Tesis sobre el tema "Pathway molecolare"

The clinical activity of the BRAF inhibitor PLX4032 (vemurafenib) in patients with BRAFV600E mutant melanoma is limited primarily by the development of resistance leading to tumor progression. Strategies to overcome primary and acquired resistance are required. In a panel of 27 genetically characterized patient-derived melanoma cell lines the sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma, such as CDKN2A, and mutations of TP53, PTEN loss, and BRAF and MITF gene amplification. To investigate the molecular basis underlying acquired resistance to BRAF inhibitor, PLX4032-resistant cells were derived from a high sensitive BRAFV600E melanoma cell line, and used as a model. The resistant variant line showed increased AKT and ERK phosphorylation and enhanced IGF-1R/PI3K signaling. Combined treatment with PLX4032 plus PI3K inhibitors resulted in significant cell growth inhibition by decreasing pAKT and pERK signaling. To explore molecular mechanisms underlying primary resistance two melanoma cell lines lacking sensitivity to PLX4032 were used as models. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA, indicating that CRAF is not involved in the activation of ERK in the resistant cell lines. Treatment with the MEK inhibitor UO126 inhibited cell growth and decreased ERK phosphorylation indicating alternative activation of MEK-ERK signaling. Genetic characterization by MLPA and analysis of pTyr signaling by MALDI-TOF mass spectrometry revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. Testing of co-inhibition of the MET, SRC and MAPK signaling pathways by the combined treatment with the MET inhibitor, SU11274 or the SRC inhibitor, BMS-354825 plus PLX4032 resulted in a significant inhibitory effect on melanoma cell proliferation, survival, migration and invasive capacity. These results support combinatorial approaches targeting MAPK pathway at different nodes and intercepting parallel signal transduction pathways as a strategy to override resistance to BRAF inhibitors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) has a key role in the innate immune system’s inflammatory response, as part of “cholinergic anti-inflammatory pathway”: a process by which acetylcholine from the vagus nerve reduces the release of the pro-inflammatory cytokine TNFα, thus allowing for a controlled response to infection. The CHRNA7 gene, in humans, is partially duplicated from exon 5-10 and forms an hybrid with four exons (D-A) of a novel gene, FAM7A. This new gene, CHRFAM7A, which is located in the opposite orientation, at 1.6 Mb, from CHRNA7, is not present on every chromosome 15 and a polymorphic variant, in linkage disequilibrium with a 2bp deletion in exon 6, in the same orientation to the CHRNA7 gene, has been described in a cohort of patients with bipolar disorders and schizophrenia. THP-1 monocytic-like cell line expresses only CHRFAM7A, which was down-regulated on treatment with LPS, by a direct transcriptional mechanism reliant on NF-kB. This effect has been confirmed in primary monocytes and macrophages cell cultures, where CHRFAM7A is expressed 200-1000 times more than CHRNA7. Here, the conventional α7 subunit was up-regulated by LPS treatment, thus suggesting the involvement of CHRFAM7A in the regulation of cell surface α7 receptors’ level (a mechanism unique to humans) and the ability of immune cells to respond to acetylcholine, released from the vagus nerve, during infection. This hypothesis seems to be supported by recent works showing that the duplicated form may have a dominant negative effect on the activity of α7 nAChR. Infact, co-expression of CHRFAM7A with α7 results in a significant reduction of the Ach-evoked currents, suggesting the presence of heteromeric non functional receptors at the plasma membrane. The promoter region that regulates the expression of CHRFAM7A is still unknown. To try to identify and characterize this region, 5'-RACE experiments were carried out to map the CHRFAM7A mRNA 5’UTR. RNA was extracted from three different cell lines: THP-1 cells, primary human macrophages and SHSY5Y neuroblastoma cell line. Multiple transcription start sites were identified, depending on the cell line used, suggesting the existence of alternative promoters. A series of constructs that recapitulate the mapping of the CHRFAM7A regulatory region, according to the transcription start sites identified, was also generated. They were cloned into a reporter vector and their functionality was tested by transient transfection both in THP-1 and SHSY5Y cell models. Through these experiments, an intronic region (-702/-208 bp from ATG codon, in exon B) and an Alu sequence (-1155/-821 bp) were identified as negative regulators of reporter gene transcription. Future experiments will allow us to identify other regulatory sites, important for proper CHRFAM7A gene expression in different tissues. Furthermore, two variants exist for CHRFAM7A gene, due to alternative splicing, that gives rise to two protein products of predicted 36 and 47 KDa, whose function is currently unknown. The N-terminally portion of each variant would lack the majority of the ligand binding domain, but the protein product retains the transmembrane region that forms the ion channel. To clarify the function of CHRFAM7A gene and to study the cellular localization of these two isoforms, CHRFAM7A (both variants) and CHRNA7 tagged proteins were generated, by cloning their cDNAs into pcDNA3.1/myc-His and pcDNA3.1/V5-His vectors. The α7 specific chaperon, RIC3, was also cloned into pcDNA3.0 vector and co-transfected with the tagged proteins, in order to increase their folding and expression. The constructs were transfected into Hela cells and characterized by immunofluorescence and western blotting experiments. The use of nicotine as therapy for chronic inflammatory diseases has often been characterised by excessive side effects due to a lack of specificity for just one receptor type. For this reason, the study of the regulation of α7 and its duplicated isoform subunits in response to pro-inflammatory stimuli, is important to understand their role in the “cholinergic anti-inflammatory pathway”, in order to discover selective nicotinic receptor agonists and to develop novel anti-inflammatory treatments.

1. Abstract The p63 transcription factor, homolog to the p53 tumor suppressor, plays a crucial role in epidermal and limb development. Dominant mutations in the p63 gene give rise to several human congenital syndromes characterized by skin, craniofacial and limb defects. One of the syndromes caused by p63 mutations is the Split-Hand/Foot Malformation-IV (SHFM-IV) syndrome, characterized by the loss of central rays of hands and feet. These developmental defects are due to failure of Apical Ectodermal Ridge (AER) development. The correct limb outgrowth and patterning is guarantee by the expression of key molecules including Fybroblast Growth Factor 8 (FGF8), p63 and the DLX5 and DLX6 transcription factors. In this context, the study of the molecular mechanisms regulating p63 stability and function is fundamental for understanding the molecular bases of the SHFM-IV pathogenesis: indeed p63 as been proposed to be one of the crucial regulators of limb and epidermal development. Little is known on the post-translational modifications and the upstream signalling pathway controlling ΔNp63α functions, one of the most expressed p63 isoform in epithelial tissues and in the AER cells. The projects performed during my PhD thesis achieved to the identification of FGF8 as a crucial regulator of ΔNp63α stability and activity in human osteosarcoma and keratinocyte cell lines. FGF8 determined also ΔNp63α protein stabilization in mice embryonic limb buds put in culture at Embryonic day 10.5 (E10.5). In particular, treatments with FGF8 of human osteosarcoma cell lines (U2OS) and human keratinocytes (HaCat), activate the tyrosine kinase c-Abl, leading to ΔNp63α phosphorylation and consequent acetylation by the p300 acetyl-transferase, promoting ΔNp63α stabilization and transcriptional activation. Moreover, I have found that p300 interacts with ΔNp63α determing its acetylation on lysine K193E, in vitro. Interestingly, this regulatory cascade is not active on the natural ΔNp63αK193E mutant associated to the SHFM-IV syndrome. Indeed, the ΔNp63αK193E mutant displays promoter specific altered DNA binding activity that results in altered expression of ΔNp63α target genes involved in limb development (like Perp, Ikkα and DLX5 gene) (Manuscript in preparation). One of the mechanism by which FGF8 promotes ΔNp63α stability and activation, is inhibiting its interaction with Pin1, a prolyl isomerase known to positively regulate p53 and p73 in response to DNA damage stress. In particular, PIN1 has an opposite effect on ΔNp63α respect to p53 and p73: it promotes ΔNp63α degradation through the proteasome pathway. Moreover, ΔNp63α mutant proteins, associated with SHFM-IV or EEC syndromes, characterized by limb defects, are not degraded by PIN1 overexpression. These data were confirmed also by in vivo experiments on PIN1 Knock-Out (KO) mice, where lack of PIN1 expression caused the accumulation of p63 in the embryonic limbs and ectoderm compared to wild-type littermates. Moreover, I found that FGF8 is a downstream target of the transcription factor Dlx5. Indeed, in the limb buds of both p63 and DLX5;DLX6 KO mice, the AER is poorly stratified and FGF8 expression is severely reduced. All these data suggest that DLX5, ΔNp63α, FGF8 and PIN1 participate in a regulatory loop essential for AER stratification, normal patterning and morphogenesis of the limb buds (1). The work performed during my PhD contributes to a better understanding of the regulatory mechanisms controlling ΔNp63α function and stability. We have identified FGF8 as a crucial upstream signal required for ΔNp63α activation and stabilization during limb development: mutations or altered expression of regulators in this pathway leads to abnormal limb development and onset of pathogenesis.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

Background. The most frequent adrenocortical tumors (ACT) are benign forms defined as adrenocortical adenomas (ACA) with an estimated incidence of about 7.3%. Malignant forms, adrenocortical carcinomas (ACC) are rare endocrine tumors with poor prognosis and incidence of 1-2 cases per million people in the population. Currenlty ACC treatments are ineffective and substantially are based on the use of mitotane (o,p'DDD) with or without traditional chemotherapeutic agents. Because of dissimilar prognosis of ACA or ACC it is important to differentiate these two forms. Many tumors stimulate the growth of blood vessels by the angiogenesis process. Recently, vascular endothelial growth factor (VEGF) over-expression in ACC and VEGF receptors (VEGFR-1 and VEGFR-2) expression were found on tumor cells; these findings suggest a possible autocrine effect of VEGF for cell growth. Sorafenib, a multikinase inhibitor, inhibits the phosphorylation of VEGFR-2 and induces in vivo tumor growth arrest. Everolimus, a rapamycin derivative, inhibits mTOR, which is involved in cell survival and located downstream toVEGFR-2. It stops production of angiogenic growth factors in several neuroendocrine tumors. To date few genetic alterations were identified in ACT involving adrenocortical tumorigenesis signaling pathways; in particular the Ras/Raf/MEK/ERK and the Wnt/β-catenin signaling pathways seem to be often altered. β-catenin constitutive activation is an alteration frequently found in ACT. It is a multifunctional molecule involved in the processes of cell adhesion together with cadherin (E-cadherin and N-cadherin). Cadherins have been implicated in the development of many cancers, but information regarding their expression in the ACT is very limited. Aim. The objective of this study is: a) to evaluate the expression of VEGF, VEGFR-1 and VEGFR-2 in normal and tumoral adrenocortical tissues; b) to examine the effect of the two drugs, sorafenib and everolimus, in vitro on cellular viability, on apoptosis and on signal pathways of 2 stabilized lines (H295R, SW13) and in ACT primary cultures and in vivo in xenografts mouse models; c) to analyze the presence of genetic alterations in key components of the Ras/Raf/MEK/ERK signaling pathway (BRAF, H-RAS, K-RAS, N-RAS genes) and of Wnt/β-catenin signaling pathway (CTNNB1 and AXIN2 genes) and to investigate the β-catenin expression in relation to the cell adhesion molecules E-cadherin and N-cadherin. Material and Methods. 24 adrenocortical carcinomas (ACC), 37 aldosterone producing adenoma (APA), 28 cortisol producing adenoma (CPA), 6 non-secreting adenomas (NSA) and 8 normal adrenal gland tissues (NA) were collected. The VEGF and its receptors (VEGFR-1 and VEGFR-2) gene expression was evaluated in 63 ACT by real-time PCR. In cell lines (SW13 and H295R) and in primary ACT cultures, cell viability was observed by incubating sorafenib and everolimus in a range of concentrations from 10 µM to 0.1 nM by MTT test. By fluorescence microscopy (TUNEL) and flow cytometric analysis (Annexin-V), apoptosis was evaluated; by western blot, the involvement of PI(3)K/Akt/mTOR and in Ras/Raf/MEK/ERK signaling pathways was analyzed. The effects of drugs, alone or in combination, were tested in vivo in ACC xenografted models. By high resolution melting analysis (HRM), mutation analysis was performed on 95 ACT on BRAF (exons 11 and 15), H-RAS (exons 2 and 3), N-RAS (exons 2 and 3), K-RAS (exons 2 and 3), CTNNB1 (exon 3), AXIN2 (exon 7). Only samples with altered melting curves were sequenced. Finally, by real-time PCR and immunohistochemistry (IHC), β-catenin, N-cadherin and E-cadherin gene expression was evaluated in 68 ACT. Results. VEGF, VEGFR-1, VEGFR-2 expression was found in both adrenocortical normal and tumoral tissues. Compared to normal adrenal glands, a significant VEGF over-expression was observed in 65% (12/18) (P = 0.049) ACC and in 61% (20/33) (P = 0.025) APA. In SW13 and H295R cell viability, sorafenib and everolimus showed a dose-dependent response, while by fluorescence (TUNEL) and by flow cytometry (Annexin V) the compounds revealed an apoptotic effect. By western blot, sorafenib induced a complete decrease in Akt, ERK1/2 and p70S6K phosphorylation, while everolimus totally abolished p70S6K phosphorylation. Out of 8 primary cultures, 3 ACC and 3 ACA significantly responded to sorafenib and everolimus treatments. In vivo experiment a significant reduction of the tumor mass and an increase in median survival (especially in xenograft subjected to combined treatment) were observed. In HRM analisys some alterations in key components of the Ras/Raf/MEK/ERK signaling pathway were found, i.e. 2 BRAF mutations and 4 H-RAS mutations. In Wnt/β-catenin signaling pathway 18 alterations in CTNNB1 gene (5 APA, 6 CPA, 2 NSA, 5 ACC) and a single mutation in AXIN2 gene in H295R cells were observed. In RT-PCR β-catenin is over-expressed in approximately 50% of ACC (12/24) and in 51% of ACA (24/47). By IHC a significant accumulation of cytoplasmic and/or nuclear β-catenin has been observed in 47% of ACC (7/15) and 33% of ACA (11/33). In all ACT expression of E-cadherin was not detected. By RT-PCR N-cadherin down-regulation has been found in 75% of ACC (18/24) and in 60% of ACA (28/47). Similar results were obtained by IHC: N-cadherin down-regulation was observed in 100% (15/15) ACC and in 55% (18/33) ACA. Conclusion. Our data underline the importance of angiogenesis in adrenocortical tumors system. Anti-VEGF strategies, such as new tyrosine kinases and mTOR inhibitors currently used in different tumors, may represent a new therapeutic tool for adrenocortical tumors. The identification of new anti-angiogenic and Wnt/β-catenin signaling targets has contributed to a better understanding of adrenocortical tumorigenesis and to generate the basis for the development of new targeted drugs (targeted therapy).
Presupposti dello studio. I più comuni tumori corticali della ghiandola surrenalica (ACT) sono tumori benigni definiti adenomi corticosurrenalici (ACA) con una incidenza stimata di circa 7,3%. I tumori maligni, i carcinomi corticosurrenalici (ACC), sono invece rare neoplasie endocrine, con prognosi infausta ed incidenza di circa 1-2 casi per milione. I trattamenti per l’ACC sono inefficaci nella maggior parte dei casi ed attualmente si basano sull’uso di mitotane (o,p’DDD) con o senza i tradizionali agenti chemioterapici. Poiché le prognosi di ACA o di ACC sono estremamente differenti è importante differenziare queste due tipologie di tumore. Numerosi tumori attraverso il processo di angiogenesi stimolano la crescita dei vasi ematici dell’ospite. Recentemente è stata osservata una over-espressione del fattore di crescita endoteliale vascolare (VEGF) negli ACC ed i recettori di VEGF (VEGFR-1 e VEGFR-2) trovati anch’essi sulle cellule tumorali, suggeriscono un possibile effetto autocrino di VEGF per la crescita cellulare. Il farmaco sorafenib, un inibitore multichinasico, inibisce la fosforilazione di VEGFR-2 ed induce in vivo l’arresto della crescita di alcuni tumori. Il farmaco everolimus, analogo della rapamicina, inibisce mTOR coinvolto nella sopravvivenza cellulare e situato valle di VEGFR-2; è inoltre in grado bloccare la produzione di fattori pro-angiogenici in molti tumori neuroendocrini. Ad oggi, le alterazioni genetiche note che colpiscono gli ACT sono limitate ed inoltre nella tumorigenesi corticosurrenalica sono coinvolte una grande varietà di vie del segnale, in particolar modo la via del segnale di Ras/Raf/MEK/ERK e la via di Wnt/β-catenina risultano spesso alterate. Un’alterazione frequentemente riscontrata negli ACT è l’attivazione costitutiva della β-catenina, una molecola multifunzionale coinvolta anche nei processi di adesione cellulare assieme alle caderine (E-caderina ed N-caderina). Le caderine sembrano essere coinvolte nello sviluppo di molti carcinomi, ma le informazioni riguardanti la loro espressione negli ACT è molto limitata. Scopo. L’obbiettivo dello studio è stato quello di: a) valutare l’espressione di VEGF, VEGFR-1 e VEGFR-2 nei tessuti surrenalici normali e/o tumorali; b) esaminare l’effetto dei due farmaci, sorafenib ed everolimus, in vitro sulla vitalità cellulare, sull’apoptosi e su alcune pathways cellulari in linee stabilizzate (H295R, SW13), in colture primarie di ACT ed in vivo in modelli murini xenotrapiantati; c) analizzare la presenza di alterazioni geniche nei componenti chiave della via del segnale di Ras/Raf/MEK/ERK (studiando i geni BRAF, H-RAS, K-RAS, N-RAS) e nella via del segnale di Wnt/β-catenina (valutando i geni CTNNB1 e AXIN-2) ed indagare l’espressione della β-catenina in relazione alle molecole di adesione cellulare E-caderina ed N-caderina. Materiali e Metodi. Sono stati raccolti 24 carcinomi corticosurrenalici (adrenocortical carcinoma, ACC), 37 adenomi secernenti aldosterone (aldosterone producing adenoma, APA), 28 adenomi secernenti cortisolo (cortisol producing adenoma, CPA), 6 adenomi non secernenti (non-secreting adenoma, NSA) e 8 campioni di surrene normale (normal adrenal, NA). L’espressione genica di VEGF e dei suoi recettori (VEGFR-1 e VEGFR-2) è stata valutata in 63 ACT tramite real-time PCR. Nelle linee cellulari (SW13 e H295R) e nelle colture primarie corticosurrenaliche, attraverso il test MTT è stata osservata la vitalità cellulare incubando con sorafenib ed everolimus in un range di concentrazioni da 10 μM a 0,1 nM. In microscopia a fluorescenza (TUNEL) e dall’analisi citofluorimetrica (Anessina V) è stato valutato il grado di apoptosi indotto dai farmaci e tramite western blotting è stato analizzato il loro possibile coinvolgimento nelle vie di PI(3)K/Akt/mTOR e di Ras/Raf/MEK/ERK. Gli effetti dei farmaci, da soli o in combinazione, sono stati testati poi in vivo in modelli murini di ACC xenotrapiantati con cellule H295R ed SW13. Attraverso l’analisi di high resolution melting (HRM) abbiamo ricercato in 95 ACT la presenza di mutazioni attivanti nei geni BRAF (esoni 11 e 15), H-RAS (esoni 2 e 3), N-RAS (esoni 2 e 3), K-RAS (esoni 2 e 3), CTNNB1 (esone 3), AXIN2 (esone 7). I campioni aventi curve di melting dubbie o alterate sono stati sottoposti tutti al sequenziamento. Infine, è stata valutata in 68 ACT l’espressione genica di β-catenina, N-caderina ed E-caderina attraverso le tecniche di real-time PCR e di immunoistochimica (IHC). Risultati. Sia i tessuti corticosurrenalici normali che tumorali esprimono VEGF, VEGFR-1, VEGFR-2. Rispetto ai surreni normali, una significativa over-espressione di VEGF è stata osservata in circa il 65% (12/18) (P = 0.049) degli ACC analizzati ed in circa il 61% (20/33) (P = 0.025) degli APA. Nella valutazione della vitalità cellulare le cellule SW13 ed H295R rispondono in modo dose-dipendente ai farmaci sorafenib ed everolimus mentre in fluorescenza ed in citofluorimetria è stato osservato un effetto apoptotico. Mediante western, sorafenib induce una completa diminuzione della fosforilazione di Akt, ERK1/2 e P70S6K mentre everolimus, abolisce totalmente la fosforilazione di p-P70S6K. Delle 8 colture primarie sottoposte ai farmaci, 3 carcinomi e 3 adenomi rispondono in modo significativo sia al trattamento con sorafenib che all’everolimus, mentre 2 casi di ACC sono resistenti ad entrambi i farmaci. In vivo è stata osservata una significativa riduzione della massa tumorale ed un aumento della sopravvivenza mediana soprattutto nei topi sottoposti al trattamento farmacologico combinato. Attraverso le indagini in HRM sono state riscontate alcune alterazioni nei componenti della via del segnale di Ras/Raf/MEK/ERK: 2 ACC con mutazioni nel gene BRAF e 1 APA, 1 CPA e 2 ACC con mutazioni nel gene H-RAS. Per quanto riguarda l’indagine genetica della via del segnale di Wnt/β-catenina sono state osservate 18 alterazioni nel gene CTNNB1 (5 APA, 6 CPA, 2 NSA, 5 ACC) ed una sola mutazione nel gene AXIN2 nelle cellule H295R. In RT-PCR l’espressione di β-catenina è over-espressa in circa 50% degli ACC (12/24) ed in circa il 51% degli ACA (24/47) mentre in IHC circa il 47% degli ACC (7/15) ed il 33% degli ACA (11/33) presenta un notevole accumulo citoplasmatico e/o nucleare di β-catenina. In tutti gli ACT non è stata rilevata l’espressione di E-caderina, mentre per quanto riguarda N-caderina, in RT-PCR circa il 75% degli ACC (18/24) ed il 60% degli ACA (28/47) possiedono una down-regolazione. Risultati simili sono stati ottenuti in IHC in cui il 100% degli ACC (15/15) e il 55% degli ACA (18/33) presentano una down-regolazione di N-caderina. Conclusioni. I nostri dati sottolineano l’importanza del sistema angiogenico nei tumori corticosurrenalici. I nuovi inibitori tirosin-chinasici e di mTOR attualmente usati in alcuni tumori come strategie anti-VEGF potrebbero rappresentare un nuovo strumento terapeutico per i tumori della ghiandola surrenale. L’identificazione quindi di nuovi target molecolari anti-angiogenetici e di target nella via del segnale di Wnt/β-catenina ha contribuito ad una migliore comprensione ed a un approfondimento della tumorigenesi corticosurrenalica generando le basi per lo sviluppo di nuovi farmaci mirati (target therapy).

Melanoma is an aggressive disease with high metastatic potential and resistance to cytotoxic agents. Early-stage melanomas can be successfully cured by surgery and, as in all solid tumours, morbidity and mortality of melanoma are a consequence of local invasion and metastatic spread. The molecular mechanisms involved in the progression of the malignancy, the genetic markers associated to metastatic melanoma dissemination and the acquisition of chemoresistance are only beginning to be defined. An understanding of the molecular biology of melanoma provides a necessary basis to enable the generation of more effective therapeutic modalities. RaLP, a new member of the SHC family of adaptor proteins was previously characterized in our laboratory as a determinant in the regulation of migration of melanoma cells in short-term assays in vitro. In this study we further characterized the role of RaLP in the progression of melanoma. We have verified that the expression of RaLP significantly correlates with the most important prognostic markers of melanoma (Breslow thickness, Clark’s level of invasion, ulceration, mitotic index and presence of metastasis in lymph nodes) and that patients with RaLP expressing tumours had reduced disease-free survival and overall survival, suggesting that RaLP can be identified as a novel prognostic molecular marker and an independent prediction factor of melanoma progression. We have shown that permanent RaLP silencing does not interfere with the proliferation of three different metastatic melanoma cell lines, while it significantly decreases their migration and this phenomenon was observed even after extended time in culture. The phenotype could be rescued by the overexpression of RaLP in the silenced cells, suggesting that RaLP is a central molecule that positively regulates migration of melanoma cells. Besides regulating migratory abilities of the melanoma cells, RaLP positively influences their invasive potential, by regulating collagen matrix digestion. We also tested cell – cell and cell – ECM adhesion abilities of melanoma cells after RaLP ablation. We observed that RaLP decreases adhesion of the cells to each other in cell – cell adhesion assays and negatively regulates adhesion of melanoma cells to different matrices in cell – ECM adhesion assay. Analyzing gene expression profiles of RaLP – proficient and – deficient cells we have shown that RaLP is involved in the regulation of the NOTCH molecular pathway. Our in vitro studies suggest that RaLP expression in melanoma might facilitate dissociation of metastatic cells from a tumour mass by loosening cell – cell adhesion, and favour invasion of the surrounding tissues. We still do not know the exact signalling cascade by which RaLP regulates cell motility and adhesion processes and additional studies are necessary to fully understand its role in melanoma progression.

La malattia di Parkinson (MdP) é il più comune disturbo neurodegenerativo del movimento, che colpisce circa 2-3% delle persone sopra i 65 anni di età. L´eziologia della malattia non è ancora definita ma casi familiari hanno fornito indizi sui meccanismi coinvolti nella malattia. In particolare, alterazioni genetiche nei geni codificanti alfa-sinucleina (aSyn) e leucine-rich repeat kinase 2 (LRRK2) sono alla base di forme autosomiche dominanti della malattia, le quali ricalcano le caratteristiche salienti della forma sporadica. Le inclusioni intraneuronali, definite corpi di Lewy e costituite prevalentemente da aSyn, sono uno dei segni distintivi della presentazione neuropatologica nella MP. I meccanismi coinvolti nella formazione, mantenimento e degradazione delle inclusioni sono tutt´ora oggetto di studio approfondito. Si ipotizza che diversi processi cellulari siano alla base di tali meccanismi, con una particolare attenzione rivolta all´autofagia. Inoltre, diversi geni associati ad aumento di rischio di MdP rivestono un ruolo nella biologia lisosomale e nell´autofagia stessa. Linee cellulari ricombinanti di neuroblastoma che sovra esprimono LRRK2 WT o G2019S riportano difetti in diversi stadi della via autofagia-lisosomi, in particolare accumulo di autofagosomi, alterazioni nella morfologia dei lisosomi e nel loro numero. Le cellule LRRK2 G2019S specificamente sono caratterizzate da inclusioni proteiche positive per pS129-aSyn. L´inibizione farmacologica dell´attività chinasica di LRRK2 in queste cellule corregge i deficit nella via autofagia-lisosomi e riduce il numero di inclusioni di pS129-aSyn. RIT2, che codifica per la proteina Rin, è un nuovo fattore di rischio genetico per la MdP che è stato scarsamente studiato finora. Si tratta di una piccola GTPasi che media la crescita neuritica tramite le vie di segnalazione delle MAPK p38 ed ERK1/2. Inoltre, RIT2 è arricchiato nei neuroni della substantia nigra pars compacta nei roditori e la sua espressione è ridotta nel cervello di pazienti di MdP. Tuttavia, non sono disponibili evidenze sperimentali sulle vie di segnalazione dipendenti da Rin e/o sulle eventuali interazioni con proteine legate causalmente alla MdP. Abbiamo appurato che i livelli di mRNA di RIT2 sono ridotti nelle cellule WT e G2019S LRRK2 ed inoltre l´attività del suo promotore è ridotta nelle stesse linee cellulari. La sovra espressione acuta di RIT2 produce una fenocopia del trattamento con l´inibitore chinasico di LRRK2, in riferimento al numero di autofagosomi, alla morfologia e numero di lisosomi e la loro attività proteolitica. In aggiunta, la sovra espressione di RIT2 riduce il carico di inclusioni positive per p S129-aSyn. Questa strategia inoltre riduce la fosforilazione di LRRK2 a carico della S1292, ma non della S935, e questo aspetto la differenzia dal trattamento farmacologico con inibitore di LRRK2. Inoltre, i livelli di espressione proteina totale di LRRK2 non vengono ridotti dalla sovra espressione di RIT2, a differenza di quanto accade in seguito ad inibizione farmacologica. La sovra espressione di RIT2 tramite vettori virali in un modello in vivo di tossicità di aSyn nel topo porta ad un miglioramento dell´attività motoria e riduce la perdita neuronale dopaminergica, indotta dall´espressione virale di aSyn. Inoltre, aSyn induce un aumento dei livelli di pS1292 LRRK2, indicativi di un´aumentata attività di LRRK2 endogena. La co-espressione con RIT2 previene tale aumento, suggerendo un´azione di RIT2 su LRRK2 stessa. Questi dati indicano che aSyn, RIT2 e LRRK2 sono alla base di meccanismi cellulari comuni, specialmente autofagia, e modulano a valle il catabolismo di aSyn. La modulazione dell´espressione di RIT2 potrebbe quindi costituire una nuova strategia terapeutica indirizzata alla patologia da aSyn, applicabile sia alla MdP familiare che idiopatica e, idealmente, alle altre sinucleinopatie.
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and affects about 2-3% of the population over the age of 65. The etiology of PD is still undefined and familial cases provided hints for mechanisms involved in the disease. In particular, alpha-synuclein (aSyn) and leucine-rich repeat kinase 2 (LRRK2) gene alterations lead to autosomal dominant PD with many commonalities to the idiopathic disorder. aSyn containing inclusions, termed Lewy bodies, are one of the neuropathological hallmarks of PD, but the mechanisms involved in formation, maintenance and degradation of these inclusions are still under intense investigation. Several cellular processes are hypothesized to mediate these mechanisms, with a prominent attention directed towards autophagy. Additionally, several PD risk genes have a role in lysosomal biology and autophagy itself. Recombinant neuroblastoma cell lines overexpressing WT and G2019S LRRK2 display defects at different stages of the autophagy-lysosome pathway (ALP), specifically accumulation of autophagosomes and abnormalities in lysosomal morphology and number. Interestingly, G2019S LRRK2 cells display inclusions staining positive for pS129-aSyn. Pharmacological inhibition of LRRK2 kinase activity in G2019S LRRK2 cells reverts ALP defects and most importantly reduces the number of pS129-aSyn inclusions. RIT2, coding for the protein Rin, is a novel PD gene risk factor and has been scarcely investigated. It is a small GTPase mediating neurite outgrowth through p38 and ERK1/2 MAPK signaling pathways. Moreover, Rin is enriched in rodent substantia nigra pars compacta neurons and is reduced in human PD brains. However, the relevance of Rin-dependent signaling pathways and/or its interactions with PD-causing proteins is currently lacking. We observed that RIT2 mRNA levels are reduced in WT and G2019S LRRK2 cells and also RIT2 promoter activity is reduced in these cell lines. Acute overexpression of RIT2 phenocopies the effects of LRRK2 kinase inhibitor treatment, specifically on the number of autphagosomes, morphology and number of lysosomes and their proteolytic function. Moreover, RIT2 overexpression strongly reduces the burden of pS129-aSyn inclusions. Interestingly, acute RIT2 overexpression reduces phosphorylation levels of S1292, but not S935 LRRK2, an aspect distinct from pharmacological LRRK2 kinase inhibition. Furthermore, total LRRK2 levels are not decreased with RIT2 overexpression, unlike after LRRK2 kinase inhibitor treatment. Viral overexpression of RIT2 in an in vivo aSyn mouse model ameliorates motor deficits and reduces dopaminergic neuron loss, induced by viral overexpression of aSyn. Viral aSyn overexpression induces an increase in pS1292 LRRK2 levels, indicating an enhanced activity of endogenous LRRK2. The co-expression of aSyn with RIT2 prevents the increase of LRRK2 kinase activity, indicating that RIT2 is acting on LRRK2. This data suggest that aSyn, LRRK2 and RIT2 might share common cellular mechanisms, in particular autophagy, and ultimately impact the degradation of aSyn. The modulation of RIT2 expression might constitute a new strategy to combat aSyn pathology, applicable to both familial and idiopathic PD and, possibly, other synucleinopathies.

Rett syndrome (RTT) is a rare and progressive neurodevelopmental disorder that occurs in 1:10,000-15,000 females. RTT is characterized by a normal early growth followed by, above all, motor and cognitive regression. RTT is caused by mutations of MECP2 gene, located on X-chromosome and subjected to random inactivation, that generate a very variable phenotype. Methyl-CpG-binding protein-2 (MeCP2) is a transcriptional factor involved in brain connectivity, neural circuits and importantly, in synaptic plasticity and deficits. However, the molecular mechanisms related with these defects are largely unknown. In previous works, we showed that c-Jun N-terminal protein kinase (JNK), a stress-activated kinase, was strictly involved in synaptic dysfunction related to neurodegenerative disease (Alzheimer’s disease and ischemic stroke) and that its specific inhibition, using the cell permeable D-JNKI1 peptide, led to a recovery of dendritic spines structure and to restoration of functionality supported by a rescue of cognitive deficits. We here proved for the fist time that JNK signalling is powerfully activated in RTT mice and acts as a key modulator of synaptic dysfunctions. The MeCP2 tm 1.1 Bird male mice (referred as Mecp2 y/-) were chosen for our evaluation because, despite not presenting mosaicism, they show an early onset and a most severe phenotype within a homogeneity of genetic background. D-JNKI1 treatment (22mg/kg) was administrated for the first time at 3rd week of age with an intraperitoneal injection and repeated after 3 weeks. Well-being and behavioural studies were effectuated with a weekly examination of food and water intake, weight and locomotor abilities. At 7 weeks, mice were sacrificed and tissues were processed for biochemical evaluations. In brain, we found a strong activation of JNK’s preferential target, c-Jun, a nuclear transcription factor. D-JNKI1 chronic treatment improved general mice wellbeing. Treated mice showed a rescue of motor deficits and an improvement of motor coordination, parameters evaluated with Rotarod and Open field behavioural tests. Since RTT is characterized by locomotor impairment, we checked in cerebellum the synaptic dysfunction evaluating AMPA and NMDA receptors levels. Isolating the post-synaptic region, we found that D-JNKI1 treatment rescued receptor levels. Moreover, PSD95 and Shank3 analysis revealed that the decrement of Mecp2 -/y mice was reported to control level thanks to D-JNKI1 treatment. Moreover, our studies were focused on inflammatory pathway triggered by JNK and we found an astrogliosis and a microgliosis activation, completely rescue by D-JNKI1 treatment. We then move to translational medicine to strengthen the JNK pivotal role in RTT by using Human RTT iPSCs. The mutant neuronal-IPSc presented JNK activation while isogenic control neuronal-IPSc did not; furthermore, we found that D-JNKI1 inhibited JNK activity. The results on iPSCs added value to the clinical relevance of the proposed treatment. RTT is a rare and incurable progressive postnatal female neurodegenerative disorder and the manipulation of JNK pathway may represent the development of an innovative strategy to tackle Rett Syndrome. JNK plays had shown a key role in both mice and human mutated neuronal-IPSc and consequently its relevance in clinical study. We now need to better characterize D-JNKI1 effect in female mosaicist model, closer to the human phenotype.

The central theme of this thesis is TAZ, a transcriptional co-activator that works - together with its homologue YAP - as transducer of the mammalian Hippo pathway. Since the beginning of my PhD training, I have been involved in the investigation of TAZ function and regulation in breast cancer stem cells. Our findings established TAZ as a molecular determinant of self-renewal and tumorigenic potential in breast cancer cells. Indeed, the vast majority of high grade breast tumors (known to be enriched with cancer stem cells) display a strong positive staining for TAZ, compared with normal mammary tissue and low grade tumors. Experiments described in the first part of this thesis show that TAZ protein is stabilized in cells that have undergone an epithelial-to-mesenchymal transition, or simply lost the apico-basal polarization proper to epithelial cells. Indeed, in polarized epithelial cells the basolateral determinant Scribble activates the Hippo kinases to inhibit TAZ; in depolarized cells (including cells in malignant carcinomas) this inhibitory mechanism is disabled and TAZ can accumulate. How TAZ overexpression modifies the cell to turn it into a cancer stem cell is the object of ongoing work. Part of these results was published in (Cordenonsi et al., 2011). During the last year, I have collaborated on a second project, dealing with TAZ regulation by Wnt signaling (Azzolin et al., 2012). We have found that Wnt signaling activates TAZ along with β-catenin. This happens because, in the absence of Wnt signals, phosphorylated β-catenin drives TAZ degradation; Wnt stimulation thus leads to the parallel stabilization of β-catenin and TAZ. Experiments presented here show that β-catenin effectively inhibits TAZ activity in the absence of Wnt signaling, and that TAZ stabilization is instrumental in both physiological and pathological Wnt biological responses.
Il tema centrale di questa tesi è TAZ, un co-attivatore trascrizionale che – insieme al suo omologo YAP – funge da trasduttore della Hippo pathway nei mammiferi. Dal primo anno di dottorato, ho partecipato allo studio della funzione e la regolazione di TAZ nelle cellule staminali del tumore al seno. I risultati ottenuti dimostrano che TAZ è un determinante molecolare della capacità di auto- rinnovamento (self-renewal) e delle proprietà tumorigeniche di cellule di tumore mammario. La maggioranza dei tumori di alto grado istologico (che contengono un numero elevato di cellule staminali tumorali) esprimono livelli di TAZ più alti rispetto al tessuto sano della ghiandola mammaria e ai tumori di basso grado istologico. Gli esperimenti descritti nella prima parte di questa tesi dimostrano che TAZ è stabilizzato – a livello di proteina – in cellule epiteliali che hanno subito una trasformazione mesenchimale, o hanno perso la polarità apico-basale tipica delle cellule epiteliali. Infatti, in cellule epiteliali polarizzate Scribble, un determinante basolaterale, attiva le chinasi della Hippo pathway ed inibisce TAZ; in cellule depolarizzate (come molte cellule nei carcinomi maligni) questo meccanismo è inattivo e TAZ si accumula. Come la stabilizzazione di TAZ modifichi le cellule per trasformarle in cellule staminali tumorali è oggetto di studi attualmente in corso. Parte di questi risultati è stata pubblicata in (Cordenonsi et al., 2011). Durante l’ultimo anno, ho collaborato ad un secondo progetto, dedicato alla regolazione di TAZ da parte di Wnt/β-catenina (Azzolin et al., 2012). I segnali Wnt attivano TAZ insieme a β-catenina. Ciò è dovuto al fatto che, in assenza di Wnt, β- catenina fosforilata porta TAZ al proteasoma; la stimolazione con Wnt determina quindi la stabilizzazione parallela di β-catenina e TAZ. Gli esperimenti qui presentati dimostrano che β-catenina inibisce l’attività biologica di TAZ in assenza di Wnt, e che la stabilizzazione di TAZ ha un ruolo funzionale nelle risposte biologiche (sia fisiologiche che patologiche) ai segnali Wnt.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy due to its diagnosis at advanced stages, when the disease has already spread beyond the ovaries. EOC is generally sensitive to first line chemotherapy, and the vast majority of patients respond to platinum (Pt)-based therapy after debulking surgery. Unfortunately, more than 80% of Pt-responsive patients relapse with a disease that progressively becomes Pt-resistant. Based mainly on clinical evidence, the process by which disease relapses is still poorly understood. The aim is to identify biomarkers of sensitivity to chemotherapy and therapeutic targets in HGS-EOC by integrating transcriptomic data, coding and non-coding RNAs. The bioinformatic analysis was applied on microarray data and RNA-seq data, embracing different classes of patients (resistant, sensitive, partially sensitive and normal). Two complementary approaches have been adopted to identify biomarkers of therapy response in microarray data: i) a classic approach and ii) a network-based approach using micrographite. The results obtained with both procedures have then been used to reconstruct a regulatory circuit involved in therapy response. The final outcome is a regulatory cell signal pathway composed of genes and miRNAs mainly involved in the therapy response. Circuit has been validated using two external and independent cohorts by quantitative real-time PCR (qRT-PCR). However, in order to complete the characterization of network as prognostic factor we decided to consider in survival analysis defect of the Homologous Recombination (HR). Approaching in survival analysis, a signature of three genes (SDF2L1, PPP1R12A and PRKG1) found to be independent prognostic biomarkers, was able to predict, at the time of diagnosis, resistance to Pt-based chemotherapy. Also, a new approach has been evaluated in order to characterize new mechanisms of chemotherapy resistance in ovarian cancers. On microarray data, we tried to stratify patients for the immunotherapy, with recent improved understanding of the immune recognition and regulation of cancer cells. In addition, using RNA-seq data and somatic DNA mutations, we went deeper in immunogenicity of ovarian cancer trying to find new elements as therapy targets, neoantigens, not associated to this tumor till now. At last, in addition, the small amount of molecular differences between Pt-r and Pt-s patients suggested the presence of potential new transcripts involved in therapy response maybe due to aberrant splicing events. To investigate this hypothesis, we used a set of RNA-seq experiments, to identify new aberrant splicing such as circular RNAs. We reported 5 circRNAs differentially expressed between tumour resistance types, and a large number of class-specific circRNAs. In particular, circ_BARD1 showed a character as prognostic factor significative in OS and PFS, in multivariate analysis with residual tumour and age as covariates. The consistency of circular RNA expression, in conjunction with the regulatory circuit, may offer new candidates for cancer treatment and prognosis, revealing that the integration of coding and non-coding RNAs data may shed light on chemotherapy resistance mechanisms in ovarian cancer.

Toll-like receptors (TLRs) play key roles in detecting pathogens and initiating inflammatory responses via the activation of specific signaling pathways. The TLRs activity must be tightly regulated to avoid excessive inflammation and consequent immunopathology, ranging from autoimmunity to cancer. MicroRNAs (miRNAs) are a new class of negative regulators involved in setting the balance of the immune response to inflammatory triggers. In this study, we identified miR-125a~99b~let-7e cluster and miR-146b as miRNAs that, after LPS engagement on human monocytes, are induced by the anti-inflammatory IL-10 and TGFβ, but are inhibited by the pro-inflammatory IFNγ. Bioinformatic analysis predicted and experimental evidence demonstrated that miR-125a-5p, let-7e-5p and miR-146b directly target the TLR pathway at multiple levels, including receptors (TLR4, CD14), signaling molecules (IRAK1, MyD88, TRAF6), and effectors (TNFα, IL-6, CCL3, CCL7, CXCL8). We showed that over-expression or inhibition of miR-125a, let-7e and miR-146b expression with lentiviral vector in human monocytes had a significant impact on the production of pro-inflammatory cytokines in response to LPS. In particular, we identified a role for miR-125a-5p and miR-146b in mediating the LPS hyporensponsiveness observed after IL-10 or TGFβ priming or during the endotoxin tolerance, the phenomenon of reduced sensitivity to subsequent challenge of LPS. The up-regulation of miR-125a-5p and miR-146b into THP-1 cells mimicked the LPS, IL-10 or TGFβ priming, whereas the inhibition of them by lentiviral vector or a pre-treatment with IFNγ reverted, partially, the tolerant phenotype. In an in vivo model of acute inflammatory response, we obtained that miR-125a-5p, miR-99b-5p and miR-146b were induced in macrophages recruited at the site of inflammation during the resolution process, and this was impaired in macrophages of IL-10 KO mice. Our studies indicated that miRNA cluster and miR-146b represent a new negative feedback mechanism of the TLR signaling pathway.

Due to their prominent role in the orchestration of a broad range of immune responses, dendritic cells (DCs) have emerged in the past decade as central target for cancer immunotherapy. Recent advance in the knowledge of DCs functions and subset specialization led to the design of novel immunotherapeutic approaches based on the possibility to target DCs directly in vivo, thus avoiding their ex vivo manipulation. A promising strategy is to use antibodies to target antigens to cell-surface molecules expressed by DCs, in order to increase T-cell mediated immune responses. The studies performed so far, have revealed that the efficacy of in vivo DC vaccination depend on several factors including the specific DC subset targeted, their maturation status and the nature and biological properties of the receptor targeted. Therefore, the identification of the most appropriate ligand/receptor pairing it is a requisite to improve the modality of delivery tumor Ags to DCs. To this aim, we screened a library of Ab fragments on mouse DCs to isolate new potential antibodies capable of targeting DCs in vivo and able to induce T-cell mediated immune responses against specific antigens. In this study, we provide the proof of principle that the phage display technology can be successfully used to isolate internalizing antibodies on mouse DCs. We further develop such technology by engineering the selected molecules to create antigen fusion proteins to use in vaccination protocols. In particular, we focus on a high affinity Ab against CD36, a multiligand scavenger receptor primarily expressed by the CD8α+ subset of conventional DCs. We characterize the antigen presenting properties of this receptor which help to delineate a novel function of CD36 in adaptive immunity. We show that targeting CD36 on DCs results in the delivery of exogenous Ags to both the MHC class-I and MHC class-II processing pathways. In addition, immunization with the recombinant anti CD36-Ag fusion Ab induces the robust activation of naïve CD4+ and CD8+ Ag specific T lymphocytes and the differentiation of primed CD8+ T cells into long term effector CTLs. Finally, we demonstrate that in vivo targeting of CD8α+ DCs with anti-CD36 Ab elicits humoral and cell mediated protection from the growth of an Ag specific tumor. Collectively, these results identify CD36 as an appropriate receptor to better elucidate the properties of the lymphoid organ resident CD8+ DCs and indicate it as a novel potential target for cancer immunotherapy.

Notch signaling mediates central cellular functions through direct cell-to-cell contact. Deregulation of Notch activity can alter cell proliferation and it is linked to many human cancers. Similarly to other oncogenes, Notch cooperates with many pathways during tumor progression. My study aimed to evaluate the effect of interactions of Notch pathway with AKT signaling and with CXCR4/SDF1a axis. Notch signaling is known to play role as the main trigger of T cell acute lymphoblastic leukaemia (T-ALL), aggressive hematologic cancer that accounts for 10-15% of pediatric and 25% of adult ALL cases. Recent evidences indicate the reciprocal regulation between the constitutively active Notch and AKT pathways in T-ALL cells. However, the mechanism of this interaction is still unclear. This prompted me to investigate the molecular mechanisms of AKT-dependent Notch1 regulation in T-ALL cells. Previous results in T-ALL cells shown that Akt signaling withdrawal induced by LY294002, a specific PI3K inhibitor, triggers Notch1 degradation via lysosomal machinery. Further, I showed that LY294002 treatment increased monoubiquitin-dependent accumulation of transmembrane Notch1 protein. Co-immunoprecipitation and densitometry analysis of confocal images revealed that Notch1 specifically co-precipitated with the ubiquitin E3 ligase, c-Cbl. This is involved a covalent attachment of ubiquitin to Notch1 and directing it to the lysosome. Finally, a confocal microscopy analysis showed Notch1 localization in the lysosome. These data suggest that AKT pathway can control the steady-state level of Notch1 rescuing it from lysosomal degradation by interfering with c-Cbl triggered mono-ubiquitination and lysosomal degradation. Notch crosstalk with the chemokine system CXCR4/SDF1awas studied in cell models of ovarian cancer (OC). OC ranks fifth in tumor mortality among women. Recent evidences support a role of Notch pathway in OC progression where its activation is related to tumor progression. Similarly, CXCR4/SDF1a correlates with OC cell survival, tumor growth and metastasis. To investigate the consequences of Notch-CXCR4 crosstalk in OC biology, a panel of OC cell lines was characterized by high expression of Notch pathway as well as by elevated levels of CXCR4 and its ligand SDF1α comparing to a normal ovarian cell line. Treatment with an inhibitor of Notch activation, DAPT, was associated with a reduced time-dependent OC cell proliferation and cell cycle arrest in G0/G1 phase. By contrast DAPT did not affect apoptosis, neither sensitize OC cell lines to other pro-apoptotic and anti-proliferative thiazolinedione’s drugs. In addition, Notch withdrawal determined a significant CXCR4 and SDF1α mRNA and protein inhibition. Further studies also displayed Notch ability to control the biological function of CXCR4 signaling, i.e. growth and migration in response to SDF1a. A confirmation of the specific role of Notch1 in the regulation of CXCR4 signaling was obtained by transient ectopic expression of Notch1 in OC cells that resulted in increased expression levels of CXCR4 and SDF1a. The evidence that Notch deregulation might affect CXCR4/SDF1a axis expression and function is relevant in the light of CXCR4 role in promoting OC cell proliferation and SDF1a-mediated dissemination of OC cells in the abdominal cavity. Altogether, these results suggest that Notch pathway might be a promising therapeutic target to hamper tumor progression and improve the efficacy of present anti-cancer therapy.

The transcriptional co-activator TAZ, known Hippo transducer together with his paralogue YAP, has recently emerged as important player in processes like organ growth and tumorigenesis. Here we focused on two aspects of TAZ biology: the first regards the role of TAZ as molecular determinant of breast cancer stem cells (CSCs); the second is the characterization of TAZ as downstream mediator of Wnt signaling. Using a bioinformatic approach, we discovered that more-malignant/CSC-enriched primary breast cancers, compared to well-differentiated/non-methastatic tumors, display an elevated activity of TAZ (Part 1), and that this correlates with a poorer prognosis. TAZ protein levels and activity are elevated in prospective CSCs and they increase during tumor evolution toward malignancy, both in vitro and in poorly-differentiated primary breast tumors. Moreover, TAZ is required to sustain self-renewal and tumor-initiation capacities of breast cancer cells. These features make TAZ a determinat of several characteristic of breast CSCs and a attractive molecule for therapy. We also studied upstream regulators of TAZ and found that, independently of the Hippo pathway, TAZ is regulated and trascriptionally-activated by the Wnt cascade (Part 2), sheding lights on the modalitites by which cells respond to the Wnt growth factors. Mechanistically, in the absence of Wnt activity, the components of the β-catenin destruction complex - APC, Axin and GSK3 - are also required to keep TAZ at low levels because phosphorylated β-catenin bridges TAZ to its ubiquitin ligase complex. Upon Wnt signaling, escape of β-catenin from the destruction complex impairs TAZ degradation and leads to concomitant accumulation and activation of β-catenin and TAZ.
Il co-attivatore trascrizionale TAZ, conosciuto per essere un trasduttore della via del segnale Hippo con il suo paralogo YAP, è recentemente emerso come importante fattore in processi quali la crescita degli organi e la tumorigenesi. In questo lavoro ci siamo focalizzati su due aspetti della biologia di TAZ: il primo riguarda il ruolo di TAZ come determinante molecolare delle cellule staminali del tumore al seno; il secondo, la caratterizzazione di TAZ come mediatore a valle della via del segnale Wnt. Usando un approccio bioinformatico, abbiamo scoperto che i tumori alla mammella più maligni e arricchiti in cellule staminali tumorali mostrano una più elevata attività di TAZ quando comparati ai tumori più differenziati e non meta-statici (Parte 1): questo correla inoltre con una prognosi più sfavorevole. I livelli proteici e l’attività di TAZ sono elevati nelle putative cellule staminali tumorali e aumentano durante la progressione dei tumori verso la malignità, sia in vitro che nei tumori primari. In più, TAZ è richiesto per sostenere il rinnovamento e la ca-pacità di formare tumori in vivo da parte delle cellule tumorali mammarie. Queste proprietà rendono TAZ un determinante di parecchie caratteristiche di cellule staminali tumorali mammarie e una molecola interessante dal punto di vista terapeutico. Abbiamo inoltre studiato i regolatori a monte di TAZ e abbiamo scoperto che, indipendentemente dalla via del segnale Hippo, TAZ è regolato dalla via del segnale Wnt (Parte 2), che ne promuove anche l’attivazione. Questo meccanismo chiarisce una delle modalità attraverso cui le cellule rispondono alle citochine del-la famiglia Wnt. Dal punto di vista meccanicistico, in assenza del segnale Wnt, i componente del complesso di distruzione di β-catenina - APC, Axin and GSK3 – sono richiesti per mantenere bassi i livelli di TAZ: questo è dovuto al fatto che β-catenina fosforilata fa da ponte tra TAZ e il suo complesso di ubiquitina ligasi. In seguito al segnale Wnt, β-catenina non è più fosforilata nel complesso di distruzi-one e ciò impedisce la degradazione di TAZ: di conseguenza vi è un contemporaneo accumulo sia di TAZ che di β-catenina.

In the yeast Saccharomyces cerevisiae, the Ras proteins are part of the cAMP/PKA signalling pathway, which plays a fundamental role in the control of many cellular processes including cells proliferation, stress resistance, metabolism, and growth. They belong to the super-family of the small GTPases that act as molecular switches by cycling between an inactive GDP-bound form and an active GTP-bound form. This process is controlled by two classes of regulatory proteins: the GEFs promote the activation of Ras by catalyzing the GDP-GTP exchange, whereas the GAPs turn off the Ras proteins by stimulating the hydrolysis of GTP to GDP. In the first section of this thesis, we investigated the localization of active Ras proteins in wild type cells and in mutants in several components of the cAMP/PKA pathway to understand how the proteins involved in this pathway influence the localization of active Ras. To this aim we used a probe in which the eGFP (enhanced green fluorescent protein) is fused to a trimeric Ras binding domain (RBD3) of the human Ras effector, c-Raf1. This RBD directly binds to the active Ras with a much higher affinity than the inactive Ras. We also investigated the influence of PKA activity on active Ras localization analyzing different mutants with either high or low/absent PKA activity. The cells of the different strains expressing the eGFP-RBD3 probe growing on glucose medium were observed under the microscope. In wild type cells, Ras-GTP was mainly localized at the plasma membrane and surprisingly in the nucleus. In cyr1∆ and gpr1∆ cells, the probe showed a similar localization as in wild type cells. In gpa2∆, hxk2∆ and hxk1∆hxk2∆ cells, the fluorescence accumulated in internal membranes and mitochondria. However, in the hxk1∆hxk2∆ cells transformed with the centromeric plasmid YCpHXK2 expressing Hxk2, the eGFP-RBD3 probe was mainly localized at the plasma membrane and in the nucleus. These results suggest that Gpa2 and Hxk2 play a role in the localization of active Ras. We also observed that the localization of active Ras is dependent on PKA activity. Indeed, in the bcy1∆ mutant, showing high PKA activity, there was a clear relocalization of active Ras to the cytoplasm and to the nucleus, while no active Ras was localized at the plasma membrane anymore. In a strain with either reduced PKA activity, the tpk1w1 tpk2∆ tpk3∆ strain or absent PKA activity, the tpk1∆ tpk2∆ tpk3∆ yak1∆ strain, active Ras was mainly localized at the plasma membrane. In the second section of this thesis, we investigated the role played by active Ras in the nucleus. To this aim, a fusion was made between the Ras2 protein and the Nuclear Export Signal (NES) from the HIV virus (HIV virus Rev protein NES) (Henderson et al., 2000), generating the NES-RAS2 strain. Our results showed that the exclusion of Ras2 protein from the nucleus did not cause a growth defect neither on fermentable nor non fermentable carbon sources and did not influence the PKA related phenotypes analyzed in our work. Cells expressing the fusion protein were only defective for the invasive growth, suggesting that nuclear active Ras2 is involved in this cellular process. These results were confirmed using also the Tlys86 strain, that is commonly used to test this phenotype. We also demonstrated that the nuclear localization of Cdc25, the main GEF of Ras proteins, is required for invasive growth and that PKA activity controls invasive growth influencing the localization of active Ras. Data in literature (Cazzaniga et al., 2008; Pescini et al., 2012) show the presence in silico of cAMP levels oscillations. In the last section of this thesis, we tested two different FRET sensors, previously used in mammalian cells, to monitor the cAMP levels (CFP-Epac1-YFP probe) and PKA activity in single cells in vivo (AKAR3 probe). We inserted the sequences coding for the CFP-Epac1-YFP sensor and for the AKAR3 sensor in a multicopy yeast expression vector and the sensors were expressed under the control of the TPI promoter in several yeast strains. We used a two-photon confocal microscope system to measure the FRET efficiency. Our preliminary results showed that in a wild type strain expressing either the Epac sensor or the AKAR3 sensor there was respectively an increase of cAMP level and PKA activity in a single yeast cell after glucose addition to glucose-starved cells.

Cardiovascular disease (CVD) is still one of the major cause of morbidity and mortality in the world, with ischemic heart disease representing the majority of deaths over the past 10 years. The high burden of the disease, both immediate and chronic, associated with the high costs for the healthcare systems, claim for the development of novel therapeutic strategies. The main issue of current pharmacological and interventional therapeutic approaches is their inability to compensate the great and irreversible loss of functional cardiomycytes (CMs). Because of the limited regenerative capacity of post-natal CMs and the difficulty to obtain and isolate heart bioptic tissue, very limited supplies of these cells are available at present for dedicated studies. Moreover, even if animal models are surely the best tool to study and understand in vivo the mechanisms of specific human pathologies in a complex organism, they are not fully predictive and representative of the human condition; from an economic point of view, animal maintenance and the related experimentations are time consuming and very expensive. In this scenario, human pluripotent stem cells (hPSCs), including human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs), play an important role in the cardiovascular research field, because they can be indefinitely expanded in culture without loosing their stemness, and differentiated into cells of the three germ layers, such as CMs. A great breakthrough in science has occurred in 2007 with the discovery of hiPSCs by the Nobel Prize Shinya Yamanaka. This has been the starting point for deriving patient-specific hiPSCs from the reprogramming of somatic cells obtained with less- or non-invasive procedures (skin biopsies, blood, urine…), useful for the generation of tissues for autologous-repair, bypassing the ethical and political debates surrounding the hESCs derivation. The researchers have made several efforts to develop strategies to efficiently direct hPSCs cardiac differentiation and the existing methods for deriving CMs involve stage-specific perturbations of different signaling pathways using growth factors (GFs) or small molecules that recapitulate key steps of the cardiac development observed in vivo. However, these strategies are accompanied by some limitations including: high intra- and inter-experimental variability, low efficiencies, presence of xeno-contaminants, undefined medium components and differences in the expression of cytokines of endogenous signaling pathways. Other strategies are based on the direct lineage conversion of somatic cells, especially fibroblasts, via the overexpression of cardiac transcription factors (TFs) combinations through integrating and non-integrating vectors. However, also these approaches are characterized by low efficiencies, combined with the risk of genomic integration and insertional mutagenesis when using integrating vectors or the need for stringent steps of purification when using non-integrating techniques. Because of the difficulty to specifically direct hPSCs cardiac fate in a robust way, combined with the scarce ability of conventional culture systems to reproduce in vitro, the environment in which cells reside in vivo, the CMs produced to date are immature and more similar to fetal cardiac cells. In 2010, Warren L. and co-workers pioneered a novel, non-integrating strategy based on repeated transfection with cathionic vehicles of synthetic modified messenger RNA (mmRNA), specifically designed to avoid innate immune response from the cell, demonstrating the possibility to both reprogram somatic cells to pluripotency and to programm hPSCs fate into terminally differentiated myogenic cells. Hence, the aim of this PhD thesis is the development of an efficient and robust method for cardiac differentiation of hPSCs by combining the mmRNA with the microfluidic technology. Repeated transfections with mmRNA encoding 6 cardiac TFs are employed to force the endogenous protein expression in the cells and to drive the differentiation toward functional maturation of CMs. The integration of cardiac differentiation within an ad hoc microfluidic platform, facbricated in BioERA laboratory, allows a more precise control of culture conditions, enabling a high mmRNA transfection efficiency, thanks to the high volume/surface ratio, and the in vitro reproduction of physiological niches. In fact, the small scale offered by microfluidics, best mimics the cellular dynamics, which occur in the soluble microenvironment in vivo. Moreover, the microfluidic technology offers the possibility to perform combinatorial, multiparametric, parallelized and highthroughput experiments at one time in a cost-effective manner, not achievable and not economically sustainable in macroscopic conventional culture systems. Chapter 1 starts with the definition of regenerative medicine and introduces the complexity of cardiac development, with the network of TFs that cooperate in this process. The state of the art regarding the derivation of CMs from hPSCs and from the transdifferentiation of somatic cells is described, together with the current limitations and challenges. Finally, the general aim of this PhD thesis is presented. Chapter 2 will focus on hPSCs (hES and hiPS) employed during this project, describing their most important characteristics. It will be also presented a monolayer-based cardiac differentiation protocol of hPSCs that, to date is considered the gold standard for the fast generation of a high yield of beating CMs in conventional culture systems. This protocol relies on the temporal modulation of Wnt pathway via the administration of small molecules. In addition, a hES line, dual reporter for 2 cardiac TFs will be described and always adopted as a tool to monitor the progression of cardiac differentiation. The results obtained in standard cultures will be showed. Chapter 3 will review the state of the art of microfluidic technology for cell culture in regenerative medicine applications. Then, the microfluidic platform fabrication will be described and employed, followed by the optimization of culture, expansion and cardiac differentiation of hPSCs with the gold standard protocol deriving form the translation from macro- to micro-scale. Chapter 4 will introduce the novel mmRNA strategy for reprogramming and programming cell fate: also in this case the state of the art will be discussed. Then, the experimental strategies developed to program cardiac differentiation of hPSCs toward a more mature CM phenotype will be presented, together with the results obtained and the related structural, functional and molecular characterizations. In this work, for the first time, it has been possible to derive CMs from hPSCs with repeated transfections of mmRNA encoding 6 cardiac TFs in microfluidics, with efficiencies higher to current methods described in literature, performed in standard systems. Finally, Chapter 5 will present the general discussion and conclusions, with the future perspectives regarding the use of mmRNA combined with microfluidic technology for deriving different CMs phenotypes, just varying the combination of TFs delivered. To conclude, the experiments developed during this project provide proof-of-principle that it is possible to program hPSCs fate toward cardiac lineage and cardiac maturation in microfluidics; moreover, thanks to the non-integrating characteristic of mmRNA, the CMs obtained are clinical-grade and could potentially be employed in the next future for clinical applications of autologous tissue self-repair and for personalized drug screening.
Le malattie cardiovascolari rappresentano ad oggi una delle principali cause di morbidità e mortalità nel mondo, tra le quali la patologia ischemica è responsable del maggior numero di decessi negli ultimi 10 anni. L’elevato impatto determinato da tali patologie, sia acute che croniche, e gli elevati costi per i sistemi sanitari, richiedono lo sviluppo di nuove strategie terapeutiche. La questione principale riguardante gli attuali approcci terapeutici, sia farmacologici sia interventistici, è rappresentata dalla loro incapacità di compensare l’elevata ed irreversibile perdita di cardiomiociti funzionali. A causa della limitata capacità rigenerativa dei cardiomiociti post-natali e della difficoltà di reperire ed isolare tessuto cardiaco bioptico, scarse sono le fonti di tali cellule disponibili per uno studio dedicato. Tra l’altro, anche se i modelli animali ancora oggi rappresentano sicuramente lo stumento migliore per studiare e comprendere in vivo i meccanismi alla base dello sviluppo di specifiche patologie umane, nel constesto di un organismo complesso, essi non sono completamente predittivi e rappresentativi della condizione umana analizzata; da un punto di vista economico, il mantenimento di tali animali e le relative sperimentazioni, richiedono molto tempo e costi elevati. In questo scenario, le cellule staminali umane pluripotenti (hPSCs), comprese le cellule staminali embrionali (hESCs) e le cellule staminali pluripotenti indotte (hiPSCs), rivestono un ruolo importante nella ricerca cardiovascolare perché possono essere espanse in coltura indefinitamente, senza perdere la loro staminalità, e differenziare nelle cellule che componogono i tre foglietti germinativi, come ad esempio i cardiomiociti. Un’importante svolta nella ricerca scientifica è avvenuta nel 2007, con la scoperta delle hiPSCs da parte del Premio Nobel Shinya Yamanaka. Ciò ha rappresentato il punto di partenza per derivare hiPSCs paziente-specifiche attraverso il reprogramming di cellule somatiche ottenute con procedure mini- o non-invasive (derivate da biopsie cutanee, sangue, urina…), utili per generare tessuti per una riparazione autologa, evitando i problemi etici e politici relativi alla derivazione delle hESCs. Notevoli studi sono stati condotti dai ricercatori nel tentativo di sviluppare strategie che efficientemente ed in maniera robusta guidino il differenziamento cardiaco delle hPSCs, basate sulla perturbazione stadio-specifica di differenti vie di segnalazione, mediante l’uso di fattori di crescita e piccole molecole, che ricapitolano i punti essenziali dello sviluppo cardiaco osservato in vivo. Tuttavia, questi metodi sono accompagnati da alcune limitazioni, quali: elevata variabilità intra ed inter-sperimentale, presenza di xeno-contaminanti, componenti indefinite nei medium di coltura e differenze nei livelli di espressione di citochine endogene. Altre strategie si basano invece sulla conversione diretta di cellule somatiche, specialmente fibroblasti, attraverso l’overespressione di una combinazione di fattori di trascrizione cardiaci mediante vettori integrativi e non-integrativi; tuttavia, anche tali approcci sono caratterizzati da basse efficienze nella generazione di cardiomiociti, associate al rischio di integrazioni genomiche e mutagenesi inserzionale nel caso dei vettori integrativi, o alla necessità di effettuare diversi step di purificazione quando si ultilizzano sistemi non-integrativi. Pertanto, a causa delle difficoltà dei sistemi convenzionali di coltura nel dirigere specificamente ed in maniera robusta il differenziamento cardiaco delle hPSCs, assieme alla scarsa capacità di riprodurre in vitro l’ambiente in cui le cellule risiedono in vivo, i cardiomiociti prodotti attualmente sono immaturi e più simili allo stadio fetale di sviluppo. Nel 2010 Warren L. ed il suo gruppo di ricerca ha sperimentato per la prima volta una tecnologia innovativa di tipo non-integrativo basata su trasfezioni ripetute con lipidi cationici di RNA messaggeri modificati sinteticamente (mmRNA) per evitare la risposta immunitaria innata da parte delle cellule; egli ha dimostrato la possibilità sia di riprogrammare cellule somatiche allo stato pluripotente, sia di programmare il differenziamento miogenico di hiPSCs. Pertanto, lo scopo di questa tesi di dottorato è quello di sviluppare un metodo robusto ed efficiente per il differenziamento cardiaco di hPSCs combinando gli mmRNA con la tecnologia microfluidica. Ripetute trasfezioni di mmRNA codificanti per 6 fattori di trascrizione coinvolti nello sviluppo e nel funzionamento cardiaco, vengono impiegate per forzare l’espressione proteica endogena delle cellule e per guidare il differenziamento verso la maturazione funzionale dei cardiomiociti. L’integrazione del differenziamento cardiaco in una piattaforma microfluidica ad hoc, prodotta nel laboratorio BioERA, consente un controllo più preciso delle condizioni di coltura garantendo un’elevata efficienza di trasfezione degli mmRNA grazie all’elevato rapporto superficie/volume e permette la riproduzione in vitro di nicchie fisiologiche. Infatti, la miniaturizzazione consente di mimare al meglio le dinamiche cellulari che avvengono in vivo nel microambiente solubile. Le tecnologia microfluidica offre la possibilità di effettuare esperimenti combinati, multiparametrici e paralleli in una sola volta e con elevato rendimento a costi ridotti, non realizzabili nei macroscopici e costosi sistemi di coltura convenzionali. Il Capitolo 1 inizia con la definizione di medicina rigenerativa e introduce la complessità dello sviluppo cardiaco ed il network di fattori di trascrizione che cooperano durante questo processo. Viene poi descritto lo stato dell’arte relativo alle strategie per l’ottenimento di cardiomiociti da hPSCs e al transdifferenziamento cardiaco di cellule somatiche, insieme alle relative limitazioni e alle problematiche attuali da risolvere. Infine viene presentato lo scopo generale di questa tesi di dottorato. Il Capitolo 2 si focalizzerà sulle hPSCs (sia hES sia hiPS) impiegate durante questo progetto, descrivendo le caratterisatiche principali di tali cellule. Verrà inoltre presentato un protocollo di differenziamento cardiaco di hPSCs in monostrato che attualmente è considerato il gold standard per ottenere velocemente un’elevata resa di cardiomiociti contrattili in supporti di coltura convenzionali. Tale protocollo si basa sulla modulazione del pathway canonico di Wnt attraverso l’applicazione di due piccole molecole. Inoltre, una linea di hES, doppio reporter per 2 fattori di trascrizione cardiaci, verrà descritta ed impiegata in tutti gli esperimenti come strumento per monitorare l’andamento del differenziamento cardiaco delle hPSC. I risultati ottenuti in colture standard verranno mostrati. Il Capitolo 3 esaminerà lo stato dell’arte della tecnologia microfluidica nelle applicazioni di medicina rigenerativa, sottolineando i vantaggi derivanti dalla combinazione della microtecnologia con la biologia cellulare. Verrà successivamente descritta la fabbricazione della piattaforma microfluidica utilizzata, con la successiva ottimizzazione della coltura, espansione e differenziamento cardiaco gold standard delle hPSCs conseguenti alla conversione dalla macro- alla microscala. Il Capitolo 4 introdurrà la nuova strategia degli mmRNA per la riprogrammazione e la programmazione cellulare: anche in tal caso verrà discusso lo stato dell’arte. In seguito, verranno presentate le strategie sperimentali sviluppate per programmare il differenziamento cardiaco delle hPSCs verso un fenotipo più maturo dei cardiomiociti, insieme ai risultati ottenuti con le relative caratterizzazioni strutturali, funzionali e molecolari. In questo lavoro, per la prima volta, è stato possibile ottenere cardiomiociti da hPSCs attraverso ripetute trasfezioni di mmRNA per 6 fattori di trascrizione cardiaci in microfluidica, con efficienze superiori rispetto ai metodi presenti attualmente in letteratura, svolti in sistemi convenzionali. Il Capitolo 5 infine presenterà la discussione e le conclusioni generali, assieme alle prospettive future riguardanti l’uso degli mmRNA combinati con la microfluidica per ottenere diversi fenotipi di cardiomiociti, variando la combinazione di fattori di trascrizione veicolati. In conclusione, gli esperimenti sviluppati in questo progetto di dottorato forniscono un proof-of-principle della possibilità di programmare con gli mmRNA il destino delle hPSCs verso il differenziamento e la maturazione di cardiomiociti funzionali in microfluidica; inoltre, essendo gli mmRNA una strategia non-integrativa , i cardiomiociti ottenuti in questo modo possono essere impiegati nel prossimo futuro per applicazioni cliniche di ricostruzione tissutale autologa e per screening farmacologici personalizzati.

The transcriptional response to oxidative stress (OS) is involved in aging. As ROS-induced damages accumulate, cell senescence or apoptosis are triggered: these two mechanisms are implicated in the progressive physiological decay of the organism. Surprisingly, transcriptional pathways involved in OS-response play a role also in the balance between proliferation and differentiation during embryonic development. Recently, it has been discovered a transcriptional network that triggers cell cycle arrest in vitro upon OS. This pathway involves two well-known aging-associated genes: p53, with its short isoform Δ40p53, and p66Shc. Here we propose the use of two complementary model organisms, Danio rerio (zebrafish) and Nothobranchius furzeri, to unravel the p53/p66Shc pathway in vivo. We report here that Δ40p53 and p66Shc are conserved in these species, partially recapitulating mammalian functions. Moreover, our findings about the spatial and temporal regulation of p66Shc expression during zebrafish embryogenesis suggest that p66Shc has a role in neural development. Furthermore, we generated the first genetic model of Δ40p53 ablation in zebrafish, exploiting the CRISPR/Cas9 technology. The structure of the p53 locus in mouse does not allow to selectively knock-out Δ40p53 without depleting also p53 activity. Our Δ40p53-/- zebrafish model shows the unique opportunity to characterize the biological functions of Δ40p53 isoform in physiological development in a context where p53 expression is maintained unaltered. Finally, we provide preliminary data showing that Δ40p53 modulates in vivo p53-dependent transcriptional response to stress.

The regulation of actin cytoskeleton operated by RhoGTPases is crucial for neuronal morphogenesis, especially for neurite elongation and branching, synaptogenesis and synaptic plasticity. Dysregulation of RhoGTPases leads to neuronal dysfunctions including intellectual disability, schizofrenia and Alzheimer disease. Rac1 is a member of the RhoGTPase family and it has been demonstrated to positively regulate dendritogenesis and dendritic spines formation and maturation. As well as other RhoGTPases, Rac1 activity is regulated principally by two kinds of molecules: GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins). Arhgap22 protein is a specific Rac1-Gap that promotes the inactivation of Rac1. Although it was previously reported that Arhgap22 transcripts is present in murine brain, its functions in neurons have not been studied yet. Here, we reported that Arhgap22 is expressed in mouse brain in a precise spatio-temporal window. Moreover, taking advantage of an animal mouse model knock out (KO) for Arhgap22, we described the effects of its silencing in hippocampal neurons. In vivo, Arhgap22 disruption led to an increase level of activated Rac1 and its downstream pathways, with a subsequent increase in dendritic spine density in CA1 region of hippocampus. Additionally, Arhgap22 lacking mice presented reduced AMPA receptors in the post-synaptic density of excitatory synapses and this alteration was reflected by the impairment in the induction and mainteinance of long-term potentiation (LTP). Arhgap22 KO mice presented also defects in cognitive tasks and decreased anxiety-like behaviours. In a nutshell, the results of this work suggested that Arhgap22 is a key regulator of Rac1 signaling and that affects the maturation of excitatory synapses, synaptic plasticity and cognitive functions.

Approximately 10-15% of breast carcinomas are classified as triple receptor-negative breast cancer (TNBC) subtype because of the lack of expression of hormone receptors. Despite the advent of new therapeutic strategies, tumor relapses remain the major challenge in TNBC management. Several studies show that treatment failure and cancer recurrence are primarily due to drug resistance acquisition and self-renewal that are specific properties of cancer stem cells (CSCs). Here I show that a fasting mimicking diet (FMD) reduces the percentage of the staminal population in mouse models of TNBC, increasing cancer free survival, and that the mechanism through which it affects CSCs is glucose dependent and mediated, at least in part, by the down-regulation of the protein kinase A (PKA) pathway. Moreover, the use of RNA-seq analysis on TNBC tumor masses, after FMD, allowed the identification of druggable escape pathways, in particular PI3K/AKT, mTOR and CCND/CDK4-6 axis, activated selectively by differentiated cells. My results show that addition of FMD to inhibitors of these pathways promotes TNBC regression, leading to complete tumor shrinkage. Notably, FMD protects also from hyperglycemia induced by PI3K pathway inhibitors, preventing side effects associated with it. Taken together, these data indicate that FMD has wide but differential effects reaching normal as well as differentiated cancer cells and CSCs, thus representing a promising strategy for the treatment of TNBC, which can be hopefully translated into the clinic.

Many enveloped viruses complete their replication cycle by forming vesicles that bud from cellular membranes of different origin, but separation of virion from host membranes is not a trivial or spontaneous step. Several enveloped RNA viruses, such as retroviruses, rhabdoviruses, filoviruses, arenaviruses, and, probably, also ortho and paramyxoviruses, solve such a problem by coopting factors that cells usually employ during the formation of vesicles within specific endosome-derived organelles: the multivesicular bodies (MVBs). Actually, enveloped RNA viruses budding and formation of MVBs intraluminal vesicles are analogous processes: in both cases membranes must curve and bud away from (rather than into) the cytoplasm. Indeed, a productive infection requires that all the components necessary for the formation of infectious particles localize to the membrane at the site where budding will take place. To that end, RNA viruses evolved two possible strategies: the presence of special sequences named Late domains (L-domains) in their structural proteins and/or their ubiquitylation. In any case proteins involved are recruited to the MVBs pathway. Much less is known about herpesviruses and, in general, enveloped DNA viruses. In fact, even if the initial steps of the herpetic infection are quite well-known, the cellular site of assembly and pericapsid’s acquisition as well as the nature of the membranes involved in viral budding are not clear yet. The aim of this work was to elucidate the molecular mechanisms behind essential steps of herpesvirus replication, such as assembly and budding from infected cells. In particular, we used the -herpesvirus HSV-1 as a model. Two recent works pointed out a possible role of MVBs in HSV-1 replication: both virus gress and intracellular trafficking and maturation of the essential glycoprotein gB require functional biogenesis of MVBs. On the basis of those considerations, we evaluated whether MVBs membranes could be the recruiting site of other main structural proteins, such as the tegumental ones, in order to identify those organelles as the pericapsid’s acquisition and final assembly site of the viral particles. Most of all, we focused on four HSV-1 tegumental proteins: VP1/2, VP13/14, VP16 and VP22. Especially, we observed that both VP1/2 and VP16 possess sequences belonging to the L-domains motifs and that both VP1/2 and VP16 localize at the MVBs membranes. We also proved the interaction between the PSAP motif of theprotein and Tsg101, its cellular partner. We supposed that the recruitment of VP1/2 to the MVBs can be due, at least partially, to such interaction. Vice versa, the reason for the localization of VP16 are not as clear. We demonstrated that VP16 does not show any direct interactions between its PPLY motif and the corresponding cellular partners, the members of the Nedd4 ubiqutin-ligases family. Moreover, our data revealed that VP16 is not even ubiquitylated, a post-translational modification that would ensure its sorting to the MVBs. Supposing that its recruitment to the MVBs was indirect and due to other viral proteins, we verified two of the interactions ascribed to VP16 and other tegument proteins in the literature, especially those related to VP13/14 and VP22. We confirmed that VP16 direct interacts with VP22, in the absence of other viral factors, but not with VP13/14. Still, that interaction did not explain the intracellular localization of VP16 that could require viral and/or cellular factors other than those examined. Last, by a deeper investigation of both VP22 and VP13/14, we demonstrated that each of those proteins is ubiquitylated and that the ubiquitylation of VP13/14 is specific for the targeting to the MVBs pathway. Concluding, our data showed that at least four HSV-1 tegumental proteins are recruited to the MVBs or own the necessary features for such an intracellular localization, that are L-domains or ubiquitin conjugation. So it is possible to suppose that MVBs could provide the appropriate platform for the tegument assembly and the acquisition of the pericapsid as well as a possible exit from the infected cell.
Molti virus dotati di envelope completano il proprio ciclo replicativo formando delle vescicole che gemmano attraverso membrane cellulari di varia natura, ma la scissione del virione da tali membrane non è un passaggio semplice o spontaneo. Per quel che riguarda diversi virus a RNA, tra cui retrovirus, rabdovirus, filovirus, arenavirus e, presumibilmente, orto- e paramixovirus, tale problema è stato risolto mediante il reclutamento di fattori normalmente utilizzati dalla cellula durante la formazione di vescicole interne a specifici organelli membranosi derivati dagli endosomi: i multivesicular bodies (MVB). In effetti, la gemmazione dei virus a RNA dotati di envelope e la formazione delle vescicole interne ai MVB sono processi analoghi: in entrambi i casi si verifica una curvatura della membrana in allontanamento dal citoplasma. Affinché un’infezione possa considerarsi produttiva, quindi, è necessario che tutti i componenti utili alla formazione della particella siano convogliati a livello della membrana in cui si verificherà l’evento di gemmazione. A questo scopo, i virus a RNA hanno evoluto due possibili strategie: la presenza di particolari sequenze note come Late domain (L-domain) all’interno delle proprie proteine strutturali e/o la loro ubiquitinazione; in entrambi i casi le proteine coinvolte vengono reclutate nel pathway dei MVB. Molto meno è noto, invece, per quel che riguarda gli herpesvirus e, più in generale, i virus a DNA dotati di envelope. Infatti, se da un lato le fasi iniziali dell’infezione erpetica sono piuttosto conosciute, dall’altro rimangono aperte alcune controversie riguardanti il sito cellulare di assemblaggio e acquisizione del pericapside nonché la natura delle membrane implicate nella gemmazione della particella virale dalla cellula infetta. Lo scopo di questo lavoro è stato quello di contribuire al chiarimento dei meccanismi molecolari dell’assemblaggio e della gemmazione degli herpesvirus, prendendo come modello il virus dell’herpes simplex di tipo 1 (HSV-1). In particolar modo, due lavori pubblicati nell’ultimo periodo hanno evidenziato un possibile ruolo dei MVB nel ciclo replicativo di HSV-1 sottolineando l’importanza di una corretta biogenesi di tali organelli per garantire la gemmazione virale e il corretto trafficking intracellulare di una proteina virale essenziale, quale la glicoproteina B. Sulla base di tali riscontri si è deciso di valutare se le membrane dei MVB costituissero il sito di reclutamento di altre importanti proteine strutturali, quali quelle del tegumento, cosìda poter identificare tali organelli come il sito di acquisizione del pericapside e di assemblaggio definitivo della particella virale. Nel presente lavoro ci siamo focalizzati soprattutto su quattro proteine del tegumento di HSV-1: VP1/2, VP13/14, VP16 e VP22. In particolar modo, abbiamo dimostrato che VP1/2 e VP16 presentano al proprio interno sequenze riconducibili a L-domain noti e che sia VP1/2 che VP16 localizzano a livello delle membrane dei MVB. Il reclutamento di VP1/2 a livello di tali organelli può essere attribuito, almeno in parte, all’interazione tra il dominio PSAP in essa contenuto e la proteina Tsg101, suo partner cellulare, con cui abbiamo dimostrato l’associazione. Viceversa, le cause della localizzazione di VP16 non sono altrettanto chiare. Infatti, abbiamo dimostrato che VP16 non presenta alcuna interazione diretta tra il dominio PPLY in essa presente e i corrispondenti partner cellulari, i membri della famiglia delle ubiquitino-ligasi Nedd4. Inoltre, in base ai nostri dati, la medesima proteina non risulta nemmeno ubiquitinata, modifica post-traduzionale che ne garantirebbe il direzionamento ai MVB. Nell’ipotesi che il suo reclutamento ai MVB sia di tipo indiretto e dovuto all’azione di altre proteine virali, abbiamo quindi verificato alcune tra le interazioni riportate in letteratura per quel che riguarda VP16 e le altre proteine del tegumento, in particolare VP13/14 e VP22. Dai nostri esperimenti è emerso che VP16 interagisce direttamente con VP22, in assenza di altri fattori virali, ma non con VP13/14. Tuttavia, nemmeno il legame con VP22 è risultato tale da giustificare la localizzazione intracellulare di VP16, che quindi potrebbe richiedere fattori virali e/o cellulari diversi da quelli analizzati. Infine, mediante studi più approfonditi su VP22 e VP13/14, abbiamo dimostrato che entrambi tali proteine risultano ubiquitinate e che, nel caso specifico di VP13/14, tale ubiquitinazione è del tipo generalmente responsabile del direzionamento di una proteina al pathway dei MVB. In conclusione, i nostri dati dimostrano che almeno quattro proteine del tegumento sono effettivamente reclutate ai MVB o possiedono le caratteristiche necessarie ad una simile localizzazione, quali la presenza di L-domain o la coniugazione all’ubiquitina. E’ quindi possibile supporre che tali organelli, oltre a rappresentare una potenziale via d’uscita dalla cellula infettata, possano fornire anche la “piattaforma” cellulare adatta al completamento dell’assemblaggio del tegumento e all’acquisizione del pericapside da parte della particella virale.

Background: A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. Recent studies have shown that red blood cells (RBCs) participate in NO formation in the bloodstream. Aim: The aim of this study was to assess the L-arginine (Arg)/NO pathway and the oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Materials and Methods: Analytes involved in Arg/NO pathway and the ratio between the oxidized and the reduced forms of glutathione, as index of oxidative stress, were measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The arginase and the NO synthase (NOS) expression were assessed by immunofluorescence staining. NOS activity was evaluated by ex-vivo experiments through the conversion of L-[15N2]arginine to L-[15N]citrulline. Results: Both MVA and CAD patients showed alterations in the ability of RBCs to produce NO, based on an increase of NO synthesis inhibitors, parallel to that found in plasma, a reduction of NOS expression and activity and an increased arginase expression. When summary scores of NO synthesis and of oxidative stress were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. Conclusions: This finding points out to an impairment of the capacity of RBCs to produce NO in pathological conditions characterized by alteration at the microvascular bed with/without no significant coronary stenosis.

The hypoxic condition determines several functional consequences that ultimately lead to cellular death and irreversible damage to cardiac myocytes. Under hypoxic condition, cells activate several protective pathways; among them, HIF-1α plays a key role in controlling cellular response to hypoxia at molecular level. However, HIF-1α regulatory mechanisms are extremely complex. On these premises, the present work was based on the hypothesis that NEU3 sialidase, a glycolytic enzyme ubiquitously expressed over the plasmatic membrane, can have a regulatory activity on HIF-1α expression in hypoxic/ischemic cardiac myocytes. The experiments performed in this in-vitro model allowed us to draw the following conclusions: 1) Endogenous NEU3 sialidase expression and activity are up-regulated in murine skeletal muscle cells (C2C12) upon oxygen starvation, leading to a signaling cascade resulting in the activation of HIF-1α. 2) Moreover, induced overexpression of NEU3 significantly increases HIF-1α expression and cell resistance to hypoxic stress, whereas NEU3 silencing causes the opposite effects and renders myoblasts more susceptible to apoptosis. 3) The hypoxia-driven activation of NEU3 sialidase can activate the EGFR prosurvival signaling pathway by controlling the content of ganglioside GM3. Furthermore, we demonstrated that NEU3 overexpression causes a reduction of ganglioside GM3, which is known to block EGFR autophosphorylation. Then resulted were extended from skeletal muscle to cardiac myocytes, particularly aiming to ascertain the role of NEU3 in activating the human cardiomyocyte response to hypoxia. Particularly, we evaluated if NEU3 activation occurred in human cardiomyocytes using two different models: 1) A model of acute cardiac ischemia achieved during aortic cross-clamp time and extracorporeal circulation in adult patient submitted to cardiac surgical procedures. 2) A model of chronic hypoxia in neonates and young patients operated for cyanogen congenital cardiac defects. In the acute model of cardiac ischemia, we harvested a sample of right atrial appendage just before and after aortic cross-clamping, during routine adult cardiac surgery procedure. However, no significant activation of NEU3 and HIF-1α was evident in cardiac sample harvested before and after aortic cross-clamping. In our opinion there are several possible explanations for the lack of NEU3 and HIF-1α increased expression in the cardiac surgery model. First, it is possible that in the in-vivo setting the mean aortic cross-clamp time was too short (mean time = 79 minutes) to elucidate the same response that we observed in the in vitro model, where the cells were incubated under hypoxic conditions for at least 12 hours. Secondly, and most important in our opinion, the technique of myocardial protection, especially cardioplegic arrest and hypothermia, by protecting the myocardium from the ischemic injury could have limited NEU3 and HIF-1α expression in our samples. To overcome these limitation, in the final part of my PhD program we evaluated HIF-1α and NEU3 expression in a human in-vivo model of chronic cardiac hypoxia, studying patients affected by cyanotic cardiac defects submitted to surgical correction. In this model of chronic hypoxia, we observed a significant increase in NEU3 expression and activity in cyanotic patients. Furthermore, a significant increase of EGFR was observed, supporting the hypothesis that this signaling pathway is upregulated by the sialidase NEU3. Indeed we observed an increase in expression of genes downstream of EGFR, both related to cellular proliferation (ERK and p38) and to apoptosis resistance (AKT and p70S6K). Finally we observed a significant activation of HIF-1α and of its downstream genes. Another important aspect of cellular adaptation to hypoxia is the metabolic switch between oxidative and glycolytic metabolism, the so-called “Pasteur effect”. In the present study we found that the glycolytic enzymes Glucose transporter Glut1, the Aldolase and the GAPDH were significantly enhanced in the cyanotic group which in turn demonstrates that the myocardium of patients affected by cyanogen cardiac defects is metabolic adapted to chronic hypoxia. In conclusion, the results of this PhD project support the hypothesis of a physiological role of NEU3 in mediating cellular response to hypoxic stress. It is interesting to underline that NEU3 activation is mediated by ganglioside GM3 on cellular membrane. Indeed, an increase in NEU3 level determines a reduction of GM3, which is a well know inhibitor of EGF receptor. On these premises, to mimic the effects of NEU3 activation, it could be possible to inhibit GM3 synthesis, in example by the selective inhibition of the sialyltransferase involved in the last passage of its synthesis. In this direction, our laboratory is performing some experiments with small chemical molecules, designed for blocking selectively the GM3 synthesis with the aim of activating the endogenous response to hypoxic stress.

Misfolded/mutated proteins are identified by the endoplasmic reticulum (ER) quality control system (ER-QC) and eliminated through the ER-associated protein degradation (ERAD) pathway. The presence of structural defects identifies these proteins as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. ER-associated E3 ubiquitin ligases are key components of the ERAD machinery, and distinct E3 ligases seem to control specific ERAD pathways. In human muscles, mutations on sarcoglycans, proteins that form a tetramer complex (alpha-beta-gamma-delta) associated to dystrophin, lead to a Limb Girdle Muscolar Dystrophy (LGMD). It has been demonstrated that the V247M alpha-sarcoglycan mutant, a type I membrane protein with a luminal defect, is ubiquitinated and degraded by the proteasome, the last ERAD component. Aim of this project is to investigate ERAD components involved in recognition, ubiquitination and retrotraslocation of the mutated protein. The attention has been focused particullary on the E3 ligases that seem to be crucial factors assuring both selectivity and specificity to ERAD pathways. Emerging literature regarding several diseases involving mutated/misfolded proteins augurs well for treatments that involved common or peculiar ER-QC and ERAD steps. Due to the fact that until now, there is no known therapy for muscular dystrophies, I believe this study is relevant both to disclose an important biological process but also to identify possible molecular targets to treat sarcoglycanopathies. A set of ER chaperones and lectins recognize and transport misfolded proteins to the site of dislocation. Among these, I have demonstrated that the chaperone GRP94 is probably involved in V247M mutant recognition, while BiP and OS9 do not seem to be implicated, however, their role is still under investigation. Usually gp78 and HRD1 are the E3 ligases involved in the ERAD-L pathway of model substrates. By using mutated variants of, or siRNAs for these E3 candidates, my results show that HRD1, but not gp78, is specifically involved in the disposal of the alpha-sarcoglycan mutant. In addition, the E2 ubiquitin-conjugase enzyme UBC6e and the cargo receptor SEL1L, well-known HRD1 partners, cooperate in the disposal of V247M alpha-SG. Immunoprecipitation experiments validated the interaction of HRD1, UBC6e and SEL1L with the alpha-SG mutant. Moreover another E3 ligase, RFP2, is involved in the degradation of V247M alpha-SG being able both to co-immunoprecipitate with the protein and to block its disposal, if a mutated variant is expressed. The driving force to eradicate misfolded membrane proteins from the ER is usually provided by the AAA-ATPase p97. My experiments demonstrated that p97, together with the ER-associated Derlin-1, form the so-called "dislocon", the proteinaceous environment for the hydrophilic luminal domain of V247M mutant to cross the ER membrane. These results describe for the first time the ERAD pathway for the disposal of the type I membrane protein V247M alpha-sarcoglycan, a pathway leaded by the E3 ligase HRD1. Moerover, recognition and delivery to proteasome of this mutant is also assisted by the E3 ubiquitin ligase RFP2. In collaboration with Dr. R. Sacchetto (Dept. of Veterinary), I also carried out a study aimed to check whether the R164H mutant of the polytopic membrane protein SERCA1a is also an ERAD client. Mutations in SERCA1a are responsible for Brody Disease, a human inherited congenital disorder that affects skeletal muscles, because of SERCA1a loss of function. A similar muscular disorder, named congenital Pseudomyotonia, has been described in the Italian Chianina cattle. In affected animals and Brody’s patients, decreased calcium ATPase activity perfectly correlates with reduced expression of SERCA1a protein. I demonstrate that inhibition of proteasome not only rescued the expression of R164H SERCA1a mutant transfected in HEK-293 cells, but also restored the enzymatic activity.
Le proteine non correttamente ripiegate e/o mutate sono riconosciute dal sistema di controllo qualità del reticolo endoplasmatico (ER) (ER-QC) ed eliminate attraverso una specifica via, chiamata degradazione associata al reticolo (ERAD). La selezione avviene attraverso l’identificazione dei difetti strutturali da parte di specifiche proteine coinvolte nel controllo qualità del reticolo. La degradazione conseguente può essere suddivisa in tre vie: ERAD-L per proteine con difetti luminali, ERAD-M per difetti nella porzione di membrana ed ERAD-C in caso di difetti citosolici. Le E3 ubiquitin-ligasi risiedenti nel reticolo sono elementi chiave di ERAD e ciascuna sembra agire prevalentemente in una particolare via piuttosto che in un’altra, assicurando quindi lo specifico riconoscimento e smaltimento di proteine aventi difetti particolari, anche se non è ancora chiaro nei dettagli come ciò avvenga. Nel muscolo, mutazioni a carico di proteine associate in un complesso tetramerico alla distrofina, chiamate sarcoglicani (alpha-beta-gamma-delta), causano nell’uomo distrofie muscolari chiamate distrofie dei cingoli. E’ stato precedentemente dimostrato che il mutante di alpha-sarcoglicano V247M, una proteina di tipo I con un difetto luminale, viene ubiquitinato e degradato dal proteasoma, tappa finale di ERAD. Scopo di questo studio è indagare i componenti del sistema degradativo atti a riconoscere questa proteina come mutante e le E3 ligasi coinvolte nella sua ubiquitinazione, processo fondamentale per l’indirizzamento al proteasoma. Il fine ultimo è chiaramente quello di individuare possibili bersagli farmacologici per lo sviluppo di una possibile terapia, non presente attualmente. Numerose patologie dovute a problemi di ripiegamento/mutazioni di proteine stanno emergendo, e con esse anche possibili trattamenti farmacologici che agiscono sui pathways di ER-QC ed ERAD. Nel reticolo le proteine malripiegate e/o mutate sono ritenute in soluzione da chaperoni e lectine, che giocano un ruolo indispensabile in ER-QC e nel delivery dei substrati ai complessi formati dalle E3 ligasi. Tra questi ho dimostrato che GRP94 è coinvolta nel riconoscimento di V247M alpha-SG, mentre BiP e OS9 non sembrano implicati, anche se ulteriori studi a questo riguardo risultano necessari. In letteratura i substrati modello di proteine di tipo I con difetti luminali vengono degradati grazie all’azione di due E3 ligasi, chiamate gp78 e HRD1. Attraverso l’uso di E3 ligasi mutate nel sito catalitico e di RNAi ho dimostrato che V247M alpha-SG è degradato grazie all’azione di HRD1 ma non di gp78. Mediante saggi di immunoprecipitazione, ho anche dimostrato che HRD1 interagisce strettamente con il sarcoglicano mutato così come UBC6e, l’ubiquitin-coniugasi partner di HRD1, e SEL1L, il recettore associato alla ligasi. Un’altra ligasi, RFP2, si è dimostrata in grado sia di interagire con il sarcoglicano mutato, sia di bloccarne la degradazione se deleta del sito catalitico coinvolto nell’ubiquitinazione. Per permettere a molte delle proteine di membrana di essere estratte dal reticolo e dirette al citosol, dove risiede il proteasoma, la AAA-ATPasi p97 sembra avere un ruolo fondamentale, grazie alla forza motrice fornita dall’idrolisi dell’ATP e il supporto dato da Derlina-1. Ho dimostrato, grazie all’uso di un dominante negativo, che l’azione di p97 è necessaria per la retrotraslocazione di V247M alpha-SG e, grazie ad esperimenti di co-immunoprecipitazione, che il mutante interagisce strettamente sia con p97, sia con Derlina-1. Questi risultati descrivono per la prima volta il pathway degradativo di alpha-sarcoglicano V247M, che, in qualità di proteina di membrana di tipo I con difetti nella porzione luminale, segue la via classica ERAD-L guidata dall’E3 ubiquitin-ligasi HRD1. Nel processo di riconoscimento e indirizzamento al proteasoma questo mutante è inoltre assistito dalla E3 ubiquitin-ligasi RFP2. In un progetto in collaborazione con la Dr. R. Sacchetto (Dip. Scienze Sperimentali Veterinarie), mi sono occupata di un’altra proteina, SERCA1a. La proteina mutata è causa nell’uomo della miopatia di Brody e recentemente un fenotipo muscolare simile, chiamato Pseudomiotonia, è stato riscontrato anche in alcune vacche Italiane di razza Chianina che presentano la mutazione R164H a carico di SERCA1a. Sia nell’uomo che negli animali, la riduzione della attività della calcio ATPasi SERCA1a è correlata alla ridotta quantità di proteina presente nel muscolo. L’espressione eterotopica di SERCA1a mi ha permesso di dimostrare che la proteina è degradata attraverso il proteasoma: l’inibizione dell’attività degradativa porta, infatti, a un aumento della quantità di proteina. Oltre ciò, studi funzionali mi hanno permesso di dimostrare che la proteina così recuperata è anche enzimaticamente attiva, indice del fatto che un suo recupero farmacologico potrebbe risultare efficace non solo nei casi di Pseudomiotonia ma anche in quelli di miopatia di Brody.

Abstract: Notch signaling is prominently involved in cell fate decision and growth regulation in metazoan tissues. Because of this, Notch is often upregulated in cancer and current efforts point to developing drugs that block its activation. Notch receptor endocytosis towards acidic compartments is a recently appreciated determinant of signaling activation. The Vacuolar H+ ATPase (V-ATPase) is responsible for acidification of endocytic organelles and recently it has been shown that mutants in V-ATPase subunit encoding genes in model organisms display loss of Notch signaling phenotypes. In the first part of my graduate studies, we aimed at discovering whether pharmacologic reduction of V-ATPase activity affected Notch signaling. We found that administration of BafilomycinA1 (BafA1), a highly specific V-ATPase inhibitor decreases Notch signaling during Drosophila and Zebrafish development, and in human cells in culture. In normal breast cells, we have found that BafA1 treatment leads to accumulation of Notch in the endo-lysosomal system, and reduces its processing and signaling activity. In Notch-addicted breast cancer cells, BafA1 treatment reduces growth in cells expressing membrane tethered forms of Notch, while sparing cells expressing cytoplasmic forms. In contrast, V-ATPase inhibition reduces growth of leukemia cells, without affecting Notch activating cleavage. However, consistent with the emerging roles of V-ATPase in controlling multiple signaling pathways, in these cells Akt activation is reduced, as it is also the case in BafA1-treated breast cancer cells. Our data support V-ATPase inhibition as a novel therapeutic approach to counteract tumor growth sustained by signaling pathways regulated at the endo-lysosomal level. The functions of Notch throughout the life of an individual are varied and complex. This complexity is not sufficiently accounted for by the limited core of known Notch signaling components and a growing body of evidence attributes it to additional factors that determine whether, when and how Notch functions within a given context. Considering this, in the second part of my graduate work, we sought to identify novel genes that might influence Notch. Thus, we performed a high content immunofluorescence-based RNA interference screen of a pharmacologically-relevant subset of the human genome. To this end, we monitored how knockdown of specific genes perturbs the localization of the Notch-1 receptor in human breast cells under resting and signaling conditions. Here we present the screen setup, the primary screen results and the candidate follow-up strategy.

Synopsis: Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life is of paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. The Notch pathway plays a key role in the regulation of cardiomyocyte proliferation during mammalian embryonic life. Moreover, it is essentially involved in the cardiac regeneration process after injury in Zebrafish. Therefore, we assessed the efficacy of Notch pathway activation to sustain cardiac regeneration in a model of myocardial infarction in mice. During early postnatal life, cardiomyocytes exit the cell cycle. We demonstrated that this event is paralleled by a decrease of Notch signaling and by the establishment of a repressive chromatin environment at Notch target genes, characterized by Polycomb Group protein 2-mediated silencing. The stimulation of the Notch pathway through Adeno-associated virus-mediated gene transfer of activated Notch1 or of the soluble form of the ligand Jagged1 prolonged the capacity of cardiomyocytes to replicate, which correlated with an increased rate of Notch target gene expression and the maintenance of an open chromatin conformation at Notch target gene promoters. However, the same vectors were ineffective in stimulating cardiac repair in a model of myocardial infarction in adult mice, despite efficient transgene expression. We identified the molecular cause of the lack of action of Notch signaling stimulation in adults in the increased DNA methylation at Notch target gene promoters, which correlated with permanent switch off of the Notch pathway. Our results confirm that the Notch pathway is an important regulator of neonata adults, due to the permanent epigenetic modifications at the DNA level at Notch responsive genes l.

In order to protect the body from a wide range of harmful environmental agents, the intestine has developed a number of barrier mechanisms to limit the entry of potential hazards. These include the physical barrier formed by the epithelial layer and the intestinal immune system that is important to induce either tolerance against food antigens and intestinal flora or inflammatory responses against dangerous microorganisms. It has been demonstrated that tolerance against commensal bacteria is strictly compartmentalized, in the sense that the systemic immune system is completely unprimed by these bacteria. It was demonstrated that the mLNs function as a “firewall” confining induction of tolerance to the mucosa while the systemic immune system remains ignorant to these bacteria. However, in these studies how the bacterial flora is excluded from the entrance in the bloodstream via the intestinal blood vessels has not been analyzed. Here, we describe a new barrier that we called the GVB (gut vascular barrier) that plays a fundamental role in controlling the spreading of molecules and bacteria to systemic sites. We found that intestinal endothelial cells (ECs) express the main components of TJs (occludin, JAM-A, CLDN-12, ZO-1 and cingulin) and AJs (VE-cadherin and junctional β-catenin), indicating the presence of a barrier that excludes bacteria from passing through the paracellular route. In addition, we observed the existence of a “gut vascular unit” (GVU) whereby ECs were associated with enteric glial cells and pericytes, whose role in the establishment of the endothelial barrier phenotype remains to be analyzed. Moreover, we show that GVB integrity could be modified by Salmonella typhimurium infection. Indeed, upon infection ECs up-regulated the expression of PLVAP, that has been previously used as a marker of immature/damaged vascular barrier in the brain, and up-regulated caveolin-1, the major component of caveolae. These changes correlated with a higher permeability of the endothelium to small molecules and to bacteria. One way by which S. typhimurium could modify the barrier properties of the intestinal blood vessels could be through the negative regulation of the Wnt/β-catenin signaling pathway. Indeed, we found that the activation of β-catenin was reduced upon Salmonella infection in vitro. Consistently, we found that Salmonella was incapable to modify ECs permeability and to spread systemically in mice where β-catenin was constitutively activated by genetic means only in vascular ECs. Furthermore, it appeared that the TTSS encoded by Salmonella pathogenicity island-2 was involved in the regulation of Wnt/β-catenin signaling pathway in ECs. Finally, preliminary results show that the microbiota could induce GVB maturation and maintenance. However, the mechanisms involved in these processes as well as the bacterial species responsible for this process have not been investigated yet.

Polycomb group proteins (PcG) are among the most important gatekeepers that ensure the correct establishment and maintenance of cellular identity in metazoans. This occurs by modifying chromatin through the activity of two Polycomb Repressive Complexes (PRC1 and PRC2) that deposit H2A ubiquitylation and H3K27 methylation respectively, in order to guarantee repression of their target genes. Although the development of PRC2 inhibitory compounds is becoming a very promising strategy for specific cancer treatment, the controversial role of PcG proteins, acting as oncogenes or tumor suppressors in a tissue/cancer specific manner, prompt us to further investigate the role PcG proteins in regulating adult tissue homeostasis. Using different genetic models, we have found that PRC1 activity is required for the integrity of the mouse intestinal epithelia. More in detail, PRC1 activity is required for the self-renewal of the intestinal stem cells (ISCs) via a cell-autonomous mechanism that is independent of Ink4a-Arf expression. Using high-throughput transcription and location analysis, we have dissected the direct transcriptional pathways regulated by PRC1 in ISC showing that PRC1 inactivation induces a loss of ISC identity as a result of a massive up-regulation of non-lineage specific transcription factors that can directly inhibit the transcriptional activity of the ß-Catenin/Tcf4 complex. Overall, we propose that PRC1 control the self-renewal of ISC by positively sustaining Wnt transcriptional activity also in the presence of oncogenic mutations that constitutively activate the Wnt pathway in intestinal tumors.

The epidermal growth factor receptor (EGFR) can be internalized through different routes. While clathrin-mediated endocytosis destines EGFR for recycling and signaling, internalization through non-clathrin endocytosis (NCE) targets the receptor for degradation. Since NCE appears to be a major negative regulator of EGFR levels, a more complete picture of this pathway would likely reveal new insights into aberrant EGFR signaling observed in many types of cancer. By combining a candidate gene approach with an unbiased proteomic approach, we have defined EGFR-NCE as molecularly distinct from other NCE pathways, relying on functional regulators not previously implicated in endocytosis. We found that reticulon 3 (RTN3), an endoplasmic reticulum (ER)-resident protein, is fundamental for NCE-mediated EGFR internalization, and that its ablation delays EGFR degradation, demonstrating that the NCE pathway is a critical regulator of the EGF-dependent cellular response. We show that, upon stimulation with high dose of EGF, RTN3 is localized in close proximity to EGFR and that it is crucial for the formation of contact sites between the ER and the plasma membrane (PM), which are needed for NCE to proceed efficiently. We also show that ER contact sites are involved in local Ca2+ release: high EGF doses induce a release of Ca2+ from the ER to the PM, which is strongly inhibited upon knockdown of RTN3. This calcium release depends on the inositol trisphosphate (IP3) cascade and is essential for the internalization of EGFR via NCE. In conclusion, we have discovered a new clathrin-independent endocytic pathway that relies on the action of RTN3. RTN3 is necessary for the formation of contact sites between the ER and EGFR-NCE sites at the PM, which are required for IP3R-dependent local calcium release and the completion of EGFR internalization through NCE.

This thesis is divided in three sections; the main project is described in the first part, while additional projects are developed in two appendixes. In the main project we studied YAP, the downstream effector of the Hippo pathway, a transcriptional co-factor that plays a fundamental role in de-differentiation, cell proliferation and transformation. While its upstream regulation has been extensively studied, its role as transcriptional co-factor is still poorly understood. We show that YAP co-adjuvates the transcriptional responses of Myc oncogene to promote cell proliferation and transformation; when both YAP and Myc are overexpressed, YAP is recruited on genomic sites pre-marked by Myc, TEAD and active chromatin and potentiate the expression of cell cycle genes regulated by Myc. In addition, we show that YAP promotes cell de-differentiation by antagonizing in cis the expression of liver-specific genes controlled by HNF4A master regulator, thus providing a mechanism on how YAP can revert the phenotype of a differentiated hepatocyte into a progenitor cell. In the first appendix we explain the mechanism of BRD4 inhibition, a promising strategy for the treatment of Myc-driven tumors. The efficacy of this strategy relies on the control of transcriptional elongation mediated by BRD4 on gene promoters, independently of the downregulation of Myc oncogene. Although the inhibition of BRD4 causes its genome-wide displacement on promoters, the effects on transcription are restricted to a subset of sensitive genes. This specificity relies on the fact that while most genes compensate the drop in elongation caused by BRD4 inhibition with further recruitment of RNA Pol2 on promoters and maintain a proficient mRNA transcription, vulnerable genes are not able to promote these compensatory effects, because RNA Pol2 recruitment on these promoters is already maximized. Our results show how the impairment of elongation genome-wide can affect specific transcriptional programs. In the second appendix we describe a new web application, Chrokit, aimed at analyzing genomic data in a fast and intuitive way. Chrokit handles a set of genomic regions of interest and performs several tasks on them, such as selecting particular subsets, computing overlaps and visualize reads enrichment of specific chromatin features interactively. The application is multiplatform and can be run on dedicated servers to maximize computational power and provide accessibility to multiple users simultaneously.

One way to regulate protein functions is by post-translational modification. Post-translational modifications have an important role in the regulation of biological activity of the protein because they allow both to extend the range of functions of a protein and to monitor the activity and determine the activation or inactivation of a protein. The most common protein post-translational modifications include ubiquitylation, phosphorylation and acetylation play an essential role in cellular functions such as cellular differentiation, apoptosis, DNA repair, antigen processing, and stress response. Under particular conditions abnormal post-translational modifications were found in many diseases like: Alzheimer’s disease, Parkinson’s disease, induction of different cancer and others. These abnormal post-translational modifications are permanent and can cause loss or alteration of protein function by changing enzyme activities or capacity aggregation (Stadtman and Levine 2000; Shacter 2000). p63 protein stability is regulated by different protein modifications such phosphorylation, ubiquitylation and sumoylation. p63 is known to be degraded by ubiquitin-mediated proteasomal degradation, the E3 ubiquitin ligase NEDD4-like, ubiquitin protein ligase Itch and ubiquitin-like protein SUMO-1 have been shown to directly interact with p63 and regulate p63 protein stability (Ghioni et al. 2005; Rossi at al. 2006; Rossi et al. 2006) suggest the importance of regulating p63 to tune its biological activity. During my PhD thesis we found three novel and distinct mechanisms that are involved in the regulation of the p63 protein levels; all these mechanisms induce p63 degradation. We demonstrated that these mechanisms are relevant in different physiological contexts and that they are involved in the regulation of p63 biological function. 1. MDM2-Fbw7 pathway contribute to reduce ΔNp63α protein levels during keratinocytes differentiation and upon DNA-damage induced by UV exposure and adriamycin treatment. 2. TRIM8 plays a role in enhancing p53 anti-oncogenic activity and at the same time down-modulate oncogenic ΔNp63α activity. 3. Hipk2 phosphorylates and promotes proteasomal degradation of ΔNp63α to enable an effective DNA-damage response induced by genotoxic drugs. All these evidences indicate that regulation of p63 protein stability is a key mechanism to control p63 activities, in particular during epithelia differentiation and in response to genotoxic agents. The knowledge and the identification of the molecular mechanisms governing p63 regulation under physiological context might be fundamental for understanding the pathogenesis of human syndromes associated to p63 mutations and the mechanism by which p63 promotes disease development. We hope that future studies focusing on the mechanisms involved in p63 protein regulation might increase our knowledge on the p63 role in tumorigenicity and in response to anti-cancer therapy to improve anti-cancer therapies.

Il tumore polmonare a piccole cellule (SCLC), detto anche microcitoma, costituisce circa il 20% dei tumori polmonari e rappresenta una delle istologie più aggressive nell’uomo in quanto è caratterizzato da una iniziale responsività alla chemio e alla radioterapia, a seguito della quale sviluppa velocemente resistenza ai trattamenti. Negli ultimi anni l’efficacia nel colpire i meccanismi molecolari che sono alla base dell’insorgenza e dello sviluppo dei tumori nell’uomo, ha spinto i ricercatori ad identificare e caratterizzare sempre nuove proteine che potessero fungere da bersaglio molecolare per la cura dei tumori polmonari. Gli scarsi dati in tal senso sui microcitomi polmonari sono dovuti alla sua elevata complessità molecolare ed alla scarsa disponibilità del materiale biologico da analizzare in una tipologia di tumore normalmente non destinato all’intervento chirurgico. La cascata cellulare regolata da KEAP1 ed NRF2 è un meccanismo conosciuto di difesa che le cellule adottano in condizioni di normalità per rispondere allo stress ossidativo e al possibile danno prodotto dalle sostanze tossiche e xenobiotiche con le quali entrano in contatto. Nelle cellule tumorali, questa cascata cellulare viene alterata ed è alla base della resistenza ai trattamenti chemio e radioterapici. La correlazione tra le alterazioni molecolari di questa cascata ed i tumori polmonari non a piccole cellule (Non Small Cell Lung Cancer, NSCLC) è ben nota e l’alterata espressione dell’NRF2 si correla alla prognosi dei pazienti e alla risposta alla chemio e alla radioterapia. D’altra parte, la crescita e la progressione dei tumori polmonari sembra coinvolgere ed intersecarsi con altre pathway ben note nel contesto dell’invasività cellulare e metastatizzazione, come quella di NOTCH, con importanti ricadute in ambito farmacologico. Attualmente, i dati riguardanti eventuali meccanismi di alterazione genetica ed epigenetica della cross-talk molecolare tra le pathway KEAP1/NRF2 e NOTCH risultano solo parziali e poco indagate nel microcitoma polmonare. Il lavoro di tesi si propone di ampliare le conoscenze dei meccanismi di intersezione tra KEAP1/NRF2 e NOTCH e delle basi genetiche ed epigenetiche della deregolazione della cross-talk nei microcitomi polmonari. Ulteriore finalità sarà quella di indagare mediante esperimenti in vitro gli effetti prodotti da queste ultime sulla risposta a terapie convenzionali e inibitori di NOTCH. Tutte le analisi sono state condotte su una collezione di linee cellulari di SCLC sulle quali è stato realizzato in primis il profilo molecolare dei geni KEAP1, NFE2L2 e NOTCH1 ed epigenetico della regione promotore del gene KEAP1. In particolare, per le determinazioni epigenetiche è stata implementata la già descritta metodica della PCR quantitativa metilazione-specifica in real-time (RT-QMSP, real time-quantitative methylation specific PCR) con saggi di pirosequenziamento. E’ stata identificata una sola mutazione missenso nel gene KEAP1 già descritta in letteratura, mentre il 42% delle linee cellulari analizzate hanno mostrato ipermetilazione a livello della regione promotrice del gene KEAP1, con una perfetta corrispondenza tra i risultati ottenuti in RT-QMSP e pirosequenziamento. Su alcune delle linee cellulari selezionate sono stati eseguiti studi di silenziamento genico del KEAP1 mediante short interfering RNA inhibition, analisi di espressione proteica mediante western blot e saggi farmacologici al fine di valutare la risposta farmacologica delle cellule in condizioni di silenziamento del KEAP1. I risultati ottenuti mostrano come 1) Il silenziamento del gene KEAP1 abbia effetto sulla cascata NRF2 mediata andando ad alterare sia l’espressione di NRF2 che quella di alcuni dei suoi geni target; 2) la soppressione dell’attività del KEAP1 mediante silenziamento genico produca una variazione significativa nella risposta delle cellule di microcitoma al trattamento chemioterapico (Etoposide, Cisplatino e loro combinazione) riducendo l’effetto di quest’ultimo sull’induzione dell’apoptosi e sull’inibizione della proliferazione cellulare. Sulla stessa linea KEAP1 silenziata si è verificato che l’inibizione dell’espressione di KEAP1 porta ad una parziale modulazione dei livelli di NOTCH e della sua attività e non sembra avere effetti sulla risposta al trattamento con il DAPT, inibitore delle γ-secretasi. I risultati ottenuti corroborano nel complesso l’ipotesi di una modulazione negativa da parte del KEAP1, di NRF2 e della sua attività nei SCLC, inoltre correlata con la resistenza delle cellule tumorali al trattamento chemioterapico con etoposide e cisplatino. Mostrano, inoltre, una prima evidenza di interazione tra NRF2/NOTCH nei SCLC ma non ancora chiaramente correlabile all’attività di KEAP1 che ad oggi non può essere considerato un marcatore predittivo di risposta di SCLC al trattamento con DAPT. Saranno necessarie ulteriori indagini allo scopo di trarre delle conclusioni definitive relativamente al ruolo dell’interazione KEAP1-indipendente/dipendente tra le pathway dell’NRF2 e del NOTCH nella modulazione del processo proliferativo ed apoptotico nel microcitoma polmonare. La robusta evidenza di un coinvolgimento della pathway KEAP1/NRF2 nei SCLC suggerisce come questa possa fornire invece già importanti indicazioni sulla resistenza all’uso di terapie convenzionali e rappresentare una indicazione terapeutica interessante per il trattamento del microcitoma polmonare.
Small Cell Lung Cancer (SCLC) represents the most aggressive pulmonary malignancy and accounts for approximately 20% of lung cancers. Despite an initial chemotherapy and radiation response, it recurs rapidly after primary treatment by the development of resistance, with only 6% of patients surviving 5 years from diagnosis. In recent years, the efficacy of targeting key “growth drivers” in cancer treatment of a small subset of lung cancers are emerged encouraging the investigation of new molecular alterations and target proteins that are selectively expressed in cancer cells. Advances have been made especially for NSCLC, but similar results have not yet been possible in SCLC because of a lack of adequate tumor tissue and the rarity of surgically resected specimens. Cells are constantly exposed to oxidative stresses and exogenous and endogenous insults against they adopt several cytoprotective mechanisms. Central to this cellular defensive machinery is the NRF2 and its negative regulator, KEAP1. In tumor cells, mainly in non-small cell lung cancer (NSCLC) it has been observed that the KEAP1/NRF2 pathway is deregulated at genetic and epigenetic levels and it is linked to response conventional therapy and patients’ outcome. Moreover, growth and progression of lung tumors could involve additional pathways and their crosstalk in the context of cell invasion and metastasis, including NRF2/NOTCH crosstalk. This interplay should have strong implications in antioxidant protection, survival of cancer cells and drug resistance to therapies. At present, KEAP1/NRF2 pathway is poorly investigated in SCLC and data concerning the mechanisms of aberrant NRF2/NOTCH crosstalk by genetic and epigenetic modulations in SCLC are lacking. We propose to investigate the molecular basis of KEAP1/NRF2-NOTCH crosstalk deregulation in SCLC and its impact on the modulation of cellular defense systems, tumor cell proliferation, and differentiation and to the response to conventional chemotherapies and NOTCH inhibitors. Genetic and epigenetic investigations of the KEAP1, NRF2, and NOTCH1 genes were firstly performed on a collection of SCLC cell lines by Sanger sequencing, real-time PCR and pyrosequencing. Despite of the rarity of KEAP1 mutations in SCLC cell lines, an unreported hypermethylation of the KEAP1 promoter region was observed in SCLC cell lines with a prevalence of 42%, suggesting this mechanism as a possible new inactivation mechanism of this pathway in SCLC, with a perfect correlation between the results obtained both from real-time PCR and pyrosequencing methodologies. Gene silencing studies were carried out on SCLC cell lines by using short interfering RNA, protein expression analysis by western blot and pharmacological assays with chemotherapeutic agents. The results obtained showed as the first instance that KEAP1 silencing affects the NRF2 and some of its target genes expression levels thus giving the first functional evidence of the driver role in SCLC of KEAP1 in the NRF2 axis deregulation. Secondly, KEAP1 suppression showed a significant impact in predicting SCLC apoptosis under cisplatin, etoposide treatment and/or their combination. In light of these results, we verified whether and how the KEAP1/NRF2 pathway modulation interferes with NOTCH signaling under the KEAP1 gene silencing condition. Results obtained proved that KEAP1 impairing controls NOTCH1 and some of its target (HES-1 and DLL3) mainly at transcripts levels and has an only partial effect on protein levels. These data partially justified the observed effects of DAPT treatments in H69V KEAP1 silenced SCLC cell lines. NOTCH1, DLL3, and HES1 transcript levels increased in KEAP1 silencing H69V cell lines, whereas none of these proteins increased in their levels under DAPT treatment and no changes in viability assay of tumor cell were observed. Additional experiments of NRF2 silencing on several SCLC cell lines are demanded to clarify the real KEAP1-NRF2/NOTCH interplay. In summary, our data provide new insights into the potential downstream effects of KEAP1/NRF2 pathway deregulation in SCLC and its consequence in terms of crosstalk with NOTCH1 activity. The findings of this present study might help to guide the understanding of SCLC biology and the development of new potential therapeutic targets for prevention of this aggressive tumor.

Genomic DNA is under constant attack from both endogenous and exogenous DNA damaging agents like reactive oxygen species which include O2, H2O2, OH, reactive carbonyl species, alkylating agents such as estrogen and cholesterol metabolites, radiations (like UV, x-rays and gamma rays) and mutagenic chemicals. Moreover, threats to DNA integrity can also come from DNA metabolism such as replication, transcription and recombination. In order to survive and faithfully transmit the genetic material to the progeny, cells must detect the damage and activate repair mechanisms and, if the damage cannot be repaired, trigger the apoptotic program. All these processes, which are collectively known as DNA damage response (DDR), are coordinated by surveillance mechanisms often called DNA damage checkpoint, which temporarily halt or slow down cell cycle progression to provide enough time for DNA repair. The failure of the DNA damage response and other mechanisms deputed to the maintenance of genome integrity leads to a condition called “Genome Instability”, consisting in the accumulation of damage, genomic aberrations, such as mutations, gross chromosomal rearrangements and chromosome loss. Genome instability is a hallmark of cancer and a driving force in tumorigenesis. We exploit budding yeast Saccharomyces cerevisiae as a model system for studies on genome maintenance pathways which are highly conserved throughout evolution from yeast to human. Despite recent advances in the field, genome integrity pathways are not yet fully understood and not all the genes involved have been identified. We developed a screening strategy, based on the overexpression of DDC2, a critical DNA damage checkpoint gene in the contest of a yeast deletion collection, in order to identify genes controlling genome integrity on the basis of spontaneous accumulation of endogenous DNA damage. We identified several genes and pathways associated with genome integrity maintenance, among which are many genes induced in peroxisome biogenesis and mitochondria structure and function, as well as several uncharacterized ORFs.

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.

Maintenance of genome stability is critical to cell survival and normal cell growth. Indeed, genome instability is a hallmark of most human cancers. To ensure the accurate transmission of genetic information to the offspring, eukaryotic cells evolved a complex network of surveillance and DNA repair mechanism, which allow the faithful transmission of genetic information throughout generations. These surveillance systems, called DNA damage checkpoints, are signal transduction cascades where the DNA damage signal is transmitted, through the action of protein kinases, to the cell cycle machinery, resulting in the temporary arrest of cell proliferation at the G1/s or at the G2/M transitions, or in the slowing down of DNA replication. Moreover checkpoint activation frequently brings about also changes in the transcritptional programme of the cell and modification in the DNA repair factors, resulting in a more efficient removal of the lesion. Since the basic checkpoint response has been shown to be conserved, from yeast to human cells, many details of this mechanism have been outlined by genetic and biochemical means in budding and fission yeasts, thanks to their genetic versatility and experimental tractability. The current model predicts that activation of the first checkpoint kinase in the cascade is not due to the DNA damage itself, but it requires recognition and initial processing of the lesion by some nucleases generating long 3’ ssDNA tails. The ssDNA regions are rapidly bound by the RPA complex, generating a structure that is responsible for the recruitment and activation of the apical kinase complex Mec1-Ddc2 and for the loading onto DNA of the 9-1-1 complex. Once activated, Mec1 phosphorylates different targets, among which Ddc2, the Ddc1 component of the 9-1-1 complex, histone H2A. Another Mec1 target is adaptor Rad9. Phosphorylation of Rad9, followed by its oligomerization, allows the recruitment and activation of the effector checkpoint kinase Rad53. This is a key step in the signal transduction cascade; it can be easily visualized as a hyperphosphorylated slower-mobility form by Western blotting and it is generally used as a marker to monitor checkpoint activation. Recent work demonstrated that histone H2B-K123 ubiquitylation, carried out by Rad6-Bre1, and histone H3 methylation on lysine 79 (H3-K79), performed by Dot1, contribute to Rad9 recruitment to chromatin. Infact, it has been demonstrated that Rad9 physically interacts with methylated H3-K79 thanks to its Tudor domain. Impairment of this recruitment-pathway prevents Rad9 and Rad53 phosphorylation in G1-arrested cells and abolishes the G1-S arrest following DNA damage. Surprisingly in M-arrested cells, deletion of Dot1 or mutation of the Rad9 Tudor domain does not completely abrogate the checkpoint function and Rad53 phosphorylation after genotoxic treatment. This evidence suggests the existence of a second pathway, partially redundant with the histone dependent branch, that, in M phase, provides an alternative way for Rad9 to be recruited in the proximity of the lesion and to be phosphorylated. We found that the replication factor Dpb11 is the keystone of this second pathway. Our data suggest that Dpb11 is held in proximity to damaged DNA through an interaction with phosphorylated 9-1-1, specifically within its Ddc1 subunit. Once recruited in the proximity of the lesion, it performs a double role: it contributes in the full activation of the apical checkpoint kinase Mec1 and it cooperates with Dot1 in the recruitment of Rad9. In particular, we discocvered that Dpb11 physically interacts with Rad9. This interaction depends upon CDK-dependent phosphorylation of Rad9 on the Ser11 residue, at the N-terminus of the protein. We also provide evidence that the Dpb11-dependent branch of Rad9 recruitment is necessary and sufficient for checkpoint activation when the histone-dependent pathway is impaired, and it allows Rad53 phosphorylation, despite undetectable Rad9 binding on the chromatin, suggesting that Rad9 complexed with Dpb11 is not tighly linked to chromatin.

The main role of MADS-box transcription factors in plant developmental processes has been well described in the model plant Arabidopsis thaliana. However, little is known about their function in crops of important agricultural and commercial value. Our study aims to investigate their role in two agronomical relevant Rosaceae crops: apple (Malus x domestica Borkh.) and strawberry (Fragaria vesca). Expression studies using qPCR and RNA seq have identified two apple Dormancy Associated MADS-box (DAM) genes. They group with the StMADS11 clade, and were named MdDAM1 and MdDAM2, the last one discovered ex novo. Real time expression studies in dormant buds collected during the chilling period and chromatin immunoprecipitation (ChIP) analyses confirmed that the genes are downregulated by exposure to cold and MdDAM1 is epigenetically repressed, as it has been demonstrated for Arabidopsis FLC and peach DAM genes. In parallel we worked on strawberry MADS-box genes of known function involved in flower development. We chose three MADS-box genes that are homologs of Arabidopsis PISTILLATA and AGAMOUS to perform gene expression and functional analysis using a RNA interference approach to obtain post-transcriptional gene silencing. The positive transgenic lines of each transformation were evaluated at the molecular and phenotypic level. Single gene mutants does not show altered flower phenotype, suggesting a different mechanism of flower development in strawberry, probably due to the peculiar flower structure.
Il ruolo fondamentale svolto dai fattori di trascrizione MADS-box nei diversi processi di sviluppo delle piante è stato descritto in dettaglio nell’organismo modello Arabidopsis thaliana. Tuttavia la loro funzione in colture di maggior valore agricolo e commerciale rimane da indagare. La presente ricerca si propone di comprendere il loro ruolo in due colture agronomicamente importanti appartenenti alle Rosacee: melo (Malus x domestica Borkh.) e fragola (Fragaria vesca). Studi dell’espressione genica attraverso Real time PCR e RNA-seq hanno permesso l’identificazione di due geni di melo appartenenti ai geni DAM (Dormancy Associated MADS-box). I due geni appartengono alla clade StMAD11 e sono stati denominati MdDAM1 e MdDAM2, quest’ultimo scoperto ex novo. Analisi di espressione con Real time PCR in gemme dormienti raccolte durante il periodo invernale e studi di immunoprecipitazione di cromatina (ChIP) hanno confermato che i geni sono silenziati in seguito all’espozione al freddo. Inoltre si è provato che solo MdDAM1 è epigeneticamente represso, come era stato in precedenza dimostrato in Arabidopsis per il gene FLC e in pesca per i geni DAM. In parallelo si è lavorato su alcuni geni MADS-box di fragola, di cui era nota la funzione, coinvolti nello sviluppo del fiore. Tra questi geni ne sono stati scelti tre, i probabili omologhi di PISTILLATA e AGAMOUS in Arabidopsis, per svolgere sia analisi di espressione, sia analisi funzionali che sfruttano l’approccio di RNA interference per ottenere silenziamento genico post-trascrizionale. Le linee transgeniche risultate positive sono state valutate a livello molecolare e fenotipico. Il silenziamento dei singoli geni non ha mostrato alterazioni nello sviluppo del fiore, suggerendo un diverso meccanismo coinvolto nello sviluppo del fiore in fragola, probabilmente a causa della sua particolare struttura.

During the production of flowers in Arabidopsis thaliana many key decisions are taken in a short lapse of time. The floral primordium has to be positioned correctly on the inflorescence meristem and it has to grow to the required dimension before flower organs are themselves positioned and differentiate. All these tasks are strictly controlled at a molecular level and the genetic networks that underlies them have been intensively studied in the last 30 years. Nevertheless we are far from having a comprehensive knowledge on this process and the genetic mechanism controlling the arise, identity of the floral primordium and the timing of its developmental phases are widely unknown. We have identified new genes potentially involved in early flower development with two approaches: (i) Analysis of the specific transcriptome of the earliest stages of flower development and (ii) Co-expression analysis using APETALA1 and LEAFY, two genes that determine the identity of the floral meristem, which is the earliest stage of flower development. We have observed that multiple REM transcription factors are co-expressed with APETALA1 and LEAFY. Characterizing insertional mutants for genes potentially involved in early flower development and REM transcription factors, we have rarely observed a phenotype in the stages under study. This is consistent with the hypothesis that genes controlling early flower development are often functionally redundant. We are implementing various methods to overcome functional redundancy implementing analysis of gene families, multiple RNA interference and gene targeting strategies.

SUMMARY (English) Very little is known about the coordination and the integration among the different regulators of the motility process. This work deals with two migration-regulatory pathways: the Rac1/WRC (Wave Regulatory Complex) pathway that drives the formation of the actin polymerization network at the front of motile cells; and RalB/exocyst pathway for which the molecular mechanisms underlying its implication in cell motility were still largely unknown at the beginning of this thesis. Rac1 and RalB are small GTPases of the Rho and Ras family, respectively. WRC and exocyst complexes are their direct effectors. In searching for connections between the exocyst and migration regulators, we found that two subunits of the exocyst, Exo70 and Sec6, interact directly in vitro with two subunits of the WRC, Abi and Cyfip, respectively. Moreover, we found that exocyst subunits can interact in vitro with the whole fully-assembled WRC complex. We also showed that these two complexes associate in vivo. Functionally, the exocyst was required for WRC complex positioning at the front of migrating cells. On the other hand, we also found that two other subunits of the exocyst, Sec8 and Exo84, interact with SH3BP1 (a RhoGAP protein) by two-hybrid assay and by co-immunoprecipitation. SH3BP1 localizes at the leading edge and this localization is dependent on the exocyst. Interestingly, in vivo, the RalB/exocyst/SH3BP1 pathway specifically targets Rac1, and not Cdc42. By a combination of approaches we concluded that SH3BP1 is required to inactivate Rac1 at the front. In our model we propose that RalB/exocyst regulates cell migration by driving to the leading edge two key signaling elements of the Rac1 pathway: its effector WRC, that stimulates actin filament nucleation, and its negative regulator SH3BP1, a GAP promoting Rac1 inactivation and GDP/GTP cycling. In conclusion, this work provides novel molecular and functional links between polarized exocytosis and actin dynamics during cell motility.

Eukaryotic cells have evolved the ATR/hCHK1, MEC1/RAD53 kinase-mediated signal transduction pathway, known as replication checkpoint, to protect and stabilize stalled replication forks in human cells and budding yeasts, respectively. rad53 mutants, exposed to high doses of the DNA replication inhibitor hydroxyurea (HU), accumulate hemireplicated, gapped and reversed forks, while treatments with low HU doses induce massive chromosome fragmentation. The aim of my work was to better understand the molecular mechanisms through which Rad53 prevents unusual alterations of the architecture of the stalled replication forks and chromosome fragility, under replication stress. We revealed that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites in unperturbed conditions, are detrimental in rad53 mutants experiencing HU-induced replication stress. Rrm3 and Pif1 ablation synergistically rescues cell lethality, chromosome fragmentation, replisome dissociation, fork reversal and ssDNA gaps formation at the forks of rad53 cells exposed to replication stress. We provide evidence that Pif1 and Rrm3 associate with stalled DNA replication forks and are regulated through Rad53-mediated phosphorylation. Our findings uncover a new replication-stress-induced regulative loop in which Rad53 down regulates the Pif1 DNA helicases at the stalled replication forks. In the second part of this thesis we examined the crosstalk between Rrm3, Pif1, the mediator of the DNA damage checkpoint Rad9 and the nuclease Dna2, during unperturbed DNA replication. The experimental evidence collected in this second part of the project, together with pioneering work previously reported from other laboratories, strongly suggests that Dna2, Pif1 and Rrm3 cooperate to finalize late stages of DNA replication.

A limitless proliferative potential is one of the hallmarks of tumour cells and can be achieved through the activation of telomere maintenance mechanisms (TMM), which rely on telomerase reactivation (TA) or, alternatively, on recombination-based processes known as alternative lengthening of telomeres (ALT). Since a substantial fraction of tumours of mesenchymal origin utilizes ALT mechanisms, they represent an interesting model to study the molecular pathways involved in the activation of TMM. With the present work, we extended our knowledge about the prevalence and the prognostic significance of the two known TMMs in different soft-tissue sarcoma histotypes (Malignant peripheral nerve sheath tumors –MPNST-, leiomyosarcoma, liposarcoma) and mixed origin tumours (Wilms’ tumour), showing the relatively low frequency of telomerase activity in soft tissue sarcomas if compared to tumors of epithelial origin, and the important role of ALT in these malignancies. Controversial results have been obtained about the clinical relevance of TMMs, whose prognostic role seems to be dependent on tumor histotypes. Specifically, ALT is a strong determinant of an unfavorable outcome in liposarcoma and leiomyosarcoma patients, whereas it failed to significantly affect the outcome of patients with MPNST (for whom TA proved to be an important predictor of poor survival). This scenario could be due, at least in part, to the lack of standardized methods to properly classify tumours with respect to their TMM status; in this context, we comparatively analysed the prognostic relevance of ALT in a series of liposarcoma as a function of the characteristic used to classify the tumour, with the final aim to identify the most suitable ALT marker. Moreover, we also proposed to identify microRNAs expressed as a function of the different TMM operating in the tumour, which could be either involved in the regulation of such mechanisms or represent surrogate markers of TA-positive or ALT-positive phenotypes. The knowledge of the specific factors/pathways by which the two known TMMs are differentially regulated in distinct tumour histotypes of mesenchymal origin might be important for a better understanding of the pathogenesis of these malignancies and for the identification of new therapeutic targets.

The goal of this thesis is the downscaling of neuronal differentiation, starting from hiPSCs, using microfluidic and transcriptional programming technologies. The microfluidic lab-on-chip platform, used for the validation of neuronal induction protocols, offers the possibility to work in a context of tight control of culture conditions, high-throughput, efficient delivery of soluble factors. Instead, the induced pluripotent stem cells (iPSCs) technology allows to reprogram somatic cells, such as skin fibroblasts, to an embryonic-like phenotype and to obtain, in this way, a clonal expansion of undifferentiated cells that can then be differentiated in the desired phenotype (e.g. neuronal lineage). The combination between cell culture in microfluidics and iPSCs provides a very important contribution in the understanding of those unknown molecular mechanisms, responsible for specific pathologies, which could be useful for finding effective therapies. This work finds its basis and strong motivation in a contest that, to date, does not provide exhaustive in vitro or in vivo models, because a large amount of neurological diseases and human brain studies are performed on post-mortem biopsies or on tissues collected at very late disease stages. The animal models, instead, could represent a possibility to understand some neurological mechanisms, but they are limited and sometimes do not fully recapitulate the patient phenotype. In this scenario, we have focused our attention on the downscaling of in vitro neuronal differentiation protocols starting from hiPSCs, taking advantage first of all of lentiviral vectors for the overexpression of a proneural transcription factors, Ngn2, After the definition of the best culture condition, we focus our attention on the regulation of the expression pattern of Ngn2 in hiPSCs. Ngn2 is characterized by an oscillatory expression, in the early stages of the neurogenesis, and becomes constantly expressed on mature neurons. So, we evaluated weather we could reproduce in vitro this oscillatory expression pattern. Furthermore, since the generation of neurons with high efficiency is influenced by the correct delivery of exogenous factors to the cells with a precise timing, we developed a protocol of neuronal induction using an automated microfluidic platform. Lastly, to avoid any genetic aberration caused by lentiviral vectors and to have a system that could be easily modulated in term of dose and frequency of administration, we induced the generation of neurons by introducing synthetic modified mRNA encoding for Ngn2 into hiPSCs and coupled this method with microfluidic technology. We focused on increasing the differentiation efficiency of neuronal differentiation, working on cellular signallings that play important roles during the in vivo development of the central nervous system.
L'obiettivo di questa tesi è il downscaling di protocolli di differenziazione neuronale, a partire da hiPSC, utilizzando tecnologie di programmazione trascrizionale, in microfluidica. La piattaforma microfluidica lab-on-chip, utilizzata per la validazione dei protocolli, offre la possibilità di lavorare in un contesto di stretto controllo delle condizioni della coltura cellulare, come ad esempio la consegna efficiente di fattori solubili. Invece, la tecnologia delle cellule staminali pluripotenti indotte (iPSCs) consente di riprogrammare le cellule somatiche, come i fibroblasti, in un fenotipo simile a quello embrionale e di ottenere, in questo modo, un'espansione clonale di cellule indifferenziate che possono quindi essere differenziate nel fenotipo desiderato (ad esempio quello neuronale). La combinazione tra coltura cellulare in microfluidica e iPSC fornisce un contributo molto importante nella comprensione di quei meccanismi molecolari, responsabili di patologie specifiche, utili all’individuazione di efficaci terapie. Questo lavoro trova le sue basi ed una forte motivazione in un contesto che, ad oggi, non fornisce modelli esaurienti in vitro o in vivo, perché una grande quantità di malattie neurologiche e studi sul cervello umano vengono eseguiti su biopsie post-mortem o su tessuti raccolti a fasi della malattia molto tardive. I modelli animali, invece, potrebbero rappresentare una possibilità di comprendere alcuni meccanismi neurologici, ma sono limitati e talvolta non ricapitolano completamente il fenotipo del paziente. In questo scenario, abbiamo focalizzato la nostra attenzione sul downscaling dei protocolli di differenziazione neuronale in vitro a partire da hiPSC, sfruttando prima di tutto i vettori lentivirali per la sovraespressione di fattori di trascrizione, nel nostro caso la Neurogenina 2, un gene proneurale. Inoltre, abbiamo usato questo approccio con una prospettiva innovativa: regolando la durata e la frequenza dell’espressione di Ngn2, attraverso la somministrazione controllata di doxiciclina, è possibile imitare, in vitro, il profilo oscillatorio di Ngn2 osservato in vivo. In vivo, infatti, i progenitori neurali sono caratterizzati da un pattern di espressione di Ngn2 oscillatorio, mentre i neuroni mostrano un'espressione sostenuta e costante di Ngn2. Inoltre, poiché la generazione di neuroni, con un’elevata efficienza, è influenzata da un corretto rilascio di fattori esogeni alle cellule, in modo time-dependent, abbiamo sviluppato un protocollo di induzione neuronale utilizzando una piattaforma microfluidica automatizzata. Infine, per evitare qualsiasi aberrazione genetica causata dai vettori lentivirali e per avere un sistema che potrebbe essere facilmente modulato in termini di dose e frequenza di somministrazione, abbiamo indotto la generazione di neuroni, in hiPSC, introducendo sintetici mRNA modificati codificanti per Ngn2 e accoppiato questo metodo con la tecnologia microfluidica. Ci siamo, quindi, concentrati sull'aumento dell'efficienza di differenziazione neuronale, lavorando sul signaling cellulare, importante per lo sviluppo in vivo del sistema nervoso centrale.

Notch pathway plays a pivotal role in regulating cell proliferation, survival, differentiation and apoptosis; accordingly, the relevance of its involvement in a variety of malignancies has been demonstrated. Particularly in T-cell acute lymphoblastic leukemia, Notch1 pathway deregulation plays a causative role. Notch1 mutations or Notch pathway abnormal activation are detectable in nearly the 60% of T-ALL patients and cell lines [Weng AP,2004]. Besides, FBW7 gene homozygous mutations were detected in 8.6% of T-ALL patients; FBW7 encodes an ubiquitin ligase that targets Notch1 for proteasome-dependent degradation [O’Neil J,2007]. Recent works indicate a role for Notch signaling in other, non-lymphoid hematological malignancies, such as acute myeloid leukemias [Thoda S,2001] and multiple myeloma [Nefedova Y,2008; Jundt F,2004; Houde C,2004]. Emerging data show that Notch interacts with various pathways playing a critical role in cancer, such as C-MYC, PI3K/Akt, PTEN and NF-κB. Besides, several evidences indicate a possible role for Notch in the control of expression and functions of chemokine receptors: this could account for possible Notch role in controlling tumor cell migration, invasion and metastases. The relevance and breadth of Notch activities in leukemias have led investigators to study the effect of Notch inhibition in cellular models, mainly through γ-secretase inhibitors (GSis). The goal is to identify a possible Notch-based therapy for such hematological malignancies. The majority of the cell lines and patient samples under study are resistant to GSi treatments, preventing the use of Notch as a major target for the development of molecularly tailored antileukemic drugs [Weng AP,2004; O’Neil J,2007]. Understanding the molecular mechanism accounting for Notch signaling effects and GSi-resistance in leukemic cells is likely to provide new insights for the development of innovative therapies. To address this goal, this thesis work aims to elucidate the mechanisms thorough which Notch interacts with its major identified partners, and to identify new components of Notch signaling pathway.

Cluster headache (CH) and bipolar disorder (BD) are pathological conditions affecting 1% and 1.5% of the general population, respectively. Albeit the pathogenesis has not yet been completely elucidated, family and twin studies have suggested a genetic basis for both disorders, with an estimated heritability of 80% for BD and up to 60% for CH. Though BD and CH are very different diseases, they show important clinical similarities, such as a temporal pattern of disturbances, dysregulation of the wake-­‐sleep cycle, neuroendocrine derangements, and more important positive clinical response to lithium and valproate treatments in a significant proportion of treated patients. In the present study, we sought to explore whether BD and CH patients responders to lithium share common molecular pathways potentially involved in predisposing to positively respond to prophylactic lithium treatment. To this aim, we carried out a transcriptome study in lymphoblastoid cell lines from 10 BD type I patients, responders to lithium, 8 CH patients responders to lithium treatment and 10 healthy subjects (CO). Expression profiles were measured by Affymetrix GeneChip Human Gene ST 1.0 covering 36,079 transcripts. Expression levels of BD and CH patients were compared with CO using a t-­‐test, in order to identify commonly dysregulated genes. Pathway analysis was performed based on the hypergeometric test for over representation of specific Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 544 and 1172 genes were differentially expressed in BD versus CO and CH versus CO respectively. Filtering criteria were based on corrected p value < 0.05 and a Fold Change (FC) ≥ |1.3|. Among these genes, 314 were commonly altered both in CH and BD compared to CO. The most significant dysregulated gene in BD and CH was RNA binding motif (RNP1, RRM) protein 3 (RBM3), a gene implicated in sleep regulation and in the temperature entrained circadian gene expression (corrected p value of 6,30x 10-­‐09 in BD vs CO and 1,88x 10-­‐09 in CH vs CO). Pathway analysis showed that Protein processing in endoplasmic reticulum pathway was one of the most significantly enriched in BD and CH when compared to CO. In conclusion, data from this pilot microarray study may provide useful and relevant information for a better understanding of the molecular underpinnings of lithium response and on the neurobiology of BD and CH.

The TGFβ pathway is critical for embryonic development and adult tissue homeostasis. Upon ligand stimulation, the TGFβ/BMP receptors phosphorylate the Receptor-activated Smads (R-Smads), which then associate with Smad4 to form a transcriptional complex that regulates gene expression through site-specific DNA recognition. Several ubiquitin ligases serve as inhibitors of R-Smads, yet no deubiquitylating enzymes (DUBs) for these molecules have so far been identified. This contributed to leave unexplored the possibility that ubiquitylation of R-Smads is reversible and engaged in regulating Smad function, in addition to degradation. Here we identify USP15 as a DUB for R-Smads. USP15 is required for TGFβ and BMP gene responses and biological effects in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes regulative ubiquitylation of R-Smads, that hits their DNA binding domain and is incompatible with promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the Smad complex. These data identify a new layer of control by which the ubiquitin system regulates TGFβ biology
La via di segnale TGFβ svolge un ruolo critico durante lo sviluppo embrionale e nell’omoestasi dei tessuti nella vita adulta. In seguito alla stimolazione da ligando i recettori per TGFβ/BMP fosforilano le R-Smads che si associano così a Smad4 per formare un complesso trascrizionale in grado di regolare l’espressione genica mediante il riconoscimento di specifiche sequenze di DNA. Molteplici ubiquitina-ligasi agiscono come inibitori delle R-Smads, ma ad oggi non sono ancora stati identificati enzimi deubiquitinanti (DUBs) per queste molecule. Questo lascia aperta la possibilità che l’ubiquitinazione delle R-Smads sia reversibile e che possa essere coinvolta nella regolazione delle funzioni delle R-Smads con modalità diverse dalla degradazione. In questo lavoro USP15 è stata identificata come una DUB per le R-Smads. USP15 è richiesta per le risposte geniche indotte da TGFβ e BMP e per gli effetti biologici mediati da questi segnali in cellule di mammifero e in embrioni di Xenopus laevis. A livello biochimico USP15 principalmente si contrappone ad una forma regolativa di ubiquitinazione delle R-Smads, la quale inibisce la loro capacità di legarsi al DNA ed è quindi incompatibile con il riconoscimeto del promotore. Tramite questo meccanismo USP15 è fondamentale nel determinare la persistenza del complesso delle Smads sui promotori dei geni endogeni. Questi risultati evidenziano un nuovo meccanismo con cui l’ubiquitina regola gli effetti biologici di TGFβ e BMP

Background: HNSCC is a heterogeneous group of tumors caused mainly by environmental factors and human papillomavirus (HPV) infections. HPV- and HPV+ HNSCC are considered distinct entities, however, they are still treated with the same therapeutic strategies. HPV-induced tumorigenesis is mainly mediated by the E6/E7 oncoviral proteins, that, among all, alter the epigenetics of the host cells. Nevertheless, epigenetic profiles of HNSCC subtypes have not been clearly profiled. Results and Conclusion: hPTMs super-SILAC analysis of HNSCC cell lines and patients’ tissue samples revealed significant differences in the enrichment levels of some hPTM in HPV+ samples compared to HPV- ones and in tumoral tissues compared to normal ones. We focused on one of these identified hPTM and demonstrated that its levels are regulated by E6 and E7. We identified a histone modifier responsible for this hPTM whose levels are upregulated by E6/E7 and are higher in HPV+ compared to HPV- HNSCC cell lines and patients’ tissue samples, as is for the related hPTM. Silencing this enzyme through shRNA in HNSCC cell lines reduced proliferation and migration rates in both subtypes. It also downregulates the expression levels of some EMT mesenchymal makers and of a crucial oncogene involved in HNSCC. RNA-seq analysis revealed that other programs are instead specifically regulated according to the subtype: immune-response related genes are mainly activated in HPV- cell lines, while genes involved in cell differentiation in the HPV+ ones. Our research paves the way to novel lines of research and identifies a promising novel epigenetic target for HNSCC treatments.

ABSTRACT MicroRNAs (miRNAs or miRs) are functional molecules that have recently emerged as important regulators of gene expression at posttranscriptional and translational levels. Recent studies showed that dysregulation of miRNA expression in heart and vasculature contributes to cardiovascular diseases (CVDs), but so far their precise pathophysiological role and their validity as therapeutic targets have not yet been confirmed. Main aim of this doctoral thesis was to determine the functional role of miRNA-199 in cardiovascular pathophysiology, to study whether its modulation might be envisaged as a new specific therapeutic tool for curing CVDs. MiRNA-199 has been recently shown to be involved in different cardiac diseases Indeed, growing evidences shows the miR-199 involvement in cardiac cellular signalling, by affecting gene expression in a cell-specific way. In this thesis, we demonstrated that miR-199 plays a key role in the cardiovascular system, being able to specifically modulate the vascular function and the cardiac response to stress. First, miR-199 resulted physiologically expressed in all the cardiac tissues and also in vessels. In pathological condition, miR-199 expression was directly related to cardiac hypertrophy (CH) in an in vivo murine model and to neurohormonal response in two different in vitro cell models. Second, functional studies performed on transgenic mice showed that in vivo overexpression of miR-199 contributed to cardiac dysfunction in pathological CH. This model is in line with in vivo loss-of-function studies on miR-199 reported by others and by us in this research. Altogether they demonstrated that oligonucleotides (antagomirs) suppressing miR-199 in vivo can restore the cardiac function in pathological CH, thus suggesting miR-199 silencing as innovative therapeutical strategy for curing LV dysfunction and eventually preventing HF. Third, miR-199 resulted to be implicated with two main neurohormonal systems by, either targeting some of their key components or affecting cardiac and vascular cells signaling pathways induced by their effectors, angiotensin II (ANG II) and endothelin-1 (ET-1). Having found that miR-199 is related to these potent vasoconstrictor peptides, we finally investigated miR-199 effects on the modulation of vascular tone. MiR-199 in vivo overexpression exerted a beneficial effect on the vascular tone, by both inhibiting the contractile response to neurohormonal stimulation and reducing the ANG II circulating levels. In vivo injection of antagomir-199 confirmed these effects at vascular level, thus indicating miR-199 as a key regulator of the vascular function. These findings provided the proof of concept for a ‘loop regulatory’ mechanism of miR-199 that relies on neurohormonal activation state and might consequently affect the cardiovascular functionality. In conclusion, the results attained throughout this doctoral research allowed us to gain significant insight in the role of miR-199 in cardiovascular pathophysiology, suggesting future studies to be performed. These findings may lead to the development of innovative therapeutical approaches for the treatment of various CVDs. RIASSUNTO I microRNA (miRNA o miR) sono molecole funzionali recentemente emerse come importanti regolatori dell’espressione genica a livello post-trascrizionale e traduzionale. Studi recenti hanno dimostrato che l’alterazione dell’espressione dei miRNA nel cuore e a livello vascolare contribuisce alle malattie cardiovascolari, ma ad oggi non è ancora stato ancora confermato il loro ruolo esatto nella patofisiologia e la loro validità quali potenziali target terapeutici. Il principale scopo di questa tesi di dottorato era determinare il ruolo funzionale del miRNA-199 nella patofisiologia cardiovascolare, per valutare se la sua modulazione possa essere considerata come nuovo metodo terapeutico specifico per la cura delle malattie cardiovascolari. E’ stato recentemente dimostrato che il miR-199 è coinvolto in diverse patologie cardiache. Infatti, un numero crescente di prove sperimentali dimostra il coinvolgimento del miR-199 nel signalling cellulare cardiaco, che avviene influenzando l’espressione genica in maniera cellula-specifica. In questa tesi abbiamo dimostrato che il miR-199 svolge un ruolo centrale nel sistema cardiovascolare, andando a modulare specificatamente le funzioni vascolari e cardiache in risposta allo stress. In primo luogo, il miR-199 è risultato specificatamente espresso in tutti i tessuti cardiaci e nei vasi. In condizioni patologiche, la sua espressione è risultata direttamente correlata i) in vivo all’ipertrofia cardiaca in un modello murino di carico sovrappressorio ventricolare ed ii) in vitro alla risposta neurormonale in 2 diversi modelli cellulari. Secondariamente, studi funzionali effettuati su topi transgenici hanno dimostrato che l’overespressione in vivo del miR-199 contribuisce alla disfunzionalità cardiaca associata all’ipertrofia patologica. Questo modello è coerente con gli studi di perdita-di-funzione in vivo riportati sia in letteratura che nella presente ricerca. In particolare, questi esperimenti hanno dimostrato come gli oligonucleotidi sopprimenti l’espressione del miR-199 (antagomir) in vivo siano in grado di ripristinare la funzionalità cardiaca nell’ipertrofica patologica, indicando quindi il silenziamento del miR-199 come potenziale strategia terapeutica innovativa per il trattamento della disfunzione del ventricolo sinistro e l’eventuale prevenzione dello scompenso cardiaco. Come terzo punto, il miR-199 è risultato essere implicato in 2 principali sistemi neurormonali, sia attraverso la regolazione di alcuni dei loro componenti chiave, che la modulazione della trasduzione del segnale cellulare cardiaco e vascolare indotta dalle loro molecole effettrici, l’angiotensina II (ANG II) e l’endotelina-1 (ET-1). Avendo dimostrato che il miR-199 è correlato a questi potenti peptidi vasocostrittori, abbiamo infine esaminato i suoi effetti sulla modulazione del tono vascolare. L’overespressione del miR-199 in vivo è apparsa avere un effetto benefico sul tono vascolare, andando ad inibire la risposta contrattile alla stimolazione neurormonale e riducendo i livelli circolanti di ANG II. La somministrazione in vivo dell’antagomir-199 ha confermato tali effetti a livello vascolare, rivelando un ruolo chiave del miR-199 nella regolazione della funzionalità vascolare. Queste scoperte hanno fornito la prova a supporto dell’esistenza di un meccanismo a ‘ loop regolatorio’ del miR-199 che agisce sullo stato di attivazione neurormonale e che potrebbe conseguentemente influenzare il funzionamento cardiovascolare. In conclusione, i risultati ottenuti nel corso di questa ricerca di dottorato ci hanno permesso di acquisire conoscenze significative sul potenziale ruolo del miR-199 nella patofisiologia cardiovascolare, suggerendo pertanto la necessità di intraprendere ulteriori studi di approfondimento. Tali scoperte potrebbero auspicabilmente portare allo sviluppo di approcci terapeutici innovativi per il trattamento di varie malattie cardiovascolari.

Il cancro colorettale (CC) è il quarto tumore più frequentemente diagnosticato e la seconda causa di morte negli Stati Uniti. Il ruolo del pathway di Hedgehog (Hh) nel carcinoma colorettale rimane controverso. In questo studio, abbiamo analizzato l'associazione tra l'espressione degli mRNA di GLI1 e GLI2, due geni bersaglio di HH e la sopravvivenza e recidiva di CC mediante microarray di espressione genica da una coorte di 382 pazienti con CC. I pazienti con una maggiore espressione di GLI1 avevano una sopravvivenza significativamente minore. Al fine di dimostrare un ruolo causale dell'attivazione del pathway HH nel pathogenesis di CC, abbiamo trattato le linee di cellule HCT 116, SW480 e SW620 CC con GDC-0449, un inibitore farmacologico di Smoothened (SMO). GGL-0449 ha marcatamente ridotto l'espressione dei geni bersaglio HH GLI1, PTCH1, HIP1, MUC5AC, indicando così che questo percorso è attivo nelle linee di cellule di CC. Inoltre, GDC-0449 ha parzialmente ridotto la proliferazione cellulare, che è stata associata a sovraregolazione di p21 e downregulation di CycD1. Ha modificato l'espressione di Snail1, il gene principale EMT, e dei marcatori epiteliali Cytokeratin-18 e E-caderina. Questi risultati sono stati confermati dal silenziamento genico di SMO. Inoltre, il trattamento con 5E1, un mAb specifico di Sonic Hedgehog, ha ridotto marcatamente l'espressione dei geni bersaglio di Hedgehog, così come ha inibito la proliferazione cellulare e la reversione verso un fenotipo epiteliale. Tali risultati dimostrano l'esistenza di un segnale autocrino mediato da Hedgehog che influenza la plasticità delle cellule e favorisce la proliferazione cellulare e la migrazione / invasione nelle linee cellulari di CC. Queste scoperte incoraggiano indagini future per caratterizzare meglio il ruolo di Hedgehog nella plasticità e invasione cellulare durante le diverse fasi della cancerogenesi colorettale

Genome integrity needs to be preserved for the propagation of genetic information. Inactivation of proteins involved in DNA damage responses (DDR) are often associated with cancer and/or developmental disorders of the nervous system, which appear particularly vulnerable to DNA distress. The Nijmegen Breakage syndrome (NBS), due to NBS1 gene mutations, is characterized by microcephaly, facial dysmorphisms and cancer predisposition. The neurological features of NBS patients have been modeled in the Nbs1CNS-Δ mouse, a Nbs1 CNS-restricted knock-out which shows microcephaly, severe ataxia and dramatically impaired cerebellar development. Strikingly, a very similar phenotype is also observed in mice with SHH/MYCN conditional KO, suggesting that Nbs1 and SHH/MYCN might be functionally linked. Prompted by this hypothesis, we generated new mouse models with CNS-restricted inactivation of Nbs1 in a transgenic SmoA1 cancer-prone context and new GCPs cultures for in vitro studies. The new SmoA1/Nbs1CNS-Δ mouse model showed that the absence of Nbs1 completely blocks the SmoA1-dependent tumor phenotype and causes severe cerebellar defects during postnatal development. This suggests an epistatic role of Nbs1 on the SHH pathway. The dramatic and degenerated phenotype of this model did not allow an appropriate analysis of the molecular interactions between Nbs1 and the SHH pathway. Therefore, we generated a new Nbs1GCP-Δ mouse model with the specific deletion of Nbs1 in cerebellar granule progenitors (GCPs). This model showed that the absence of Nbs1 caused defects in cerebellar development and this was associated with downregulation of the SHH pathway both in vivo and ex vivo. This was also confirmed in an inducible cell autonomous context. Given that the primary cilium is an essential structure for SHH signaling and given the emergent link between DDR proteins, centrosomes and ciliogenesis, we examined whether Nbs1 might be required for primary cilia formation and regulation, which in turn could affect SHH signaling. Accordingly, we provided strong evidence that loss of Nbs1 determines severe defects in ciliogenesis. Moreover, we showed that either the ciliary phenotype or the associated downregulation of the SHH pathway are rescued by loss of p53. In conclusion, our data support a novel function for Nbs1 in cilia-dependent SHH signaling and raise the possibility that the Nbs1-p53 axis, in addition to their conventional role in DNA damage, may regulate ciliogenesis both in physiological and pathological conditions in the cerebellum.

Hedgehog (Hh) pathway is essential for embryonal development and tissues homeostasis; its alteration causes several human cancers, including medulloblastoma (MB), the most common brain malignancy in childhood. Because of its crucial role in tumorigenesis, the regulation of the Hh pathway and the identification of new Hh modulators emerged as a field of great interest in tumor biology. My Ph. D. project was focused on two main goals: Aim 1 | Identification of new molecular mechanisms involved in Hh signalling pathway regulation and MB tumorigenesis. To address this aim, we focused on the identification of new interactors of the known Hh negative regulator and tumor suppressor RENKCTD11. Through mass spectrometry analysis, we identified the transcription factor SALL4A. SALL4A plays a key role in maintaining pluripotency and self-renewal of embryonic stem cells, regulating different signalling pathway. SALL4A expression is inhibited in the post-natal period in many adult tissues, but it is reactivated in different tumors and is often related to worse prognosis and lower survival rate. Our data demonstrate that SALL4A is a substrate of RENKCTD11, that induces its poly-ubiquitylation and its consequent proteasome-mediated degradation. In order to investigate its biological role in Hh signalling, we demonstrated that SALL4A enhances GLI1 activity working in complex with HDAC1, a well known Hh activator. Of note, we observed that the proliferation ability of human MB cell lines increases in presence of SALL4A, whereas their migration rate is reduced after its genetic depletion; in vivo, SALL4A genetic depletion leads to a reduction of Hh-dependent tumor growth. Our findings identify SALL4A as a previously unknown regulator of Hh pathway able to promote, in complex with HDAC1, GLI1 activity and to contribute to Hh-dependent tumorigenesis. Hence, SALL4A stands as a new molecular target involved in the onset and progression of Hh-dependent tumors and represents an interesting focus in cancer research. Aim 2 | Pharmacological targeting of Hedgehog pathway. Given the primary role of Hh signalling in tumorigenesis and in the maintenance of cancer stem cell niches, this pathway is now considered an attractive therapeutic target in cancer. In recent years, many efforts have focused on the development of drugs that can block the activating effect of SMO receptor. However, several studies underlined some limitations of the inhibitors identified so far, linked in particular to the poor selectivity, the onset of drug resistance events and the activation of GLI1 mediated by other oncogenic pathways. These evidences raise the need to identify new and more effective Hh inhibitors able to overcome drug resistance and to counteract tumor growth. To this end, my research activity was aimed at identifying, characterizing and optimizing new molecules able to block the oncogenic potential of Hh. In particular, by combining the most profitable pharmacophores for targeting SMO and GLI1 by synthetic isoflavones, we designed and synthesized the isoflavone 22, a small molecule that acts as a multitarget Hh inhibitor blocking both SMO and GLI1 activity at the same time. Compound 22 is able to inhibit Hh-dependent tumor growth in human and murine MB cells at sub-micromolar concentration, as a consequence of the reduction in GLI1 expression levels. Isoflavone 22 remarkably shows a strong anti-tumor effect also in vivo by suppressing cell proliferation and promoting apoptosis. Molecular modeling further corroborated the multitarget mechanism of action of compound 22, showing that the drug is able to fit the ligand binding site in both SMO and GLI1. Overall, these results reveal a valuable form of targeted therapy to increase the efficacy and to decrease the toxicity of individual anticancer agents; our findings discover the first multitarget Hh inhibitor that impinges Hh-dependent tumor growth and stands as new potential weapons against Hh-driven tumors.

KCASH2 gene participates in the modulation of the Hedgehog (Hh) pathway, which plays a role in development, embryogenesis and tumorigenesis. In particular, KCASH2 protein prevents the transcriptional Gli1 activity, interacting with the E3 ubiquitin Ligase Cullin 3, mediating histone-deacetylase 1 (HDAC1) ubiquitin-dependent degradation, which would otherwise deacetylate Gli1 enhancing their transcriptional activity. In the present work, we generated KCASH2 knockout mice (KCASH2KO) to evaluate in vivo effect of KCASH2 deletion on the Shh pathway and cerebellar development. KCASH2 expression was present in differentiated cerebellar granule cells, and histological analysis highlighted a larger thickness of the internal granule layer (IGL) in the IV-V cerebellar lobules in the adult KCASH2KO mice cerebellum, confirming the KCASH2 loss determines a prolonged proliferation of granule cells precursors (GCP) during post natal development. Behavioural experiments have highlighted a significant alteration in cerebellum functions; indeed that the loss of KCASH2 induces in male KCASH2KO a delayed learning and in female KCASH2KO a diversity in procedural navigational strategy compared to WT siblings. Our observations demonstrate that KCASH2 acts in vivo as negative regulator of Hh pathway and suggest, for the fist time, the role of the Hh pathway alteration in such behavioural disorders related to cerebellar function. During the characterization of the KCASH2KO mouse, we unveiled a potential physiological role of KCASH2 in spermatozoa development and function. We have demonstrated that KCASH2 is expressed in Leydig cells and in Epididymal Epithelium cells and we have observed anomalies in KCASH2KO mice spermatozoa morphology (increased atipicity) and sperm mobility (dyskinesia).