Thèses sur le sujet « BIO/12 »

TP53 is a tumor suppressor gene whose product is involved in cellular growth inhibition, apoptosis and senescence. Two new classes of proteins recently discovered, p63 and p73 proteins, share with p53 a high structural homology and overlapping yet specific functions. p63 and p73 can be expressed as TA forms that have the Transcriptional Activation Domain, behave as p53-like proteins and show variability at the C-terminus due to alternative splicing. Indeed p63 and p73 can be also expressed from an alternative promoter or by N-terminal alternative splicing and the products are ΔNp73 or ΔTAp73 forms that are truncated at the N-teminus and act as dominant negative proteins of the other p53 family members. Although p53 family proteins share the main functional domains and activate the transcription of a subset of common genes, unlike p53, p73 and p63 do not have clear features of tumor suppressors. Actually their activity is more complex and still not well defined. The entire p73 network of proteins, in fact, is involved in neuronal differentiation, in the apoptotic response to damaging agents (cisplatin, IR, doxorubicin) and in tumorigenesis. TP73 gene is transcriptionally regulated by E2F1, in the G1/S transition and in the DNA damage or oncogenes activated apoptotic response. The p73 protein functions are modulated by posttranslational modifications and protein-protein interactions in different physiopathological cellular contexts. The aim of this PhD thesis has been the characterization of posttranslational modifications that regulate p73 transcriptional functions upon DNA damage and in physiological contexts.

L’ossido nitrico (NO) ha un ruolo stabilito nella difesa contro le infezioni batteriche e esercita molteplici attività modulatorie sia sulle risposte infiammatorie che immunologiche. Comunque, la rilevanza di NO sulle funzione delle cellule dendritiche (CD) è stata poco investigata. In questo studio, abbiamo trovato che l’aggiunta di un donatore di NO, S-nitrosoglutathione (GSNO), a CD derivate da monociti maturate con lipopolisaccaride (LPS) o con il ligando solubile di CD40 è legata ad una diminuita capacità di attivare cellule T allogeniche vergini ma con una più prominente polarizzazione di tipo Th1, con un’aumentata secrezione di interferon- (IFN-) e un rilascio ridotto di interleuchina (IL-)5. La presenza di GSNO durante la maturazione delle CD causa una ridotta espressione di superficie di CD86, mentre l’espressione di CD80, di CD83 e delle molecole MHC rimane inalterata. In più, GSNO induce una diminuizione dipendente dalla dose di IL-10 ed aumenta il rilascio di fattore  della necrosi dei tumori (TNF-) da parte di CD mature. In parallelo, si osserva una marcata riduzione di produzione della subunità p40 di IL-12 ma una non significativa perturbazione della produzione della forma bioattiva di IL-12 p70. Infine, GSNO riduce significativamente il rilascio di IP-10/CXCL10 e RANTES/CCL5 ma no di IL-8/CXCL8 da parte delle CD mature. Malgrado GSNO possa rafforzare la capacità delle CD mature di indurre polarizzazione di tipo Th1 dei linfociti T, i nostri dati suggeriscono che induce diverse funzioni anti-infiammatorie, eventualmente riducendo la proliferazione ed il reclutamento dei linfociti T.
Nitric oxide (NO) has an established role in the defense against bacterial infections, and exerts multiple modulatory activities on both inflammatory and immune responses. However, the relevance of NO on dendritic cell (DC) functions has been poorly investigated. In this study, we found that addition of the NO donor S-nitrosoglutathione (GSNO) to monocyte-derived DCs matured in the presence of LPS led to a decreased capacity to activate naive allogeneic T cells but a more prominent Th1 polarization, with increased IFN- secretion and reduced IL-4 and IL-5 secretion. The presence of GSNO during maturation of DCs caused a reduced expression of surface CD86, whereas CD80, CD83 and MHC molecule expression was not affected. Moreover, GSNO induced a dose-dependent decrease of IL-10 and enhancement of TNF- release. In parallel, a marked reduction of IL-12 p40 subunit in the supernatant of mature DCs, but no significant perturbation of the bioactive IL-12 p70 production was observed. Finally, GSNO significantly reduced the release of IP-10/CXCL10 and RANTES/CCL5, but not IL-8/CXCL8 by DCs. Although GSNO can strengthen the capacity of mature DCs to induce type 1 polarization of T lymphocytes, our data suggest that it elicits distinct anti-inflammatory functions, eventually reducing T lymphocyte proliferation and recruitment.

Gli anticorpi ricombinanti oggi rappresentano una potente tecnologia che può essere utilizzata per diversi scopi, che vanno dall’utilizzo come reagenti immunocitochimici, alla medicina per applicazioni cliniche sperimentali e sono tra le più sicure applicazioni per l’individuazione di malattie autoimmuni infiammatorie e tumorali. Oggi è possibile ottenere anticorpi monoclonali mediante procedimenti d’ingegneria genetica, rendendo del tutto inutile l’utilizzo di animali e cellule somatiche. Ciò è possibile attraverso il clonaggio e l’espressione su fagi filamentosi di geni VH-VL fino all’isolamento di frammenti immunoglobulinici (single chain fragment variable, scFv), capaci di legarsi unicamente all’antigene d’interesse. Inoltre, è possibile assemblare delle collezioni di fagi modificati attraverso mutagenesi nei geni delle regioni ipervariabili, chiamate librerie fagiche anticorpali, che consentono di selezionare in vitro anticorpi monoclonali ricombinanti umani sotto forma di scFv, specifici per una larga varietà di antigeni. Recenti studi sono stati mirati all’utilizzo di scFv a singolo dominio VH per applicazioni terapeutiche, mirati a migliorare la stabilità e la solubilità degli scFv a livello intracellulare. Nel nostro lavoro si è cercato di approfondire e migliorare la selezione anticorpale attraverso la metodica del phage display, creando una valida alternativa di screening rispetto all’utilizzo dei fagi filamentosi, sfruttando il ciclo litico del fago T7. Si è formata una libreria scFv-VH su fago litico T7 e testata la sua validità contro l’antigene in precedenza utilizzato per selezioni con fagi filamentosi, l’Ubiquitina bovina. Gli studi effettuati dimostrano che dalla libreria fagica anticorpale su fago T7 scFv-VH creata, è possibile isolare anticorpi monoclonali con gli stessi risultati, ma in tempi molto rapidi rispetto all’utilizzo della metodologia di selezione su fagi filamentosi M13.
Recombinant antibodies represent a powerful technology which can be used for different goals, such as immunocytochemical reagents or experimental clinical applications. They constitute one of the most reliable and safest applications for identifying autoimmune inflammatory diseases and cancer diseases. At present, it is possible to obtain monoclonal antibodies through genetic engineering procedures, without the use of animals or somatic cells which, thanks to these new technologies, comes to be unnecessary. This is possible through the cloning and the expression on filamentous phages of VH-VL genes, and through the isolation of immunoglobulin fragments (single chain fragment variable, scFv) from antibodies’ libraries built on filamentous phages. Furthermore, it is possible to assembly phages collections, modified through mutagenesis on hypervariable regions genes (called antibodies phages libraries), which allow to select in vitro human recombinant monoclonal antibodies in shape of scFv, specific for a wide range of antigens. Recent studies aim to use single domain VH scFv for therapeutic applications and for improving the stability and the solubility of scFv at intracellular level. In our research we try to deepen and develop antibody selections through the phage display method, by presenting a valid screening alternative to the use of filamentous phages and by taking advantage of the phage T7 lytic cycle. A new library scFv-VH on phage has been assembled on lytic phage T7 and its validity has been tested on the antigen bovine Ubiquitine, previously used for filamentous phages selections. The studies carried out demonstrate that, by employing our antibody phage library on phage T7 scFv-VH, it is possible to isolate monoclonal antibodies and achieving results comparable to the ones obtained by using the traditional methodology of selection on filamentous phages M13, but in very short time.

Motor neuron diseases (MNDs) form a heterogeneous group of pathologies characterised by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND, even if the particular step in RNA metabolism that is vulnerable in motor neurons remains unknown. FUS, a nuclear protein supposed to have several functions in DNA and RNA metabolism, forms cytoplasmic aggregates in cells affected by amyotrophic lateral sclerosis (ALS), and mutations disturbing the nuclear import of FUS cause the disease. We engineered mouse motorneuronal NSC34 cells to express wild-type FUS, as well as variants mutated in the C-terminal region and associated to familial ALS (R514G, R521G), a combination of the two single mutants (R514G/R521G), and a truncation mutant associated to a juvenile and aggressive form of familial ALS (R495X), and we showed that our cellular model well recapitulates the FUS-ALS phenotype of mislocalisaton and aggregation. It is extremely likely that the FUS cytoplasmic aggregates are cytotoxic because they trap important factors; the nature of these factors, however, remains to be elucidated. In this study we showed that mutated FUS colocalise with Stress Granules upon oxidative stress induction. Most importantly, mis-localised, aggregated FUS colocalises and associates with SMN, the causative factor in spinal muscular atrophy (SMA). SMN is known to have a crucial role in the biogenesis and localisation of the spliceosomal snRNPs, which are essential assembly modules of the splicing machinery. Our results indicate that FUS and SMN work on the same pathway, as FUS binds to SMN and to spliceosomal snRNPs downstream of the SMN function. Pathogenic FUS mutations do not disturb snRNP binding. Instead, cytoplasmic mislocalisation of FUS causes partial mis-localisation of snRNAs to the cytoplasm, which in turn causes a change in the behaviour of the alternative splicing machinery. FUS, and especially its mutations, thus have a similar effect as SMN1 deletion in SMA, suggesting that motor neurons could indeed be particularly sensitive to changes in alternative splicing and that such alterations could represent a common pathogenic patway for ALS, SMA and – perhaps – other MNDs.

Scopo dello studio: Analizzare dal punto di vista immunoistochimico l’attività rigenerativa e delle cellule satellite a livello delle fibre muscolari e della giunzione muscolo-tendinea (MTJ) dei muscoli della gamba nel piede torto congenito idiopatico, al fine di studiare il ruolo di tali strutture nella fisiopatologia della deformità. Materiali e metodi: Sono stati ottenuti 8 campioni di muscolo e MTJ dal materiale di scarto di 8 pazienti con età compresa tra i 4 ed i 7 anni, che sono stati sottoposti ad intervento chirurgico di allungamento del tendine di Achille e trasposizione del tendine del tibiale anteriore dal primo al terzo cuneiforme, al fine di correggere la recidiva del piede torto congenito. I campioni di tessuto ottenuti sono stati valutati dal punto di vista morfologico ed immunoistochimico. Nello specifico, dopo adeguato trattamento sono stati incubati con i seguenti anticorpi: anti-MSTN, anti-BMP2, antiPAX7, anti-miogenina e anti-CD44. La differenza dei risultati tra le fibre muscolari e la MTJ è stata analizzata dal punto di vista statistico. Valori di p<0,05 sono stati considerati statisticamente significativi. Risultati: L’espressione dei marcatori studiati è stata valutata mediante la conta del numero di cellule positive su 10 campi ad alto ingrandimento o High Power Field (HPF) sia a livello del tessuto muscolare che della MTJ. Il numero medio delle cellule positive alla miostatina a livello del tessuto muscolare è stato di 103,6±76,5, mentre a livello della MTJ il numero medio delle cellule positive alla miostatina è stato di 84±73,2; la differenza non risultava statisticamente significativa (p=0,78). Il numero medio delle cellule positive alla BMP2 a livello del tessuto muscolare è stato di 148,4±151,5, mentre a livello della MTJ il numero medio delle cellule positive alla BMP2 è stato di 42±64,1; la differenza risultava statisticamente significativa (p<0,05). Il numero medio delle cellule positive al PAX7, al CD44 e alla miogenina a livello del tessuto muscolare è stato rispettivamente di 86,6±26,5, 21±11,1 e 40,8±22,8. A livello della MTJ invece, il numero medio delle cellule positive al Pax7, al CD44 e alla miogenina è stato rispettivamente di 178,8±49,1, 147,4±55,1 e 105,6±48,8; per tutti e tre gli anticorpi la differenza era statisticamente significativa (p<0,05). Conclusioni: I nostri dati sono al momento preliminari in quanto non abbiamo potuto compare i risultati con campioni di controllo sani della stessa età dei bambini con PTC. Il presente studio tuttavia suggerisce come sia il muscolo che la MTJ presentino possibili anomalie a livello dei pathway che regolano la miogenesi e la 4 rigenerazione muscolare. Mentre un’alterazione della normale interazione tra MSTN e BMP signalling potrebbe giocare un ruolo importante nell’induzione dell’ipotrofia muscolare nel PTC e nell’infiltrazione di tessuto adiposo, la MTJ potrebbe avere un ruolo compensatorio nei confronti della stessa ipotrofia.

L’acronimo NAFLD (“non-alcoholic fatty liver disease”) comprende sia la condizione di steatosi epatica semplice sia la steatoepatite non-alcoolica (NASH: “non-alcoholic steatohepatitis”) caratterizzata da necroinfiammazione e dalla possibile presenza di fibrosi epatica. È stato dimostrato che la NASH può talvolta progredire fino a determinare l’insorgenza di cirrosi e/o di epatocarcinoma cellulare (HCC). Negli ultimi anni, numerosi studi hanno evidenziato un sostanziale aumento nel numero di casi con HCC correlato alla NAFLD, sia in presenza che in assenza di cirrosi epatica. Indipendentemente dall’eziologia, durante il processo di epatocarcinogenesi, numerose vie di trasduzione del segnale e di controllo di espressione genica risultano essere alterate. Tuttavia, i meccanismi molecolari alla base della progressione della NAFLD verso un suo fenotipo pro-carcinogenico non risultano ancora del tutto chiariti. Tra le numerose proteine coinvolte nell’insorgenza e nella progressione dell’HCC, la proteina FAK (Focal Adhesion Kinase) è spesso trovata overespressa o iper-fosforilata in pazienti affetti da tumore, suggerendo un ruolo chiave per questa proteina nell’induzione del fenotipo neoplastico. Recentemente, anche la metil-transferasi EZH2 è stata associata al processo di epatocarcinogenesi e, in particolare, un recente studio ha evidenziato una correlazione positiva tra l’over-espressione di FAK ed EZH2 e l’aggressività del tumore in pazienti affetti da neoplasia endometriale, suggerendo un potenziale collegamento tra le due proteine. In questo studio, pertanto, abbiamo volto l’attenzione al ruolo di FAK ed EZH2 in modelli in vitro ed in vivo di NAFLD o di HCC. Gli esperimenti condotti sia sul modello in vitro che sul modello in vivo di NAFLD, hanno mostrato un aumento significativo della proteina FAK, della sua fosforilazione in tirosina 397 e della sua proteina target Paxillina. Tuttavia, il silenziamento di FAK in una linea cellulare di epatoblastoma umano è risultato essere associato con l’attivazione del processo di de novo lipogenesis e con un conseguente aumento dell’accumulo lipidico intra-cellulare. D’altro canto, gli esperimenti condotti su un modello murino di progressione della NAFLD verso l’HCC hanno evidenziato che livelli di FAK e della sua fosforilazione in tirosina 397 aumentavano parallelamente alla progressione del danno epatico fino all’insorgenza dei noduli tumorali. Abbiamo inoltre dimostrato, per la prima volta, il coinvolgimento di EZH2 nella NAFLD, riportando una correlazione inversa tra l’espressione di EZH2 e il grado di severità della malattia. Infine, un modello murino di epatocarcinoma è stato utilizzato per caratterizzare il ruolo di FAK nell’epatocarcinogenesi. In particolare, i nostri dati dimostrano che il silenziamento di FAK riduce drasticamente la crescita del tumore che esprime anche livelli più bassi di EZH2. Allo stesso modo, studi in vitro hanno evidenziato l’azione anti-proliferativa/pro-apoptotica del silenziamento di FAK in cellule di HCC. Entrando nel merito del potenziale meccanismo molecolare che potrebbe connetter le due proteine di nostro interesse, nelle cellule silenziate per FAK abbiamo riscontrato una riduzione del trascritto, della localizzazione nucleare e dell’attività di trimetilazione di EZH2. Sulla base di ulteriori risultati abbiamo dimostrato che p53 e E2F2/3 sono coinvolti nella regolazione della trascrizione di EZH2 mediata da FAK. Pertanto, in conclusione, in questo studio abbiamo dimostrato il ruolo chiave di FAK nella NAFLD e nell’epatocarcinoma, e abbiamo fornito evidenze dell’esistenza di una inter-connessione tra le proteine FAK ed EZH2, suggerendo p53 e E2F2/3 come possibili mediatori.
NAFLD is one of the most common liver disease worldwide and it encompasses a wide range of liver injuries, ranging from simple steatosis (non-alcoholic fatty liver “NAFL”) to non-alcoholic steatohepatitis (“NASH”). NASH can be sometimes associated with hepatic fibrosis and may potentially progress to irreversible cirrhosis and in some cases to hepatocellular carcinoma (HCC). The number of HCC new cases with a NAFD-dependent aetiology has strongly increased during the last decade but the molecular mechanisms regulating NAFLD-related hepatocarcinogenesis remain to be explored yet. Among all the factors involved in HCC onset and progression different epigenetic mechanisms and signalling pathways, affecting cell homeostasis (e.g. cell proliferation, apoptosis, migration and invasion) may play a major role, but their action in NAFLD is still obscure and their connection with hepatocarcinogenesis is unknown. Interestingly, the focal adhesion tyrosine kinase (FAK) is often found overexpressed or hyper-phosphorylated in HCC patients suggesting a key role of this protein in the control of cancer cells behaviour. Similarly, the methyltransferase EZH2 has been recently associated with the process of hepato-carcinogenesis. Further, a recent study reported a positive correlation between FAK and EZH2 expression and their association with tumour aggressiveness in endometrial cancer. Therefore, a potential direct/indirect interplay between these proteins might affect the development of different tumours, including HCC. In this study, we point to investigate the role of FAK and EZH2 in in vitro and in vivo models of diet-induced NAFLD and of HCC. Our results demonstrated an increased expression of Tyr-397 phosphorylated FAK and of its target paxillin in vivo and in vitro NAFLD. Moreover, the silencing of FAK also promoted increased lipid accumulation via the activation of the de novo lipogenesis pathway in HepG2 cells. Interestingly, in a model of NAFLD-induced HCC, both total and pTyr397 FAK correlated with disease severity. We reported the first evidence of EZH2 connection to NAFLD observing a down-regulation of EZH2 in our in vitro and in vivo models. Furthermore, the pharmacological inhibition of EZH2 worsened liver steatosis and inflammation. Results obtained from human HCC xenografts on NOD/SCID micrevealed a crucial role of FAK in HCC development and progression. Accordingly, we found that silencing of FAK reduced cell proliferation and invasion, and induced apoptosis in HCC cells. Additionally, we demonstrated that the silencing of FAK critically affected EZH2 transcription, nuclear localization and H3K27 tri-methylation activity. Importantly, we found that p53 and E2F2/3 are key mediators of FAK-dependent effects on EZH2 expression/activity. In conclusion, we demonstrated a master role of FAK in NAFLD and HCC and provided strong evidence of its connection with EZH2, introducing a new protein network active in the control of cancer cells’ proliferation, in which p53 and E2F may act as mediators.

La proteina Translationally Controlled Tumor Protein (TCTP) svolge un importante ruolo come fattore di sopravvivenza nelle cellule tumorali ed è overespressa nelle cellule di carcinoma mammario scarsamente differenziato. TCTP è un target specifico della Diidroartemisinina (DHA). La DHA è il principale metabolita dell’Artemisinina, il principio attivo estratto dalla Artemisia annua L. La DHA è attualmente utilizzata come farmaco antimalarico. Recentemente, è stata inoltre dimostrata l’efficacia della DHA come antitumorale. In questo studio abbiamo valutato l’effetto antitumorale della DHA su lineee cellulari di carcinoma della mammella presentanti fenotipo altamente aggressivo, come le linee cellulari MDA-MB-231 ed SKBR3. I nostri risultati mostrano che la DHA inibisce la crescita delle cellule tumorali della mammella ed induce apoptosi, attraverso la riduzione dei livelli di espressione della forma fosforilata della TCTP. Inoltre, abbiamo dimostrato che la DHA aumenta l’efficacia dei farmaci comunemente utilizzati nella terapia antitumorale, come la Doxorubicina e il Trastuzumab. I dati ottenuti da questo studio suggeriscono che la fosfo-TCTP può rappresentare un nuovo bersaglio terapeutico per il trattamento del cancro della mammella. Inoltre, il trattamento combinatoriale con la DHA e i convenzionali chemioterapici potrebbe rappresentare una nuova potenziale strategia terapeutica per il cancro della mammella.
Translationally Controlled Tumor Protein (TCTP) is a survival factor in tumor cells overexpressed in poorly differentiated breast cancer cells. TCTP is a specific target of Dihydroartemisinin (DHA). DHA is a metabolite of Artemisinin, the active principle of Artemisia annua L. DHA is an anti-malaria drug with antitumor properties. We studied the effect of DHA on human breast cancer cell lines (such as MDA-MB-231 and SKBR3 cells) with more aggressive phenotype. Our results show that DHA inhibits breast cancer cells growth and induces apoptosis by reducing the levels of the phosphorylated form of TCTP. We also show that DHA improves the antitumor effect of the conventional chemotherapy drugs, such as Doxorubicin and Trastuzumab. Altogether, these results suggest that phospho-TCTP is a novel therapeutic target for breast cancer cells. DHA in combination with conventional chemotherapeutics is a novel strategy to treat breast cancer

Caspase 8 is a well characterized protein that plays a pivotal role transducing the extrinsic apoptotic pathway. A hallmark of tumor cell is resistance to apoptosis; this feature is the result of several molecular aberrations in signalling pathways involved in the control and execution of cell death. In this context, Caspase 8 can represent a good target as its loss of function may severely impinge on apoptosis. Surprisingly, its expression is lost only in a small percentage of tumors, indicating that the retention of Caspase 8 expression in some tumors may be well tolerated and suggesting that it may even positively contribute to tumour progression. Consistent with this introduction, in the first part of this this project we show evidences supporting the idea that Caspase 8 expression may promote cell transformation in hepatocarcinoma and in glioblastoma cellular models. Interestingly, we could show that Src kinase, aberrantly activated in these cancer cellular models, may drive Caspase 8 phosphorylation on Tyr380. Using a cancer cellular model characterized by Src constitutive activation engineered to express either Caspase 8-wt or Caspase 8-Y380F we provide evidence that Caspase 8 expression and phosphorylation on Tyr380 but not its enzymatic activity promote in vitro cell transformation, Rac activation and resistance to anoikis. To further investigate the requirement for Caspase 8 expression in tumor progression we focused our studies on glioblastoma cellular models. Glioblastoma multiforme (GBM multiforme) is the most aggressive primary brain tumor in the adult nervous system and it is associated with a poor prognosis. Interestingly in this tumor a major role is played by the ability of cancer cells to strongly modulate tumor microenvironment by secreting interleukines and cytokines that overall sustain cancer cell survival and promote neo-angiogenesis. In the second part of the project we provide evidence that the inhibition of Caspase 8 expression, obtained by RNA interference, severely downregulates the mRNA levels and the production of IL-6, IL-8, IL-1β, MCP-1 and VEGF-A by glioblastoma cell lines. Consistently it impairs in vivo angiogenesis and in vitro proliferation and capillary tube-like network formation on matrigel triggered by conditioned media of cultured cells. How Caspase 8 is able to modulate the expression of these factors has not been elucidated yet. A possible molecular mechanism contemplates Caspase 8 interaction with NF-kB pathway. Several studies suggest an important role for nuclear factor kappa light chain enhancer of activated B cells (NF-kB) signalling in glioblastoma and implicate NF-kB activation as an important driver of the malignant phenotype that confers a negative prognosis in patients and resistance to therapeutic treatments, supporting the importance of a role of NF-kB to promote aggressiveness, invasion, neo-angiogenesis in this tumor. Importantly, previous studies have shown that NF-kB transcription factor may promote the expression of IL-6, IL-8, IL-1β, MCP-1 and VEGF-A. Moreover, it has been suggested that Caspase 8 may promote NF-kB activity. We demonstrate that Caspase 8 supports NF-kB nuclear translocation also in glioblastoma cell lines and provide evidence for a possible role of its phosphorylation on Tyr380.

HIV-1 has evolved effective strategies to evade the host immune response, most of which are mediated by a set of viral “accessory” proteins: VIF, VPR, VPU and NEF. The majority of these proteins interfere with multiple intracellular pathways by usurping the cellular Cullin Ring Ubiquitin Ligase systems (CRLs) and by promoting the ubiquitination and the degradation of host antiviral factors. The HIV-1 accessory protein VPU interacts with the Cullin1 complex to enhance the degradation of the transmembrane protein BST2, a factor involved in the retention of viral particles and the inhibition of viral spread. On the other hand, VPR associates to the Cullin4 Complex to stimulate the degradation of the Uracyl DNA Glycosylase UNG2, a protein that, in addition to its role in the progression from S to G2 phase, is able to cause mutations in the viral genome. Recent findings demonstrate that the proautophagic protein AMBRA1 interacts with multiple Cullin RING complexes and regulates their activity (Antonioli et al., 2014), including both Cullin 4 and Cullin 1 adaptors. Moreover, AMBRA1 was identified among VIF interactors in a HIV proteomic study, suggesting the possible involvement of AMBRA1 in the activity of different HIV-1 accessory proteins. In this work, we found that AMBRA1, in addition to VIF, it also interacts with other two HIV-1 accessory proteins: VPR and VPU. Moreover, we show that the HIV-1 accessory proteins VPR and VPU promote the interaction of AMBRA1 with Cul4 and Cullin1 complexes, respectively. Importantly, AMBRA1 silencing impairs HIV-induced degradation of both UNG2 and BST2. Finally, we show that VPU expression results in increased basal and starvation-induced autophagy, and induces the colocalization of BST2 with LC3 positive vesicles, which mediate its delivery to the lysosomes. Altogether, these data provide new insights about the role of the proautophagic protein AMBRA1 and on how HIV-1 exploits the autophagy process for its life cycle, further highlighting the complex interplay between autophagy and the innate immune response.

Macroautophagy is the major regulated catabolic mechanism used by eukaryotic cells to degrade long-lived proteins and organelles. It involves the formation of cytosolic doublemembrane vesicles, called autophagosomes, that sequester portions of cytoplasm and then they fuse with lysosomes to form autolysosomes. Autophagy has a well-documented role in the maintenance of tissue homeostasis and in the response to stressful environments; moreover, this process is often dysregulated in various human diseases, such as neurodegeneration and cancer. In this context, autophagy has been identified as a crucial process in oncogenesis and in tumour progression. Allelic loss of the essential autophagy gene Beclin 1 occurs in human cancers and renders mice tumour-prone. The regulation of the Beclin 1/Vps34 complex lipid kinase activity is a critical step in autophagy signaling pathway. Ambra1 (Activating molecule in Beclin 1-regulated autophagy) has been shown to be an important member of this complex and to be involved in autophagosome formation. This evidence prompted us to investigate a possible role for Ambra1 as a haplo-insufficient tumour suppressor gene. We show that monoallelic deletion of Ambra1 promotes tumorigenesis. We found that Ambra1+/gt mice have a significantly higher probability than Ambra1+/+ mice to develop a malignancy, showing approximately three folds increase of spontaneous tumorigenesis in a number of organs, such as liver, spleen, lymphonodes, and lung. In lung, Ambra1 hemizygous tumours show traits of lung papillary adenocarcinoma. We have previously shown that Ambra1 deficiency during embryogenesis in vivo and in vitro induces an increase in cell proliferation. Therefore, we have also investigated whether the observed tumours could be related to a direct impairment of cell growth control by autophagy. The first evidence that we found was a general increase of the organ size, especially of liver, kidney, heart and spleen of the heterozygous animals in comparison with the wt mice, thus suggesting a role for Ambra1 in cell growth control. Moreover, we elucidate this aspect also in vitro by analysing the proliferation rate and the markers of cell cycle in Ambra1 defective systems. In principle, the demonstration of a haplo-insufficient tumour suppressor phenotype for Ambra1’s reduced function in mice may have direct implications for analysing the molecular pathogenesis of human cancer. Therefore, we isolated mouse embryonic fibroblast (MEFs) from embryos wt, heterozygous and knockout for the Ambra1 gene trap mutation and we evaluated the cell growth rate by BrdU incorporation assay and by cell counting. Both analyses revealed a marked increase in the proliferation rate of Ambra1+/gt and Ambra1gt/gt MEFs when compared with wild-type cells, indicating that the loss of the only one allele of Ambra1’s alleles is sufficient to increase cellular proliferation. Since the molecular mechanism responsible for these results could be due to a deregulation of the cell cycle, we decided to examine the main cell cycle regulators. First of all we focussed our attention on analysing the levels of the four main cyclins: D, E, A, B. During our analysis we have found that in Ambra1 defective systems, unlike the cyclin D and E, high levels of the cyclin A and B were present. These proteins, called mitosis cyclins, are more expressed in the S- and M-phase of cell cycle. Therefore, our finding correlates with the hyperproliferative phenotype specific for Ambra1 deficient cells. Moreover the transcription of the cyclin A gene is under the control of p107 hyperphosphorylation (Zerfass et al., 1996), a protein belongs to the Retinoblastoma protein family. The increased levels of cyclin A could explain the hyperphoshorylate state of p107 that we found in our experimental systems. Moreover, since activities of cyclin/CDK complexes is also mediated by their binding to other proteins, we decided to examine whether the Ambra1 dosage was correlated to the expression of these proteins. In particular, we analyzed the levels of two main members of the Cip/Kip family, p21 and p27. In our studies we found a deregulation of a number of cell cycle regulatory proteins: A and B cyclins, p107, p21 and p27. This may explain the hyperproliferative phenotype observed in Ambra1-defective systems in vitro. In summary, we identified a novel haplo-insufficient tumour suppressor gene. This strongly support for the idea that Ambra1 could play an important role in the regulation of tumour development and that its activity is tightly regulated in coordination with cell growth. The detailed mechanism by which Ambra1 contributes to tumour suppression is still unknown.

The medical treatment of colorectal cancer (CRC) has evolved greatly in the last years, involving combined chemotherapy protocols and, more recently, new biologic agents. Nevertheless, prognosis remains adverse for patients with metastatic disease and a significant portion of early-stage patients develop recurrence after chemotherapy. Clinical trials are now directed to evaluate new drug combinations and treatment schedules in order to overcome the mechanisms of chemoresistance. By the use of two patient-derived colon cancer cell lines, CC09 (BRAFV600E) and R511 (wt BRAF), and the established colon cancer cell line HT29 (BRAFV600E ; PIK3CAP449T), we found that the tyrosine kinase receptor HER3 is strongly involved in the mechanisms of resistance to 5FluoroUracil (5-FU) and Oxaliplatin drugs. By the use of a monoclonal antibody targeting HER3, named U3-1287, we found down-regulation of HER3 phosphorylation, HER3 internalization and degradation in all cell lines. Functionally, U3-1287 inhibits tumor cell proliferation inducing growth arrest in the G1 phase of the cell cycle, and reduces tumor mass in a CC09-derived xenograft model. Even though U3-1287 administration is higly efficient in abrogating the HER3-dependent activation of PI3K pathway in colon cancer cells, we also found that it induces a compensatory mechanism, involving the increase of HER2 receptor expression that in turn activates MAPK pathway. To overcome U3-1287-induced activation of MAPK, we used a combination therapy with U3-1287 antibody and the MEK-inhibitor Trametinib. We show that Trametinib alone induces the phosphorylation of HER3 receptor that in turn activates PI3K pathway; the combination therapy results in the complete abrogation of both PI3K and MAPK pathways, and in a significant reduction of cell survival in vitro in all three cancers cell lines. These data identify a new combination strategy that, independently of the genetic background of the cells, may overcome the mechanisms of resistance to chemotherapy in HER3-overexpressing colon cancers.

Autophagy is a self-degradative process involved in the turnover of cellular components in response to nutrient starvation or to organelle damage. During this process, portions of cytoplasm are sequestered by double-membrane vesicles, the autophagosomes, and degraded after fusion with lysosomes. Different protein complexes participate to autophagosome formation including various components of the class III phosphatidylinositol-3-OH kinase complex (Ambra1, Beclin 1, Vps34, Vps15, UVRAG) and most of the Atg genes. Here, I show that Ambra1 also plays a role in the regulation of the late steps of autophagy via the interaction with the lysosomal protein Spinster. Spinster is trans-membrane protein known to regulate the endosomal pathway in Drosophila neurons. Recent studies have shown that Spinster is able to bind the antiapoptotic protein Bcl2 and is involved in the execution of a caspase-independent cell death associated to autophagic vacuole formation. In this study, I demostrate that Spinster is involved in the regulation of autophagosome maturation. Spinster localizes with LC3, a marker of the autophagosomal compartment. Notably, Spinster overexpression induces autophagy, while its down-regulation leads to an alteration of autophagolysosomal acidification causing a block of autophagic degradation. Finally, I showed that Ambra1 regulates Spinster activity by regulating its levels of ubiquitination. Taken together, my data show that Spinster is a positive regulator of the autophagic process and that Ambra1 has a novel role in the regulation of autophagosome maturation.

La frataxina è una proteina mitocondriale, la cui ridotta espressione è responsabile di una malattia neurodegenerativa ereditaria, l’atassia di Friedreich (FRDA). La frataxina è una proteina che lega il ferro ed è coinvolta nella biogenesi dei gruppi ferro-zolfo (ISC), gruppi prostetici che svolgono funzioni cellulari essenziali come la fosforilazione ossidativa, la catalisi enzimatica e regolazione dei geni. La frataxina è richiesta per lo sviluppo, poiché la sua assenza è letale in embrioni di topo e provoca l’arresto nello sviluppo del nematode C. elegans. Una parziale espressione della frataxina permette lo sviluppo e la sopravvivenza dell’organismo, e determina una progressiva degenerazione di tessuti specifici. Sebbene molte evidenze suggeriscano che la frataxina agisca da chaperone nel compartimento mitocondriale, è stato recentemente dimostrato l'esistenza di un pool funzionale di frataxina extramitocondriale in vari tipi di cellule umane. Lo scopo del mio lavoro nella prima parte è stato di indagare sul possibile ruolo fisiologico della frataxina extramitocondriale nel compartimento citoplasmatico studiando l’interazione della proteina con un possibile patner ISC-dipendente. E' stato dimostrato che la forma extramitocondriale della frataxina interagisce direttamente con l’aconitasi citosolica/ proteina regolatoria del ferro-1 (IRP1), una proteina bifunzionale che alterna la funzione enzimatica di aconitasi e la funzione di “RNA-binding” attraverso il meccanismo dello “switch” del cluster ferro-zolfo. Inoltre il difetto dell’aconitasi citosolica e la conseguente attivazione di IRP1 come proteina che lega l’RNA, che si verifica nelle cellule dei pazienti affetti da atassia di Friedreich, viene revertito con l'azione della frataxina extramitocondriale. La frataxina, inoltre, protegge le cellule tumorali dallo stress ossidativo e dall’apoptosi, ma agisce anche da soppressore tumorale. Le basi molecolari di questo apparente paradosso non sono ad oggi note. Nella seconda parte del mio lavoro ho osservato che l'espressione della frataxina è aumentata in diverse linee cellulari tumorali in risposta allo stress ipossico, una condizione spesso associata alla progressione del tumore. Inoltre, l'aumento della frataxina in risposta all'ipossia dipende dai Fattori di espressione Ipossia-Inducibili (HIF) e modula l’attivazione del soppressore tumorale p53. E’ stato mostrato per la prima volta in vivo l’ aumento di frataxina in campioni chirurgici di glioblastoma umano e campioni umani di carcinoma di colon. Questi risultati mostrano che la frataxina partecipa alla risposta allo stress indotto da ipossia nei tumori, ciò implica che la modulazione della sua espressione potrebbe svolgere un ruolo determinante nella sopravvivenza e/o nella progressione delle cellule tumorali.
Defective expression of frataxin is responsible for the degenerative disease Friedreich’s ataxia (FRDA). Frataxin is an iron-binding protein involved in the biogenesis of iron–sulfur clusters (ISC), prosthetic groups allowing essential cellular functions such as oxidative phosphorylation, enzyme catalysis and gene regulation. Frataxin is a protein required for cell survival since complete knock-out is lethal. Partial expression of the frataxin allows the development and survival of the organism, yet results in progressive degeneration of specific tissues. Although several evidence suggest that frataxin acts as an iron-chaperone within the mitochondrial compartment, it was recently demonstrated the existence of a functional extramitochondrial pool of mature frataxin in various human cell types. The aim of my work in the first part was to investigate for a physiological role of extramitochondrial frataxin in the cytoplasmic compartment searching for ISCdependent interaction. The extramitochondrial form of frataxin was demonstrated to directly interact with cytosolic aconitase/iron regulatory protein-1 (IRP1), a bifunctional protein that alternates between an enzymatic and a RNA-binding function through the “iron–sulfur switch” mechanism. Importantly, the cytosolic aconitase defect and consequent IRP1 activation occurring in FRDA cells was found to be reversed by the action of extramitochondrial frataxin. Frataxin protects tumor cells against oxidative stress and apoptosis but also acts as a tumor suppressor. The molecular bases of this apparent paradox are missing. The aim of my work in the second part was to investigate the pathways through which frataxin enhances stress resistance in tumor cells. Frataxin expression was found to be upregulated in several tumor cell lines in response to hypoxic stress, a condition often associated with tumor progression. Moreover, frataxin upregulation in response to hypoxia is dependent on HypoxiaInducible-Factors (HIFs) expression and modulates tumor suppressor p53 activation. Importantly, this work shows for the first time an in vivo increase of frataxin in human glioblastoma and colon carcinoma tumor samples. These results show that frataxin participates to the hypoxia-induced stress response in tumors, thus implying that modulation of its expression could play a critical role in tumor cell survival and/or progression.

The denitrosylating enzyme GSNOR profoundly impact s on cellular signal transduction by controlling cellular concentrations of protein-SNOs. Excessive S-nitrosylation is a well-documented phenomenon known as nitrosative stress, which increases during age. In this phD thesis we provide evidence that GSNOR physiologically may undergo epigenetic silencing during aging probably due to DNA methylation of its promoter, carried out by TET proteins. It is plausible to speculate, therefore, that nitrosative stress occurring upon GSNOR deficiency, could compromise mitochondrial function, resembling, in such a way, aging-like conditions. By promoting mitochondrial defects, indeed, nitrosative stress has been associated with several features related to aging and to pathological states typical of advanced age, such as cancer. Coherently, GSNOR depletion has been recently demonstrated to contribute to development of hepatocellular carcinoma (HCC) and , being downregulated in almost the half of HCC cases, GSNOR deficiency characterizes a large subclass of this tumor. In this work we demostrate that hepatocarcinoma HepG2 cells stably downregulating GSNOR (shGSNOR HepG2) exhibit defective and fragmented mitochondria and that this condition is associated with the upregulation of succinate dehydrogenase (SDH) in order to sustain ATP production. Therefore, GSNOR deficiency, compromising mitochondrial function, prompts mitochondria to a higher vulnerability and this molecular adaptation enhances cell sensitivity of shGSNOR HepG2 cells to even low doses of different SDH-directed drugs (mitocans). In particular, we demonstrate that shGSNOR HepG2 cells undergo necroptosis via PARP1/RIP1 pathway. As a matter of fact, pharmacological inhibition of both proteins, as well as reverse genetics experiments completely rescue cell viability. We show that non-apoptotic cell death depends on caspase 3 Snitrosylation, which leads to its inactivation, and on the capability of SDHtargeting mitocans to produce ROS as side-effect of their mechanism of action. Indeed, pre-treatment with antioxidants fully abolishes cell demise. In addition, we revealed that SDH upregulation is associated with the cytosolic degradation of the mitochondrial chaperone TNF-associate protein 1 (TRAP1) via the ubiquitin-proteasome pathway. In particular, we provide the first evidence that TRAP1 is S-nitrosylated and that this modification is mandatory for its degradation. TRAP1 silencing by siRNA, indeed, recapitulates the molecular and cellular phenotype of shGSNOR HepG2 cells, and increases cell sensitivity to SDH-targeting mitocans. Overall, our results indicate that GSNOR is implicated in aging process, being epigenetically regulated during age and impacting on mitochondrial homeostasis, the first determinant of cell senescence/aging. Furthermore, excessive S-nitrosylation due to GSNOR downregulation elicits TRAP1 Snitrosylation and degradation, which in turn leads to increased SDH levels and enhanced sensitivity to SDH -targeting mitocans. Being GSNOR depletion distinctive of the 50% of HCC cases, our results argue for this class of molecules as new promising drugs to selectively eradicate HCC.

Cell organization is governed and maintained via specific interactions between its constituent macromolecules. In this thesis I investigate the role of the topological characteristics of protein interaction networks in promoting cell organization. Comparison of the experimentally determined protein interaction networks in different model organisms has revealed little conservation of the specific edges linking ortholog proteins. Nevertheless, some topological characteristics of the graphs representing the networks – namely non random degree distribution and high clustering coefficient – are shared by networks of distantly related organisms. I have used ProtNet, a stochastic model representing a computer stylized cell to ask questions about the dynamic consequences of the topological properties of the static graphs representing protein interaction networks. By using a novel metric of cell organization, I show that natural networks, differently from random networks, can promote cell self-organization. Furthermore the composition of the ensemble of protein complexes that form in pseudo-cells, that self organize under the interaction rules of natural networks, are more robust to perturbations. This analysis carried out by using networks with a variety of topological characteristics led me to conclude that self organization is a consequence of the high clustering coefficient, while the scale free degree distribution is probably a relic of the evolutionary processes that lead to interactome evolution and has little functional relevance.

Cancer is a highly complex disease which is caused by an upset of balance between cell growth and death. During cell transformation, abnormality in the machinery of apoptosis often occurs, resulting in a chemotherapy-resistant phenotype of cancerous cells. Endoplasmic reticulum stress (ER stress) and autophagic process are now representing serious candidates as alternative pathways to induce cell death in chemotherapy-resistant tumor cells. If on the one hand they induce cytoprotective functions to reestablish normal cellular homeostasis, on the other hand they contribute to an efficient cell killing when the stress is prolonged and unresolved. Therefore, harnessing stress-induced survival response, such as ER stress and autophagy, may represent a new anticancer strategy to induce cell death in apoptosis-resistant tumors. In line with this hypothesis, it has been recently shown that some chemotherapeutic agents overcome apoptosis-resistance through induction of both autophagy and ER stress. Thus, understanding the link between ER stress/apoptosis and autophagy/apoptosis induction may offer the opportunity to find out new targets to design more effective therapeutic regimes to treat cancer malignancies. Here, we show that: i) the down-regulation of the transcription factor E2F1, a key regulator of proliferation and cell death, represents a critical event in ER stress-induced apoptosis, unveiling E2F1 inactivation as a novel therapeutic strategy to increase the response of tumor cells to ER stress based anticancer treatments; ii) oncogenic activating mutation in B-RAF confers resistance to autophagy in response to both classical inducers (serum starvation and rapamycin), and to ER stress-induced apoptosis (fenretinide and velcade), suggesting that autophagy is required for efficient melanoma cell killing; iii) Ambra1, an essential regulator of autophagy, plays a role in the regulation of apoptosis, and consequently in the interplay between autophagy and apoptosis, underlining the functional link existing between these processes.

A non-coding GGGGCC (G4C2) repeat expansion in the C9orf72 gene is the major genetic determinant of Amyotrophic Lateral Sclerosis (ALS), a late‐onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons (Cozzolino et al., 2012). A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic by sequestering RNA-binding proteins, whose decreased availability may then affect various steps of post-transcriptional gene regulation, such as alternative mRNA splicing, translational regulation, mRNA transport, or mRNA decay (La Spada and Taylor, 2010). In samples from C9orf72 patients as well as patient-derived iPSC, RNA foci containing the sense as well the antisense RNA repeat sequence are likewise detected (Gendron et al., 2013; Lagier-Tourenne et al., 2013; Mizielinska et al., 2013), indicating that the sequestration of RNA-binding proteins and hence a dysregulation in one of the steps of RNA metabolism might well play a role in ALS. The precise step that is affected, however, remains ill defined, and thus our understanding of C9orf72 toxicity in the disease pathogenesis is still elusive. To get insights into these mechanisms, we built a cellular model based on the overexpression of normal and expanded G4C2 repeats, and we observed that the expression of (G4C2)31 pure repeats is sufficient to induce the formation of intra-nuclear RNA foci in mouse motor neuron-like NSC34 cells as well as in human HeLa cells. We then used an in vitro-transcribed biotinylated RNA containing (G4C2)31 repeats to identify C9orf72 RNA binding proteins. Through pull down assays and mass spectrometry analysis we were able to identify many different factors involved in posttranscriptional gene regulation, such as members of the hnRNP family, which regulate alternative splicing, and translational regulators, including initiation and elongation factors. Since a significant, although not complete, sequestration of some of these factors into RNA foci was observed, we analysed whether alternative splicing process and protein translation could be affected in cultured cells expressing the expanded repeats. Indeed, we observed that the expression of (G4C2)31 repeats alters the alternative splicing pattern of a splicing reporter (pSMN2), through the interaction with the splicing factor hnRNP H. Moreover, the expression of (G4C2)31 is able to activate a stress response that lead to a general reduction of translation. Indeed, (G4C2)31 repeat widely affects the overall distribution of Pura and its binding partner FMRP, two well-known regulators of translation, that accumulate into intra-cytosolic granules which are positive for stress granules markers. However, translational repression is not achieved through eIF2a phosphorylation-mediated ternary complex inhibition, while C9orf72 repeats strikingly induce an abnormal nuclear accumulation of polyadenylated mRNAs, and this is associated to the nuclear relocalization of the cytosolic form of poly(A) binding protein (PABPc). Thus, our observations suggest that nuclear accumulation of mRNAs, as a consequence of C9orf72 repeats ability to impair nuclear mRNA export, might contribute to ALS pathogenesis. Overall, our results indicate that G4C2 repeats affect post-transcriptional gene regulation, thus supporting the concept that a selective vulnerability of motor neurons to alterations in mRNA metabolism may be a common denominator in ALS, as well as in other motor neuron disease.

Il TNF receptor-associated factor 2 (TRAF2) è un membro della famiglia di proteine Tumor Necrosis Factor (TNF) receptor associated factor (TRAF) che si associano e mediano la trasduzione del segnale tra i membri della superfamiglia dei recettori TNF. In seguito all’attivazione del recettore TNF, TRAF2 è reclutata al sito del recettore determinando l’attivazione di cascate di segnale a valle che, a seconda del tipo di stimolo, possono condurre all’apoptosi o alla sopravvivenza della cellula. Abbiamo caratterizzato la struttura secondaria e terziaria della proteina in funzione della temperatura utilizzando spettroscopia nell’UV in cuvetta (in particolare Dicroismo Circolare, fluorescenza statica e dinamica), mentre la struttura quaternaria e lo stato di aggregazione della proteina sono stati studiati effettuando esperimenti di light scattering. La stabilità della proteina e le transizioni di unfolding/refolding sono state studiate in dettaglio utilizzando alta pressione, agenti denaturanti chimici (guanidina) e temperatura. Abbiamo scoperto che in soluzione, a temperatura ambiente, la proteina esibisce una struttura oligomerica compatibile con quella trovata nel cristallo, in particolare quest’ultima si è dimostrata essere un trimero costituito da tre identiche sub unità. Abbiamo anche dimostrato che la stabilità della struttura quaternaria di TRAF2dipende strettamente dalla presenza di 3 segmenti di α-elica che sono tenuti insieme nella proteina nativa foldata. Abbiamo anche condotto uno studio per monitorare l’interazione di TRAF2 con le membrane modello (GUV) poiché è stata precedentemente avanzata l’ipotesi che i lipid rafts delle membrane cellulari svolgessero un ruolo cruciale nella funzione di TRAF2. Siamo stati in grado di stabilire utilizzando tecniche di spettroscopia a correlazione di fluorescenza (FCS), il coefficiente di diffusione della proteina all’interno di diverse tipologie di membrane, sia sintetiche, sia estratte da tessuti reali. Abbiamo inoltre utilizzato la Generalized Polarization e l’analisi dei fasori spettrali del Laurdan per studiare come TRAF2 influenza la fluidità delle membrane. I nostri risultati dimostrano che TRAF2 esercita una effetto meccanico sul doppio strato lipidico in quanto la sua aggiunta alle GUV induce la formazione di vescicole intraluminali.
The TNF receptor-associated factor 2 (TRAF2) is a member of the Tumor Necrosis Factor (TNF) receptor associated factor (TRAF) protein family which associate with, and mediate the signal transduction from members of the TNF receptor superfamily. Upon TNF receptor activation, TRAF2 is recruited at the receptor site resulting in the activation of downstream signaling cascades that, depending on the kind of stimulus, can lead to apoptosis or to cell survival. We have characterized both the protein secondary and tertiary structure as a function of temperature using conventional UV spectroscopy in cuvette (namely Circular Dicroism, steady-state and dynamic fluorescence) while the quaternary structure and aggregation state have been investigated by light scattering experiments. The stability and the protein unfolding/refolding transition have been also studied in detail using high hydrostatic pressure, chemical denaturing agents (guanidine) and temperature. We found that in solution, at room temperature, the protein exhibits an oligomeric structure compatible with that found in the crystal, which has been demonstrated to be a trimer of identical subunits. We also demonstrated that the TRAF2 quaternary structure stability is strongly dependent on the presence of 3 α-helices segments that are stuck together in the native, folded protein molecule. We also carried on a study to monitor the interaction of TRAF2 with model membranes (GUV) since it has been suggested that lipid rafts play a crucial role in the TRAF2 function. We were able to evaluate the diffusion rate of the protein within different kind of membranes, both synthetic and extracted from real tissues, using Fluorescence Correlation Spectroscopy (FCS) techniques. We also used Laurdan Generalized Polarization and Spectral Phasor Analysis to study how TRAF2 affects the membrane fluidity. Our results demonstrated that TRAF2 exert a mechanical effect on the lipid bilayer since its addition to GUVs induces the formation of intraluminar vesicles.

Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus which belongs to the Flaviviridae family. With almost 170 million people infected worldwide, HCV is a leading cause of chronic hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Poynard, T., et al., 2003). Major improvements in patient treatment were achieved by the approval of the first direct acting antivirals (DAAs) in 2011 (Welsch, C., et al., 2012). Nonetheless, HCV variants resistant to DAAs may arise during treatment as well as a heavy burden of side effects, thus affecting therapy outcome. Cellular models of HCV replication and infection have recently allowed the characterization of molecular details of HCV life cycle. HCV RNA replication occurs in association with ER-like cellular membranes and requires several viral non-structural (NS) proteins including; NS3, NS4A, NS4B, NS5A, and NS5B, as well as host cell factors (Moriishi, K., and Matsuura, Y., 2003; Gosert R., et al., 2003). The site of virus assembly is yet unknown, but recent data haves proposed the recruitment of HCV RNA from the replicase complex to lipid droplets mediated by the HCV core and the non-structural proteins as an early event in virion assembly (Miyanari, Y., et al., 2007). Since HCV is a relative small virus, in order to accomplish genome replication and formation of new viral particle, it needs to interact with and subvert cellular machineries for its own purpose. A large number proteinprotein interaction has been described between HCV and host cells. However to date, most of them are only descriptive and their functions in HCV life cycle remain to be characterized (Tellinghuisen and Rice, 2002). NS5A is an important component of the viral replication complex and also participates to HCV particle assembly (Kim, S., et al., 2010). While no known enzymatic function has been ascribed to NS5A, it is known to interact with host cell proteins to affect a variety of processes, including innate immunity, host cell growth and proliferation (He, Y., et al., 2006). The molecular mechanism by which NS5A contributes to HCV life cycles remains largely uncharacterized. To get insights on NS5A function, we performed a screening to search for cellular proteins interacting with NS5A in HCV replicon cells, by means of a tandem affinity purification approach coupled to mass spectrometry (TAPMS/MS). NS5A protein complexes were isolated from HCV replicon cells, resolved by monodimensional gel (SDS-PAGE) and revealed by SYPRORuby staining. Protein bands were submitted to trypsin digestion and identified by MALDI-TOF/TOF mass spectrometry. 24 proteins were identified as NS5A binding proteins, including 14 factors not previously reported. Among them, we functionally characterized the interaction of NS5A with the kinase protein Mob1b, an important regulator of the Hippo pathway, and the mitochondrial protein LRPPRC. Here we show that individually knockdown of Mob1b and LRPPRC significantly reduces new HCV particle production and, to lesser extent, intracellular HCV RNA replication. We also found that Mob1b relocalizes to NS5A-positive structures and Mob1b downregulation results in a decrease in NS5A phosphorylation. Finally, we show that the Hippo signaling pathway is perturbed in HCV replicon cells, as indicated by the increased nuclear localization of the Mob1b target transcription factor Yap. Furthermore we observed that NS5A interaction with LRPPRC occurs at mitochondria-ER synapses (MAM) and that LRPPRC downregulation causes a significant increase in different the inflammation-related genes: IFNβ, Mx1, IL 6 and TNFα. Altogether, our results indicate that, through NS5A interactions, HCV hijacks the cellular functions of Mob1b and LRPPRC to ensure its life cycle.

Autosomal recessive leukodystrophy with ataxia and deafness (AR-LAD) is a rare white matter disease that slowly develops cerebellar ataxia and deafness. As disease progresses, the patients develop a fatal cardiomyophathy that strongly recalls that observed in patients affected by laminopathies, a group of disorders caused by mutations in A-type lamins (Lamin A/C), the major components of nuclear lamina. Linkage analysis and positional cloning experiments allowed us to identify the causative gene as RNF220 and two different homozygous missense mutations in highly conserved residues in exon 8 (c.1088G>A and c.1094G>A) (Dr. Bertini, unpublished data). RNF220 gene encodes for a RING finger ubiquitin ligase with a predicted molecular weight of 62.7 kDa highly expressed in liver, kidney and brain. Ubiquitin ligases catalyze the covalent attachment of ubiquitin to the substrate and play a key role in the ubiquitin-mediated proteolysis, however to date the role of RNF220 and the effects of pathological mutations associated with AR-LAD still need to be demonstrated. Our study has been focused on the functional characterization of RNF220, particularly we proposed to: 1) study the expression and subcellular distribution of RNF220, 2) verify if RNF220 may be involved in protein degradation via UPS and eventually determinate the spatial relationship between RNF220 and the different compartments associated with the proteasome, 3) investigate the involvement of RNF220 in the maintenance of nuclear lamina integrity, 4) investigate the possible interaction of RNF220 with molecules involved in the autophagic pathway. The results of this study have shown that: 1) RNF220 is an ubiquitin ligase with a nuclear distribution, 2) RNF220 co-localized with the 20S subunit, the catalytic core of the 26S proteasome, in nuclear speckles where is likely involved in protein degradation via the ubiquitin system, 3) RNF220 is a key player in the maintenance of nuclear integrity as shown by alteration/delocalization of Lamin A/C in AR-LAD fibroblast and in COS-1 cells ectopically expressing RNF220, 3) RNF220 forms a complex with Ambra1, a novel autophagic molecule, and co-localized with it in the nucleus, suggesting a RNF220 implication in autophagic pathway. Taken together, our results suggest that RNF220, the defective protein of AR-LAD is a key player of a novel degradation complex at nuclear level, connecting autophagic and proteosomal pathways and playing a crucial role in maintaining nuclear integrity.

La transglutaminasi 3 è una proteina solubile che appartiene a un’importante famiglia di enzimi dipendenti dal Ca2+, che catalizzano diversi tipi di modificazioni post-traduzionali. La più studiata e nota reazione catalizzate dalle transglutaminasi è il legame crociato tra proteine. La generazione di legami covalenti ε(γ-glutamil)lisina tra polipeptidi adiacenti oppure intracatena, permette la stabilizzazione di complessi proteici durante vari processi cellulari. La famiglia delle transglutaminasi include nove membri, di cui tre (TG1, TG3 e TG5) sono espressi nella pelle durante il differenziamento dei cheratinociti. Questo processo è noto come cornificazione e rappresenta un modello esclusivo di differenziamento e morte cellulare programmata. Durante la cornificazione, la TG3 è attivata tramite proteolisi, che le permette di cooperare con la TG1 nell’assemblaggio e il rinforzo dello strato corneo. La TG3 catalizza la formazione di legami crociati di diverse proteine strutturali, tra cui la loricrina, le small proline-rich proteins e la tricoialina. Nel nostro lavoro, abbiamo caratterizzato un nuovo modello di topo knockout per TGM3 (TG3KO), il gene codificante la TG3. Nonostante i topi non esprimenti la TG3 presentino un fenotipo apparentemente normale, abbiamo scoperto che nella loro epidermide c’è un’alterazione dell’espressione dei marcatori tardivi del differenziamento cheratinocitico. Avendo anche dimostrato che i corneociti estratti dall’epidermide dei topi TG3KO sono più suscettibili alla disintegrazione cellulare con ultrasuoni (sonicazione) rispetto ai WT, abbiamo ipotizzato che i topi TG3KO potessero essere più sensibili all’apoptosi indotta da irradiazione UVB, per via di una ridotta capacità filtrante dello strato corneo. Con i nostri esperimenti, abbiamo quindi provato che I’assenza della TG3 causa una maggiore formazione di dimeri di pirimidine a seguito dell’irradiazione con UVB di topi neonati di 5 giorni di età. L’esteso danno al DNA fa sì che, di conseguenza, nell’epidermide dei topi TG3KO sia presente anche un maggior numero di cellule apoptotiche. Tali dati sono stati confermati dal rilevamento di un cospicuo livello di caspasi 3 attiva e di cellule TUNEL-positive nell’epidermide dei topi TG3KO rispetto ai WT. In conclusione, i nostri risultati indicano che il legame crociato dei precursori dell’involucro corneo mediato dalla TG3 contribuisce in maniera importante alla funzione protettiva dello strato corneo dagli UVB. Questa nuova evidenza potrebbe anche spiegare il motivo per il quale la TG3 è inclusa tra i fattori prognostici e i candidati soppressori tumorali dei tumori umani della testa e del collo.
Transglutaminase (TG3) is a soluble protein that belongs to an important family of Ca2+-dependent enzymes responsible for protein cross-linking. Transglutaminases stabilize protein assemblies by catalysing the formation of intra- or intermolecular Nε(γglutamyl)lysine bonds between adjacent polypeptides. TGs family includes nine members, among them three (TG1, TG3 and TG5) expressed in the skin during keratinocyte differentiation. This process is an exclusive model of terminal differentiation and programmed cell death, also known as cornification. During cornification, TG3 undergoes to proteolytic activation resulting in high cross-linking activity enzyme. It cooperates with TG1 for the assembly and the reinforcement of the stratum corneum, by catalysing the cross-linking of several structural proteins, including loricrin, small proline-rich proteins, and trichohyalin. In our study, we characterized a novel TG3-deficient mouse. TG3-depleted mice have normal gross morphology, but we identified an altered expression of the late differentiation markers in the TG3KO epidermis. Since CEs isolated from TG3KO epidermis are more susceptible to sonication than WT, we hypothesized that TG3KO mice could be more sensitive to apoptosis induced by UVB than WT mice, due to the decreased UVBfiltering capacity of the stratum corneum. We found that the skin of TG3-deficient mice is high sensitive to the formation of cyclobutane pyrimidine dimers after UVB irradiation of new-born mice skin, leading to increased level of UVB-induced cell death. This data have been confirmed by a stronger activation of the caspase 3 and a higher amount of TUNEL-positive cells in irradiated TG3KO skin. Our results indicate that TG3 strongly contributes to the protective function of the stratum corneum from UVB, by reinforcing CEs adding specific crosslinks. This novel finding could explain the reason for including TG3 among candidate tumour suppressor genes in human head and neck cancers.

The heat-shock response, a fundamental defense mechanism against proteotoxic stress, is regulated by a family of heat shock transcription factors (HSFs). In humans HSF1 is considered the central regulator of heat-induced transcriptional responses. The main targets for heat shock factor-1 (HSF1) are specific promoter elements (HSE) located upstream of heat-shock genes encoding cytoprotective heat-shock proteins (HSP) with chaperone function. In addition to its cytoprotective function, HSF1 was recently hypothesized to play a more complex role in human cells. The zinc-finger AN1-type domain 2a (ZFAND2A) gene, also known as AIRAP, was recently identified as a novel human canonical heat shock gene, strictly controlled by HSF1. Little is known about AIRAP gene regulation in human cells. In this study we demonstrate that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, potently induces AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated proteins accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of heat shock factor-2 (HSF2), a factor implicated in differentiation and development regulation. We demonstrated that, differently from heat-mediated induction, in endothelial cells AIRAP expression is regulated at the transcriptional level by a mechanism involving both HSF1 and HSF2 transcription factors, via the formation of HSF1/HSF2 heterotrimeric complexes. These complexes were recruited to a specific heat shock element in the AIRAP promoter. Furthermore we show that, while HSF1 is critical for AIRAP gene transcription, HSF2 negatively regulates AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor during proteotoxic stress. AIRAP function is still not defined; however, the fact that AIRAP is abundantly expressed in primary human and cancer cells at bortezomib concentrations comparable to plasma-levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment.

Mitochondria play a pivotal role in controlling cellular homeostasis. The mitochondrial respiratory chain is severely compromised in different human mitochondrial-associated diseases (HMAD), resulting in oxidative stress and reduced ATP production, ultimately leading to degeneration of affected tissues. Similarly, in the nematode Caenorhabditis elegans, severe mitochondrial disruption leads to deleterious effects such as developmental arrest or lethality. On the other hand partial mitochondrial disruption in different species, including C. elegans, prolongs lifespan. Different and distinct reproducible phenotypes are therefore associated in the nematode C. elegans with different levels of mitochondrial stress and were used in the first part of my study as a readout for an RNAinterference screening aimed at developing different HMAD models, which mimic different phases of the disease progression. I then systematically characterized some of the new models with a special emphasis on neuronal deficits evaluation. Investigating the molecular mechanisms underlying the transition in animal phenotypes (from mild to severe mitochondrial stress), and in particular the characterization of the adaptive beneficial pathways extending lifespan in response to mild mitochondrial stress, may lead to the identification of genes likely relevant for the prevention or delay of neurodegenerative or age-related diseases associated with progressive mitochondrial deterioration. With this aim in mind, in the second part of my thesis I assessed neuronal structure and functions during animal aging and investigated the involvement of specific neuronal genes (chemosensory-related genes and globins) in lifespan specification, upon mild mitochondrial stress. I demonstrated that a moderate mitochondrial alteration leads to increase animal’s lifespan through neuronal hormesis. Finally, taking advantage of an automated microscopy platform (the Cellomics ArrayScan VTI HCS Reader) coupled with the typical phenotypic readouts observed in C. elegans in response to different level of mitochondrial stress, I optimized and validated the conditions to carry out an in vivo, high-content screening (HCS) aimed at identifying interventions inducing beneficial mitochondrial stress responses. This platform will offer the double opportunity to screen for potential HMADs therapeutics as well as for general anti-aging drugs

Copper (Cu) is a redox transition metal existing in two ionic forms, Cu+ and Cu++. In biological systems the electrochemical properties of copper ions make it an important cofactor for many enzymes that catalyze a wide range of biochemical processes such as oxidative phosphorylation, antioxidant defense, catecholamine synthesis, iron homeostasis and connective tissue cross-linking. However, the same properties if uncontrolled, may damage biological systems through the formation of reactive oxygen species that can adversely modify proteins, lipid and nucleic acids thus affecting the overall cell physiology including the function of organelles like mitochondria (Jomova and Valko, 2011). To maintain the control on copper homeostasis, biological systems have evolved a class of proteins called copper chaperones characterized by the presence of one or more copper binding domains (CXXC) (Palumaa, 2013). Copper enters the cells by the high affinity Copper transporter 1 (Ctr1); it is a plasmamembrane pore, responsible for the uptake of at least 80% of copper. Copper itself modulates Ctr1 abundance: elevated intracellular copper levels stimulate endocytosis and degradation of Ctr1; moreover copper regulates Ctr1 expression through Sp1 (Specific protein 1). This mechanism may prevent the accumulation of potentially toxic levels of copper (Wang et al, 2011). Intracellular copper levels are also controlled by the activity of ATP7A pump, copper chaperone devoted to the metallation of Cu-dependent enzymes of the secretory pathway and to the efflux of excess Cu from the cell (Palumaa, 2013). It was shown that ATP7A activity is affected by glutaredoxin 1 (Grx1) a protein belonging to the thioredoxin family, which catalyzes protein deglutathionilation by interacting with the Cu-binding domains in the presence of the metal thus promoting their function (Lim et al., 2006; Singleton et al., 2010). Grx1 has a mitochondrial homologue, glutaredoxin 2 (Grx2), which is the first [Fe-S] Grx discovered; it has a modification in the active site (Ser for Pro) that allows the enzyme to complex a redox inactive [2Fe-2S]2+ cluster that bridge two Grx2 molecules to form a dimeric holo Grx2 complex. In this condition the enzyme is inactive; the increase of intracellular oxidative stress determines the disruption of the complex and the subsequent activation of Grx2. The present study is aimed at further investigating the effect of Grx1 on copper metabolism, in an experimental model represented by SH-SY5Y human neuroblastoma cell line constitutively over-expressing Grx1 (SHGrx1 cells). In the first part of my work, I focused my attention on the intracellular Cu content of SH-SY5Y and SH-Grx1 cells, under basal conditions and after CuSO4 treatment (50 or 150 μM, for 24 hours). The SH-Grx1 cells show a higher copper content than control cells, particularly after copper overload. In order to understand this issue, expression level of Ctr1 has been measured, showing that it is higher in SH-Grx1 cells. This effect is accompanied by a higher expression of Sp1 transcription factor. However, SH-Grx1 cells seem to be more resistant to copper induced cell toxicity. CCS (the cytosolic copper chaperone), which is expected to be degraded in copper loaded cells (Bertinato and L’Abbè, 2003), decreases more in SHGrx1 than in SH-SY5Y cells. SOD1 activity doesn’t change between the two cell lines. It has been demonstrated that upon copper exposure, mitochondrial copper overload occurs, accompanied by mitochondrial damage (Arciello et al., 2005). By comparing the two cell lines, under basal conditions, I observed that the mitochondrial copper content is comparable; however, after copper exposure, it increases more in the SH-SY5Y cells than in SHGrx1, suggesting that Grx1 restrains copper translocation to mitochondria, possibly sequestering the metal in the cytosol, thus protecting cells from copper-induced toxicity. To investigate this issue, I used ImmobilizedMetal-Affinity-Chromatography to select Cu-binding proteins from SHGrx1 total cell lysates and by Western blot analysis I identified Grx1, in the fraction eluting at 40 mM imidazole. Eventually, I measured Grx1 activity in SH-Grx1 cells, after treatment with 150 μM CuSO4 for 24 hours, and I found that Grx1 activity seems to be lower than under basal conditions. Given these results, I hypothesize that copper binds to the cysteines localized in the catalytic domain of Grx1, affecting its activity. In the second part of my work, I analyzed the effect of Grx2 overexpression on copper metabolism in SH-SY5Y cells, with particular focus to mitochondrial functionality. These cells seem to be more susceptible to copper induced toxicity and accumulate more copper than control cells, but at a lesser extent than SH-Grx1 cells. In fact, the increase of intracellular copper content observed in SH-Grx2 cells treated with CuSO4 (150 µM) is not sufficient to induce CCS degradation. Despite SH-Grx2 cells are more sensible to copper overload, they accumulate less copper in mitochondria than control cells when exposed to exogenous copper. This data needs to be further investigated in order to understand the mechanism underlyng this phenomenon. I observed that the protein level of COX17, a mitochondrial copper chaperone involved in copper transfer to cytochrome c oxidase (COX), is lower in mitochondria of SH-Grx2 cells; this is accompanied by a reduction of COX enzyme activity and protein content. In fact, it is known from literature, that COX17 downregulation affects COX assembly and so its enzyme activity (Oswald et al., 2009). However, this aspect must be examine in depth in order to clarify how Grx2 may modulate COX17 protein level and COX activity. The last part of my work focused to study the effect of sodium selenite, used in cancer theraphy, on SH-SY5Y neuroblastoma cells. It was demonstrated that sodium selenite induces Grx1 transcription, translation and activity (Wallenberg et al, 2010). Interestingly, selenite was shown to revert the acquired resistance to cisplatin, another well-known chemotherapic agent, used against a wide variety of solid tumors (Siddik et al, 2006). Cisplatin is known to enter the cells via Ctr1 (Ishida et al, 2002) and lower Ctr1 levels have been reported to be associated with innate or acquired cisplatin resistance (Lin et al., 2002; Holzer et al., 2004b, 2006a; Larson et al., 2009). Therefore, experiments were planned in order to understand whether sodium selenite treatment of SH-SY5Y cells, by increasing the level of Grx1; may lead to the Sp1-mediated induction of Ctr1 level. The results obtained support this hypothesis and may thus represent an important base for further investigations.

La terapia antiossidante rappresenta una nuova frontiera per prevenire e curare una serie impressionante di gravi malattie umane, e la ricerca di farmaci antiossidanti adeguata è in aumento. Le nanoparticelle di ossido di cerio (nanoceria) sono considerate redox-attive perchè presentano la coesistenza di due stati di ossidazione, Ce3 + e Ce4+, e i difetti Ce3+, compensati dalle vacanze di ossigeno, sono più abbondanti in superficie. La nanoceria esercita eccezionali effetti antiossidanti in vivo, poichè agisce come un antisenescenza e anti-infiammatorio ben tollerato, e può essere considerata potenzialmente uno strumento terapeutico innovativo. Tuttavia, il meccanismo antiossidante che genera questo effetto biologico è ancora poco chiaro. In questa tesi di dottorato, è stata effettuata una analisi su due linee cellulari di leucociti, indotti in apoptosi attraverso meccanismi redox-dipendenti o indipendenti; questo studio ha consentito di dimostrare una relazione diretta di causa-effetto tra l’effetto antiossidante e prosopravvivenza della nanoceria. La nanoceria drogata con samario, che diminuisce la quantità di Ce3+ e aumenta il contenuto di vacanze dell’ossigeno, riduce questi effetti, a dimostrazione che le reazioni redox tra Ce3+ / Ce4+ sono responsabili dell'eccezionale proprietà in vivo della nanoceria.
Antioxidant therapy is the novel frontier to prevent and treat an impressive series of severe human diseases, and the search for adequate antioxidant drugs is fervent. Cerium oxide nanoparticles (nanoceria) are redox-active owing to the co-existence of Ce3+ and Ce4+ oxidation states and to the fact that Ce3+ defects, and the compensating oxygen vacancies, are more abundant at the surface. Nanoceria exert outstanding antioxidant effects in vivo acting as well tolerated anti-age and anti-inflammatory agents, potentially being innovative therapeutic tools. However, the biological antioxidant mechanisms are still unclear. Here, the analysis on two leukocyte cell lines undergoing apoptosis via redox-dependent or independent mechanisms, allowed demonstrating a direct cause-effect relationship between the cell anti-radical and pro-survival effects of nanoceria. Sm doping, which decreases Ce+3 and increases oxygen vacancy contents, blunts these effects, demonstrating that Ce+3/Ce+4 redox reactions are responsible for the outstanding in vivo properties of nanoceria.

Il Morbo di Parkinson (PD) è una patologia degenerativa del Sistema Nervoso Centrale caratterizzata dalla progressiva perdita dei neuroni dopaminergici del pathway nigrostriatale. Sebbene finora siano stati condotti diversi studi riguardo il PD, la sua eziologia è ancora poco nota. Le forme familiari o ereditarie infatti, rappresentano soltanto il 5-10 % dei casi di PD, mentre il restante 95% è rappresentato dale forme idiopatiche o sporadiche,. Per tale ragione il PD viene considerata una malattia multifattoriale dove l’esposizione a fattori ambientali come pesticidi altri inquinanti gioca un ruolo fondamentale nell’insorgenza della malattia. E’ stato ormai ampiamente dimostrato che lo stress ossidativo e la disfunzione mitocondriale siano fortemente coinvolte nella morte neuronale nel PD. Inoltre, difetti nella rimozione delle proteine danneggiate e/o organelli, dovuto probabilmente a disfunzioni a carico del sistema ubiquitina-proteasoma e dell’autofagia, rappresentano un ulteriore evento coinvolto nell’eziopatogenesi del PD. In particolare, nei cervelli PD sono state osservate alterazioni del processo autofagico e in accordo con tali evidenze è stato recentemente dimostrato che il trattamento con la rapamicina, un noto induttore di autofagia, risulta protettivo contro tossine che causano il PD e che l’inibizione dell’autofagia basale è in grado di causare degenerazione nei topi. Diversi approcci terapeutici sono stati sviluppati finora per rallentare la sintomatologia del PD ma nessuno di questi trattamenti messi a punto è in grado di prevenire o curare la degenerazione neuronale. Negli ultimi anni l’attenzione di molti ricercatori si è focalizzata sullo studio di composti di derivazione naturale, come i polifenoli, in quanto il loro uso farmacologico potrebbe rappresentare un promettente approccio terapeutico/preventivo in patologie correlate allo stress ossidativo. Infatti, in aggiunta alla loro primaria attività antiossidante, è stato dimostrato come tali composti svolgano altre differenti attività biologiche, pertanto il loro utilizzo potrebbe risultare una strategia terapeutica vincente in patologie multifattoriali e complesse come il PD. Sulla base delle nostre conoscenze, questa tesi di Dottorato ha avuto lo scopo di caratterizzare gli effetti neuroprotettivi di un composto di derivazione naturale, il campferolo, in modelli in vitro di PD. Abbiamo dimostrato che il campferolo protegge le cellule SH-SY5Y e i neuroni corticali primari dalla tossicità del rotenone. Inoltre il campferolo è in grado di diminuire significativamente la produzione di specie reattive dell’ossigeno e l accumulo di proteine mitocondriali carbonilate, oltre a preservare la vitalità e la funzionalità della rete mitocondriale. Abbiamo dimostrato che l’evento principale reponsabile degli effetti antiapoptotici e antiossidanti del campferolo è l’induzione dell’aumento del turnover mitocondriale per mezzo dell’autofagia. Infatti analisi in microscopia elettronica e a fluorescenza hanno evidanziato un incremento del tasso di fissione mitocondriale e degli autofagosomi contenenti mitocondri. Inoltre la proteina LC3 (autophagosome-bound microtubule-associated protein light chain-3) aumenta durante il trattamento col campferolo e l’inibizione chimica o genetica del processo autofagico abolisce gli effetti protettivi di tale composto. La protezione mediata dal campferolo è inoltre specifica per altre tossine mitocondriali (1-metil-4-fenilpiridinio, paraquat) usate per riprodurre le tipiche caratteristiche patogenetiche del PD, ma risulta completamente inefficiente contro altri stimuli che non danneggiano il mitocondrio (H2O2, 6-idrossidopamina, staurosporina). L’evidenza inoltre che il campferolo è in grado di preservare l’attività fisiologica dei neuroni striatali in fettine di cervello di ratto, ci suggerisce un protezione più generale di tale composto nel PD. Complessivamente tali evidenze forniscono ulteriori evidenze che il campferolo possa essere identificato come un composto in grado di incrementare il processo autofagico con nuove potenziali applicazioni terapeutiche.
Parkinson Disease (PD) is a neurodegenerative disorder characterized by degenerative loss of neurons in the nigrostriatal dopaminergic pathway. So far, although several studies concerning PD have been performed, its aetiology remain still unclear. Familiar or hereditary forms account only for the 5 - 10% of the PD cases, while the remaining 95%, defined idiopathic or sporadic, rely on environmental factors without any certain genetic involvement. Therefore, PD can be considered a complex multifactorial pathology, where genetic background should be considered a predisposing factor towards environmental insults such as pesticides and chemicals. By now oxidative stress and mitochondrial injury have been extensively indicated to be involved in PD neuronal demise and are tightly related each other. Moreover the unsuccessful removal of damaged proteins and/or organelles, probably due to the impairment of either the ubiquitin-proteasome system or autophagy, is a further event associated with PD aetio-pathogenesis. In particular, autophagy has been observed to be deregulated in PD brains and in line with this evidence, it has been recently demonstrated that rapamycin, a well-known autophagic inducer, protects from PD toxins and that suppression of basal autophagy causes neurodegeneration in mice. Several therapeutical approaches to slow down PD symptoms have been developed but none of them is currently able to prevent neuronal degeneration. In the last few years the attention of many researchers has been focused on natural-occurring compounds, such as polyphenols, because their use has been suggested to be a promising therapeutic/preventing strategy in oxidative-related diseases. In fact, in addition to their primary antioxidant activity, they could display a wide variety of other biological functions and these properties could be helpful in a complex and multifactorial pathology such as PD. On the basis of this knowledge, this PhD thesis is aimed to elucidate the processes underlying neuroprotection of a natural occurring compound kaempferol in in vitro models of PD. We demonstrate that kaempferol protects SH-SH5Y cells and primary cortical neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Kaempferol induced-autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenylpiridinium, paraquat) used to reproduce the typical features of PD, but is inefficient against apoptotic stimuli not directly affecting mitochondria (H2O2, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in PD. Overall, this data provide further evidence for kaempferol to be identified as an autophagic enancher with potential therapeutic capacity.

ΔNp63α è un fattore trascrizionale ampiamente studiato nel cancro alla mammella. Studi recenti indicano che esso è implicato nella tumorigenesi e nell’auto-rinnovamento delle cellule staminali di suddetto cancro. Anche se il profilo trascrizionale di p63 è stato ampiamente caratterizzato, la nostra conoscenza sui suoi possibili interattori implicati nella regolazione del cancro alla mammella è limitata. In questo lavoro abbiamo inizialmente effettuato un saggio di doppio ibrido per cercare dei putativi interattori della proteina che potessero avere un ruolo nella tumorigenicità del cancro al seno. Fra questi, abbiamo identificato Setdb1, una istone lisina metiltrasferasi, che interagisce specificamente con l’isoforma alfa troncata della proteina, ΔNp63α, interazione che contribuisce alla stabilità di p63. Setdb1 risulta essere amplificato nei tumori primari della mammella e la sua assenza rende le cellule tumorali meno prone alla crescita. Abbiamo inoltre trovato trenta geni che risultano aumentare dopo il silenziamento di Setdb1 o p63 (quindi geni che sono repressi da entrambe le proteine) e l’espressione di quattro di essi risulta essere positivamente correlata alla sopravvivenza dei pazienti di cancro al seno. Questi risultati suggeriscono che p63 e Setdb1 potrebbero agire come oncogeni in questo tipo di cancro andando a reprimere dei geni che si comportano da soppressori dei tumori.
ΔNp63α has been widely studied in breast cancer, and recent studies indicate that it is involved in both breast tumorigenesis and self-renewal potential of breast cancer stem cells. Although the p63 transcriptional profile has been extensively characterized, our knowledge about p63 binding partners, potentially involved in the regulation of breast tumor progression, is limited. Here, we performed the yeast-twohybrid approach to identify p63α interactors involved in breast tumorigenesis. We found that Setdb1, a histone lysine methyltransferase, interacts with ΔNp63α and that this interaction contributes to p63 protein stability. Setdb1 is often amplified in primary breast tumours and its depletion confers growth disadvantage to breast cancer cells. Also, we identified a list of thirty genes repressed by ΔNp63 in a Setdb1dependent manner and the expression of four of them is positively correlated to survival of breast cancer patients. These results suggest that p63 and Setdb1 may have diagnostic and prognostic potential via the repression of common target genes.

Neuroectoderm is one of the earliest cell lineages that establish at gastrulation. Neuroectodermal tumors arise from cells originating from the primitive neuroectoderm, which include glial cells, neurons, neural crest cells, parenchymal cells of the pineal gland and primitive embryonic cells of brain and retina. Both melanocytes and glial cells are derived embryologically from the neuroectoderm. Their malignant transformed counterparts, melanoma and glioma cells, respectively, may share common antigens. Numerous tumor-associated antigens have been identified in melanomas but only a few in gliomas. We undertook a study of how invasiveness of tumor cells can be regulated in gliomas as well as in melanomas, focusing our attention on two genes, Sox2 and PDE5, which have been hypothesized to play a role in melanoma invasiveness. In particular, we modulated PDE5 expression in glioblastoma cell lines and Sox2 in a melanoma animal model to evaluate the invasiveness potential of this tumor cell types in vitro and in vivo.

La Leucemia Mieloide Acuta (AML) è una malattia aggressiva, geneticamente eterogenea, derivante da un accumulo di mutazioni cariotipiche, genetiche o epigenetiche, che portano ad un ridotto differenziamento (classe di mutazioni II, i cui target sono fattori di trascrizione, come PML-RARα) e aumentata capacità di auto-rinnovamento dei progenitori emopoietici (classe di mutazioni I, di solito coinvolgono tirosin chinasi come FLT3-ITD, soppressori tumorali come p53, ed oncogeni come N-Ras). L'intero spettro di anomalie sottostanti la patogenesi di AML non è stato completamente chiarito. Un’ulteriore caratterizzazione di molecole potenzialmente rilevanti e di marcatori prognostici potrebbe migliorare la valutazione dei rischi e le strategie terapeutiche per i pazienti con AML. L'E3 ubiquitina ligasi WWP1 è un potenziale oncogene trovato deregolato in tumori umani solidi e la sua sovra-espressione è stata associata ad un aumento della proliferazione e della sopravvivenza delle cellule tumorali. L'attività oncogenica di WWP1 è data dall'osservazione che un certo numero di suoi bersagli proteici sono molecole importanti per la regolazione della proliferazione cellulare, del differenziamento e dell’apoptosi. E’ quindi probabile che le azioni a favore di sopravvivenza e crescita esercitate da WWP1 in tumori epiteliali solidi derivino dalla regolazione di rilevanti vie di segnalazione, e questi meccanismi potrebbero essere condivisi anche dalle leucemie. Con l'obiettivo di indagare il ruolo di WWP1 nella patogenesi delle leucemie mieloidi, abbiamo valutato il profilo di espressione di WWP1 in una coorte di pazienti con de novo AML, e abbiamo trovato un aumento dei livelli del mRNA di WWP1 nei campioni ottenuti da pazienti affetti da leucemia, rispetto a quelli ottenuti da donatori sani, usati come controllo. È degno di nota il fatto che l'inattivazione di WWP1 in linee cellulari di AML portata ad arresto del ciclo cellulare, diminuzione della vitalità cellulare, e promuove il differenziamento delle cellule leucemiche, indicando che la funzione di WWP1 è rilevante nelle AML. L'utilizzo della linea cellulare NB4 come modello di sistema AML, ha posto particolare attenzione sulla regolazione della proteina di fusione PML-RARa. Sono stati così trovati down-regolati i livelli proteici dell’oncogene a seguito del silenziamento dell’E3 ubiquitina ligasi, con conseguente riattivazione dei geni bersaglio di RARα coinvolti nel differenziamento granulocitico (ad esempio, C/EBPε). Abbiamo così osservato l’induzione di caratteristiche morfologiche e funzionali di differenziamento nelle cellule NB4 silenziate per WWP1, rispetto al gruppo di controllo. In clinica, l'induzione di autofagia come meccanismo terapeutico della clearance di PML-RARα ci ha portato a indagare il possibile coinvolgimento della ligasi nel processo autofagico. Abbiamo così scoperto che la deplezione di WWP1 porta ad aumentato flusso autofagico nelle cellule AML, contribuendo al loro fenotipo di differenziamento. Inoltre, la morte cellulare non-apoptotica osservata nel nostro sistema, ha evidenziato un ruolo importante per l'autofagia nell’eliminazione dei blasti leucemici. p27, inibitore delle chinasi ciclina-dipendente, si è mostrato essere il bersaglio a valle del meccanismo di azione di WWP1: i suoi livelli proteici sono sovraespressi quando WWP1 viene silenziato in tutte le linee cellulari testate, ed essendo coinvolto nella fase G0/G1 del ciclo cellulare, contribuisce all’induzione dell’arresto della crescita osservata nella linea cellulare NB4. Inoltre, esperimenti in vivo hanno mostrato che la sopravvivenza dei topi trapiantati con cellule NB4 trasdotte con sh-WWP1 è stata maggiore rispetto ai topi che hanno ricevuto NB4 trasdotte con sh-Scr, ricapitolando così il fenotipo d’inibizione della crescita delle cellule AML osservato in vitro. Insieme, questi meccanismi puntano ad un effetto biologico di WWP1 nel sostentamento della leucemia, indicando in tal modo possibili bersagli cellulari per nuove molecole terapeutiche.
Acute Myeloid Leukaemia (AML) is a genetically heterogeneous aggressive disorder resulting from accumulation of multiple karyotipic, genetic or epigenetic changes that lead to impaired differentiation (class II mutations, targeting transcriptional factors such as PML-RARα) and increase self-renewal ability (class I mutations, usually targeting tyrosine kinases like FLT3-ITD, tumour suppressors like p53, and oncogenes like N-Ras) of hematopoietic progenitors. The whole spectrum of abnormalities underlying AML pathogenesis has not been fully elucidated. Further characterization of potentially druggable and prognostic relevant molecules might improve riskassessment and therapeutic strategies for AML patients. The E3 ubiquitin ligase WWP1 is a potential oncogene found deregulated in solid human cancers and its overexpression has been associated with increased solid cancer cell proliferation and survival. The oncogenic activity of WWP1 is empathized by the observation that a number of its protein targets are molecules relevant for the regulation of cell proliferation, differentiation and apoptosis. Hence it is likely that pro-survival and growth promoting actions exerted by WWP1 in solid epithelial tumours arise from the regulation of relevant signalling pathways, and these mechanisms could be shared also in leukaemias. With the aim to investigate the role of WWP1 in the pathogenesis of myeloid leukaemogenesis, we have evaluated the expression profile of WWP1 in a cohort of patients with de novo AML, and found increased levels of WWP1 mRNA in the leukemic samples as compared to healthy donor controls. Noteworthy, inactivation of WWP1 in AML cell lines led to cell cycle arrest, decreased cell viability, and promoted differentiation of leukaemia cells, indicating WWP1 to be functionally relevant in AMLs. The use of the NB4 cell lines as an AML model-system, brought the attention on PML-RARα fusion protein regulation upon WWP1 knockdown. We found oncogene down-regulation at protein levels, with subsequent reactivation of the RARα target genes involved in granulocytic differentiation (e.g. C/EBPε). As a result, we observed induction of morphological and functional differentiating features upon WWP1-silencing in the NB4 cells as compared to the control group. The induction of autophagy as therapeutic mechanism of PML-RARα clearance in clinic brought us to investigate the possible involvement of the ligase in autophagy. We indeed found that depletion of WWP1 increased the autophagic flux of AML cells, thus contributing to the differentiation phenotype. Moreover, the non-apoptotic cell death observed in our system, highlighted a role for autophagy for the clearance of AML blasts. The cyclindependent kinase inhibitor p27 resulted our candidate as downstream target of WWP1: it is overexpressed at protein level when WWP1 is silenced in all the AML cell lines tested, and its is involved in G0/G1 cell cycle arrest, contributing to the growth arrest induction observed in NB4 cell line. Moreover, in vivo experiments showed that the survival of the mice transplanted with sh-WWP1 transduced NB4 cells was higher as compared to mice which received sh-Scr transduced NB4 cells, thus recapitulating the growth inhibition phenotype of AML cells seen in vitro. Together, these mechanisms point to unveil WWP1 biological effect in leukaemia sustainment, thus pointing at possible molecular targets for new therapeutic molecules.

Friedreich’s ataxia (FRDA) is a rare debilitating, life-shortening, autosomal recessive inherited disease that leads to progressive damage to the nervous system. Onset is usually around the puberty and patients develop a progressive loss of motor coordination, inability to walk, slurred speech, and a cardiac hypertrophy that often leads to premature death. The particular genetic mutation – expansion of an intronic GAA triplet repeat in the FXN gene – leads to reduced expression of the mitochondrial protein frataxin involved in iron-sulfur cluster biogenesis. The subsequent frataxin insufficiency causes mitochondrial dysfunction and oxidative damage with ultimately cell death, particularly in peripheral sensory ganglia. No therapy to prevent or slow down the progression of the disease has been found yet. Since there is an inverse correlation between the amount of residual frataxin and the severity of disease progression, therapeutic approaches aiming at increasing frataxin levels are expected to improve patients’ conditions. We have recently proven the therapeutic relevance of increasing frataxin levels by preventing its degradation. Indeed, we have recently shown that a significant amount of frataxin precursor is degraded by the ubiquitin-proteasome system before its functional mitochondrial maturation and we have described the therapeutic potential of small molecules that promote frataxin accumulation by docking on the frataxin ubiquitination site, thus interfering with its ubiquitination and degradation. In light of these data, inhibition of frataxin E3 ubiquitin ligase, the enzyme responsible for frataxin ubiquitination, could represent another attractive strategy to prevent frataxin degradation. We therefore pursued the identification of such enzyme by performing a functional screening of an E3 ubiquitin ligase small interfering RNA library. HIT2 was identified and validated as a candidate from this screening. Consistently, knockdown of HIT2 promotes frataxin accumulation in cells. Most importantly, silencing of this candidate gene results in frataxin accumulation also in cells derived from FRDA patients, suggesting the therapeutic potential of strategies aimed at inhibiting this E3 enzyme. Additionally, we demonstrated that HIT2 directly interacts with frataxin and its overexpression increased frataxin protein ubiquitination in a catalytic activity-dependent manner, both in cells and in in vitro assay, indicating that this enzyme may actually represent the frataxin E3 ubiquitin ligase. These findings suggest that HIT2 could be a novel important therapeutic target for Friedreich’s ataxia.

Antimicrobial materials are essential in many medical applications. In the recent years a lot of research has been dedicated to nitric oxide-releasing nanomaterials and in particular mesoporous particles in order to improve the storage and delivery performances of nitric oxide carriers for a wide range of applications due to their high surface area-to-volume ratio and easy penetration. In this report the principles underlying in this process are first reported with particular attention towards the mesoporous silica nanoparticles. Next, the process dependence on such parameters as size, shape, and porosity of the silica nanoparticles, and then the surface functionalization with NO donors were also taken into consideration. Porous scaffolds hold great potential for wound healing and skin tissue engineering. Over the past couple of decades, nanoparticles (e.g. silica, gold, and silver nanoparticles) have been extensively explored in wound-healing applications as efficient antimicrobial agents and the use of these nanoparticles has raised concerns. Therefore, there is a real need for the development of scaffolds with controlled release of different antimicrobial and anti-inflammatory agents such as nitric oxide. In this project, we investigate the efficacy of silica nanoparticle in releasing the nitric oxide in the effective concentration for anti-bacterial effects. It is supposed that silica nanoparticles as biocompatible scaffolds, enable the incorporation of nitric oxide with programmable release. This approach will allow the creation of customised antimicrobial structures for a broad range of tissue engineering applications, with particular emphasis in wound-healing applications. The incorporation of a uniform, continuous layers of nanoparticles/antimicrobial (NO@Si) agents will be verified by FTIR analysis. Scanning Electron microscopy will be used to investigate morphological features of the particles. The antimicrobial efficacy of the antimicrobial scaffolds against a range of gram positive and gram negative bacterial (e.g. Staphylococcus aureus and Pseudomonas Aeruginosa) will be determined on broad range of mentioned bacteria isolates. Cytotoxicity analyses of the antimicrobial scaffolds toward dermal fibroblasts will be performed using MTT assay of cell viability and proliferation.

Oggi l’infarto del miocardio, e la conseguente perdita della piena funzionalità del tessuto cardiaco, rappresenta la maggiore eziologia per lo scompenso cardiaco. Nonostante la terapia primaria aggressiva, la prognosi rimane grave in pazienti con infarto e grave disfunzione ventricolare sinistra. Sarebbe quindi altamente desiderabile poter influenzare la guarigione della lesione cardiaca per mantenere la struttura e la funzione del cuore. Il Fattore XIII (FXIII) viene attivato dalla trombina nella fase finale della cascata della coagulazione ed ha un ruolo prominente nel crosslinking della fibrina solubile verso un coagulo insolubile stabile. Evidenze sperimentali in modelli murini suggeriscono che il fattore XIII potrebbe svolgere un ruolo chiave nella guarigione dopo l’infarto miocardico. Per quantificare il reale contributo del FXIII in questo processo, e per esplorare il suo possibile ruolo prognostico, abbiamo monitorato i livelli FXIII-A in 350 pazienti con infarto miocardico acuto durante i primi sei giorni (D0-D5) più un controllo a 30-60 giorni (D30). Ad un anno il follow-up è stato effettuato per tutti i pazienti. Qui confrontiamo i livelli di FXIII a quelli dei marcatori cardiaci standard per prevedere il danno causato dall’infarto del miocardio e il relativo outcome clinico, ma anche per valutare il potenziale terapeutico di questa molecola. Il risultato principale della nostra ricerca traslazionale è stato quello di attribuire una prognosi più infausta per i pazienti con un più alto consumo di FXIII nei primi giorni post-IMA rispetto al sottogruppo di pazienti in cui il consumo di FXIII (valutato come livelli residui dello stesso) è stato trascurabile. Questa interessante osservazione è indipendente dalla quantità di Troponin_T e CK-MB, attribuendo così al FXIII un ruolo di fattore prognostico indipendente per l’infarto. Inoltre, varianti comuni del gene FXIII (Val34Leu, Pro564Leu, Tyr204Phe, Val650Ile, Glu651Gln) influenzano in modo significativo l'attività molecolare. Abbiamo così analizzato tutte le varianti citate coinvolte nel processo di guarigione del muscolo cardiaco e nel determinare l'estensione del danno, fattori che sono direttamente collegati all’insufficienza cardiaca e, di conseguenza, alla sopravvivenza. Diversi livelli e dinamiche del FXIII-A potrebbero essere utilizzati come i primi indicatori prognostici durante l’infarto miocardico acuto, assieme ai marcatori tradizionali di ischemia eseguiti di routine, nei pazienti con sospetta o conclamata insufficienza cardiaca in modo da da personalizzare il trattamento. Inoltre potrebbero rappresentare un metodo alternativo per studi su biomarcatori finalizzati a una più pratica valutazione della progressione di patologia.
Today, myocardial infarction and the consequent loss of fully functional myocardium is the major aetiology for heart failure. Despite aggressive primary therapy, prognosis remains serious in patients with large infarction and severe left ventricular dysfunction. Thus, it would be highly desirable to influence healing of the cardiac wound to maintain structure and function of the heart. Factor XIII (FXIII) is activated by thrombin in the final step of the clotting cascade coagulation and it has a prominent role in cross-linking soluble fibrin to a stable insoluble clot. Experimental evidence in mouse models suggests that Factor XIII might play a key role in myocardial healing after infarction. To quantify the real contribution of FXIII in this process, and to explore its possible prognostic role, we monitored the FXIII-A subunit levels in 350 acute MI patients during the first six days (d0-d5) plus a control at 30-60 days (d30). A one-year follow-up was performed for all the patients. Here, we compare FXIII levels to those of standard cardiac markers to predict the damage due to the infarction and the clinical outcome, but also to evaluate the therapeutic potential of this molecule. The main result of our translational research was to attribute a poor prognosis for patients with higher consumption of FXIII in the early days post-MI when compared with the subgroup of patients in whom FXIII consumption (rated as residual levels of the same) was negligible. This interesting observation is independent of the amount of Troponin_T and CK-MB, thus attributing to FXIII a role as an independent prognostic factor in heart attacks. Furthermore, common FXIII gene variants (Val34Leu, Pro564Leu, Tyr204Phe, Val650Ile, Glu651Gln) significantly influence molecular activity. So, in addition, we have analyzed all mentioned variants involved in the healing process of the heart muscle and in determining the extention of the damage, factors that are with directly related to heart failure and, consequently, to survival. Different FXIII-A dynamics and levels could be utilized as early prognostic indicators during acute MI, toghether with traditional markers of ischemia routinely performed, in patients with suspected or overt heart failure to personalize treatment. Furthermore, they could be an alternative method for biomarker studies aimed to more practical assesment of progression.

Chemoreception is the process that allows animals to respond to the chemical stimuli in their environment. In insects, this is mediated by specialized neurons expressing a variety of dedicated receptors and carrier proteins: olfactory (OR), gustatory (GR) and Ionotropic (IR) receptors, odorant binding (OBP) and chemosensory (CSP) proteins. How the evolution of these genes correlates with adaption to new ecological niches is still a debated topic; when these genes arose during arthropods evolution is also an unresolved question. To tackle the first of these issues I have studied these gene families in Drosophila suzukii Matsumura (Diptera: Drosophilidae), an invasive pest that, unlike other Drosophila, oviposits in fresh fruits. I have initially contributed in curating D. suzukii’s genome and transcriptome, and then annotated its entire repertoire of chemosensory genes. Analysis of these genes on a 14 Drosophila phylogenetic framework revealed that ORs, OBPs, and GRs are characterized by high turnover rates and uneven distribution of duplication and gene loss events on the phylogeny: these peculiar evolutionary patterns are consistent with a change in selective pressures. D. suzukii is characterised by loss of function of key ORs that bind volatiles typically released by fermenting substrates, providing with a rare example of ecological adaptation due to genes loss. I further present my work in annotating and studying genes involved in the metabolism and perception of an aggregating pheromone that may play an active role in D. suzukii’s peculiar biology. To inquire the origin of chemosensory genes in insects and other arthropods, I have screened various recently released genomes: I identified multiple lineage-specific expansions of GRs in each of the arthropod clades, an interesting loss of GRs in most crustaceans, and the first genomic evidence of ORs in the Palaeoptera. My postgraduate work has contributed in identifying receptors and ligands that will be used in downstream D. suzukii control applications, and has shed some light in the evolution of chemosensory genes in arthropods.

The objective of an effective therapy against cancer is to destroy every single cancerous cell, including cancer stem cells, in order to allow the survival of the patient. Some anti-cancer therapies tend to “educate” the host’s immune system to recognize the remaining tumour cells, in order to minimize the risk of relapse. Chemotherapy and immunotherapy are often hardly compatible. In fact, most of the chemotherapeutic agents induce DNA damage, in order to trigger apoptosis of the cancer cells. These treatments often result into the induction of massive immune system effector’s depletion. Etoposide and mitomycin C, two widely used drugs, induce non-immunogenic cell death while, other compounds, such as anthracyclines, are able to induce immunogenic cell death. In addition, there are reports suggesting that anthracyclines are able to induce immunogenic apoptosis, have a direct effect on cancer and also they are able to mediate the immune response. It has been demonstrated that those compounds are able to stimulate the exposure of Calreticulin (CRT) on the plasma membrane of tumour cells in a pre-apoptotic step. One of the peculiar biochemical aspects of the immunogenic cell death is this pre-apoptotic translocation of CRT from the Endoplasmic reticulum (endo-CRT) to the cell surface (ecto-CRT). Calreticulin is a Ca2+-binding molecular chaperone expressed in the endoplasmic reticulum, where it takes part in calcium homeostasis. It can be found also in other cellular compartments, such as the nucleus and the plasma membrane, and it is a multi functional protein able to interact with the α-subunit of integrins and with proteins involved in the ER-stress response, such as PDI and ERp57. In addition, it has been shown that CRT expression on the surface of damaged cells might function as an “eat-me” signal, which elicits cellular recognition and removal by macrophages or dendritic cell. Type 2 Transglutaminase (TG2) belongs to a family of Ca2+-dependent enzymes capable of covalently modifying proteins by cross-linking them via the formation of ε(γ-glutamyl)lysine bonds. TG2 is a peculiar member of the family, which could be externalized on the cell surface and thus it mediates the interaction of integrins with fibronectin and cross-links proteins of the extra-cellular matrix. On the basis of its sub-cellular localisation TG2 may also act as a protein disulphide isomerase (PDI) at mitochondrial level or as a G-protein at plasma membrane level. In fact, it has been shown that α-subunit of the etherotrimeric G-proteins is TG2. Interestingly, it has been have shown that the β-subunit of these G-proteins is CRT. CRT interacts with TG2, when TG2 bounds GDP. We hypothesised that TG2-CRT interaction might modulate not only their intra-cellular activities but also their relationships with the plasma-membrane and, possibly, CRT exposition on cell surface. In order to address our hypothesis we assessed the presence of CRT on the plasma membrane of the human neuroblastoma cell line Sk-n-BE(2), which does not express detectable levels of TG2, in respect to the TGA cell line, transfected to achieve high TG2 expression levels. We used different approaches, such as flow cytometry, surface protein purification, to analyse cell surface exposition of CRT in these two cell lines. Our results indicate that, at steady state, the SK-n-BE(2) cell line express about 2 fold more CRT on cell surface, as compared to TGA cells, thus suggesting a role for TG2 in CRT exposure. In addition, we showed that pre-treatment of the cells with cystamnine, a pan inhibitor of TG2 transamidasic activity, lowered the amount of cell surface exposed CRT. On the basis of these results we hypothesised that TG2, when bound to GDP and acting as a G-protein, might bind CRT and prevent its translocation on cell surface. TG2-dependent modulation of CRT translocation is detectable even upon anthracyclines treatment. In fact, when we analysed the presence of CRT on cell surface after doxorubicin treatment, we spotted the same differences detected at steady state. This aspect might be of relevance, as doxorubicin not only induces high level of CRT exposure but also has been show to induce apoptosis and that TG2 might be involved in the modulation of this process (Fesus and Szondy, 2005). It is know that anthracyclines induce ER-stress. On the basis of this evidence we would like to assess whether the various TG2’s biological activities could be involved in cell death regulation cancer. During the last years, many groups demonstrated that TG2 can not only carry out different activities in relation to its sub-cellular localisation (Jones et al., 1997; Malorni et al., 2009; Rodolfo et al., 2004; Siegel et al., 2008) but also that different localisation of the enzyme may result in both pro-survival or pro-death action (Fesus and Szondy, 2005). We previously showed that TG2 may localise on mitochondria, where it’s involved in the maintenance of the organelle’s homeostasis and in the regulation of the mitochondrial pathway of apoptosis. Mitochondria have also a prominent role in the decoying and integration of cellular stress signals, such as DNA damage and ER-stress, and anthracycline, like other chemotherapeutic drugs, are not only DNA damaging agents but also powerful induces of ER-stress. This side of the picture might be very relevant to our project as CRT is an ER resident protein, involved in the activation of the ER-stress response. In addition, during metastasis formation, tumour’s cells are subjected to high levels of stress that involves both ER and mitochondria. We then decided to investigate the possible involvement of TG2 in the overall regulation of ER-stress induced cell death, in our neuroblastoma cell model, by treating cells with two different compounds: tunicamycin and thapsigargin. The choice of these drugs has been dictated by their different modes of action. In fact thapsigargin, is a specific inhibitor of SERCA ATPase (Sarco/endoplasmic reticulum Ca2+ ATPase) and causes the depletion of Ca2+ stores from the endoplasmic reticulum and the increase of the cytosolic Ca2+ concentration. Tunicamycin is an inhibitor of one of major post-translational modifications that take place in the endoplasmic reticulum, the protein’s glycosylation process. We started our investigation by performing time and dose response curve, for the two drugs, and analysing cell death induction by means of flow cytometry. After treatment with 4 and 8 µg/ml of tunicamycin for 24 and 48h, the TGA cell line seems to be less sensitive to cell death induction as compared to the SK-n-BE(2) cell line, thus suggesting TG2 over-expression as protective against tunicamycin induced cell death. Treatments were also carried out with thapsigargin at 2 and 4 µg/ml for the same times. After 24h of treatment there are no differences in cell death levels between the two cell lines for both the doses used while, after 48 h of treatment, there is a clear protection of TGA cells at the highest dose we used. These data suggest TG2 over-expression as protective against thapsigargin induced cell death, and highlighted a dose-dependent effect. The results obtained with thapsigargin were somehow un-expected because thapsigargin causes an increase in the cytosolic calcium concentration, that we hypothesised to be high enough to activate the TG2 transamidasic activity and then to induce apoptosis. We then decided to investigate whether the two cell lines displayed a different response to the ER-stress. To this aim we performed western blot analysis of the expression of ER-stress induced factors, like GRP78 protein, and of cell death markers, like PARP. The results obtained demonstrated that there were no differences between the two cell lines despite the inducer used. As we do not see any difference in the upstream regulation of cellular response to ER-stress, we decided to investigate whether there was any difference in the induction of cell death. It is well known that the ER-stress dependent induction of apoptosis relies on the activation of the pro-apoptotic members of the Bcl-2 family Bax and Bak, and on their action on mitochondrial and ER membranes. In addition, the Ca2+ release induced by Thapsigargin might have an effect on both TG2 activation and mitochondrial release of cytochrome c. In keeping with these considerations, we decided to analyse what happens in the two cell lines after treatment with 4 µg/ml of thapsigargin for 48 hours, by means of an immuno-fluorescence approach. The results obtained showed that cytochrome c release and caspase-3 activation seem to happen at the same extent in the two cell lines. The only difference that we might spot is on the number of fragmented nuclei we observed, again confirming a protection of the TGA cells. Besides these observation it remains still unclear how TG2 over-expression might protect cells from thapsigargin induced cell death. The data obtained by this kind of approach were purely descriptive and not quantitative, so we decided to investigate these events by means of western blot analysis. To this aim we treated the cells in the same way as before and we performed sub-cellular fractionation in order to obtain cytosolic and mitochondrial fraction. The western blot analysis of PARP cleavage, a marker of cell death, revealed a more extensive processing of this protein in the SK-n-BE(2) cell line, in respect to the TGA. This result indicates a more proneness of these cells to cell death induction after thapsigargin treatment, in keeping with what we previously observed by FACS analysis. The analysis of Bax and Bak activation and translocation as well as of the cytochrome c release, does not revealed, in the two cell lines, such striking differences to justify the less sensitivity of the TGA cell line to cell death. On the contrary, the TGA cell lines showed an even marked translocation of Bax and Bak to the mitochondria, suggesting a massive induction of cell death. These results indicates that, upon thapsigargin treatment, the onset of the ER-stress response and the induction of cell death takes places in a similar way in the two cell lines and they does not justify the minor sensitivity displayed by the TGA cell lines. Recently, in vitro experiments have demonstrated that caspase-3 might be a protein substrate for the cross-linking activity of TG2. Following thapsigargin treatment, the increase of the cytosolic Ca2+ concentration activates TG2, which might act on caspase-3 leading to the formation of polymers with a molecular weight of about 64 kDa. This polymerisation inactivates the caspase and leads to the inhibition of cell death. In order to verify whether this hypothesis was true in our model system, we performed a western blot analysis of caspase-3 activation, after thapsigargin treatment, by means of an antibody able to recognize the processed forms of caspase-3. After 48 hours of treatment with 4 µg/ml of Thapsigargin, we observed the decrease of the 32 kDa signal, corresponding to the pro-caspase, in both cell line at the same extent. When we checked for the appearance of the two active forms of caspase-3 at 17 and 10 kDa, we could detect their appearance only in the SK-n-BE(2) cell line. On the other hand the TGA cell line show the appearance of a faint signal corresponding to the 10 kDa active form of caspase-3, but we detected the appearance of a strong signal at a molecular mass of about 34 kDa, as well as the increase of the signal at about 27 kDa. The molecular mass of these anti-caspase 3 positive bands suggests that they are polymers of the processed caspase-3 forms and that they are dependent on the thapsigargin induced activation of the TG2 cross-linking activity, even if slightly visible also in the SK-n-BE(2) cell line. These data are also supported by the measurement of the TG2 activity we performed upon thapsigargin treatment. In fact, upon thapsigargin treatment, we detected TG2 cross-linking activation in both cell lines even if more pronounced in the TGA cell line. The more evident formation of the caspase-3 oligomers, as well as the quite complete disappearence of the signal corresponding to the 17 kDa form, in the TGA cell line could explain the reduced sensitivity to cell death induction displayed by these cells. As a final verification we checked out if also the tunicamycin treatment might induce this polymerisation of the caspase-3 and then explain the slight protection observed in the TGA cell line. Even in this case we detected the polymerised form of caspase-3 in TGA cells only but at a minor extent in respect to the thapsigargin treatment. The data we obtained support the hypotesis that during ER-stress, the increase in cytosolic Ca2+ concentration is able to activate TG2as a cross-linking enzyme, which in turn acts on casp-3. The polymerisation of casp-3 inhibits its proteolytic activity thus protecting TG2 over-expressing cells from apoptosis.

Antibiotic resistance occurs when bacteria causing an infection survive after being exposed to a drug that, under normal conditions, would kill it or inhibit its growth. As a result, these surviving strains multiply and spread due to the lack of competition from other strains sensitive to the same drug. Carbapenems and 3rd generation cephalosporins resistant Enterobacteriaceae represent one of the most critical group against which there is an urgent need to develop new antibiotics. These bacteria are common pathogens causing severe infections such as bloodstream infections, pneumonia, complicated urinary tract infections and complicated intra-abdominal infections. According to the Centers for Disease Control and Prevention (CDC) definition, CRE are defined as any Enterobacteriaceae which are resistant to any carbapenem or are documented to produce a carbapenemase. There are three major mechanisms by which Enterobacteriaceae become resistant to carbapenems: enzyme production, efflux pumps and porin mutations. Of these, enzyme production is the main resistance mechanism. Gram-negative bacteria generally develop resistances through the production of β-lactam-hydrolyzing enzymes, called β-lactamases. In the present study, two classes of these enzymes were analyzed and characterized also in association with a new molecule with inhibitory activity. In the first part of this thesis, the role of the Ω-loop (159-182 residues) in GES class A β-lactamase is well investigated. Recent studies report that, in these enzymes, carbapenemase activity is attributed to a single amino acid substitution at position 170. In our study, we decided to investigate other positions of the Ω-loop, in particular residue 174, occupied by a proline residue. This residue is well conserved in class A β-lactamases. Our kinetic and computational data have demonstrated that not only 170 residue is important in the carbapenemase activity, but also residue 174. Being Ω-loop a mobile structure of class A β-lactamases, we suppose that any mutation in this region could modify the substrate hydrolysis of the enzyme, not only toward carbapenems. The second part of the thesis is addressed to the involvement of some luecines located in the Loop 10 of NDM-1 and the effect of a new boronic acid molecule (taniborbactam) as inhibitors of metallo-β-lactamases. We have pointed the attention on residues L218, L221 and L269 that give hydrophobicity to the enzyme. Our results have demonstrated that the replacement of these leucines reduces the enzyme catalytic activity toward β-lactams. In addition, we studied the effect of Y229W substitution in our laboratory variant (L209F) with a drastic reduction in β-lactamase activity. This substitution is able to restore the catalytic activity in the L209F mutant enzyme. Actually, one of the most problem is related to the absence of inhibitors for metallo-β-lactamases in clinical therapy. For this reason, in collaboration with VenatoRx Pharmaceuticals (Malvern, PA, USA), we tested a new boronic acid, VNRX-5133, combinated with cefepime (taniborbactam), in NDM-1 and some engineered NDM-1 mutants able to hydrolyze β-lactams very efficiently. Taniborbactam was also tested in NDM-1- producing Enterobacteriaceae clinical strains. Our results revealed that the addition of VNRX-5133 restored cefepime activity in both NDM-1 producing recombinant strains of E. coli and clinical isolates of Enterobacteriaceae. The development of new non-β-lactam inhibitors would provide a new opportunity for the treatment of bacterial infections with existing antibiotics.

Bacteria live in a hostile habitat and are continuously exposed to a multitude of environmental stress that can compromise the survival of the cells in the absence of an efficient adaptation system. To survive and adapt to these “hard” conditions, bacteria have developed a global response system that modulates the expression of specific genes and, consequently, the cellular metabolism, allowing the microorganisms to respond to the modification of the environment. This inducible system is termed SOS response, which is mainly triggered by lesion to DNA. The SOS response entails the induction of multiple proteins that promote the integrity of DNA and cell survival. SOS response includes more than 50 genes that carry out diverse functions in response to DNA damage. The role of SOS response is not limited to DNA damage reparation; it also promotes processes that allow the bacteria to resist other stressed abuses. In this manner, microorganism develops tolerance towards the stimuli that triggered the response; this is the mechanism by which bacteria acquire resistance to antibiotics. Many antibiotics trigger the SOS response, either directly through DNA damage or indirectly, e.g., damaging protein or cell wall and membrane or producing toxic intermediates. The resistance and low susceptibility to antimicrobics is a consequence of an overexpression of SOS inducing proteins. The SOS response also contributes to spreading the resistance by promoting horizontal dissemination of antibiotic resistance genes. Stress-induced by antibiotics can promote acquisition and expression of resistance genes by mobile DNA elements, promoting resistance development Only two proteins regulate the SOS expression: a transcriptional repressor LexA and a recombinase enzyme RecA. In a physiological state, LexA inhibits specific gene expression by binding specific SOS-boxes sequences, which are included in the SOS gene promoter region. When cells undergo stress pressure, particularly when lesion on DNA occurred, single-strand DNA (ssDNA) is formed. RecA binds to ssDNA to form a nucleoprotein filament. This complex is a flexible structure and represents the active form of RecA; it can perform three separate functions: homologous recombination, SOS induction and SOS mutagenesis. RecA induces the SOS response by promoting the autocatalytic cleavage of LexA and release from the SOS box, thus inducing the expression of the SOS genes. Our laboratory identified several potential inhibitors for RecA and developed a high throughput assay for screening potential inhibitors of the SOS response. Nautiyal et al. identify the suramin as a potent inhibitor for all biochemical activities for RecA. This work has investigated the mechanism of interaction between RecA and suramin, elucidating the binding site and the inhibition mechanism. Surprisingly the suramin has a dual effect on RecA activity: at high concentration inhibits the ATPase activity of RecA, as previously demonstrated, whereas, at low concentration, it leads to an increase in ATPase activity. Moreover, in the presence of suramin, RecA ATPase activity becomes independent from ssDNA. The activity of suramin has been further investigated in the RecA-LexA interaction. As previously observed, the activity of suramin depends on its concentration; in fact, suramin performs as an activator at low concentrations (<1 µM) even in the absence of ssDNA, while acts as an inhibitor at higher concentrations. To better understand how suramin modulates the RecA activity, molecular docking and molecular dynamic simulations were performed. This study can provide further information regarding a new potential binding site for modulating RecA activity.

The ubiquitin‐proteasome system (UPS) and autophagy, the two major intracellular protein degradation systems, play a critical role in the regulation and maintenance of cellular homeostasis. The proteasome is known to degrade the majority of intracellular proteins, including cyclins, metabolic enzymes, antigen, transcription factors, and tumour suppressor proteins. Autophagy, or self‐eating, is a lysosomal degradation pathway in charge of recycling dysfunctional organelles and aggregated proteins. Impairments in the functionality of proteolytic pathways favour the accumulation of misfolded and abnormal proteins, resulting in the deposition of toxic aggregates that characterize diverse pathologic conditions such as cancer and neurodegenerations. For many years UPS and autophagy have been thought as separated pathways whereas an increasing number of data recently elucidated their intimate correlation. This PhD thesis is focused on understanding the role of UPS and autophagy in such diseases aimed at better clarifying their interplay. The potential of natural occurring compounds as proteasome modulators in the treatment and prevention of cancer is widely documented, with epigallocatechin‐3‐gallate (EGCG) being the most studied polyphenol. In particular, owing to EGCG instability under physiological conditions, its degradation pattern was monitored and an equally active metabolite has been isolated and evaluated for its ability to modulate both proteasome functionality and apoptotic pathways. Furthermore, the marine sponge metabolite petrosaspongiolide M (PM), a natural proteasome inhibitor, has been considered, describing the molecular mechanism of interaction of PM with the immunoproteasome, a proteasome isoform with a prevalent role in immune response. This natural compound was also able to impair autophagy, with p62 serving as the link between the two proteolytic processes. Among polyphenols‐based anticancer approaches, curcumin represents a promising but poor bioavailable compound. Considering that metal complexes offer an opportunity for the design of bioactive compounds with anticancer properties, and that ruthenium is a valid non toxic alternative to Cisplatin, three ruthenium(II)curcumin complexes containing different arene moieties have been synthesized and analyzed for their ability to modulate proteasome functionality, comparing their efficacies with that of free curcumin in isolated proteasome complexes and in cultured colon cancer cells. A stable and effective curcumin derivative, in terms of proteasome inhibitory ability, antioxidant capacity, DNA binding ability has been identified, thus proving that the complexation of curcumin with ruthenium(II) is a good starting point for the development of curcumin‐based anticancer drugs. Successively, a series of ruthenium(II) arene complexes with the 4‐(biphenyl‐4‐carbonyl)‐3‐methyl‐1‐phenyl‐5‐pyrazolonate ligand, and different ancillary ligands have been synthesized and characterized, identifying the hexamethylbenzene−ruthenium complexes as the most efficacious, in terms of antiproliferative activity in four human cancer cell lines, through the induction of apoptosis. Finally, regarding the interplay of UPS and autophagy in cancer, the effect of the hunger hormone ghrelin on both proteasome and autophagy has been analysed. Interestingly, this endogenous compound triggers apoptosis in colon cancer cells, via proteasome inhibition and autophagy induction, with p53 protein having an interactive role. Aging and neurodegenerative conditions, including Alzheimer disease (AD) are characterized by alterations in the normal cellular homeostasis with deregulation of the proteolysis. UPS and autophagy interplay in AD has been studied using human SH‐SY5Y neuroblastoma cells stably transfected either with wild‐type β‐amyloid precursor protein (AβPP) gene or mutant Val717Gly AβPP gene as experimental model. The APPmut clone produces and releases significantly higher amounts of Aβ42, the amyloid peptide which is more prone to aggregation. The over‐expression of the APP correlated with an increase in oxidative stress and with a reorganization of the cellular proteolytic machineries. Additionally, HDAC6‐increased expression has been identified as the cellular attempt to activate compensatory autophagy in such altered scenario. The existence of an amyloid (Aβ42) threshold level beyond which proteasome‐dependent proteolysis becomes definitely dysfunctional has been proved. Moreover, these cellular models have been used to demonstrate a role of the APP in affecting the downstream effects of proteolysis inhibition. The occurrence of APP wild‐type form or the APP Val717Gly mutated form was observed to impair both proteasome or autophagy activities upon treatment with proteasome or autophagy inhibitors. Collectively, our results provide evidences on the key role of UPS and autophagy in both neoplastic and neurodegenerative diseases and gain insight into the interplay between the two pathways in proteinopathies, thus representing a real contribution in the development of new strategies to modulate the two pathways for therapeutic purposes.

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.