Littérature scientifique sur le sujet « MEDICINA MOLECOLARE »

A tutt'oggi non esiste una tradizione disciplinare che vada sotto il nome di "filosofia della medicina molecolare". Il presente lavoro tratteggia le basi per un ampio e sistematico progetto sui fondamenti di questa disciplina, individuando le possibili aree di analisi e le eventuali strategie di approccio: informazione genetica e patologia, organismi modello dell'uomo, transizione da medicina molecolare a medicina clinica e loro teorie dell'evidenza e della spiegazione. Dove possibile, gli autori suggeriscono anche eventuali strategie di soluzione, tracciando cosě la strada lungo la quale delineare la formazione della filosofia della medicina molecolare come vera e propria disciplina teorica.

Le tecniche di fecondazione artificiale - la cui finalità prima doveva essere il superamento della sterilità di coppia- sono divenute in realtà occasione privilegiata per ottenere "materiale umano" su cui sperimentare. Gli embrioni rimasti in soprannumero o ottenuti con fecondazioni in vitro apposite, possono essere utilizzati in studi di biologia cellulare, di genetica molecolare, di citogenetica, e biochimici. In questo articolo, l'Autore, dopo una accurata analisi della situazione attuale, valuta se esista o meno l'esigenza di utilizzare gli embrioni umani nella sperimentazione, tenendo presente che, in quanto individui umani fin dalla fecondazione, essi esigono lo stesso rispetto dovuto a chi è già nato.

La distinzione tra medicina ufficiale e medicine alternative (più propriamente denominate non-convenzionali o complementari) va inquadrata sul piano storico ed epistemologico, come parte di un continuo e mai concluso confronto tra diversi paradigmi scientifici e medici. Il ricorso a pratiche mediche di origine orientale o comunque di matrice extra-scientifica è in espansione in tutti i Paesi europei e negli Stati Uniti e ciò pone un’ampia serie di problemi sociosanitari, deontologici, etici, metodologici. La crisi attuale della medicina occidentale deriva sostanzialmente dalla difficoltà di conciliare il progressivo dominio dell’approccio scientifico-molecolare e tecnologico con la crescente necessità di riscoprire gli aspetti più personali ed individualizzati della cura, che sono particolarmente importanti nelle malattie croniche e multifattoriali. Le medicine non convenzionali possono probabilmente contribuire a colmare alcune lacune metodologiche e concettuali lasciate dalla super-specializzazione e dal prevalere del pensiero razionalista rispetto a quello empirico negli ultimi secoli e particolarmente negli ultimi decenni. Il modo più corretto di guardare al fenomeno è quello di esaminarne le ragioni d’essere, individuarne le molte possibili distorsioni, sfruttarne le potenziali positività in vista di una possibile integrazione di diversi approcci terapeutici. Tale eventuale integrazione di alcune pratiche non convenzionali richiede un maggiore impegno nella ricerca scientifica e nella qualifica delle varie figure professionali.

Santoro M, Grieco M, Melillo RM, Fusco A, Vecchio G. Molecular defects in thyroid carcinomas. Role of the RET oncogene in thyroid neoplastic transformation. Eur J Endocrinol 1995;133:513–22. ISSN 0804–4643 Tumors are believed to arise as a result of an accumulation of mutations in critical genes involved in the control of cell proliferation. Thyroid neoplasms represent a good model for studying the role of these mutations in epithelial cell multistep carcinogenesis because they comprise a broad spectrum of lesions with different degrees of malignancy. Recent reports have described the involvement of specific genetic alterations in different types of thyroid neoplasms. Papillary carcinomas are characterized by the activation of the receptor tyrosine kinases RET and TRK-A proto-oncogenes. Ras point mutations are frequently observed in tumors with follicular histology and a high prevalence of p53 point mutations have been found in anaplastic carcinomas. A definition of molecular defects characterizing thyroid tumors will be helpful in establishing sensitive and specific detection strategies and, in addition, to define genetic and environmental factors important for their pathogenesis. Giancarlo Vecchio, Dipartimento di Biologia e Patologia Cellulare e Molecolare "L, Califano", Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli "Federico II", via S Pansini 5, 80131 Napoli, Italy

Salvatore D, Celetti A, Fabien N, Paulin C, Martelli ML, Battaglia C, Califano D, Monaco C, Viglietto G, Santoro M, Fusco A. Low frequency of p53 mutations in human thyroid tumors; p53 and Ras mutation in two out of fifty-six thyroid tumours. Eur J Endocrinol 1996;134:177–83. ISSN 0804–4643 Objective: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene known to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. Desing: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. Methods: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5–9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. Results: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. Conclusions: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours. Alfredo Fusco, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia, via S. Pansini 5, 80131, Napoli, Italy

Palmieri G, Lotrecchiano G, Ricci G, Spiezia R, Lombardi G, Bianco AR, Torino G. Gonadal function after multimodality treatment in men with testicular germ cell cancer. Eur J Endocrinol 1996;134:431–6. ISSN 0804–4643 We evaluated gonadal function in 63 patients with testicular cancer both within 1 month of unilateral orchiectomy before further treatment (pretreatment) and 3 years after treatment discontinuation (post-treatment). Sixteen patients underwent orchiectomy alone (group 1), nine patients underwent infradiaphragmatic radiotherapy (group 2) and 28 patients received four cycles (group 3) and 10 patients received six cycles (group 4) of cisplatin-based chemotherapy (cisplatin, vinblastine and bleomycin—PVB, or cisplatin, etoposide and bleomycin—PEB), Pretreatment semen analyses showed reduced sperm cell density, motility and impaired morphology of spermatozoa in all four groups (p>0.05). At the same time elevated estradiol and decreased serum follicle-stimulating hormone (FSH) levels in 28.5% of subjects were correlated with high serum beta human chorionic gonadotropin concentrations. Semen analyses revealed the lowest values for all parameters after infradiaphragmatic radiotherapy. Sperm cell count, motility and morphology were significantly better in patients treated with orchiectomy alone or with a conventional dose of chemotherapy than in the groups that received radiotherapy or high doses of chemotherapy (p < 0.05). We also observed a correlation between serum FSH values and sperm cell density for both pretreatment and post-treatment in every group of patients (p<0.05). Persistent subclinical Leydig cell dysfunction in groups treated with radiotherapy or high doses of chemotherapy was expressed by increased basal luteinizing hormone levels (78% of patients in group 2 vs 60% of patients in group 4) (p < 0.05) and by normal testosterone serum values (89% of patients in group 2 vs 80% of patients in group 4). Spermatogenesis and Leydig cell function are, therefore, persistently impaired in the majority of testicular cancer patients treated with radiotherapy or with more intensive chemotherapy. G Palmieri, Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Facoltà di Medicina e Chirurgia, Università "Federico II", Via S Pansini 5, 80131 Napoli, Italy

La ricerca di base ha identificato i due principali difetti legati alla patologia policistica: a) le cellule cistiche proliferano eccessivamente e b) queste cellule secernono del fluido che ingrossa le cisti. Le principali strategie in studio nell'ADPKD consistono nell'utilizzo di farmaci in grado di interferire con i meccanismi cellulari legati a questi difetti. Una delle strategie esplorate è stata l'inibizione del sistema mTOR. Purtroppo, due trial clinici hanno fallito nel mostrare un'attività protettiva di questa classe di farmaci. La somatostatina è un'altra molecola sotto intensa validazione clinica. Al momento, i dati suggeriscono una sua possibile azione di contrasto sulla malattia ADPKD, ma i dati sono ancora preliminari per conclusioni clinicamente significative. Il Tolvaptan è un antagonista recettoriale della vasopressina che è stato ampiamente studiato: un trial clinico di numerosità adeguata ha suggerito un possibile effetto positivo di questa molecola nella riduzione della crescita dei volumi renali e nel raggiungimento di target clinici significativi. Il prossimo futuro vede in campo nuovi trial clinici esplorativi di molecole già valutate nel recente passato e di nuove strategie terapeutiche. Per la numerosità dei pazienti arruolati attira l'attenzione della comunità scientifica lo studio HALT, che esplorerà il ruolo dei farmaci antagonisti del sistema renina-angiotensina nel rallentamento della progressione dell'ADPKD. Inf ne, una categoria di farmaci precedentemente inesplorati riguarda gli inibitori del recettore dell'Epidermal Growth Factor. La ricerca clinica nell'ADPKD è straordinariamente attiva in questo periodo e questa considerazione permette un cauto ottimismo sulle possibili prospettive terapeutiche in questa patologia rimasta a lungo orfana. Qualche ombra sulla prospettiva dei risultati futuri nella ricerca clinica in questo campo proviene dalla constatazione in un numero considerevole di trial di disegni metodologicamente non adeguati.

Le cellule tumorali circolanti (CTC) rappresentato una popolazione cellulare identificata nei pazienti con tumori solidi, che sono principalmente implicate nel processo di sviluppo di metastasi. La peculiarità di queste cellule è legata al fatto che sono in grado di fornire informazioni circa l'eterogeneità del tumore e, pertanto, possono essere utilizzate non solo come dei biomarker ma anche come dei bersagli terapeutici. Le CTC hanno quindi un ruolo chiave nella biopsia liquida che, a differenza della biopsia tissutale, è non-invasiva e fornisce informazioni in tempo reale circa le caratteristiche molecolari della neoplasia. Inoltre, la biopsia liquida consente di definire monitorare la risposta ai trattamenti oncologici, l'eventuale persistenza di residuo microscopico di malattia e di identificare precocemente lo sviluppo o la presenza di meccanismi di resistenza. Alla luce di ciò, le CTC possono rappresentare un elemento centrale per la terapia personalizzata nei pazienti oncologici. L'obiettivo di questo studio è quello di effettuare una caratterizzazione molecolare multi livello ovvero su diversi campioni biologici dello stesso paziente e/ stesso campione biologico prelevato a diversi stadi della patologia. Nel nostro studio sono stati arruolati 190 pazienti; in 19 di essi è stato possibile identificare una conta di CTC > a 4. In questo lavoro riportiamo quindi i principali punti di forza e possibili limiti delle tecniche utilizzate e soprattutto le implicazioni cliniche/terapeutiche delle analisi genetiche conseguenti all'isolamento delle CTCs.
Circulating tumor cells (CTCs) represent a subset of cells found in the blood of patients with solid tumors, which are mainly involved in the process of metastatization. CTCs preserve primary tumor heterogeneity and mimic tumor properties, and may be considered as clinical biomarker, preclinical model, and therapeutic target. As such, CTCs can play a pivotal role as being a component of liquid biopsy which has potential in analyzing the genomic landscape of patients with cancer, supervising treatment responses, monitoring minimal residual disease, and managing non-invasive therapy resistance. Compared with traditional tissue biopsy, liquid biopsy is noninvasive and real-time. Therefore, CTCs can be used to tailor treatment in oncology patients. The aim of this work is to obtain through the detection of CTCs a multi-level molecular characterization using different tumor samples from the same patient or the same sample but taken throughout different stages of the disease. In our study 190 patients were enrolled; among them, 19 had a CTC count > 4. We report the results of these patients highlighting the strengths and the pitfalls of the techniques utilized as well as the clinical implications of the genetic analyses which followed the identification and isolation of CTCs.

Human serum paraoxonase (PON1) is an HDL-associated enzyme involved in the protection of lipoproteins from oxidation. Endothelial dysfunction has been documented in subjects with diabetes mellitus and is probably related to increased oxidation of circulating LDL. A genetic polymorphism of the PON1 (Gln192Arg) has been suggested to influence its anti-oxidant capacity, with the Arg allele associated with lesser protection of LDL against the accumulation of lipid peroxides. The first step of the present study was to test a fluorescent 5’ exonuclease assay to detect the point mutation in the paraoxonase gene (Q192R). This assay takes advantage of 5’ exonuclease activity of Taq DNA polymerase to cause the cleavage of two allelic-specific probes, which hybridized to template DNA during the PCR and that determine an increase of reporter fluorescence in solutions. This assay was applied to 120 sample of genomic DNA, extracted from whole blood, and we had a perfect allelic discriminations between sample wild-type, mutant and heterozygote . This results show the complete concordance of Real Time fluorescent 5’ Exonuclease Assay with a standard Polymerase Chain Reaction and restriction analysis and is an assay easy to use, rapid and accurate for the study of single nucleotide polymorphisms that permits to analyse a great number of samples in a short period of time. The second step was to evaluate, in vivo, the contribution of the PON1-192 polymorphism to the protective effect of HDLs. Three hundred and forty seven subjects in the upper and lower decile of sex-specific HDL cholesterol distribution were selected from participants in a cardiovascular disease prevention study. PON1 genotypes were determined by PCR amplification and restriction analysis and, at the same time, by Real Time PCR. Blood pressure, height, weight, smoking and alcohol habits, as well as the presence of familial or personal history of CHD were recorded. Plasma lipids and blood glucose were measured by routine enzymatic methods. As a measure of antiatherogenic HDL effect, carotid atherosclerosis was assessed by ultrasonography. Allele and genotype frequencies did not differ significantly between low and high HDL groups. Similarly, no significant difference was observed among genotypes in all variables studied. Subjects with Gln/Arg or Arg/Arg had more carotid abnormalities than Gln/Gln with an adjusted odds ratio (OR) of 3.27 (95% CI, 1.61-6.64, P=0.001) for abnormal carotid score. Stepwise logistic regression analysis showed that in the whole population age and presence of low-HDL were the only independent predictors for abnormal carotid score. In low-HDL, age was the only independent predictor entered into the model (OR 1.09/year, P<0.0001); in high-HDL, age entered first (OR, 1.07/year, P=0.001), followed by the presence of Gln/Arg or Arg/Arg (OR, 2.94, 95% CI, 1.47-5.91, P=0.002). In conclusion, in subjects with elevated concentration of HDL cholesterol, the presence of carotid atherosclerosis is significantly associated with the arginine variant in position 192 of thePON1 gene. Since there is a relative dearth of data on the association between the genetic polymorphism of PON1 and endothelial function in subjects with diabetes mellitus, we have also investigate the possible association between the PON1 Gln192Arg polymorphism and the brachial artery endothelial function in subjects with diabetes mellitus. Sixty-nine patients with type2 diabetes and 41 sex- and age-matched controls have been enrolled. The clinical and biochemical characteristics and Gln/Arg polymorphism were determined and endothelial function has been evaluated as flow mediated vasodilation (FMD) of the brachial artery after forearm ischemia. In controls no difference was observed in FMD between subjects homozygous for the Gln allele and those carrying the Arg allele. In diabetic patients, FMD was lower among those carrying the Arg allele (1.90±1.65% vs. 3.69±2.40%, p=0.02). This difference became more marked after exclusion of subjects with hypertension (2.01±1.27% vs. 5.72±3.24%, p=0.01). In multiple regression analysis, hypertension and PON1 gene polymorphism were independently and significantly associated with FMD. In conclusion Gln>Arg polymorphism of PON1 influences endothelial dysfunction in diabetic subjects, especially in those with normal blood pressure.

2013/2014
2013/2014
La sindrome di Bernard-Soulier (BSS) è una rara piastrinopenia ereditaria causata da alterazioni a livello del complesso glicoproteico GPIb-IX-V, presente sulla membrana piastrinica e responsabile della adesione delle piastrine in seguito a danno vascolare. La BSS si trasmette come malattia autosomica recessiva (BBSA1) e i pazienti affetti presentano piastrine giganti e severi episodi di sanguinamento. Tuttavia in tempi recenti sono state descritte delle famiglie con una forma dominante nota come BSSA2. In questi pazienti la piastrinopenia è moderata e le piastrine presentano un volume leggermente aumentato. Finora sono state individuate solo 5 varianti in eterozigosi nel BSSA2:, 4 nel gene GP1BA e 1 in GP1BB. Fatta eccezione per p.Ala172Val del gene GP1BA che è relativamente frequente nella la popolazione Italiana, le altre 4 sono state descritte in singole famiglie. I pochi casi di cui disponiamo, soprattutto per la forma recessiva non ci permettono di avere informazioni sui meccanismi patogenetici e sulla sua evoluzione nel tempo. Per questo motivo è stato istituito un Consorzio Internazionale per lo studio della BSS grazie al quale è stato possibile raccogliere i dati clinici e molecolari di 132 famiglie. Tutte le informazioni sono state inserite in un database (BSS Consortium database) attualmente gestito dal nostro laboratorio e consultabile dai gruppi di studio che hanno aderito al Consorzio. Inoltre per aumentare le informazioni sulle varianti identificate nel BSSA1 abbiamo incrementato i dati molecolari delle famiglie del Consorzio con i dati di altre 79 famiglie descritte in letteratura, raggiungendo un totale di 211 famiglie. Tutte le mutazioni identificate in queste famiglie sono state poi inserite in un database pubblico disponibile in rete (LOVD: Leiden Open Variation Database). La raccolta e l’elaborazione dei dati ci ha permesso di chiarire alcuni aspetti clinici e molecolari della malattia. Tuttavia data l’eterogeneità genetica e l’elevata espressione fenotipica gli studi genotipo-fenotipo si sono rivelati difficili da eseguire. Nonostante le molte informazioni acquisite, il database risulta ancora incompleto e limitato; per questo motivo è necessario raccogliere nuovi casi e inserire assieme alle varianti anche i relativi studi funzionali che si rivelano indispensabili per poter definire l’effetto delle varianti sul complesso GPIb-IX-V. Nell’ambito invece dello studio e caratterizzazione della forma meno grave di BSS (BSSA2) sono stati selezionati 120 pazienti piastrinopenici senza diagnosi caratterizzati da piastrine grandi. In questi pazienti sono stati analizzati i geni GP1BA, GP1BB e GP9 e sono state identificate 11 diverse varianti: 1 nonsense, 2 mutazioni di framshift, 1 mutazione nel codone di inizio e 5 varianti missense. Gli studi funzionali eseguiti sulle varianti missense per stabilire il loro ruolo patogenetico sono ancora in corso. Tuttavia se gli studi dovessero confermare la loro patogenicità 11 pazienti su 120 risulterebbero BSSA2 e questa forma dovrebbe essere considerata una tra le piastrinopenie ereditarie più frequenti in Italia. In conclusione grazie a questo studio è stato possibile raccogliere la più ampia casistica di pazienti affetti da BSSA1 fin’ora descritta e ottenere numerose informazioni sia sulla clinica che sulle mutazioni coinvolte. Il BSS Consortium database permetterà ai clinici che hanno partecipato allo studio di osservare nel tempo l’andamento della malattia nei pazienti e di ottenere informazioni utili per stabilire un corretto protocollo per la presa in carico dei pazienti. Infine la caratterizzazione di nuove forme di BSSA2 rappresenta il punto di partenza per descrivere al meglio la malattia BSSA2 sia dal punto di vista clinico che molecolare. In futuro sarà quindi indispensabile estendere il BSS Consortium database anche alla forma BSSA2.
XXVII Ciclo
XXVII Ciclo
1979

Classicamente, la segnalazione dipendente dall’attivazione del cAMP mediata dal recettore dell'ormone follicolo-stimolante (FSHR) e dal recettore dell'ormone luteinizzante (LH) (LHCGR) favorisce la crescita del follicolo ovarico umano e la maturazione degli ovociti. Tuttavia, esistono dati in vitro contraddittori che suggeriscono un diverso significato fisiologico della segnalazione di cAMP mediata da FSHR, mostrando allo stesso tempo l'attivazione di eventi steroidogenici e pro-apoptotici. Questi segnali possono essere alterati dagli estrogeni che inducono eventi anti-apoptotici attraverso i loro recettori nucleari e all'azione non genomica di un recettore degli estrogeni accoppiato a proteine G (GPER). Lo scopo del progetto è chiarire il ruolo degli estrogeni/gonadotropine e dei loro recettori di membrana nella regolazione della fisiologia ovarica e nella selezione del follicolo dominante. In questo studio è stato dimostrato che GPER forma dimeri sia con FSHR che con LHCGR sulla superficie cellulare di cellule HEK293 che sovraesprimono i due recettori e di cellule primarie della granulosa luteina umana (hGLC). Gli eteromeri FSHR/GPER riprogrammano i segnali del cAMP e di morte, in stimoli proliferativi fondamentali per sostenere la sopravvivenza degli ovociti. Nelle cellule della granulosa umana, i segnali di sopravvivenza mancano quando è presente un rapporto FSHR:GPER elevato, che influisce negativamente sulla maturazione del follicolo e si correla con l'accoppiamento preferenziale alla proteina Gαs, l’attivazione della via cAMP/PKA e la capacità di risposta ad FSH dei pazienti sottoposti a stimolazione ovarica controllata. Gli eteromeri FSHR/GPER, invece, mediano segnali anti-apoptotici e proliferativi dipendenti da FSH tramite il dimero Gβγ e la compromissione della formazione degli eteromeri o il knockdown GPER aumenta la morte cellulare e la steroidogenesi FSH-dipendenti. Al contrario, il complesso GPER/LHCGR non influenza la produzione di cAMP indotta da LH e hCG e non compromette l'attivazione della via cAMP/PKA. Ciò si evince dalla simile fosforilazione di CREB ed ERK1/2 e dalla stessa produzione di progesterone in hGLC trattate con siRNA contro GPER e quelle trattato con mock. È interessante notare che GPER riduce l’accoppiamento LHCGR/Gαq e di conseguenza impedisce il rilascio intracellulare di Ca2+ e l'accumulo di IP1, dopo stimolazione con LH e hCG, in cellule HEK293 che co-esprimono LHCGR e GPER rispetto alle cellule che esprimono solo LHCGR. Inoltre, è stato dimostrato che in presenza di GPER la cinetica dell'internalizzazione di FSHR attraverso endosomi precoci e tardivi è ridotta, suggerendo la sua capacità di bloccare FSHR a livello intracellulare e riducendo il riciclaggio di FSHR sulla membrana. Infatti, l'internalizzazione di FSHR è necessaria affinché GPER inibisca la risposta del cAMP indotta da FSH. Secondo i nostri risultati, gli estrogeni sono selettivamente coinvolti nella regolazione dei segnali pro e anti-apoptotici, nell'internalizzazione dei recettori attraverso i complessi FSHR/GPER e nella modulazione della cascata di segnali mediata da LHCGR. I nostri risultati indicano come la maturazione degli ovociti dipenda dalla capacità del GPER di modulare i segnali selettivi di FSHR e LHCGR, indicando che gli eteromeri dei recettori delle gonadotropine possono essere un marker della proliferazione cellulare.
Classically, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone (LH) receptor (LHCGR) -driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signalling, showing at the same time the activation of steroidogenic and pro-apoptotic events. These signals can be impaired by estrogens inducing anti-apoptotic events via nuclear receptors and non-genomic action of a G protein-coupled estrogen receptor (GPER). The aim of the project is to better understand the role of estrogens/gonadotropins and their membrane receptors in regulating ovarian physiology and the selection of the dominant follicle. In this study it was demonstrated that GPER heteromerizes both with FSHR and LHCGR at the cell surface of HEK293 cells overexpressing the two receptors as well as human primary granulosa lutein cells (hGLC). FSHR/GPER heteromers reprogram cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gβγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. On the other hand, GPER/LHCGR complex does not affect the LH and hCG-induced cAMP production and do not compromise the activation of the cAMP/PKA pathway, as it is indicated by similar CREB and ERK1/2 phosphorylation and same progesterone production in hGLC treated with siRNA against GPER, and the mock-treated one. Interestingly, GPER displace the LHCGR/Gαq coupling and consequently impedes the intracellular Ca2+ release and IP1 accumulation in LHCGR-GPER co-expressing HEK293 cells upon LH and hCG treatment compared to LHCGR-expressing cells. Also, it was demonstrated that in presence of GPER the kinetic of FSHR internalization through early and late endosomes is reduced, suggesting its ability to blockade FSHR at the intracellular level and reduce FSHR recycling on cell membrane. Indeed, FSHR internalization is necessary for GPER to inhibit FSH-induced cAMP response. According to our results, estrogens are selectively involved in the regulation of pro- and anti-apoptotic signals and receptor internalization through FSHR/GPER complexes and in modulation of LHCGR-mediated signaling cascade. Our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR and LHCGR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

2006/2007
ABSTRACT HMGA proteins are members of the high mobility group (HMG) non-histone, architectural chromosomal proteins. The HMGA family consists of: A1a, A1b and A2, the first two being isoforms produced by alternative splicing. These are small proteins, about 100aa residues long, characterized by three basic stretches of AT-hooks which bind to the AT-rich sequences on the DNA minor groove. They are normally present in rapidly proliferating cells (embryo) whereas upon differentiation the levels of these proteins decrease until they are nearly absent from adult cells. Overexpression of these proteins has been correlated with various types of neoplastic transformations. The interaction network of HMGA has been an object of study for years in our laboratory and one of the most intriguing protein-protein interactions studied is the one between HMGA and nucleophosmin. Nucleophosmin (NPM, B23, numatrin or NO38) is one of the most abundant phosphoproteins, mainly localized in the nucleoli but it also shuttles between the nucleus and the cytoplasm. This versatile protein has been proven to be involved in various cellular functions and related to both proliferative and growth-suppressive roles in the cell. The first evidence of the interaction HMGA2-NPM in our lab has been obtained by in vitro assays (affinity chromatography). The step ahead was to see if this interaction occurs in vivo and if it does, to try to find ts functional significance. Thus,immunoprecipitation (IP) experiments were performed.The first one gave the proof thet interaction between HMGA2 protein and NPM (transfected and endogenous) occurs. The second IP gave the evidence that interaction occurs between HMGA1a and NPM too. This in vivo protein-protein interaction was to be given a functional significance. We tested by siRNA HMGA1a treatment if downregulation of HMGA1a expression influences the expression of the SOD2 gene (regulated by NPM). The effect of the HMGA1a silencing has proven to be very slight on the SOD2 gene (a 14% of expression reduction). EMSA experiments have been performed in order to test the effect of NPM on the DNA binding properties of the HMGA2 protein with the DNA probes E3 and HCRII. In both cases it has been observed that NPM increases the DNA binding affinity of the HMGA2 protein.
1979

2006/2007
The advent of molecular “-omics” technologies enabled an unprecedented view into the inner molecular mechanisms of cancer and enhanced optimism towards a patient-tailored vision of medicine. The successful application of these molecular approaches in the discovery of candidate biomarker has accelerated the shift towards personalization of medicine. Indeed, biomarkers hold great promise for refining our ability to establish early diagnosis and prognosis, and to predict response to therapy. The develoment of clinically useful biomarkers would be impossible without access to human biological specimens and associated patient data, since they complete the molecular information gained from laboratory research. Furthermore, with the advances of sensitive molecular technologies, human bio-specimens can be now successfully used for wide analysis at all molecular levels (DNA, RNA and proteins), in addition to conventional cytologic and histologic investigations. However, despite the hundreds of reports on tumor markers, only a few markers have proven clinically useful. The insufficient experience in clinical application of molecular methods combined with the high complexity of clinical material represent the major obstacles for the development of clinically useful biomarkers. Thanks to the possibility to have access to the fresh and archival samples from the hospital, our laboratory can investigate the potential of technological innovations and the current technical pitfalls directly on clinical material. The work in my thesis is strictly correlated to this activity. In particular, the first part is focused on the technical optimization of molecular methods for gene expression analysis in biological fluids and especially in urine samples. In this context we validated a new experimental kit for total RNA extraction from urine samples and tested the potential of a colorimetric approach for PCR product detection. The major part of the study is focused on the technical optimization of molecular methods for gene expression analysis in archival material. This activity is in step with one of the main objectives of the European project called “Archive tissues: improving molecular medicine research and clinical practice-IMPACTS”, in which my laboratory and other 20 European centres are directly involved. In this phase the comparison of the experiences between laboratories and their active collaboration are essential for a more rapid validation of protocols dedicated to RNA (but also DNA and protein) analysis. In particular, we investigated some molecular aspects involved in the pre-analytical phase (tissue fixation procedures) and analytical phase (RNA extraction, RNA quantification and integrity assessment, qRT-PCR) of tissue processing. The final objective of this activity will be the definition of common technical guidelines for a reliable quantification of molecular biomarkers for diagnosis, prognosis and therapy directly in human archival samples. Finally, my thesis includes the clinical application of molecular methods for the quantification of candidate biomarkers in two archival case studies (a breast cancer and an adrenal gland cancer case study). In the breast cancer case study we showed that a panel of seven genes (involved in different cell pathways) is associated to patients’ survival. The adrenal gland tumor case study is part of a preliminary study about the angiogenetic process in rare human cancers.
1979

Recently we have described the synthesis of a new class of asymmetrical nitrido complexes, based on the chemical properties of the substitution labile [Tc(N)X2(PNP)]2+ complex (PNP=aminodiphosphine, X=Cl, OH, H2O), which represent an interesting opportunity in design both perfusion and target-specific radiopharmaceuticals. According to this strategy, the strong electrophylic [Tc(N)(PNP)]2+ moiety efficiently reacts with bifunctional ligands (XY) carrying coordinating atoms π-donors such as S, O or N, to afford asymmetrical nitrido heterocomplexes of the type [Tc(N)(XY)(PNP)]0/+. In this work, studies to demonstrate the effective applicability of this technology to the preparation of perfusion and target-specific agents are reported, through synthesis and biological evaluation of: 1) 99mTc-nitrido complexes as potential myocardial imaging tracers, 2) 99mTc-nitrido complexes as potential target-specific agents, for imaging CCK-B receptor, 3) 188Re-nitrido complexes for therapeutic targeting.

2007/2008
The development of new blood vessels is a complex and highly regulated process that requires the coordinated action of different growth factors. Vascular Endothelial Growth Factor (VEGF) is a powerful inducer of angiogenesis, acting through the metabolic activation, proliferation and migration of endothelial cells. The major angiogenic member of the VEGF family is VEGF-A, a gene which is transcribed to give rise to at least 4 major splicing variants, of 206, 189, 165 and 121 amino acids in humans. The angiogenic effect of the most abundant isoform (VEGF165) is basically mediated by its interaction with VEGFR2 (KDR) and VEGFR1 (Flt-1) as well as with the co-receptor Neuropilin-1 (NP-1). The shortest isoform (VEGF121) binds VEGFR1 and VEGFR2 only, but not NP-1. NP-1, together with PlexinA1, also acts as a receptor of Semaphorin 3A (Sema3A), a factor also involved in vascular patterning, besides its very well known role in axonal guidance. The aim of this project has been the detailed analysis of the peculiarity/specificity of the effect of VEGF165, VEGF121 and Sema3A in endothelial cells. We first exploited an Adeno Associated Virus (AAV)-based gene delivery to unravel the in vivo effect of these cytokines. In particular, we observed that the prolonged expression of the main isoform of VEGF, VEGF165, acted as a powerful inducer of angiogenesis, stimulating the development of both a larger capillary network and the formation of an impressive new set of arterioles. Most surprisingly, an unexpected effect of VEGF165 was also the recruitment to the sites of its expression, of a vast set of mononuclear cells. These cells derived from the bone marrow and expressed a broad set of monocytic markers (CD45+, CD11b+); their presence correlated with the formation of arterioles and the maturation of the VEGF-induced vasculature. Strikingly, VEGF121 (the shorter form unable to bind NP-1) was, on the contrary, unable to induce maturation of the newly formed capillaries to mature arteries and to recruit cells, an observation that suggested that these monocytes might be essential in blood vessel maturation. We found that cell recruitment both in vitro and in vivo strictly depends on NP-1 and that Sema3A, a NP-1 activator, acts as a powerful recruiter of these cells. The expression of VEGF and Sema3A receptors by CD11b+ cells together with the VEGFR2 interaction with NP-1, indicate that these cells could be target of a peculiar VEGF and/or Sema3A induced signalling pathway. Taken together, these results prompted us to develop an in vitro model to unravel the signalling features elicited by the VEGF165, VEGF121 and Sema3A on endothelial cells. In particular, we wanted to dissect these pathways through a proteomic approach involving the detection of the phosphoproteome of endothelial cells expressing specific receptor after treatment with the various ligands of interest. Due to the large amount of growth factors required for this kind of experimentation, we first established a Baculovirus-based system to generate the recombinant factors. For this purpose, we engineered suitable plasmids encoding VEGF165, VEGF121 and Sema3A endowed of a cleavable histidine tag at the N-terminus. Once the recombinant protein expression conditions were set up, we moved to optimize protein purification by affinity chromatography, together with the removal of the tag. The functionality of baculovirus-expressed VEGF165 and VEGF121 was ascertained by the analysis of VEGF receptor phosphorylation, as well as proliferation assays on endothelial cells; a cell contraction assay confirmed that the recombinant Sema3A produced was as active as protein expressed in mammalian cells. To dissect the differential signalling induced by each of the recombinant factors, we explored their transduction pathways in endothelial cell lines expressing the main receptors (KDR and NP1). We set up the lysis conditions and bi-dimensional SDS-PAGE, together with the optimal phospho-proteome staining. The findings obtained, together with phospho-enrichment approaches, strongly supported that peculiar signalling pathways exist in endothelial cells, selectively triggered by VEGF165, VEGF121 and Sema3A, as demonstrated by the differential pattern of phosphorylated proteins induced by the recombinant proteins. Furthermore, we first demonstrated a close similarity in the phospho-tyrosine proteome induced by VEGF165 and Sema3A, at least in the cellular system examined.
1980

L’aterosclerosi è una patologia multifattoriale ed estremamente complessa. Alcuni studi mostrano un possibile coinvolgimento di agenti microbici, ma i risultati sono contrastanti. Questo studio ha valutato la presenza di anticorpi anti-Chlamydia pneumoniae e anti-Simkania negevensis nei sieri di pazienti in attesa di sottoporsi a endoarterectomia carotidea. Inoltre, dopo l’intervento è stato possibile ricercare la presenza del DNA di questi microrganismi all’interno del tessuto patologico rimosso con intervento di routine. Per la valutazione sierologica è stata utilizzata una metodica immunoenzimatica home-made, per quella molecolare una nested PCR. Per questo studio sono stati arruolati 50 pazienti. I risultati ottenuti hanno evidenziato una positività per le IgG anti-S. negevensis del 28% e del 10% per le IgA; per C. pneumoniae le percentuali evidenziate sono del 20% per le IgG e dell’8% per le IgA. I dati molecolari della PCR ottenuti sono 34% di positività per S. negevensis e 26% per C. pneumoniae. I risultati di questo studio avvalorano l’ipotesi che questi microrganismi possano sviluppare uno stato quiescente o persistente di infezione, nel quale però è possibile riscontrare, mediante PCR, il DNA degli stessi all’interno del tessuto patologico.
Atherosclerosis is a multifactorial and complex pathology. Some studies show a possible involvement of microorganisms, but the results are contradictory. The aim of this study is to evaluate the presence of anti- Chlamydia pneumoniae and anti-Simkania negevensis antibodies in sera of patients waiting for carotid endarterectomy (CEA) surgery. In addition, after the surgery it was possible to search DNA of microorganisms into the removed pathological tissue. For the serological evaluation it was used a home-made immunoenzymatic technique, for the molecular evaluation a nested PCR. For this study, 50 patients were enrolled. The obtained results pointed out a positivity of the 28% for IgG anti-Simkania negevensis and of the 10% for IgA; for C. pneumoniae the percentages were of 20% of IgG and 8% for IgA. Molecular data of the PCR obtained were: 34% of positivity for S. negevensis and 26% for C. pneumoniae. The results of this study support the hypothesis that these microorganisms can develop a quiescent or persistent status of the infection, in which it is possible to observe, through technique of PCR, DNA of them into the pathological tissue.