Graduation day

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 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 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. Besides regulating migratory abilities of the melanoma cells, RaLP positively influences their invasive potential, by altering collagen matrix digestion. We also observed that RaLP decreases adhesion of the cells to each other and to different matrices. 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.

Epigenetic control of developmental genes has emerged as a key mechanism in the acquisition of developmental competence. Recent studies have provided fundamental insight into the problem of lineage specification by comparing global changes in chromatinand transcription between embryonic stem cells (ESCs) and neural stems cells (NSCs),points of departure and arrival for neural commitment, respectively. In particular, the observation that lineage-specific genes repressed in ESCs by Polycomb-mediated histone H3 lysine 27 trimethylation (H3K27me3) are demethylated and derepressed in differentiated cells posited the existence of a H3K27-specific demethylase. In order to gain insight into the epigenetic mechanisms that enable lineage specification, we investigated the early stages of neural commitment using as a model system the neural differentiation of mouse ESCs. We identified Jmjd3 as a H3K27me3 demethylase that is specifically upregulated at the onset of neural differentiation. This study revealed that Jmjd3 controls the expression of key regulators and markers of neurogenesis and is required for commitment to the neural lineage. To elucidate the role of Jmjd3 in vivo, we have used a genetic loss-of-function approach. Mice lacking Jmjd3 die at birth from respiratory failure. A detailed characterisation of this neurodevelopmental phenotype demonstrated that the defect in respiratory rhythmogenesis upon loss of Jmjd3 is due to an abnormal maturation of the preBötzinger complex, one of the two principal sites generating respiratory rhythm in mammals.

Sumoylation has emerged as an important player in several biological processes involved in the maintenance of genome stability. A number of proteins implicated in DNA metabolism-associated processes, such as DNA replication, repair and chromosome segregation, were shown to sumoylated. Moreover, alterations in protein sumoylation and desumoylation, caused by deregulation of enzymes involved in the SUMO pathway, were associated with cancer development. Work done previously in our laboratory and by other groups suggested an important role for sumoylation in promoting DNA damage tolerance. However, the molecular mechanisms by which DNA damage regulates sumoylation and how balance between sumoylation and desumoylation impinges the cellular response to genotoxic stress are questions that need to be clarified.
Different scenarios could account for regulating sumoylation, one of which envisages a control over the activity of the desumoylating enzymes. Budding yeast has two desumoylating enzymes, Ulp1 and Ulp2, which have distinct cellular localization and enzymatic activities. Starting by investigating whether Ulp1 and/or Ulp2 undergo any type of functional regulation, I found the nuclear SUMO protease Ulp2 to be phosphorylated both in a cell cycle-dependent manner and in response to replication stress or DNA damage. The present work aimed at studying the molecular mechanisms through which Ulp2 promotes genome integrity, both in physiological conditions and in conditions of replication stress. By examining the genome-wide localization of Ulp2 by ChIP-on-chip I observed that Ulp2 is bound to centromeric regions throughout the cell cycle and this enrichment is extended to pericentromeric regions in cells arrested at metaphase. Lack of Ulp2 affects the chromatin structure at centromeres and nearby regions. Following replication stress, Ulp2 is bound to most of the origins of replication that are active under our experimental conditions and is required for efficient origin firing. Also under conditions of replication stress Ulp2 is important for the maintenance of chromatin structure and for the binding of histone variants that are known to play a role in damage tolerance to origins of replication. Thus, Ulp2 appears to play key roles in regulating the status of chromatin to make it suitable for efficient replication and chromosome segregation.

In this study, we investigated the role of individual class I histone deacetylases (HDACs) namely Hdac1, -2 and -3 using retroviral RNA interference to define their specific contribution to control an inducible gene expression program, namely inflammatory gene expression in 3T3 fibroblasts and primary macrophages. In addition to genes showing the expected transcriptional de-repression, we observed 8/20 genes in a test set being down-regulated following individual HDAC depletion. The requirement for HDACs function in gene induction as opposed to the more commonly observed role as transcriptional repressors may either underlie an indirect consequence of impaired HDAC mediated repression or a direct involvement of Hdac1 and Hdac3 in inducible gene activity. Therefore, we extended both the in vitro and in vivo analyses using conditional knockout (KO) mice. Genetic deletion of Hdac3 indicates that Hdac3 is required for the activation of 45% of the lipopolysaccharides (LPS)-induced genes. Global analysis of histone H4 acetylation showed that transcriptional down-regulation in Hdac3-/- cells did not correlate with increased histone acetylation, suggesting the possible involvement of indirect or secondary effects. We found that the LPS-inducible, Hdac3-dependent genes include a large group of interferon-β (IFNβ)-inducible genes (eg. IP10, Irf1) and another group (eg. Il-6) that may be regulated by AP-1 family proteins. In addition, gene expression analyses identified interferon-signalling pathway as being impaired in Hdac3-/- cells. Basal and inducible Ifnβ transcriptions require cJun/AP-1 and the decreased amount of AP-1 family proteins in Hdac3-/- cells may explain the lack of Ifnβ activation and the increased acetylation in genomic regions.

Methylation on lysine 4 of histone H3 (H3K4) by the histone methyltransferases (HMTs) of the trithorax group is associated with the activation and maintenance of transcriptional programs, but virtually nothing is known about the function of these key epigenetic regulators in differentiating cell compartments of adult animals. Here I present the first characterization of the role of the H3K4 HMT Mll2 (Mixed lineage leukemia), also known as Mll4 or Wbp7 in adult tissues focusing on the ontogeny of B cells as the best characterized system for the dissection of lineage commitment and terminal specification. Expression analysis in sorted populations revealed that both Mll2 and its closest homolog Mll1 are expressed throughout B cell maturation, and the hypothesis I set out to test was whether they form a redundant regulatory circuit or whether their selective ablation would uncover functional requirements for specific B cell subsets.
To this end I used a conditional knock out mouse line in which the Mll2 gene is ablated according to the ‘knock-out-first’ approach, (Glaser et al., 2006; Testa et al., 2004; Testa et al., 2003), an innovative engineering strategy that allows to knock out the gene by transcription trapping, restore it using FLP-mediated recombination and knock it out again using Cre-mediated recombination. In order to knock out Mll2 specifically in mature B cells I selected the CD21-Cre3A mouse line that expresses Cre recombinase under the CD21 promoter (Kraus et al., 2004). In this line Cre-mediated deletion follows the expression of CD21, which takes place at the immature B to mature B cell transition (Kraus et al., 2004) (Takahashi et al., 1997). Flow cytometric analysis of B cell subsets in compound mutants performed on over 50 pairs of mice (homozygous versus heterozygous Mll2 flox mice carrying the CD21-Cre transgene) revealed that upon Mll2 deletion, size of marginal zone (MZ) (CD19high - CD21high - CD38high - CD1dhigh - CD23low) B cell compartment on average was reduced by about 50% with respect to the control animals.This was confirmed by histological analysis of the spleen of control and mutant mice. Other B mature cell compartments, including follicular, B1 and germinal center B cellsfrom different lymphopoietic organs were not affected in mutant animals, indicating aspecific requirement for Mll2 in the establishment and/or maintenance of the MZ B celllineage. A gene expression profiling did not reveal aberrant expression of key regulators ofthe homeostasis of MZ B cell compartment, nor remarkable differences between transcriptomes of Mll2 deficient and proficient cells. However in vitro analysis of the dynamics demonstrated higher, by about 75%, rate of proliferation of mutant MZ B cell compartment. Similarly significant increase in the proliferating fraction was also observed in mutant follicular and immature/transitional B cells. This higher turnover of splenic B cell populations upon loss of Mll2 may reflect the presence of a stimulus promoting cell proliferation as part of a feed back loop triggered by the decrease in MZ B cells. Moreover functional analysis of MZ B cells revealed that upon loss of Mll2 cells are not capable to switch class of immunoglobulin in a response to NP-Ficoll immunization indicating a crucial role of Mll2 in the establishment of a new transcriptional program. These findings are in agreement with previously published results demonstrating requirement of Mll2 for the activation and timing of lineage-specific transcriptional programs in both sperm and oocyte germ layers (Andreu-Vieyra et al., 2010; Glaser et al., 2009). Together, present results could suggest that Mll2 plays a role also in B lymphophoiesis and therefore we consider a candidacy of Mll2 as a putative lymphopoietic regulating factor.

MicroRNA (miRNAs) are endogenous non-coding RNAs of 22 nucleotides ~ in length that function as post-transcriptional regulators of gene and protein expression. MiRNAs show altered expression profiles in several human pathologies, including cancer. They can act as tumour suppressors or as oncogenes, depending on the characteristics of their target genes. More than half of the mammalian miRNAs, including several of the miRNAs implicated in breast cancer, are localized within the introns of protein-coding genes, and usually transcribed together with their host gene. It is therefore possible, at least in principle, to identify novel intronic cancerregulated miRNAs by examining the expression profile of their host genes by means of microarrays. For this purpose, we analyzed the regulation of 253 miRNA host genes in five large breast cancer microarray data sets comprising more than 950 samples, examining their association with different clinical and pathological parameters. We found that MCM7 and SMC4 were the most frequently and significantly overexpressed genes in high grade tumours. These genes contain two well known cancer-associated miRNA clusters: miR25-93-106b and miR-15b/16-2 respectively. In addition we found that six the genes containing miR-218-1, miR-342, miR-483, miR-548f-2, miR-1245 and miR-1266, were significantly downregulated in high grade tumours. We validated this latter observation by Real Time PCR on an independent cohort of 36 formalinfixed paraffin-embedded samples. MiR-342 and miR-483, the most significantly downregulated miRNAs in grade 3 tumours, were also found to correlate with bad prognosis in grade 2 tumours.

Encouraging clinical studies are recently showing how a number of anticancer compounds work through a synthetic lethal mechanism by targeting pathways that are specifically essential for the viability of cancer cells but not of normal cells.
We applied the concept of synthetic lethality by performing yeast high-throughput screens to define the chemical-genetic profile of three chemotherapeutic drugs: cisplatin, ecteinascidin, 5-fluorouracil.
By means of the DDR-library, a collection of mutants defective in the DNA damage response, we identified the DNA repair pathways required to survive to the under investigation compounds: DSBR through HR, NER and PRR for cisplatin, DSBR, NER for ET-743, DSBR through HR for 5-FU. Importantly, we also defined the pathways whose absence leads to resistance: MMR and NHEJ for cisplatin, NER, MMR and NHEJ for 5-FU. By exploiting the complete knockout collection YKO-library, we analyzed in detail the yeast proteins involved in cisplatin and ET-743 response. Ecteinascidin-screen revealed that the lack of factors belonging to Swi/Snf complex, Swr1 complex and Mms4-Mus81 endonuclease causes hypersensitivity, while the absence of Slx5-Slx8 dymer leads to resistance. Through the cisplatin-screen, besides confirming the results obtained through the DDR-library, we determined the involvement of these proteins in CDDP survival: Rts1, B-type regulatory subunit of protein phosphatase 2A, Wss1, SUMO-dependent isopeptidase and Irc21, whose function had never been defined.

Jmjd3, a JmjC family histone demethylase, is quickly induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases trimethylated lysine 27 in histone H3 (H3K27me3), a histone mark associated with transcriptional repression and involved in lineage determination, differentiation and tissue homeostasis. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to innate immunity and inflammation remains unknown. To define this role, we generated gene-targeted mice lacking histone demethylase using standard protocol to see the effect of this specific Histone demethylase depletion on all tissues and in particular in immune system to understand the biological functions of jmjd3 in inflammatory responses.. Strikingly, transcription of most Jmjd3 target genes was unaffected by its deletion, a few hundred genes including IL12b and Ccl5 showed mild to moderate mRNA changes associated with impaired transcription; however, no gene was completely dependent on Jmjd3 for induction. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and the transcriptional effects of Jmjd3 absence in the window of time analyzed were uncoupled from measurable effects on this histone mark. Overall, these data demonstrate that Jmjd3 participates in fine-tuning the transcriptional output of LPS-activated macrophages in a manner that is largely independent of H3K27me3 demethylation.

MicroRNAs (miRNAs) negatively regulates gene expression in many cellular processes. Originally, miRNAs were discovered as developmental regulators in worms and even in higher organisms miRNAs display a tissue- or lineage-specific pattern of expression, underlying a direct role in tissue differentiation. Conversely, a widespread down-regulation of miRNAs (and in particular tissue specific miRNAs) often occurs in human tumors, which correlates with advanced diseases in breast and ovarian cancer and favors tumor progression in vivo. We, therefore, hypothesized that differentiation associated miRNAs could also act as negative regulator of proliferation and behave as a tumor-suppressor pathway. We tested this hypothesis by exploiting the ability of the adenoviral oncogene E1A to overcome the proliferative block of terminally differentiated (TD) myotubes by mimicking cancer-related proliferative and dedifferentiative pathways. By using genome-wide approaches, we deeply characterized the transcriptional reprogramming induced by E1A on TD-cells at both transcriptional (mRNA) and post-transcriptional (microRNA) level, focusing on the role of differentiation-associated microRNA in the control of cell-cycle exit and differentiation. We isolated two independent genetic components in the oncogene-induce reprogramming. On one hand, E1A induces the up-regulation of genes essential for proliferation through the interference with the Retinoblastoma (Rb) tumor suppressor pathway. Concomitantly, E1A downregulates miRNAs accumulated over differentiation by a mechanism which is largely Rb-independent and involves tissue specific transcription factors and Myc. Importantly, the two components (messages and miRNAs) are functionally coupled, since microRNA negatively regulate proliferation and inhibit oncogene induced cell-cycle re-entry by interfering with the expression of Rb-dependent proliferative genes.

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions by controlling the number of free actin growing ends and organizing filaments into higher order structures, respectively. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. We combined biochemical, structural and genetic approaches to dissect the molecular mechanism responsible for the distinct Eps8 activities. We show how diverse portions within the Cterminal region of Eps8 contribute to actin binding and mediate Eps8 functions. An N-terminal amphipathic H1 helix is responsible for high affinity interactions with the barbed end unit of filamentous actin and is crucial for the capping activity of the protein. A four helix bundle represents the second interaction surface which mediates contact with the filament side and is crucial for the F-actin bundling activity. Within this context, the two actin binding surfaces are spatially coordinated by a flexible linker that contributes to the high affinity interaction of Eps8 to filaments ends, ensuring a bimodal topological arrangement of Eps8 wrapped around actin filaments and accounting for its dual modes of organizing F-actin, via capping and cross-linking, respectively. Single-point mutagenesis validated this mode of binding, further permitting us to dissect Eps8 capping from bundling activity in vitro and in vivo. Thus, Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caernorhabditis elegans strains.

In budding yeast, the protein phosphatase Cdc14 is a critical regulator of exit from mitosis. Cdc14 activity is regulated by changes in its subcellular localization. The phosphatase is sequestered in the nucleolus from G1 up to metaphase by binding to a competitive inhibitor called Cfi1. During anaphase, Cdc14 is released from its inhibitor by the sequential activation of two signaling networks, the FEAR network and the MEN. Several observations suggest that phosphorylation of Cdc14 and/or Cfi1 is responsible for the dissociation of Cdc14 from Cfi1. Three kinases play a relevant role in regulating Cdc14 release: the polo-like kinase Cdc5, the Clb2-Cdk complex and the MEN kinase Dbf2. The aim of my project was to assess the relevance of the phosphorylation of Cdc14 and Cfi1 for Cdc14 release and to investigate the contribution of the mentioned kinases to this process. By modulating the kinases of interest, we found that a correlation exists between the release of Cdc14 from the nucleolus and the phosphorylation of both Cdc14 and Cfi1. Our results suggest a role for Cdc5 in promoting the phosphorylation of Cdc14 and for Clb-Cdk and MEN in promoting the phosphorylation of Cfi1. Moreover we propose that Cfi1 phosphorylation by Clb-Cdk or MEN serves as a priming step to build up the Cdc5- mediated phosphorylation events. Our data also suggest that Clb2-Cdk is not sufficient to promote Cfi1 phosphorylation and that phosphorylation of Clb2 by Cdc5 could enable the protein to perform its function in the FEAR network, likely to promote Cfi1phosphorylation.

Inflammation is a fundamental response to the loss of cellular and tissue homeostasis with many important physiological roles, including host defence, tissue remodelling and repair, and the regulation of metabolism. Macrophages are not only crucial mediators of the inflammatory response, but they are also able to integrate lipid metabolism and inflammatory signalling. After an inflammatory stimulus, the expression of several hundred genes is either induced or repressed in macrophages.
Although in the last decades the signalling pathways leading to the activation of an inflammatory response have been very well characterized, the knowledge about the tuning of this complex transcriptional network is still limited.
Using a candidate approach we demonstrated that Jarid1b is transcriptionally up-regulated with a slow kinetics by LPS stimulation under the control of the transcription factor Hif1?. Jarid1b is a member of the JARID1 family of H3K4 demethylases, and it has been proposed to act as transcriptional repressor.
Depletion experiments in primary macrophages suggested that Jarid1b is necessary for the activation of the master regulator of cholesterol metabolism, LXR, and the expression of its target genes, likely through the control of cellular oxysterols pools.
We also generated Jarid1b knock-out mice, and performed global expression analysis in Jarid1bdeficient macrophages. The obtained data together with genomic binding analyses showed a very limited transcriptional regulatory function for Jarid1b in this cellular system. We also collected preliminary data on a possible role for Jarid1b, independent of its reported histone demethylase enzymatic activity, in regulating RNA methylation levels.

Stem Cells (SCs) may play a critical role in cancer development, yet the direct demonstration is still lacking. The characterization of mechanisms thatsustain and regulate Cancer Stem Cells (CSCs) may help elucidating their role in tumors. Using a MMTV-ErbB2 breast cancer mouse-model, we had previously shown that CSCs, through multiple rounds of symmetric divisions and extended self-renewal, drive tumor growth and the expansion of the CSC pool. Critical for this property is the lack of functional p53, that drives CSCs to symmetric divisions. Here we show that symmetric divisions and extended self- renewal in CSCs are due to the activation of c-myc as direct consequence of the loss of functional p53. In particular, c-myc is upregulated in transformed and p53-/- immortalized SCs, and enforced expression of c-myc in normal mammary SCs induces symmetric divisions and extended self-renewal, but does not transform. We show that mammary progenitors expressing c-myc acquire in vitro self-renewal properties (mammosphere formation and expansion) and in vivo regenerative potential (formation of normal mammary gland upon transplantation). Our data suggest that c-myc is critical for the maintenance of CSCs in ErbB2-mammary tumors and their aberrant self-renewing properties. This is achieved through its binary e ect on CSCs (induction of symmetric divisions) and progenitors (reprogramming into CSCs). Recent studies demonstrate that c-myc over-expression in epithelial tissues induces an abnormal transcriptional pattern, similar to that of embryonic SCs, and that the same is activated in epithelial cancers and CSCs. This suggests that c-myc may be a general regulator of CSCs and their altered self-renewing properties.

Expression of uPAR has been extensively correlated with the malignant progression and metastasis of cancer; however, little evidence for a causal connection betweenincreased uPAR expression and these processes has been documented to date. A complete functional alanine scan of human uPAR, pinpointed the extracellular matrix protein vitronectin (VN) as the critical uPAR-interactor required to induce cell adhesion, migration, and signaling in vitro, identifying this molecular interaction as a possible target for anti-cancer therapy (Madsen et al., 2007). The same study helped to determine the binding epitope in uPAR responsible for its interaction to VN in an integrin-independent fashion. The composite epitope is fully conserved in mouse and included three amino acids (W32, R58, I63) in domain 1 (D1) and 2 amino acids (R92, Y93) in the linker region between D1 and D2. We substituted these residues with alanine by site directed mutagenesis and analyzed the biological activity of resulting receptor variants in CHO Flp-In cells as compared to wild-type muPAR. All the mutant receptors displayed a deficiency for the binding to VN, preserving the receptor binding to its natural ligand uPA. They also failed to induce muPAR-induced cell morphology changes. These changes include the formation of actin-rich lamellipodia, loss of stress fibers, reduced cell-cell contact as well as a complete failure to form colonies when seeded at low density. The mouse uPARW32A (muPARW32A) was chosen for further experiments since it did not show folding problems. Moreover the W32 position is not a part of the receptor chemotactic epitope and is not involved in the receptor cleavage.
Additional experiments using recombinant soluble muPAR confirmed the impaired VN-binding and a normal uPA-binding. To determine if uPAR and/or VN are involved in tumor formation and progression, and more specifically, if the direct uPAR/VN-interaction is important inthis process, we exploited a xenograft mouse model. For this purpose muPAR or muPARW32A or a muPAR variant lacking of the D1, required for both VN and uPA binding (muPA), were expressed in HEK293 Flp-In GFP positive cells and injected in the fourth mammary fat pad of immunodeficient mice.
In parallel the cells were tested for some in vitro assays. We demonstrated that HEK293 cells expressing muPAR are more proliferating and less apoptotic than the other cells. The interaction with VN is required to increased cell proliferation and to prevent from apoptosis. Moreover, muPAR-VN interaction induced cell spreading and migration. muPAR expressing cells formed palpable tumors earlier than cells expressing the mutant receptors and the tumor growth was significantly faster. Despite the expression of the muPARW32A didn’t affect the timing of the primary tumor formation, it drastically slowed down the growth of the primary tumor mass.
Finally we conducted in vitro studies to determine the molecular mechanisms underlying the possible role of the uPAR/VN-interaction in vivo. From these tests emerged that VN may act as an adhesion “bridge” between different cells expressing uPAR and VN-integrins or cells expressing both uPAR, suggesting a possible role of the uPAR/VN-interaction not only in cell-ECM interactions but also in cell-cell adhesion events including the extravasation of metastatic cancer cells.

Viruses need to hijack cellular machineries both for their replication and propagation and to overcome cellular defenses. Since they often reconvert the functions of host proteins for their purposes, viruses are also powerful tool to better understand some molecular mechanisms underlying cellular processes. Gam1 is an early protein of the avian adenovirus CELO and it possesses a BC-box domain to interact with the host adaptor heterodimer ElonginB/C and, hence, to act as a substrate receptor. It reconstitutes active Cullin2- and Cullin5-based E3 ligase complexes to ubiquitylate the host SAE1 protein, a subunit of SUMO E1 activating enzyme, inducing its proteasomal degradation. Von Hippel-Lindau (VHL) protein is a cellular substrate receptor that associates to Cullin2 and ElonginB/C to specifically target the ? subunits of HIF (Hypoxia-Inducible Factor) transcriptional factors for degradation. VHL is a tumor suppressor protein and its loss leads to the von Hippel-Lindau syndrome, characterized by the onset of renal cell carcinoma and other highly vascularized tumors. We analyzed the possible effects on VHL due to the hijacking of host Cullin2 E3 ligase complexes by Gam1. Interestingly, we observed that Gam1 leads to VHL proteasomal degradation and to the consequent stabilization and activation of HIF-1. Further experiments revealed that VHL protein degradation was not dependent on Gam1-related E3 ligase activity. Rather, the simple binding of Gam1 and other cellular and viral BC-box proteins to ElonginB/C was enough to induce VHL degradation, probably due to the reduced availability of free ElonginB/C complex that is essential for VHL stability. Indeed, since unbound VHL undergoes misfolding, we are currently investigating the possible involvement of heat shock proteins and chaperonedependent CHIP E3 ligase in affecting VHL stability upon BC-box proteins overexpression, as suggested by our preliminary data. If verified, this will be an additional mechanism uncovered by the initial contribution of a viral protein.

Kinetochores are large protein assemblies built on chromosomal loci named centromeres. Four distinct modules accomplish the main functions of kinetochores. The first module, in the inner kinetochore, contributes a sturdy interface with centromeric chromatin. The second module, the outer kinetochore, contributes a microtubule-binding interface. The third module, the spindle assembly checkpoint, is a feedback control mechanism that monitors the state of kinetochore-microtubule attachment to control progression of the cell cycle. The fourth module discerns correct from improper attachments, preventing the stabilization of the latter and allowing the selective stabilization of the former. The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome bi-orientation on the mitotic spindle. Here, I report work showing that the small-molecule Reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the RZZ complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using Reversine, I also demonstrated that MPS1 is required for the correction of tensionless chromosome-microtubule attachments. An important conclusion from this work is that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. My studies describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle assembly checkpoint, and suggest that these processes are co-regulated and are likely to involve the same catalytic machinery.

Integrins are the major family of cell surface adhesion receptors responsible for the regulation of the physical contact and biochemical communication between the cell and the surrounding extracellular matrix (ECM). Binding of the extracellular domains of integrins, to components in the ECM triggers a series of molecular events commonly referred to as “outside-in” signaling, leading to context-dependent changes in cell morphology, migration and proliferation. In this prevailing paradigm of cell adhesion induced signaling the primary functions of the integrin is to provide the physical transmembrane bridge connecting the intracellular signaling machinery and cytoskeleton to the extracellular environment. We now present evidence that most, if not all, cell adhesion receptors trigger integrin-dependent outside-in signaling independently of direct contacts between the integrins and their ligands in the ECM. The urokinase-type plasminogen activator receptor (uPAR/CD87) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the outer membrane leaflet by a GPI-anchor. Through an extensive structure-function analysis of uPAR, VN, b1 and b3 we document that cell adhesion induced by the uPAR/Vninteraction triggers integrin-mediated, but ligand independent, cell spreading and signaling. This signaling is fully active on VN lacking functional integrin binding sites and by integrin mutants deficient in ligand binding, but is crucially dependent on an “active” conformation of the integrin as well as its binding to intracellular adaptor proteins including talin and kindlin. This novel paradigm of ligand-independent integrin signaling is not restricted to uPAR as it poses no identifiable constraints to the adhesion receptor with respect to ternary-structure, ligand type or means of membrane anchorage. In full accordance with a general validity of this paradigm, we show that cell adhesion physically mediated by a signaling-incompetent b3 integrin is effectively translated into cell spreading and signaling by the b1 integrin. Our results show that integrins are active in transducing adhesion-induced signaling in the absence of their cognate ligands, suggesting that the bi-directional signaling capability of these receptors may have evolved primarily to allow for tightly regulated inside-out signaling.

Development requires the coordination of signalling pathways driving proliferation, differentiation and migration. Adaptor proteins play an important role in this regulation by linking receptors to their downstream targets. The aim of this PhD project was to characterise the function of ShcD/RaLP, the newest member of the Shc adaptor proteins, as its physiological role is still unknown. To this end, RaLP knockout (KO) and heterozygote (HT) embryonic stem cells (ESCs) were derived to dissect the role of RaLP in development. RaLP expression was tightly regulated during the formation of the epiblast and then re-expressed during neuronal commitment of ESCs. We found that the loss of RaLP resulted in an impairment of the establishment of epiblast stem cells (EpiSCs) due to defective proliferation capacity upon differentiation. Furthermore, Cdx2 expressing cells emerge during ESC commitment to EpiSC fate and the absence of RaLP resulted in an enrichment of this population and enhanced levels of MAPK/Erk1/2 activation. Our data suggests that RaLP plays an important role in the switch of key pathway/s involved in determining EpiSC identity. We also showed that RaLP is implicated in the differentiation of neural stem cells to neuronal fate.
In summary, our studies evidenced that RaLP is involved in EpiSC formation and maintenance as well as neurogenesis, and this dual role is unique for this member of the Shc family, providing an insight into the mechanisms that the cell has evolved to regulate signalling pathways through adaptor proteins in a cell type specific manner.

The correct partitioning of the genomic content during cell division in eukaryotes is required for proper cell physiology. The presence of an abnormal number of chromosomes, a condition known as aneuploidy, impinges on cell fitness and eventually leads to tumorigenesis. To avoid aneuploidy, eukaryotic cells have developed a molecular safety device known as the Spindle Assembly Checkpoint (SAC). By delaying anaphase onset until all chromosomes are bioriented, the SAC ensures the fidelity of chromosome segregation. To do this, the SAC inhibits the Anaphase Promoting Complex/Cyclosome (APC/C), a protein complex responsible for mitotic exit. The Mad2 protein is a crucial component of the SAC. Mad2 exists as two different and interacting conformers (open, O, and closed, C). Once biorientation is achieved, the SAC is promptly switched off and anaphase occurs. The timely silencing of the SAC requires the protein p31comet. p31comet selectively binds to C-Mad2 and prevents the O-C dimerization. Moreover, p31comet has been shown to activate the APC/C in vitro. How p31comet is regulated in mitosis and how it ensures SAC silencing are poorly investigated issues. Here, we report a characterization of p31comet in cells. We show that, upon SAC activation, p31comet is phosphorylated by the mitotic kinases Mps1 and Aurora B, and in a Mad2-dependent manner. The function of this phosphorylation still remains elusive. We also report new p31comet interactors, and demonstrate that p31comet physiologically regulates APC/C activity, by regulating its association to the inhibitory Mitotic Checkpoint Complex. We also find that p31comet regulates the checkpoint slippage and the mitotic turnover of the protein Cdc20. Finally, we prove a previously unreported role for protein synthesis in the maintenance of the SAC.

To date, few lymphoma determinants have been, causatively linked to the pathogenesis of B-cell tumors. To identify novel genetic determinants of B-cell lymphomas, we made use of a cell-type and stage-specific transposon-based, insertional mutagenesis system dependent on the Sleeping Beauty (SB) transposase and the T2/Onc2 transposon, engineered to cause overexpression of protooncogenes or disrupt tumor suppressor genes. To restrict transposon mutagenesis to the mature Bcell compartment, we used conditional Rosa26-SB mice, in which SB expression, driven by the Rosa26 promoter, occurs upon Cre-mediated deletion of a loxP-flanked stop cassette. SB expression was induced in mature and germinal center (GC) B-cells using CD21-Cre or C?1-Cre transgenes. The conditional SB system was also used in mouse models of Bcl6- and Bcl2-driven lymphomagenesis. Insertional mutagenesis in GC B-cells was was sufficient to promote clonal Bcell tumors. Histological evaluation revealed close resemblance to human diffuse large B-cell lymphoma and follicular lymphoma. Also, we found that transposon mutagenesis efficiently cooperated with deregulated Bcl6 and Bcl2 to promote tumorigenesis. High throughput sequencing of transposon insertions from 29 lymphomas identified 240 genes lying within Common Insertion Sites (CIS). Bioinformatic analysis revealed enrichment for genes belonging to the NF-κB signaling network, commonly deregulated in human B-cell neoplasms. Analysis of gene-copy gain or loss in human lymphomas, occurrence of somatic mutations in human cancers of different type and expression in human B-cell neoplasms contributed to select, the strongest candidates to represent novel cancer determinants. Among them, we found genes controlling B-cell differentiation, survival, proliferation, chromatin remodeling and protein glycosylation.

Nucleophosmin (NPM1) is an abundant nuclear-cytoplasmic shuttling protein that is involved in different cellular processes. Recently, it has been demonstrated that NPM1 forms a stable complex with the tumor suppressor p19/Arf and it is absolutely required for its correct localization and stabilization in the nucleolus. Moreover, it has been found that about one-third of primary adult Acute Myeloid Leukemia (AML) patients bear mutations in the last exon of the NPM1 gene, leading to an aberrant cytoplasmic localization of the protein (NPMc+). Interestingly, the accumulation of NPMc+ in the cytoplasm appears responsible for the delocalization of proteins that, under normal conditions, interact with wild type NPM1 in the nucleus. In particular, NPMc+ binds, delocalizes and inactivates the p19/Arf tumor suppressor, as well as the Fbw7ϒ F-box protein that is involved in the proteasome-dependent degradation of the c-Myc oncoprotein. These data prompted us to explore the possibility of blocking the aberrant activity of NPMc+ using NPMmutant-specific antibodies. We isolated, from the ETH2-Gold phage display library, a recombinant antibody in scFv format that univocally targets the mutated region at the C-terminal end of NPMc+. The specificity of the selected antibody was evaluated in vitro by performing western immuno-blot analyses, immunofluorescence and immunoprecipitation assays. Furthermore, we found that the scFv can be successfully expressed in mammalian cells as an intrabody and specifically interacts with the native NPMc+ in vivo, thus giving the premises for its further development as a potential innovative therapeutic tool.

Within the medical nanotechnology scenario, optical fluorescence techniques have a pivotal role thanks to their non-invasive capability to address biological questions in three dimensions with a remarkable capability to distinguish fine details, tremendously improved up to the nanoscale level in the last decade.
This Thesis concerns the study and the development of advanced fluorescence optical methods for the improvement of the imaging capability and a better exploitation of its potentialities.
In particular, following a fluorescent probe approach, a nanostructured polyelectrolyte system has been designed to study the fluorescence quenching effect induced by specific quencher molecules on the fluorescence emission process. The system allows probing quencher metal ions at microrange concentrations, significantly higher with respect to the current fluorescent quenching based technologies.
As well, following an optical approach, the interferences effect induced by structuring the illumination light with different masks have been studied in order to improve some imaging features.
In the Two-Photon Excitation and in the Confocal Microscope, the insertion of a ring-shaped mask in the illumination pathway is proposed to enhance the signal to noise ratio at the high spatial frequencies, thus improving their capability to practically resolve fine details of the image. In the Widefield Microscope, the insertion of a periodic grid to structure the light has been investigated in order to confer a 3D optical sectioning capability comparable to that of the confocal microscope, with major advantages in terms of the efficient use of the light, simplicity of construction, speed of imaging acquisition, versatility and low cost.

Cluster-assembled nanostructured Titanium Oxide (ns-TiOx) deposited by Supersonic Cluster Beam Deposition (SCBD) recently proved to be a very promising biomaterial, allowing the adhesion and proliferation of cancer and primary cells, with no need of additional coating with extra-cellular matrix proteins, and the adhesion of proteins, such as streptavidin, with no need of additional coatings of polycations. The intrinsic nanostructure of this material, with fine granularity, high porosity and specific area, coupled to the chemical reactivity of the surface is likely to be a key element in determining the biological affinity of the material with nanometer-sized biomolecules, such as proteins. However, little is known of the specific role played by each of these surface properties in the interaction of proteins with nanostructured biocompatible materials. For understanding the role of different surface properties we used atomic force microscopy (AFM) to study morpho-chemical nature of ns-TiOx biocompatible surfaces, in particular we have characterized the adhesive properties of ns-TiOx against nanoprobes carrying chemical groups similar to those involved in protein-surface adhesion processes, and we have characterized the electric charging of ns-TiOx surfaces in aqueous medium at different pH, and how this is affected by surface roughness.
AFM Force-Spectroscopy measurements have been used to characterize local adhesive properties of ns-TiOx surfaces. In order to achieve this goal we have developed a patterning strategy based on the combined use of SCBD and Nanosphere Lithography (NSL), for the production of sub-micrometer patterns of ns-TiOx on glass and other substrates. With this methodology one can have both target and reference material in the same investigation area. Results indicated that atoms on the surface of ns-TiOx can form coordinate bond with protein molecules thereby aiding in irreversible protein adsorption at the same time retaining complete biological activity.
To further understand how protein adsorption is affected by the buffer medium and by the surface properties of the substrate, we have measured the point of zero charge (PZC) of nanostructured cluster-assembled TiOx. As each kind of protein has different isoelectric point (IEP), hence their adsorption is greatly affected by pH of the buffering medium and concentrations of ions in the solutions. To this purpose, colloidal probes were developed to measure attractive and repulsive forces of a silica micro-sphere against metal oxide surface as a function of pH. Estimated PZC values for TiOx (rutile) and ns-TiOx is 4.9 ± 0.5 & 3.0 ± 0.5, the latter being significantly smaller than PZC typically measured on crystalline surfaces. These results can open up new avenues towards understanding adsorption characteristics of various proteins on metal oxide surfaces.

In this dissertation I consider the ethical foundations of clinical research and propose a shift towards a new framework. Since the beginning, the ethical foundations of clinical research have been focused on the protection of human subjects, with a strong emphasis of the importance of the informed consent process and on issues of vulnerability,coercion, and exploitation. This is understandable, as the ethical guidelines regulating clinical research worldwide (Nuremberg Code, Helsinki Declaration, Belmont Report) were born in the aftermath of the WWII. But the historical context has changed, and the emphasis on the protection of subjects may be anachronistic today and have deleterious repercussions on the clinical research enterprise, by placing the regulatory bar too high. My case-study is the first Phase 0 clinical trial performed at NCI-NIH, the ABT-888 study. Phase 0 trials are clinical studies, which have no intention to treat and are performed before the traditional Phase 1 studies, with the aim of proving in humans a molecular mechanism of action which has been demonstrated in the animal models. In this work I first analyze the traditional topics in the ethics of clinical research, such as informed consent, the possibility of therapeutic misconception or misestimation, the vulnerability of terminally ill subjects. I then consider the arguments in favor and against the so-called “duty to participate in clinical research”, providing an extensive overview of the ethical debate. Considered the pros and cons, I conclude that there are sufficiently solid ethical arguments to support a shift in our attitude towards participation in research from the traditionally conceived super-erogatory view to a, even if imperfect, moral duty. I then switch to the policy level, and propose some practical implementations of the ethical arguments in favor of the duty in research based on the libertarian paternalistic view developed by Sunstein and Thaler. To conclude, the ethical foundations of clinical research need to be reshaped: we all have an interest in the advancement of medical research, and participation in research should be seen both as a duty and a right. Trust should become one the fundamental value of these new foundations, which also have practical implications, for the rewriting of Helsinki Declaration.

This thesis discusses the pleiotropy theory of ageing in evolutionary biology in some of its most important aspects. In short, the theory says that natural selection fosters the early-life survival and reproductive performances of an organism even if this would necessarily impose a cost on later-life survivorship and reproduction thereby leading to the evolution of that peculiar pattern of age-correlated decline in biological functioning that characterizes ageing. The theory predicts that the level of external/environmental mortality should positively correlate with the rate of ageing in a population. And it is often invoked to suggest how the phenomenon of cellular senescence, which indicates the state of somatic cells unable to perform further division, may have an impact on ageing.
Using the notions of the semantic view of theories it is shown that the pleiotropy theory of ageing is actually a family of related mathematical models. Among these, two main models are identified: the genetic variant, which is a population-genetic model; and the optimality variant, which represents an instance of optimality thinking in evolutionary biology. Relevant similarities and differences between the two models are discussed. It is then shown that the current ways of interpreting Gompertz’s mortality model - which is a simple empirical model to which mortality data can be fitted - in order to test the above mentioned prediction of the theory does not make it a genuine truth-making structure for the theory thereby exposing the test of the prediction to potential unreliability. Independently from the semantic view of theories, it is then discussed how to provide new methodological foundations to the hypothesis that cellular senescence may be a case of the pleiotropy theory.

Normal mitotic cells duplicate their genome once each cell cycle to ensure its correct transmission. The biochemical process leading to genome duplication has been highly conserved during evolution and can be described by the well-established replicon model (Jacob and Brenner 1963; Newlon and Theis 1993; Gilbert 2004). According to this model, sequence elements (replicators) genetically determine where DNA replication initiates by interacting with trans-acting regulatory factors (initiators, such as the ORC and the MCM complexes) that are cell cycle regulated (Ohta, Tatsumi et al. 2003). Despite the early successes in the identification of microbial eukaryotic origins (Aladjem and Fanning 2004; Gilbert 2004), the available methods for origin identification in mammalian genome and the main results so far generated by their application are sometimes controversial and have actually led to the detailed characterization of just few origins (S. Otha 2003). Only recently the application of genome wide techniques for the hybridization to tiling micro-arrays of neo-synthesized DNA has allowed the identification of a larger number of replication origins, but only in a small portion of the human genome (Lucas, Palakodeti et al. 2007; Cadoret, Meisch et al. 2008). To better understand the regulation of DNA replication, I developed a new method to map replication origins on the basis of their chromatin structure. In this work, I first isolated origin-rich DNA and then it was hybridized onto a chromosome 19 microarray: 217 replication origins were identified, characterized by an open chromatin structure. Among them, I selected 101 replication origins (with a validation rate of 95%), all containing a CCAAT box motif and bound by the NFY transcription factor. 77% of the newly identified origins were located near transcription start sites of expressed genes. I finally used massive sequencing of the same origin-rich DNA and identified 1,846 putative replication origins along the entire human genome. Most of them were located close to gene TSSs, confirming the likelihood of a correlation with gene transcription.

Kinetochores are highly conserved proteinacious structures that assemble on centromeresfollowing DNA replication. Kinetochores attach sister chromatids to the mitotic spindle and orchestrate chromosome segregation. An erroneous kinetochore can lead to aneuploidy, a hallmark of cancer. An amenable organism to study kinetochore assembly and activity is the budding yeast Saccharomyces cerevisiae. Cnn1p, a non-essential kinetochore protein of S. cerevisiae, was recently co-purified with conserved kinetochore subunit Nnf1p. This project aims to genetically, biochemically and functionally analyse Cnn1p to understand its role in chromosome segregation. We determined that Cnn1p localises at low levels only to centromeres in an Ndc10p and Ndc80p dependent fashion. Cnn1p affects chromosome segregation as its overexpression causes chromosome loss. A strong interaction of Cnn1p with members of the Ndc80 complex was shown via yeast two-hybrid analysis and co-purifications, potentially implicating the Ndc80 complex as a direct interaction partner of Cnn1p. The possibility that Cnn1p might be a checkpoint protein was excluded by performing a viability assay in nocodazole. Cnn1p strongly genetically interacts with essential kinetochore proteins Nnf1p, Okp1p, Spc105p and Dam1p. Deleting CNN1 increases mono-orientation of nnf1-17 mutants at semi-permissive temperature, yet after treating cells with and releasing them from nocodazole this is not the case, indicating that Cnn1p may be involved in kinetochore assembly and/or integrity rather than in microtubule capture per se. This function is further supported by the fact that Cnn1p is phosphorylated by Clb5p-Cdk1p in S-phase (Loog & Morgan 2005) when yeast kinetochores assemble. The function of Cnn1p is therefore likely in establishing and/or maintaining kinetochore integrity.

Recent findings suggest that the increased tumor suppression activity of p53 can promote aging (Rodier et al., 2007; Serrano and Blasco, 2007). P53 mutant mice characterized by chronical p53 activation (p44-Tg and p53m/+ model) exhibit increased cancer resistance but a shortened lifespan in association with early aging-associated phenotypes (Maier et al., 2004; Tyner et al., 2002). The lifespan determinant p66Shc was the first mammalian gene whose mutation was demonstrated to extend lifespan by conferring resistance to oxidative stress-induced apoptosis without increase in cancer risk (Migliaccio et al., 1999; Trinei et al., 2002). Ideed, p66Shc is a mitochondrial red-ox enzyme that, in response to a variety of oxidative stresses, generates reactive oxygen species (ROS) as mediators of apoptosis (Giorgio et al., 2005). Indirect evidences suggest that p53 is implicated in the p66Shc apoptotic-signalling pathway and that p66Shc is an oxidative-stress downstream target of activated p53 (Trinei et al., 2002). Since the p53-p66Shc pathway is specifically involved in the propagation of pro-apoptotic oxidative signals (p53-dependent apoptosis in response to gamma-irradiation or adryamicin proceeds normally in p66ShcKO cells), our working hypothesis is that distinct p53-pathways might regulate tumour suppression and aging and p66Shc might be a selective downstream-target of p53 not involved in tumor suppression (Trinei et al., 2002). To further characterize the p53-p66Shc-ROS-signalling pathway we investigated whether the p53-p66Shc-ROS pathway, besides apoptosis, regulates a specific transcriptional program involved in physiological aging. In summary I found that: i) p53 and p66Shc repress a mitotic signature of genes in H₂O₂ treated MEFs as well as physiologically in thymus; ii) the up-regulation of the mitotic signature genes in p66ShcKO thymus correlates with a retarded aging-associated involution and senescence of the organ; iii) the p66Shc deletion in the progeric p44-Tg mouse model rescues the boosted repression of the mitotic signature as well as the accelerated thymus involution; iv) p66Shc might regulate the transcriptional-inhibitory function of p53 on the mitotic signature genes through a ROS-dependent effect on the life span determinant Sirt1.

The TOCA (Transducer of Cdc292 dependent actin assembly) family of F-BAR (Bin, Amphiphysin, Rvs) -containing proteins binds to and remodels lipid bilayers via their conserved F-BAR domains, and regulates actin dynamics via their N-WASP (Neuronal Wiskott–Aldrich syndrome protein) binding SH3 (Src homology 3) domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) Loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 (WASp homolog) and WVE-1 (WAVE (WASP-family verprolin) homolog), and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell-cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1 (Adherens junction marker 1). iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C.elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1 (Abl-interactor 1). Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP-dependent actin-dynamics.

Giuliani C, Troglio F, Bai Z, Patel FB, Zucconi A, Malabarba MG, Disanza A, Stradal TB, Cassata G, Confalonieri S, Hardin JD, Soto MC, Grant BD, Scita G "Requirements for F-BAR proteins TOCA-1 and TOCA-2 in actin dynamics and membrane trafficking during Caenorhabditis elegans oocyte growth and embryonic epidermal morphogenesis". PLoS Genet. 2009 Oct;5(10):e1000675

In Saccharomyces cerevisiae the conserved and essential Rio1p kinase regulates 20S rRNA processing during ribosome synthesis. However, shutting-off Rio1p expression leads to an accumulation of the Rio1p-depleted population in G1 and in metaphase, suggesting an involvement of Rio1p in mitosis. In Aspergillus nidulans growth of a sudD (rio1) temperature-sensitive mutant was rescued by overexpressing SUDA (SMC3), which encodes cohesin subunit Smc3. These observations and the recent identification of Rio1p, kinetochore proteins, and cohesin components in immunopurifications of kinetochore proteins Mtw1p and Nnf1p (De Wulf lab), further indicated a possible involvement of Rio1p in chromosome segregation by acting at kinetochore or cohesin level. The aim of my project was to analyse the functional involvement of the kinase at this cell-cycle stage. We found that Rio1p localises both to centromeres and cohesin binding sites along the chromosome arms. Epistatic analyses performed both with a strain reduced in Rio1p function and with a strain containing enhanced Rio1p levels, revealed strong, positive interactions between Rio1p and some kinetochore complexes and with most components of the cohesin pathway, except for Esp1p with which it negatively interacted. To functionally dissect the involvement of Rio1p in the regulation of budding yeast mitosis, we constructed a yeast strain in which two TEV protease cleavage sites were introduced in the Rio1p protein sequence. The strain was then transformed with a high-copy plasmid expressing TEV protease from the galactose-inducible GAL1 promoter. Cells in which Rio1p was conditionally cleaved showed a 15 minute delay in G1 and a 30-40 minute delay in metpahase. The later was not the result of spindle assembly checkpoint response. A retarded degradation of Pds1p-3HA and Scc1p-3HA confirmed the metaphase delay and points to a delayed cleavage of cohesin complex. Taken together our genetic and cell cycle analyses propose a hitherto unknown role of Rio1p in regulating mitosis and the correct timing of sister chromatid separation.

The aim of this thesis is to investigate two important networks involved in the cell cycle progression: the spindle assembly checkpoint (SAC), and the regulation of the phosphatase Cdc14. The SAC is active at the metaphase-to-anaphase and its target is APCCdc20, which triggers anaphase onset via Securin and Cyclin B degradation. By inhibiting Cdc20, the SAC delays sister chromatids separation until the very last of them attached to the spindle. First, we focused our attention on the interaction between Cdc20 and Mad2, a key component of the SAC. Studying this interaction in vitro, we found that the basal rate of Mad2 binding to Cdc20 is slow, and Mad2 dimerization accelerates it via a catalytic step. Interestingly, we showed that catalysis does not modify the equilibrium of the Mad2:Cdc20 interaction, and suggest that the release of Mad2 from Cdc20 is an energy-driven process. Second, starting from the in vitro results, we developed a mathematical model describing SAC activation and disengagement. The model reproduced the main systems level proprieties of the SAC: a sharp and irreversible threshold set at one unattached kinetochore, robustness to variations in proteins concentration, and fast dynamics of activation and inactivation. Lastly, we studied the regulation of Cdc14 in budding yeast where the phosphatase is known to have a pivotal role in mitotic exit. During a regular cell cycle, Cdc14 is kept sequestered and inactive in the nucleolus. We clarified that its release in anaphase requires Cdc5 and either one between MEN and CDKs kinase. Once activated, Cdc14 triggers a negative feedback loop, which culminates in Cdc5 degradation by APCCdh1, and thus in Cdc14 re-sequestration in the nucleolus. We showed that, in the presence of high levels of mitotic Cyclins, the negative feedback loop is blocked in an oscillatory regime where we could observe periodic cycles of Cdc14 release and sequestration. Both the SAC and Cdc14 contribute to induce the irreversible switch from a mitotic to a G1 state of the cell cycle via the inactivation of mitotic CDKs. At the metaphase-to-anaphase transition, the SAC assures that the APCCdc20 starts Cyclin B degradation in coordination with sister chromatids separation. Soon after, Cdc14 allows the completion of mitotic Cyclins degradation and the dephosphorylation of CDKs substrates.

Simonetta M*, Manzoni R*, Mosca R, Mapelli M, Massimiliano L, Vink M, Novak B, Musacchio A, Ciliberto A. “The influence of catalysis on Mad2 activation dynamics”. PLoS Biol. 2009 Jan 13;32(1):e10. *equal contribution

Homeodomain proteins constitute a large class of eukaryotic DNA-binding proteins that regulate transcription of a broad range of developmentally important genes. These proteins share a 60 amino acid DNA-binding domain which has been conserved in sequence, structure and mechanism of DNA-binding. While monomeric homeodomain proteins exhibit a limited ability to discriminate between different DNA sequences, their specificity is significantly enhanced through the cooperative binding to DNA with other DNA binding partners. Pbx1 (pre-B-cell leukemia homeobox 1) and Prep1 (Pbx-regulating protein 1), both belonging to the TALE (three amino acids loop extension) family of homeodomain proteins, forms a strong and stable complex with each other, independent of DNA binding. Pbx1 contains a nuclear localisation signal and carries Prep1 into the nucleus. Prep1 and Pbx1 form trimeric complexes with HoxB1 on target enhancers. HoxB1 is known to play an important role in development. The aim of this project was to characterise the structural and functional properties of the homeodomain proteins Prep1 and Pbx1 and their interaction with DNA in order to reach an understanding of the structural basis underlying the functional DNA target specificity. In a broader perspective, the determination of these structures will be valuable in the characterisation and understanding of the functions of this class of transcription factors during processes of development and cancer. I have therefore set up methods and conditions to purify crystallisable Prep1/Pbx1 and Prep1/Pbx1/DNA complexes. I have optimised protein constructs, expression conditions, purification methods and characterized the DNA binding in order to select the optimal DNA oligonucleotides for co-crystallisation and performed crystallisation screenings of a range of DNA oligonucleotides with Prep1and Pbx1.

Villaescusa JC, Buratti C, Penkov D, Mathiasen L, Planagumà J, Ferretti E, Blasi F. "Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation". PLoS One. 2009;4(4):e5213

Inflammation is a response of a tissue to injury, often caused by invading pathogens. Inflammation is considered one of the most important factors contributing to tumorigenesis and tumor progression. Here we have shown that both TLR4 and MyD88 are essential for DMBA-croton oil induced mouse skin tumorigenesis. By contrast, TLR2 and TLR9 do not seem to contribute to skin tumorigenesis. Absence of TLR4 or MyD88 leads to reduced inflammation after croton oil/TPA treatment, suggesting that inflammation plays an important role in TLR4/MyD88 mediated tumor development. Both bone marrow derived and radio-resistant cells are required for carcinogenesis as bone marrow chimeras where TLR4 is missing in either one cell types do not develop carcinomas. Eliminating skin-colonizing bacteria with antiseptics or blocking bacterial lipopolysaccharide (LPS, a ligand for TLR4) in the skin does not reduce inflammation after croton oil/TPA treatment, suggesting that skin-colonizing bacteria are not involved in TLR4-dependent tumorigenesis. In contrast, blocking of high mobility group box-1 protein (HMGB1), one of the endogenous ligands for TLR4, inhibits the recruitment of inflammatory cells and croton oil/TPA-induced inflammation, which shows that TLR4 signaling is triggered by HMGB1 protein released in situ. These results suggest that the initial release of HMGB-1 triggers an inflammatory response that is dependent on TLR4 and leads to tumor development.

Avogadri F, Mittal D, Saccheri F, Sarrafiore M, Ciocca M, Larghi P, Orecchia R, Rescigno M. "Intra-tumoral Salmonella typhimurium induces a systemic anti-tumor immune response that is directed by low-dose radiation to treat distal disease". Eur J Immunol. 2008 Jul;38(7):13337-47.

Acute promyelocytic leukaemia (APL) occurs as a consequence of a chromosomal translocation involving the retinoic acid receptor alpha (RARα) and, in most of the cases, the promyelocytic leukemia protein (PML). Other proteins, however, were found translocated near to the receptor in APL and, strikingly, all the identified RARα partners bear a self-association domain that has been shown to be responsible for altered transcriptional properties of the fusion protein. It is known the dominant negative behaviour of PML-RARα on both PML and RARα coming from the remaining wild type alleles. Indeed, upon the expression of the chimeric oncogene, PML-RARα homo-oligomers are formed, which constitutively repress the transcription from the Retinoic Acid Responsive Elements (RAREs), together with the deregulation of PML protein, due to NBs delocalization. In the present study we analyze more in details the contribution of the PML oligomerization domain (a Coiled-Coil region), and of the recruitment of the Corepressor complexes to leukemogenesis. We used two chimeric proteins containing alternatively the homologous PML Coiled-Coil domain (CC-RAR) or a heterologous oligomerization domain derived from the yeast GCN4 transcription factor (GCN4-RAR). We evaluated their transforming potential in hematopoietic progenitors and in transduction/transplantation experiments in wild type mice. We thus demonstrated that, despite the transforming potential of both the oligomerization constructs, only the presence of the PML Coiled-Coil domain triggers RARα to exert its full leukemogenic potential. Additionally, we found that the PML-RAR/AHT mutant, unable to recruit the N-CoR/HDAC complex, and thus impaired in blocking myeloid differentiation, is otherwise able to trigger an APL-like leukemia in mice. Therefore we proved that other mechanisms of transcriptional repression, beyond HDACs recruitment, are involved in the transcriptional inactivation of RARα target genes. In conclusion, the present structure-function study of the chimeric oncogene PML-RARα has expanded our comprehension of the structural determinants of PML-RARα oncogenic action, and is beginning to unravel alternative views on the mechanism of action of the fusion protein, with respect to the actual knowledge regarding both the PML and RAR moieties.

Villa R, Pasini D, Gutierrez A, Morey L, Occhionorelli M, Viré E, Nomdedeu JF, Jenuwein T, Pelicci PG, Minucci S, Fuks F, Helin K, Di Croce L. "Role of the polycomb repressive complex 2 in acute promyelocytic leukemia". Cancer Cell. 2007 Jun;11(6):513-25.

Eps15 and Eps15R are homologous proteins involved in clathrin and non-clathrin mediated endocytosis of RTKs. I aimed to understand the physiological role of these proteins taking advantage of knockout (KO) mice. Eps15KO mice were viable and had an increased number of red blood cells (RBCs), with decreased size and mean corpuscular hemoglobin. Since Eps15KO RBCs lost transferrin receptor (TfR) protein expression faster than wild type (WT) once, lack of Eps15 might impair TfR endocytosis and/or trafficking. Eps15KO mice showed also an increased number of splenic marginal zone (MZ) B cells. In competitive repopulation of the immune system, Eps15KO cells have an advantage over WT once in producing MZ B cells and thymic T cells, but a disadvantage to form B1 B cells. Since the development of these populations is regulated by Notch signaling, it is possible that in the immune system Eps15 is involved in the Notch pathway. Eps15RKO mice were postnatal lethal and lethality was not explained but, based on the function of Eps15/R orthologue in lower organisms and of their mammalian interactors, a role for Eps15R in synaptic vesicle recycling is compatible with the observed lethality. Combined lost of Eps15/R let to an embryonic lethal phenotype similar to that of Numb, Notch and epsin1-2 KO mice. Since Eps15/R interact with Numb and Epsins and that endocytosis and Notch signaling are linked mechanisms, it is possible that Eps15/R interact with Notch pathway during organogenesis. This project pointed out the possibility that Eps15/R are involved in different physiological processes that require them to act in non-redundant and redundant cellular processes. Further experiments will be necessary to elucidate the exact nature of these cellular processes.

Offenhäuser N, Castelletti D, Mapelli L, Soppo BE, Regondi MC, Rossi P, D'Angelo E, Frassoni C, Amadeo A, Tocchetti A, Pozzi B, Disanza A, Guarnieri D, Betsholtz C, Scita G, Heberlein U, Di Fiore PP. "Increased ethanol resistance and consumption in Eps8 knockout mice correlates with altered actin dynamics". Cell. 2006 Oct 6;127(1):213-26.

Starting from two datasets of cancer related genes, we have analyzed genomic and network properties of ~600 genes mutated in different cancer types. Genes mutated in cancer are prevalently singletons at the genomic level and encode for central hubs of highly interconnected modules within the protein–protein interaction networks (PIN). We have compared the duplicability rate and the network topology of cancer genes with the rest of human genes, showing that genes related to cancer progression have particular genomic and network properties. According to these results, we depict cancer genes as fragile components of the human gene repertoire, sensitive to dosage modification. In contrast to what has been observed in yeast, where all essential genes are prevalently singletons that encode for highly interconnected hubs, in the human PIN the nodes with similar characteristics are rare and probably enriched in candidate cancer genes. We have then extracted from the human protein interaction network genes with similar properties, which may be involved in cancer progression.

Rambaldi D, Ciccarelli FD. "FancyGene: dynamic visualization of gene structures and protein domain architectures on genomic loci". Bioinformatics. 2009 Sep 1;25(17):2281-2.
Rambaldi D, Giorgi FM, Capuani F, Ciliberto A, Ciccarelli FD. "Low duplicability and network fragility of cancer genes". Trends Genet. 2008 Sep;24(9):427-30. Review.
Fumasoni I, Meani N, Rambaldi D, Scafetta G, Alcalay M, Ciccarelli FD. "Family expansion and gene rearrangements contributed to the functional specialization of PRDM genes in vertebrates". BMC Evol Biol. 2007 Oct 4;7:187.

Histone deacetylases (HDACs) are members of an ancient enzyme family, that catalyze the removal of acetyl groups from lysine residues in both histone and non-histone proteins. Among the 18 HDACs encoded in the mammalian genome, many studies suggest that HDAC1, HDAC2 and HDAC3 are critically involved in cell proliferation, but it is still unclear their individual contribution. To address this point, we analyzed the proliferation of HDAC1, HDAC2 and HDAC3 depleted cells. We show here that in the absence of HDAC1, cells can arrest either at the G1 phase or at G2/M transition of the cell cycle, resulting in the loss of mitotic cells, cell growth inhibition and increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation, unless we concurrently knocked down HDAC1. Given the defective cell cycle progression of HDAC1 depleted cells, we asked whether and how its cell cycle related function are regulated. Our data revealed that HDAC1 is subjected to a hyperphosphorylation in mitosis. A mitotic hyperphosphorylation of HDAC2 has already been reported and was confirmed also in our analysis. In vitro phosphorylation assays show that HDAC1 and HDAC2 are phosphorylated by Aurora kinases, mitotic-specific serine-threonine kinases that orchestrate proper mitotic progression and are found deregulated in many cancers. Treatment of synchronized cells with different inhibitors of Auroras as well as siRNA experiments confirm that Aurora kinases are responsible for HDAC1 and HDAC2 phosphorylation in vivo, while inhibition of other mitotic kinase cascades, such as the Plk-1 pathway, has no effect. S406 HDAC1 and S407 HDAC2 were identified as phospho-acceptors sites by mass spectrometry analysis and confirmed by site directed mutagenesis in vitro and in vivo. A phosphopeptide-specific antibody recognizing phosphorylated S406 HDAC1 (anti-pS406-HDAC1) revealed a stronger HDAC1 hyperphophorylation in prophase that quickly diminished in prometaphase. In metaphase a crown-like distribution of pS406-HDAC1 was also reported. Our data globally suggest a mechanistic link between HDACs and Aurora kinases in mitosis. Since both HDACs and Aurora kinases are involved in human tumorigenesis and their inhibitors represent promising anticancer drugs, elucidating this relationship could provide new insights for the application of combinatorial approaches in cancer therapy.

1) Zupkovitz G, Grausenburger R, Brunmeir R, Senese S, Tischler J, Jurkin J, Rembold M, Meunier D, Egger G, Lagger S, Chiocca S, Propst F, Weitzer G, Seiser C. "The cyclin dependent kinase inhibitor p21 is a crucial target for Histone deacetylase 1 as regulator of cellular proliferation.". Mol Cell Biol. 2009 Dec 22.
2) Senese S, Zaragoza K, Minardi S, Muradore I, Ronzoni S, Passafaro A, Bernard L, Draetta GF, Alcalay M, Seiser C, Chiocca S. "Role for histone deacetylase 1 in human tumor cell proliferation". Mol Cell Biol. 2007 Jul;27(13):4784-95.

There is a large body of literature that describes the geometry and the physics of filopodia using either stochastic models [1, 2] or partial differential equations and elasticity theory [3]. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures [4]. In this thesis, using a combination of in-vivo and in-vitro experiments, and in-silico modeling we characterized a network of interacting molecules that differentially regulate filopodia formation in multiple cell lines. The components of our network are the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi1 and IRSp53, respectively; VASP and Capping Protein, which exert antagonistic functions in controlling filament elongation. We connected the protein network to a simple system of actin polymerization, and determined most of the parameters governing the interactions of this minimal system to build a mathematical model. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which we experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular context, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia.

The control of cell growth, proliferation and differentiation on biomaterials surfaces is of fundamental importance for regenerative medicine, prosthetics, or cell-based assays. The microfabrication of cell-on-chip platforms based on a new family of poly (amidoamine) hydrogels, are promising for in vitro and in vivo applications. Hydrogels present characteristics that mimics biological environments, such as the cross-linked nature of the extracellular matrix, the tissue properties (high water content), and the permeability to oxygen and metabolites. Hydrogels based on poly (amidoamine) results in an optically transparent, biocompatible and fully biodegradable substrates, recommended for body implants that are minimally invasive, and naturally eliminated by human body. In my PhD work I intended to use microfabricated hydrogels for fine-tuning the contact guidance of cells. As microfabrication tools I set up reaction injection moulding for producing features down to 100 µm and developed a novel approach relying on electron beam lithography. This innovating microfabrication consists in the ability of directly writing patterns on already cross-linked hydrogels, with the capability of producing structures at sub-micrometric scale. The exposure to the electron beam produces particular modifications enabling the control of physico-chemical properties of irradiated area. I obtained a selective attachment of proteins as a function of the electron-beam dose; an exclusive adhesion and growth of neural cells on the exposed surfaces; and the control of neurite outgrowth guidance along a microfabricated network. These results offer new perspectives to build physiological microenvironments or cell-on-chip platforms, based on a novel class of microfabricated hydrogels.

A paradigm in transcriptional regulation is that a graded increase in transcription factor (TF) concentration is translated into a digital on/off transcriptional response by cooperative TF binding to adjacent cognate sites in cis-regulatory sequences. This digital type of response underlies the sharp definition of boundaries among body parts during development. Here we show that NF-kB, a key TF controlling the inflammatory and immune transcriptional responses, is instead an analogical transcriptional regulator relying on the usage of homotypic clusters of high-affinity binding sites. We show that a widespread feature of NF-kB target genes is the homotypic clustering of high-affinity binding sites in their promoter-proximal regions. Contrarily to what expected, we observed that gradually increasing concentrations of NF-kB in the cell nucleus are translated into gradually increasing levels of transcriptional activity of NF-kB inflammatory target genes. We provide a thermodynamic interpretation of the experimental observations by combining quantitative measurements and a minimal physical model of an NF-kB dependent promoter. We show that NF-kB does not bind cooperatively to adjacent sites in a cluster; conversely, independent binding of NF-kB to adjacent sites promotes RNA Pol II recruitment in an additive fashion, thus resulting in a graded transcriptional response. These findings reveal a novel paradigm in the usage of clustered TF binding sites, which may be relevant in all biological conditions in which the transcriptional output must be proportionate to the strength of an environmental input.

Licciulli S, Luise C, Zanardi A, Giorgetti L, Viale G, Lanfrancone L, Carbone R, Alcalay M. "Pirin delocalization in melanoma progression identified by high content immuno-detection based approaches". BMC Cell Biol. 2010 Jan 20;11(1):5.
Giorgetti L, Bongiorno G, Podestà A, Berlanda G, Scopelliti PE, Carbone R, Milani P. "Adsorption and stability of streptavidin on cluster-assembled nanostructured TiOx films". Langmuir. 2008 Oct 21;24(20):11637-44.
Giorgetti L, Zanardi A, Venturini S, Carbone R. "ImmunoCell-Array: a novel technology for pathway discovery and cell profiling". Expert Rev Proteomics. 2007 Oct;4(5):609-16.

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