por onde passam pequenas moléculas e íons que mantêm a homeostasia celular. A junção GAP é formada seis proteínas, as conexinas. Na célula endotelial encontram-se as conexinas 37, 40 e 43. Nesse estudo, estimulamos a angiogênese em córnea de camundongos, através da cauterização com cristal de nitrato de prata. Foram utilizados camundongos heterozigotos para o gene da conexina 43 (Cx43+/-) e camundongos selvagens (Cx43+/+). As córneas foram analisadas 2 e 6 dias após a cauterização atravéspor meio da morfologia vascular, detecção das Cx37, Cx40, Cx43, PCNA por meio de Western Blot e avaliação ultraestrutural das células endoteliais. Como resultado obtivemos uma menor área de preenchimento vascular nos animais Cx43+/- em 2 e 6 dias após a lesão corneal, porém, em relação a extensão dos vasos não foi observado diferenças entre os grupos. Uma menor proliferação celular foi verificada através da detecção do PCNA, nos animais heterozigotos, somente após 2 dias da lesão corneal. Não houve alteração da Cx37 e Cx40 entres os grupos. A Cx43 parece ser uma conexina importante para a célula endotelial durante o processo de angiogênese. / The GAP junctions are intercellular streams responsible for the communication between close cells, which allow small molecules and ions to pass through them maintaining the cellular homeostasis. The GAP junction is formed of six proteins, the connexin. In the endothelial cell, there are the connexin 37, 40 and 43. In this study, we stimulated the angiogenesis in the mice\'s cornea through its cauterization using silver\'s crystal glass. It was used heterozygote mice to the gene of connexin 43 (Cx43+/-) and wild mice (Cx43+/+). The corneas were analyzed 2 and 6 days after the cauterization through the vascular morphology, detection of Cx37, Cx40, Cx43, PCNA through Western Blot and ultrastructural evaluation of the endothelial cells. As a result, we obtained a smaller area of vascular fillness in the animals Cx43+/- with 2 and 6 days of corneal injury, however, in regard to the extensions of the vessels, it wasn\'t observed any changes between the groups. A smaller proliferation of cells was verified, through the detection of PCNA, in the heterozygote animals only 2 days after the corneal injury. There wasn\'t any modification of the Cx37 and Cx40 between groups. The Cx43 seems to be an important connexin to the endothelial cell during the process of angiogenesis.