Altieri Fabio

Unravelling the ERp57/PDIA3, a multifunctional protein disulfide isomerase involved in cellular stress response, cell signalling and tumour progression: potential therapeutic target in several human diseases (01-01-2013 - 31-12-2017)
ERp57/PDIA3 is a member of the protein disulfide isomerase family mainly present in the endoplasmic reticulum, where it carries out a major role as a key molecular player in the quality control and correct folding of newly synthesized glycoproteins. Many studies suggest that ERp57, through its specific enzymatic activity and wide binding capabilities, may play a variety of functions, especially outside endoplasmic reticulum. ERp57 can prevent oxidation of thiol residues and aggregation of target proteins which might result in a loss of functional and/or structural properties. ERp57 may be engaged in cellular signalling pathways involving reactive oxygen species: it may rescue the functional activity of target proteins that undergo to redox modification and/or control the formation of macromolecular complexes involved in the adaptive response to oxidative stress. ERp57 has also a critical role in embryonic development, since its deficiency in knockout mice is lethal at embryonic stage, and is involved in differentiation of certain cancer cells and in several disease states. Considering the association of ERp57 with receptors or with signal transduction proteins and its status as a receptor itself, this protein appears to have an important role in signal transduction and gene regulation. ERp57 can be considered a marker of various disease states and an important target for developing novel strategies in the treatment and early diagnosis of several human diseases. Aims of this research project are: i) to identify proteins associated with ERp57, ii) to elucidate cellular processes in which it plays a key role, and iii) to find specific substances able to bind ERp57 and modify its properties to selectively control cellular processes and signalling pathways involving ERp57.

Antonelli Guido

Deciphering the identity of Torque Teno virus (TTV) as marker and potential determinant of immunity (01-01-2009 - 31-12-2017)
Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is an effective therapeutic option for several hematological malignancies whose success is limited by acute graft-versus-host disease (GVHD) and infections. In allografted patients, after the significant damage of the immune system, due the immunosuppressive treatment, stem cells reconstitute the immune function. Recent reports suggest that a good approximation of functional immune reconstitution could be obtained by monitoring Torque Teno virus (TTV), a disease-orphan virus ubiquitous in the human population that has been very recently shown to be the virus more represented into the human virome. Nevertheless, TTV is one of the less characterized viruses and, in particular, the molecular mechanisms through which TTV interacts with the immune system are nearly completely unknown. On these bases, we designed a project whose goal is to verify whether TTV viremia could represent a surrogate marker of immune reconstitution and whether the dynamics of TTV load could also influence the recovery of immune response other than being a marker that reflects the state of the immune system. To this end, first we will develop cell-based assays allowing to study molecular interactions between TTV and some innate immune pathways including interferon; then, we will try to extrapolate the obtained findings in ex vivo setting; finally, we will try to verify whether TTV could represent a unique surrogate marker, representing the synthesis of several immunological markers, able to witness the level of immune activation, the process of immune-reconstitution, the risk of GVHD or infections and, finally, the overall clinical outcome.

Barnaba Vincenzo

Interplay amongst chronic immune activation, apoptosis, cross-presentation, immune-regulation, and autoimmunity (01-01-2013 - 31-12-2017)
Hepatocellular carcinoma (HCC), with over 560,000 new cases each year, is driven by multiple, non-mutually exclusive factors, including viruses (HBV, HCV), chronic inflammation, DNA damage, epigenetic and genetic changes. Vigorous acute inflammatory processes can favor an immune effector response potentially able to induce tumor regression. Conversely, a status of pre-existing chronic inflammation can participate to the development of cancer, by the production of growth and angiogenic factors eventually promoting cancer-cell survival, implantation and growth, as in the case of liver cirrhosis by both HBV and HCV, where the phenomena of necrosis, cell renewal and even neoplastic transformation might simultaneously occur. In addition, chronic inflammation can affect the immune-surveillance directly via its own intrinsic mechanisms (i.e., expansion of Treg cells, T cell exhaustion, etc), and indirectly by the incapacity to limit the immunosuppressive effects by tumors. Wnt/-catenin pathway is frequently activated in HCC, and recent studies indicate that this pathway is critical for the differentiation, polarization and survival of mature T lymphocytes. The expression of HBV-HBx protein in hepatocytes and HCV core protein in HCC cell lines, induces the activation of Wnt/-catenin signaling that may interact with tumor-infiltrating T cells, ultimately affecting T cell activation and function. Aims of our proposal is to define: (i) the impact of chronic low-level inflammation in HCV-induced HCC development; (ii) the role of Wnt signaling pathway in T cell activation and differentiation in tumoral microenvironments; (iii) the capacity of tumor cells producing Wnt ligands to favor microenviromental cues influencing tumor-infiltrating T cell function and phenotype.

Bernardini Maria Lina

Critical role of the inflammasome activation/evation induced by Shigella Flexneri and Pseudomonas aeruginosa: analysis of bacterial triggersand host cell responses (01-01-2013 - 31-12-2017)
Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18 and pyroptosis. Inflammasome activation and subsequent pyroptosis or release of pro-inflammatory cytokines are required to remove the replicative niche of intracellular pathogens. Therefore, pathogenic bacteria respond to this attack by modulating their recognition by PRRs and subverting the mechanisms of inflammasome formation. In this proposal we will analyze the impact of the inflammasome activation during the infectious process of Shigella flexneri and Pseudomonas aeruginosa. These two bacteria have been identified as prototypes of infectious agents inducing an acute inflammatory disease, i.e. shigellosis, and a chronic infection, i.e. that occurring in patients of cystic fibrosis chronically infected by P. aeruginosa. Shigella spp. are enteroinvasive bacteria that invade the colonic and rectal mucosa where their presence is characterized by an intense inflammatory response. P. aeruginosa is an ubiquitous microorganism, acting as the major driver of chronic respiratory infection in cystic fibrosis patients. IL-1β release, which is the hallmark of inflammasome activation, is a key event in the pathogenesis of Shigella infection. Likewise, IL-1β is associated with the severity of inflammation during acute and chronic phases of P. aeruginosa infection in CF patients. We plan to characterize the host cell molecules and bacterial triggers involved in the inflammasome activation/evasion induced by these two pathogens.

Boffi Alberto

Bacterial globins as regulators of thiol redox equilibrium in bacteria (01-01-2013 - 31-12-2016)
The present proposal addresses newly discovered pathways for oxygen/redox sensing in parasitic microorganisms of the genus Lehismania by both Structural Biology and Proteomic approaches. Based on our previous work on redox metabolism in Lehismania, a complex switch over point has been envisaged/hypothesized that entails a cross-talk between disulfide stress metabolism (trypanothione pathway) and dioxygen (or ROS) sensing. A unique globin-coupled sensor (GCS) made of a tandem neuroglobin-like gene coupled to an adenylate cyclase sequence, has been found by in silico analysis of proteins encoded by different Leishmania strains. Such unusual GCS system, unmatched in both bacterial and eukaryotic genomes, may represent the key redox metabolic switch in parasitic microorganisms. These species (including most relevant pathogens, such as trypanosomes) are in fact commonly characterized by the presence of a unique thiol redox metabolic pathway and a restricted heme proteins toolbox. Thus, in the framework of the present project, we propose to analyze the expression of GCS from L. major, one of the model strains, and L. infantum and L. donovani, responsible for visceral Leishmaniasis, under different oxidative stress conditions. In parallel, we aim at characterizing the ligand binding properties, enzymatic activity and fine structural properties of this most unusual biosensor. Given the exclusivity of this pathway among living beings, we will also investigate possible mechanisms of inhibition in the framework of novel drug target discovery.

Bozzoni Irene

RNA-RNA and RNA-protein interactions: role of small non-coding RNAs in gene expression control (01-01-2014 - 31-12-2017)
The field of interest applies to the study of muscle differentiation and disease. The main objective of this project is to deepen our knowledge on the molecular networks controlling normal muscle differentiation, and to identify their alteration in pathology. The state of art in this field is thoroughly advanced since well-established master regulators (transcriptional factors and miRNAs - Buckingham and Vincent, 2009; Zhao et al., 2005) have been deeply characterized and integrated in regulatory circuitries controlling muscle development and differentiation. However, recent discoveries point to the hierarchically relevant role of a previously disregarded class of transcripts, named long non-coding RNAs (lncRNAs), in the control of gene expression. The main objective of this project is to deepen our knowledge on the role of nuclear lncRNAs in the molecular networks controlling cell differentiation; in particular, in consideration of the long lasting expertise of the proposer team on muscle differentiation, the project intends to re-evaluate and re-design established molecular circuitries in the light of the contribution of this class of RNA. This is a very new and innovative field of research that holds promise for a significantly increase in our understanding of basic molecular processes and should constitute a vast and largely unexplored territory for the development of novel therapeutics and diagnostics.

Cacchione Stefano

The role of nucleosomes in the stability of human telomeres (01-01-2014 - 31-12-2017)
The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. In humans, telomeres consist of thousands of TTAGGG repeats bound by a six-protein complex named shelterin. The complete replication of telomeric repeats is assured by the enzyme telomerase, which is active only in germinal and stem cells. In somatic cells, telomeres shorten with each replication round till they reach a critical length leading to a cell cycle arrest known as replicative senescence. Mutations in the growth arrest pathway could allow cells to bypass senescence and continue dividing. Most telomeres uncap, resulting in a crisis stage characterized by chromosome end-fusions and massive apoptosis. To acquire unconstrained proliferative capacity, cancer cells must escape senescence and/or crisis by reactivating telomerase or by alternative mechanisms of telomere maintenance. Despite the essential role telomeres play in cancer development and in aging, our knowledge of the chromatin structure of protected and deprotected telomeres is still limited. In this research project we will address two main issues: 1. The structural and epigenetic changes of human telomeres switching from a protected to a deprotected state. 2. The role played by nucleosomes in telomere capping and how they affect shelterin binding.

Caiafa Paola

Does PARylated PARP-1 introduce an epigenetic mark on chromatin? (01-01-2013 - 31-12-2017)
In a scenario where the molecular mechanism that maintains the unmethylated state of CpG islands is still an open question, the presence of PARylated PARP1 could explain how some CpG islands are protected from anomalous methylation. It has been demonstrated that PARylation controls the unmethylated state and expression of Dnmt1, thrombomodulin, p16 genes and of the imprinted locus Igf2/H19 whose altered expression occurs in tumorigenesis. These demonstrations stem from our previous suggested models where ADP-ribose polymers present on PARP1 interact non-covalently with DNMT1 preventing its enzymatic activity. In absence of PARylated PARP1, DNMT1 is free to methylate DNA. Conversely, persistence of high levels of PARylated PARP1 stably inhibits DNMT1, preventing DNA methylation. CTCF has been identified as an important player in this mechanism as it is able to activate PARylation of PARP1 by itself. In this case the maintaining of the unmethylated state is reached by formation of CTCF-PARP1 complex, PARylation of PARP1 and, in turn, inhibition of DNMT1 activity. Our aim is to validate the hypothesis that PARylated PARP1, by itself or through the binding with CTCF, introduces an epigenetic code on chromatin marking unmethylated DNA regions of genes involved in different pathologies. CTCFL, the antagonist of CTCF upregulated in cancer cells, will be also taken into consideration. Moreover, we will identify binding sites for CTCF that could be involved in seeling the ends of chromatin loops and the possible involvement of PARylation in long-range looping interaction between DNA sequences involved in the control of expression.

Camilloni Giorgio

DNA recombination of repeated sequences and genome instability: epigenetic implications (01-01-2014 - 31-12-2017)
The mechanisms and molecular basis of cancer cell response to DNA Topoisomerase 1 inhibitors, have been determined and studied in detail. Topo 1 can be blocked during its catalytic reaction (breaking and rejoining a single DNA strand), into a covalent cleavage complex with DNA by using camptothecin like inhibitors. The collision of both replication or transcription machineries with these intermediates leads to highly toxic DNA strand breaks, especially for replicating cells. The rational combination of Topo 1 inhibitors with other anticancer drugs is considered as an efficient strategy to improve the cytotoxic effect toward tumor cells. In this project we want to investigate, at molecular level, the possibility to increase the efficiency of Topo 1 inhibitors, in damaging DNA. This result could be obtained by reducing histone content or increasing histone acetylation levels combined together with Topo 1 inhibitors. Both conditions determine an accessible chromatin structure, that in principle, could facilitate Topo 1 reaction with DNA and, as a consequence, could increase the toxic power of Topo 1 inhibitors. Our recent studies on histone content in yeast and mammal cells, gave comparable results on basic chromatin structure. Our long experience in topoisomerase biology and chromatin structure prompted us to investigate whether the conditions of chromatin opening may result in an enhanced toxicity of camptothecin in yeast cells. Results of these studies will be preparatory to the employment of a similar approach on human cells.

Carnevale  Daniela

Ricerca: Characterization of immune reservoirs involved in the adaptative cardiac remodeling to pressure overload

Ruolo della risposta immunitaria nel rimodellamento cardiaco adattativo nel sovraccarico di pressione e nello scompenso cardiaco. (01-01-2014 - 31-12-2017)
L’insufficienza cardiaca – ancora oggi una delle cause principali di assenza dal lavoro e di mortalità – è lo stage finale di una serie di fasi di rimodellamento del cuore che, genericamente, sono ascrivibili a un sovraccarico di pressione o di volume sul ventricolo sinistro. L’evoluzione della patologia consiste nel passaggio da una fase iniziale di tipo adattativo a una più tardiva, caratterizzata da un deterioramento del muscolo cardiaco, che non riesce più a pompare il sangue efficientemente. In modo da poter progettare terapie preventive, per decenni la cardiologia molecolare si è occupata di analizzare i meccanismi alla base di queste fasi di rimodellamento. Varie evidenze suggeriscono un potenziale ruolo del sistema immunitario nell’evoluzione della patologia; ciononostante, i meccanismi correlati restano ancora oscuri. Con questo suo progetto, Daniela mira proprio ad analizzare, in modelli sperimentali di scompenso cardiaco, come gli attori del sistema immunitario partecipano alle varie fasi del rimodellamento e con quali meccanismi regolano la transizione dalla fase iniziale a quella patologica.

Cenci Giovanni

Ricerca: Identification of new factors required for telomere capping in Drosophila

Identification of new factors required for telomere capping in Drosophila (01-01-2014 - 31-12-2017)
Telomeres are nucleoprotein complexes that protect chromosome ends from incomplete replication, degradation and fusion. In most organisms, telomeres are extended by telomerase and contain G-rich repeats. Drosophila telomeres are elongated by transposition of specialized retroelements rather than telomerase activity, and are assembled independently of the terminal DNA sequence. In organisms with telomerase, telomeres are protected by the CST and shelterin complexes, which specifically bind the telomeric DNA sequences. We have recently identified a Drosophila telomere-capping complex, terminin, which binds telomeric DNA in sequence independent fashion and shares functional analogies with CST and shelterin. Here, we further extend our comprehension on the molecular mechanisms of Drosophila telomere protection and unravel new functions in Drosophila telomere homeostasis. This grant proposal has three principal goals: (1) investigate the roles of the E2 variant ubiquitin-conjugating enzyme encoded by a gene dubbed pendolino (peo), at Drosophila telomeres; (2) examine the telomeric role of Drosophila TAT-like (Dtl); (3) characterize the unanticipated role of Separase in telomere maintenance. Because many proteins involved in Drsophila telomere maintenance are conserved in mammals, our studies might provide useful information on human telomere biology.

Cerboni Cristina

Ricerca: Expression of the ligands of the activating NK cell receptors NKG2D and DNAM-1 in human cytomegalovirus infected cells: role of the DNA damage response pathway.

Expression of the ligands of the activating NK cell receptors NKG2D and DNAM-1 in human cytomegalovirus infected cells: role of the DNA damage response pathway. (01-01-2014 - 31-12-2017)
NK cells play a crucial role in the resistance against viral infections, by exerting their effector functions and by participating in modulating adaptive immune responses. NK cell activation is regulated by multiple activating and inhibitory receptors, and individuals with defects in NK cell function often exhibit enhanced susceptibility to viruses, particularly to human cytomegalovirus (HCMV). This virus evolved several strategies to evade cytotoxic lymphocytes, including the specific down-modulation of ligands of the activating receptors NKG2D and DNAM-1, both expressed on NK cells, CD8+ T cells and gamma-delta T cells. Despite this, we believe that there is still a window of opportunity to develop an adequate immune response, during which activating ligands are expressed as a consequence of viral-induced stress response, and before immune-evasion events occurs. Based on this idea and on our recent work demonstrating that expression of NKG2D and DNAM-1 ligands is in part dependent on the activation of the DNA damage response, on ATM/ATR kinases and on the induction of cellular senescence, we will evaluate if these pathways are involved in the modulation of ligand expression upon HCMV infection, using the in vitro model of human primary fibroblasts infected with the AD169 strain. The role of specific immediate-early viral proteins (IE1/IE2) will be also investigated, since they are vital proteins for HCMV replication as well as potent transactivators of cellular genes. Finally, to study how these pathways influence NK cell recognition of HCMV infected cells, we will analyze NK cell cytotoxic activity and IFN production.

Cervone Felice

Plant innate immunity: signalling and recognition of Damage-Associated Molecular Patterns (DAMPs) (01-01-2014 - 31-12-2017)
Plants are continually exposed to pathogens and, in most cases, successfully defend themselves. Knowledge of the mechanisms underlying their defence ability paves the way to strategies for crop protection. The basic mechanism of the “plant innate immunity” is the recognition of pathogens and the activation of the signalling pathways that lead defence responses. In most cases this is based on the detection of the so-called pathogen-associated molecular patterns (PAMPs). The cell wall plays an instrumental role in the plant innate immunity. Cell wall-derived oligolacturonides (OGs) are formed during attempted attacks by pathogens and trigger the typical responses elicited by PAMPs. OGs are regarded as DAMPs (Damage-Associated Molecular Patterns) and the plant innate immunity elicited by DAMPs is the focus of this proposal. Cell wall-mediated signalling will be investigated utilizing the widely studied reference plant Arabidopsis. The formation of OGs takes place during the degradation of homogalacturonan of the plant cell wall by microbial polygalacturonases (PGs); accumulation of OGs is favoured when PGs interact with specific plant inhibitors (polygalacturonase-inhibiting proteins or PGIPs). Micromolar concentrations of OGs activate the expression of defense genes via a signal transduction pathway that functions independently of the known pathways involving salicylic acid (SA), jasmonate (JA), and ethylene (Et). The characterization of this signalling pathway is one of the key activities of this proposal, which continues a very successful 3-year-project financed by the Cenci-Bolognetti Foundation (2010-13) in which we have achieved the important result of identifying the receptor of OGs. The project has two main objectives: A) Dissection of the OG signalling pathway. B) Mechanism of release and recognition of OGs. The wide spectrum of approaches used to achieve the scientific objectives will constitute an excellent environment for research training of young scientists.

Colonna Bianca

Defining the contribution of the VirF protein to the regulative circuitry and to the genome plasticity of Shigella and enteroinvasive E.coli (01-01-2013 - 31-12-2017)
Like in other life threatening human pathogens also in Shigella and enteroinvasive E.coli (EIEC) the regulation of virulence genes occurs at diverse levels and is modulated by environmental stimuli from the host. The decision to activate the invasive programme is an exceptionally complex process that involves a multitude of signals rooting in all layers of gene regulatory hierarchy. A central factor in this process is the VirF protein, an AraC-like positive activator, whose expression is activated as soon as Shigella senses to have entered the host environment. The recent discovery that VirF, besides activating the virulence gene cascade, is able to control several housekeeping genes reveals how crucial this regulator is in reprogramming the transcriptional profile of cell. The aim of this project is to dissect relevant steps of the multilayered regulatory system that has shaped the genome of Shigella and EIEC, turning them into successful pathogens. Major focus will be on transcriptional and post transcriptional events and on the interplay of a diversified group of molecules, including sRNAs, positive activators, nucleoid associated proteins, and polyamines. This should contribute towards a thorough understanding of the strict connections between genome organization, evolutionary strategies and fine tuning mechanisms which allow Shigella and EIEC to maintain and activate the virulence factors required for the colonisation of the host environment.

Cruciani Fulvio

Sequence diversity and evolution of human endogenous retroviral LTRs: the role of ectopicgene conversion (01-01-2013 - 31-12-2017)
Recent data suggest that the long terminal repeats (LTR) of human endogenous retroviruses are not just "junk" DNA, but might play important evolutionary roles in the regulation of the host gene expression. A human solitary LTR12 element from the Y chromosome containing a high number of SNPs has been found in a re-sequencing analysis from our laboratory. This finding is in line with the observation of an excess of putative SNPs in some LTR12 elements, as reported in public databases. The possible role of gene conversion in contributing to this relatively high SNP content remains to be investigated. To evaluate the extent of LTR nucleotide diversity in humans and the role of ectopic gene conversion in shaping the observed diversity pattern, we will undertake a comparative re-sequencing analysis of 60 LTRs (33 LTR12 elements, and additional 27 LTRs representative of different groups) in one hundred unrelated males. To this aim we will exploit the haploid nature of the human Y chromosome and the availability of a stable and reliable Y chromosome phylogeny. Focusing on the LTR sequence divergence may allow us to gain a further insight into the evolution of these elements, whose impact on the host genome expression pattern in mammals is still under study.

Cutruzzolà Francesca

Inhibition of Pseudomonas aeruginosa biofilms: new molecular strategies targeting cyclic-di-GMP metabolism (01-01-2015 - 31-12-2018)
Cyclic-di-GMP (c-di-GMP) is considered one of the most important regulators of bacterial adaptation strategies such as biofilm formation, persistence, cytotoxicity and development. The biological importance of c-di-GMP is connected to its amazing capacity to interact with a large repertoire of proteins and nucleic acids using different binding modes; its conformational plasticity is likely at the basis of its extraordinary success as a signal molecule. C-di-GMP dependent modulation of protein function appears to occur via restriction of the conformational space accessible to these multi-domain or multi-subunit proteins. Activation/deactivation of individual proteins in a complex c-di-GMP signaling cascade is thus likely to be controlled by a combination of c-di-GMP affinity and binding mode(s), producing a wide variety of allosteric control mechanisms, few of them have been characterized in detail biochemically. In this project we focus on the biochemistry of c-di-GMP by studying the protein domains involved in its synthesis (GGDEF domain) and degradation (EAL domain), which are supposed to interact to each other allosterically. We will address the crucial question of how events occurring at the single-domain level are controlled by domain-domain interactions, integrating advanced biochemical and molecular biophysics methodologies with structural biology (including bioinformatics and biocrystallography). We expect to obtain the structural determinants adopted by these conserved domains to deal with c-di-GMP and with the other partner domain(s), to ultimately being able to predict the mode of action of a given GGDEF-containing protein in different bacterial species.

De Biase Daniela

Bacterial infections of the gastrointestinal tract represent a serious global health concern. Childhood diarrhoea in the developing countries and among travellers is a common pathology, affecting millions of people. The major goal of this research project is to shed light on the interplay that in the typical enteropathogenic Escherichia coli (EPEC) strain E2348/69 occurs at the level of the horizontally acquired chromosomal islands LEE (locus of enterocyte effacement) and AFI (acid fitness island). This interplay, which appears to be essential for timely activation of acid resistance genes in the stomach and virulence genes in the intestine, remains poorly defined in EPEC at both genetic and molecular levels. Our analysis will be also extended to the virulence plasmid pMAR2, which carries the genes for the bundle-forming pili. Further, we will pursue a biochemical and structural characterization of the acid resistance regulator GadX and of the GadA/B decarboxylases with the aim of providing a more clear picture of the events that at the molecular level lead to the intracellular activation of glutamate-dependent AR. The role played by the AFI genes in terms of their ability to affect bacterial motility, aggregation and biofilm formation will be also investigated. Impairment in any of the above abilities is also linked to the pathogenic process. We expect that our work will also contribute to spot useful targets for therapeutics or vaccines.

Involvement of Escherichia coli acid resistance genes in virulence expression and in the adaptation to changing environmental conditions (01-01-2013 - 31-12-2017)

De Stefano Maria Egle

Developmental alterations of autonomic and central neurons in a mouse model of Duchenne muscular dystrophy (02-01-2013 - 31-12-2017)
Duchenne muscular dystrophy (DMD) is a severe X-linked myodegenerative disease caused by defective expression of full-length dystrophin (Dp427) following mutations in the DMD gene. Dp427 is a cortical cytoskeletal protein expressed by skeletal, cardiac and smooth muscles, and by populations of central and autonomic neurons. Along Dp427, tissues other than muscles express shorter dystrophin isoforms, derived by internal promoters usage. Characteristic of the disease is progressive and lethal muscle degeneration; however, DMD patients also experience different forms of cognitive impairment, neurological and autonomic disorders, altered scotopic electroretinogram and red-green color vision defect. Differently from muscles, DMD-related damages to nervous system are established during embryonic development and are not progressive after birth, suggesting a role for all dystrophins in nervous system development and differentiation. Little is known, however, on how and to what extent lack of Dp427 and its isoforms expressed in brain, retina and autonomic neurons induce the described physiological alterations. This project aims at clarifying in part this aspect, based on: a) data on the role of dystrophin in dynamics of axon growth and regeneration, which we previously acquired by using genetically dystrophic mdx mice (a well-characterized DMD animal model) and their wild-type controls; b) a recent re-evaluation of the localization of Dp427 in the mouse retina, which describes this protein more diffusely expressed than what was previously reported and, therefore, more involved than it was thought before in retina development and physiology. The project will be articulated in three main sections, which intermingle to each other. In the first, we plan to further investigate the role played by Dp427 in the signal transduction downstream the NGF-TrKA pathway in autonomic neurons in vitro. Similarly, a role in axon-dendritic elongation in cultured hippocampal neurons, a brain region notoriously affected in both DMD patients and mdx mice, will be analyzed. In parallel, we will examine some of the developmental aspects of wild-type and mdx mouse retina (i.e. large scale gene expression, histology, protein localization) in order to uncover whether the physiological alterations reported in the retina of dystrophic mice could stem from developmental failures, as occurring in other districts of the nervous system. Specifically, based on previous published data reporting that lack of Dp427 modulates, directly and/or indirectly, the expression of genes involved in neuron survival and differentiation, we will explore whether genes important for retinal development and synaptogenesis could also be differentially expressed. At this aim, mice at different pre- (E) and post-natal (P) ages (E18, P5, P10, 6-7 weeks) will be used. Levels and localization of the protein products of those genes that will result affected, will be also investigated, along with an accurate histological and ultrastructural study of the retinas. Finally, we will evaluate whether early functional expression of dystrophin in mdx mice after exon skipping, both in vitro and in vivo, could ameliorate some of the neuronal structural and functional alterations we previously described.

Della Torre Alessandra

Genetic and phenotypic characterization of species and “molecular forms” of the Anopheles gambiaecomplex (Diptera: Culicidae), afrotropical malaria vectors (01-01-2014 - 31-12-2018)
Anopheles gambiae s.s. is responsible of most of the 200 million annual malaria cases in sub-saharan Africa. Due to its medical relevance it was the first insect, after Drosophila, whose genome was entirely sequenced. Our group has traditionally focused on the study of its striking degree of genetic heterogeneity leading to novel entities: in fact, we described a novel taxon, recently named A. coluzzii. This species shows the highest capacity in breeding in made-made larval sites, which allows the expansion of its spatial/temporal range in regions/seasons not exploited by its siblings. Last year, an "Anopheles gambiae Genome Variation Mapping Project” has been launched by the “Malaria Genome Epidemiology Network” coordinated by the Welcome Trust SANGER Institute. The project aims to exploit next generation sequence data to i) perform genome-wide association studies for various phenotypes (e.g. insecticide resistance) and ii) to evaluate levels of genomic differentiation within and between A. gambiae and coluzzii. A specific interest has been shown in including in the pipeline individuals karyotyped for paracetric inversion polymorphisms to allow i) evaluation of their impact on the ability to produce high quality SNP calls, ii) understanding genetic structure of major inversions, and iii) development of PCR-tools for molecular karyotyping. This is an extraordinary opportunity for our group, which has an internationally recognized leadership in A. gambiae cytogenetics, to be involved in very high level genomic research on our model organisms.

Di Marcotullio Lucia

Identification of novel Hedgehog/Gli pathway antagonists in brain tumors treatment (01-01-2015 - 31-12-2017)
Tumors of the nervous system arise from aberrant developmental processes as a consequence of genetic and/or epigenetic defects affecting cell growth and differentiation. Childhood is the most specifically affected by these tumors (i.e. medulloblastoma, MB), representing a dramatic challenge for the development of new therapeutic strategy. MB is an aggressive brain tumor caused by disruption of the development program of the cerebellum; MB has poor prognosis and is resistant to standard and advanced chemotherapeutic protocols. This is probably due to the presence, in tumor mass, of cancer stem cells (CSCs) that exhibit an increased resistance to conventional tumor treatments. In the last few years, several publications have suggested the Hedgehog (Hh) pathway as a 'druggable' therapeutic target in cancer, since its critical role in tissues development, proliferation and in the maintenance of CSCs in tumors. Studies from our laboratory have recently identified new endogenous Hh/Gli inhibitors (i.e. KCASH/REN family) lost in MB and acting through specific post-translational modification (ubiquitylation and acetylation). This made us to wonder whether these molecular events might act as Hh antagonists also in a context of CSCs in order to correctly promote tumor suppression. The main objectives of this proposal are: 1. Identification of KCASH/Cul3 interactors and characterization of KCASH/REN family members in the control of the behavior of neural stem/progenitor cells and in tumorigenesis; 2. Discovery of novel Gli inhibitors and their pharmacological effects on Hh-dependent activity in brain cancer stem cells.

Di Mauro Ernesto

Spontaneous generation and evolution of genetic information (01-01-2012 - 31-12-2016)
We aim to reconstruct the initial reactions leading to the generation of genetic information. Starting from the consideration that it will be not be possible to know how life started but that it is nevertheless possible to approximate its first steps, we will extend the observations made so far that have brought us to establish three principles: 1) all the nucleic bases necessary for building extant nucleic acids can be synthesized non-enzymatically, from simple components, with high efficiency and in plausible prebiotic conditions. 2) Nucleic bases may react, within the same prebiotic physico-chemical frame, into nucleosides and nucleotides. 3) Polymerization mechanisms exist leading from spontaneously activated precursors to long nucleic polymers. We plan to explore the conditions that could extend the mechanisms identified so far, passing from the abiotic non-enzymatic synthesis of homogenous polymers to the generation of complex sequences. The experimental outline is the following: 1) confirm and optimize the conditions leading to the spontaneous polymerization of 3’,5’ cyclic GMP (as first reported in refs # 1 and 2). 2) Confirm and optimize the conditions for the spontaneous polymerization of 3’,5’ cyclic CMP (as observed in preliminary results, see below). 3) Extend to 3’,5’ cyclic AMP and 3’,5’ cyclic UMP the discovered mechanism of non-enzymatic polymerization. 4) Develop the conditions for mixed polymerizations of 3’,5’ cGMP and 3’,5’ cCMP. 5) Confirm and optimize the conditions for non-enzymatic terminal ligation of oligonucleotides (see refs 3-5). 6) Confirm and optimize the RNA “terminal sequence swapping mechanism” detected in preliminary observation (see below). The key question is: is it possible to spontaneously non-enzymatically generate RNA sequence complexity, based on intrinsic properties of the RNA structure?

Di Santo Roberto

New azole derivatives as antiprotozoal agents (01-06-2015 - 31-12-2017)
The primary objective of this project is to develop innovative small-molecules useful as therapeutic tool for the treatment and prevention of HIV/AIDS. The innovative aspect of this strategy is that targeted activity is the relatively unexplored reverse transcriptase (RT)-associated ribonuclease H (RNase H) function, which selectively degrades the RNA of the RNA-DNA hybrid produced by the retrotranscription process. The primary objective will be reached through the development of compounds with selective inhibiting activity against RNase H domain of HIV-1 RT enzyme. This group has already identified: i) a class of diketo acids that inhibits both HIV-1 RNase H and IN activities; ii) compounds binding to a novel RT site that are able to selectively inhibit RT-associated RNase H, iii) amino acid residues within the catalytic core of RNase H interacting with the inhibitos. This is a strong base for rational drug design of specific Rnase H inhibitors.

Dimitri Patrizio

Functional analysis of CG40218, a Drosophila melanogaster gene encoding a BCNT-like protein required for chromosome organization (01-01-2014 - 31-12-2017)
The midbody (MB) has become the focus of intense investigation through the identification of a growing number of molecular pathways that take place on it and contribute to its functions in cytokinesis. Cytokinesis failure results in tetraploid and polyploid cells, that can in turn originate genetically unstable states, recognized as a hallmark of cancer. Sparse evidence implicate some chromatin remodeling proteins in midbody organization and function in human cells. Among those, Pontin and Reptin are implicated in many cancer-relevant processes, while Tip60 is a haplo-insufficient tumour suppressor. Our preliminary studies suggest the existence of a global process of reorganization whereby P400/Tip60 and SRCAP chromatin remodeling proteins translocate from the nucleous to the mitotic apparatus during cell division and assume new roles in cytokinesis. We also suggest that dysfunction of these factors contribute to cytokinesis errors. To test our hypothesis, we will use human HeLa carcinoma cells, highly suitable for high-resolution microscopy in the following experiments: 1) Assess the dynamic localization of P400/Tip60 and SRCAP complex proteins in intact cells and purified midbody preparations. 2) Characterize the effects of RNAimediated depletion of P400/Tip60 and SRCAP proteins on midbody formation, cytokinesis and recruitment of key midbody players (e.g. AURORA-B, MKLP1, MKLP2, PRC1, CYT-K). 3) Identify interactors of four major midbody-associated remodelers (CFDP1, BAF53a, P400, Tip60). We expect to gain novel informations on critical events underlying midbody formation and cytokinesis, with a potentially relevant impact on cancer.

D’Amati Giulia

Disease due to mitochondrial tRNA mutations: cellular models to evaluate novel therapeutic strategies (01-01-2013 - 31-12-2016)
Pathogenic mutations in mitochondrial transfer RNA (mt tRNA) genes are responsible for a wide range of pathologies, for which no effective treatment is currently available. Evidence produced by researchers involved in this project suggest that short peptides (SPs) derived from the non-catalytic C-terminal domain (CTD) of human mitochondrial Leucyl tRNA syntethase (mt LeuRS) can be used to correct mt defects due to mutations in mt tRNA genes. The main objective of this project is to evaluate the capability of mt aminoacyl-tRNA synthetases (aaRSs) and SPs derived from their CTD to correct mitochondrial dysfunction caused by mutations in mt tRNA. To this end we will use two cellular models: human transmitochondrial cybrids and the yeast S. cerevisiae.Specifically, we aim to: . evaluate the specificity in the suppressive effect of aaRSs (cross-suppression); . investigate the therapeutic effect of the LeuRS CTD and of short peptides derived from it; . identify the shortest peptide with suppressing activity and study its mechanism of action. aaRSs and their fragments will be over expressed in human cells carrying mutations in mt tRNAIle or tRNALeuUUR and yeast strains with human equivalent mutations. The suppressive effect will be tested by viability assays, oxygen consumption and tRNA steady-state levels. We expect to make a significant step towards the development of peptide-based compounds for therapeutic applications.

Fanti Laura

Circadian rhythms and Stress: functional role of period gene (01-01-2015 - 31-12-2017)
The ability to react to environmental changes is essential for survival and reproduction. Therefore, organisms develop mechanisms to sense external changes and activate appropriate cellular responses. Among the genes involved in the stress response are the heat-shock coding genes. Heatshock proteins protect genomes subject to environmental changes by helping other proteins maintain the right conformation and avoid denaturation. Our recent results show that one of the stress proteins, HSP90, affects the silencing pathway mediated by Piwi-interacting RNA, causing the activation of transposable elements and consequently the induction of morphological mutants through an insertional mutagenesis. In addition, flies exposed to heat-shock during the pupal stage frequently show an extensive range of morphological abnormalities, some induced by transposon insertions. This project proposes to test a different kind of stress induced by the dysfunction of the biological clock. To this end, we plan to investigate the mechanisms of stress and the related effects produced by mutations in period, a master gene involved in the biological clock in Drosophila melanogaster.

Giannini Giuseppe

Functional interactions between the MRN complex and N-Myc in neuronal development and carcinogenesis (01-01-2015 - 31-12-2017)
Genome integrity needs to be thoroughly preserved for the propagation of genetic information to subsequent generations. Indeed, inactivation of proteins involved in DNA damage responses are strictly associated with cancer and/or developmental disorders of the nervous system. The Nijmegen Breakage syndrome (NBS) due to NBS1 gene mutations is characterized by microcephaly, facial dysmorphisms and cancer predisposition. With MRE11 and RAD50, NBS1 forms the MRN-complex, a pivotal regulator of DNA double strand break repair, whose role in replication stress is also emerging. Interestingly, microcephaly is also a feature of N-Myc haploinsufficiency in the human Feingold syndrome. Microcephaly and strongly impaired cerebellar development with proliferation defects of the granule cell precursors (GCPs) are shared by the CNS-restricted N-Myc and NBS1 KO mice. We have now shown that N-Myc transcriptionally regulates the MRN complex and requires it to restrain replication stress associated with the expansion of primary GCPs. Based on these and other preliminar observations, we raise the hypothesis that N-Myc and the MRN complex are linked in a feedforward circuit essential for a safe expansion of specific neuronal cell compartmens, but also for N-Myc-associated cancer, such as neuroblastoma and medulloblastoma. Therefore, we designed a number of approaches based on animal and cellular models already developed in our laboratory to characterize the biological relevance and the molecular mechanisns of N-Myc-MRN interplay. Hopefully our work will shed light on the yet unexplained neuronal phenotypes of DDR-defective syndromes such as NBS, and potentially provide a proof of concept for new therapeutic approaches to neuroblastoma and medulloblastoma.

Gianni  Stefano

Ricerca: PDZ domains in cancer

I domini PDZ e il Cancro. (01-01-2014 - 31-12-2017)
Le interazioni tra proteine sono spesso mediate da specifiche porzioni (o domini) che fungono da collante. Tra questi, i domini denominati “PDZ” rappresentano la forma più abbondante di porzioni che regolano il riconoscimento molecolare. Una perturbazione della funzione dei domini PDZ è alla base di vari effetti devastanti per la vita, come avviene nel cancro indotto dagli virus oncogeni. Con il suo progetto di ricerca Stefano si propone di studiare i meccanismi con i quali i domini PDZ riconoscono il loro specifico partner fisiologico, al fine di disegnare un meccanismo antitumorale di protezione dai virus oncogeni.

Hiscott John

Ricerca: New immunotherapic approaches to cancer and infectious diseases

 

Iebba  Valerio

Ricerca: Comunicazioni tra un ceppo aderente-invasivo di Escherichia coli (AIEC) e il nucleo cellulare dell’ospite: ruolo dei meccanismi epigenetici ospite/batterio coinvolti nel danno al DNA cellulare e nei sistemi di riparazione.

Comunicazioni tra un ceppo aderente-invasivo di Escherichia coli (AIEC) e il nucleo cellulare dell’ospite: ruolo dei meccanismi epigenetici ospite/batterio coinvolti nel danno al DNA cellulare e nei sistemi di riparazione. (01-01-2014 - 31-12-2017)
AIEC è un nuovo ceppo patogeno del batterio Escherichia coli isolato nel 100% delle lesioni intestinali iniziali del morbo di Crohn, una malattia infiammatoria le cui cause sono ad oggi sconosciute. AIEC può indurre lesioni al DNA dell’organismo che infetta (ospite) attraverso la genotossina Colibactina: ciò comporta l’alterazione del ciclo cellulare e la proliferazione batterica. Analizzando le caratteristiche cellulari e la regolazione dell’espressione genica, Valerio si propone di esaminare come AIEC possa trasportare la Colibactina nel nucleo ospite, e come esso possa modulare gli enzimi di riparazione del DNA, favorendo la propria sopravvivenza all’interno delle cellule dell’organismo.

Imperi Francesco

Exploring the Pseudomonas aeruginosa cell envelope as a source of novel protein drug targets (01-01-2013 - 31-12-2016)
Lo scopo del progetto è quello di identificare nuovi e accessibili bersagli molecolari per lo sviluppo di farmaci anti-Pseudomonas aeruginosa, uno dei batteri più temuti in ambito nosocomiale perché causa di gravi infezioni che, generalmente, risultano difficilmente trattabili con le normali terapie antibiotiche. A questo scopo, Francesco analizza le caratteristiche di proteine presenti sulla superficie cellulare di P. aeruginosa che rivestono un ruolo cruciale nella patogenicità batterica, per esempio proteine essenziali per la crescita cellulare e/o la formazione del biofilm.

Limatola Cristina

Molecular and functional approaches to investigate the neuroprotective and neuromodulatory roles of chemokines and their receptors in the central nervous system (01-01-2014 - 31-12-2017)
In the last few years, we demonstrated that CX3CL1 protects hippocampal neurons from glutamate-induced excitotoxicity and that this protective mechanism requires the presence of extracellular adenosine, acting through adenosine receptor 1 (A1R, Limatola, 2005; Lauro, 2008, 2010). More recently we also demonstrated that CX3CL1 is neuroprotective in an in vivo model of cerebral ischemia in mice and rats and that CX3CL1 activity again requires the presence of A1R, being its protective effects absent in A1R-/- mice or in the presence of the A1R antagonist DPCPX (Cipriani et al., 2011). Recently, we also demonstrated for the first time that CXCL16 has neuroprotective roles in the nervous system, reducing Glu-induced excitotoxicity, in hippocampal cultures, with mechanisms requiring A3R and astrocyte-derived CCL2 (Rosito et al., 2012). In the present project we plan to further develop these studies investigating the effects of CX3CL1 and CXCL16 on glial cells (astrocytes and microglia) focusing our attention on 1) Glu transporter activity, expression and localization, 2) glutamine synthase expression, 3) microRNA (miRNA) modulation, 4) release of soluble factors. Similarly to what performed on CX3CL1, we also want to investigate the protective effects of CXCL16 in in vivo models of cerebral ischemia and its potential neuromodulatory action on glutamatergic and GABAergic synaptic transmission, on synaptic plasticity, ans the potential role of the transmembrane chemokines CX3CL1 and CXCL16 in functional rescue of synaptic transmission upon ischemic conditions.

Londei Paola

From leaderless to leadered mRNAs: mRNA features modulating ribosome binding and translation initiation in Archaea (01-01-2014 - 31-12-2016)
It has long been known that ribosome architecture and the basic mechanism of protein synthesis are highly conserved in evolution. For this reason, ribosomes have been traditionally regarded as a static factory that passively carry out translation, while the mechanisms controlling translation have been mainly searched for among the translation initiation factors and the specific sequence motifs of mRNAs. Recently however, studies performed in both bacteria and eukaryotes have revealed the existence of “specialized” ribosomes which selectively translate specific classes of mRNAs. These specialized ribosomes may be diversified from the common ribosome pool in their protein composition, or they may contain rRNAs with sequence variants or structural alterations. The specialized ribosomes may operate, for example, under stress conditions or in tumor cells, subtly modifying the cellular proteome to accommodate the arising physiological needs. The main goal of this project is to investigate whether specialized ribosomes can be found in Archaea, the third domain of life, and, if so, to identify the mRNA classes preferentially translated by the ribosomal variants under defined physiological conditions. Furthermore, we mean to investigate whether IF6, a translation factor conserved in archaea and eukarya, has a role in promoting the biosynthesis of ribosomal variants or in controlling ribosome loading of specific mRNAs. The results will reveal whether translational regulation by means of specialized ribosomes is ubiquitously distributed in the three domains of life and whether eIF6 is a conserved player in this process in the archaeal/eukaryal lineage.

Lucarelli Marco

The influence of epigenetics, DNA repair and cell cycle pathways on the gene therapy approach of Small Fragment Homologous Replacement (SFHR) (01-01-2013 - 31-12-2015)
The aim of the project is to clarify the molecular mechanisms of recognition of cell invasion by exogenous DNA, possibly underlying a gene targeting approach called Small Fragment Homologous Replacement (SFHR). This approach is able to stably modify a genomic sequence by homologous replacement of a small DNA fragment, by a still poorly understood molecular mechanism. The modification is inheritable, physiologically expressed and long-term maintained. SFHR potential is currently limited by a low and variable frequency of correction. This project will focus on the relationship between SFHR and chromatin structure (modifying during cell cycle), DNA methylation (a main effector of both chromatin remodelling and host defence), DNA repair and cell cycle pathways. To clarify these mechanisms, the reciprocal influence of these 4 main biochemical pathways and the SFHR will be studied, in a reporter cellular system of mouse embryonic fibroblasts, in a differentiated human cellular model of Cystic Fibrosis and in a Spinal Muscular Atrophy mouse embryonic stem cell model. Drugs acting on specific target mechanisms and single-gene targeting will be also used to dissect the pathways and to manipulate SFHR efficiency. The results will clarify the molecular mechanisms of cell invasion by exogenous DNA and its genomic integration, as well those of SFHR. The selection of strategies to increase SFHR efficiency will open up new perspective for SFHR therapeutic applications for in vivo and ex vivo treatments of inherited diseases, using differentiated and stem cells as targets.

Mai Antonello

Targeting DNA and histone methylation for cancer therapy: identification and development of new DNMT and EZH2 inhibitors. (01-01-2014 - 31-12-2017)
DNA methylation and histone methylation (in particular H3K27me2/3, through the action of EZH2, a component of PRC2) are highly studied epigenetic mechanisms for regulation of gene expression. Down-regulation and depletion of both these epigenetic targets led to tumor suppressor gene re-expression and/or cell growth arrest in a variety of cancer cells. In addition, mutations of EZH2 were reported in lymphomas and myelodysplastic and myeloproliferative disorders. Starting from the structure of the non-nucleoside DNMT inhibitors SGI-1027, we identified MC3343 as more potent than SGI in DNMT1 and DNMT3A assay, and as potent as SGI in cancer cells but less toxic. In this Project, we will develop new series of MC3343-related molecules, also with computer-aided techniques, in order to obtain more potent and selective derivatives to test in in vitro (including cancer stem cells, CSCs) and in vivo cancer models. Recently, we reported novel low micromolar EZH2 inhibitors, showing strong pro-apoptotic induction, and antiproliferative activity at submicromolar concentrations when tested against colon and glioblastoma cancer stem cells. As a part of this Project, we will prepare novel analogues of such molecules to increase the potency and selectivity against EZH2 and to improve their anticancer properties. The new compounds will be tested against human DNMT1 and DNMT3A, and against EZH2/PRC2 through HTS, and the most potent inhibitors will be tested in cancer cells to assess growth inhibition, apoptotic effects, and differentiation. Selected compounds will be tested in colon, glioblastoma, and medulloblastoma CSCs, in collaboration with internal and external Sapienza research groups. In vivo studies on mouse cancer models will be performed with the most potent identified compounds.

Maione Rossella

Transcriptional regulation through long-range chromatin interactions in muscle cells: the CDKN1C-KCNQ1 locus (01-01-2013 - 31-12-2016)
We have recently reported a novel transcriptional mechanism by which the muscle regulatory factor MyoD, the master regulator of myogenesis, induces the expression of the cdk inhibitor p57, a critical regulator in many developmental and pathological processes. p57 is an imprinted gene and its expression, restricted to specific cell types, is subject to a complex epigenetic control not yet completely understood. We found that, during differentiation, MyoD induces the coordinate activation of p57 and of the co-imprinted gene kcnq1. Moreover, we demonstrated that the induction of p57 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a repressive chromatin loop involving p57 promoter. The present project has two main objectives. The first is the identification of the molecular mechanisms by which KvDMR1 controls the restriction and the induction of p57 during skeletal myogenesis. Specific tasks will be: 1) the analysis of the functional interaction between MyoD and CTCF at KvDMR1, in regulating the dynamics of the chromatin loop. 2) the analysis of the role of the long noncoding RNA Knq1ot1 in the differentiation-dependent regulation of p57. 3) the analysis of the epigenetic determinants of KvDMR1 accessibility to MyoD binding The second objective is the elucidation of the epigenetic regulation of kcnq1, a gene coding for a subunit of a voltage-dependent potassium channel and playing an important role in the physiology of skeletal and especially cardiac muscle. This analysis will be performed in an in vitro cell system of cardiac differentiation based on the direct reprogramming of fibroblasts into cardiomyocytes. We plan the following approaches: i) analysis of kcnq1 expression during differentiation ii) analysis of kcnq1 promoter function iii) analysis of the functional interaction of cardiogenic bHLH proteins with KvDMR1 iv) mapping of the long-range chromatin interactions of kcnq1 promoter

Mangoni Maria Luisa

Fighting microbial infections: a multidisciplinary startegy to develop short-sized native peptide-based antimicrobials (01-01-2014 - 31-12-2017)
The increasing emergence of resistant microbes to the available antibiotics is a major concern worldwide, leading to a search on antimicrobial peptides (AMPs) as potential agents to overcome bacterial resistance and also to neutralize the toxic effect of bacterial cell wall components (e.g. the lipopolysaccharides in Gram-negative bacteria) which cause septic shock syndrome. For many years our research group studied extensively the structure-function relationships of short-sized AMPs that we have isolated from amphibian skin. These studies have led to the identification of peptides with potent microbicidal activity against important human pathogens. Among them are the highly pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus aureus, both colonizing mucosal surfaces, such as the conjunctiva, the cornea or the respiratory epithelium particularly in cystic fibrosis patients. However, there are two major tasks to be reached in order to make such peptides good candidates for therapy. The first one is to minimize enzymatic degradation, and the second one is to design a good delivery system to the site of the infection. In line with the above, the present proposal has two major goals: (i) To screen for the best frog-skin AMPs/synthetic analogs (containing both native and non gene-coded residues) to be further developed as novel compounds against infections associated with lung and/or ocular surfaces; and (ii) To screen for the best formulations for improving the stability profile of the selected AMPs and for optimizing their delivery and release kinetics in the desired site of the infection (i.e. lung tissue).

Mazzoni  Cristina

Ricerca: Study of RNA damage in yeast as a model for age-related degenerative diseases

Montanari  Arianna

Ricerca: Towards a therapy for mithocondrial tRNA disorders

Verso una terapia per le malattie mitocondriali. (01-01-2015 - 31-12-2017)
Le malattie mitocondriali sono un gruppo di patologie ereditarie dovute alla disfunzione delle centrali energetiche delle nostre cellule (i mitocondri). Private di “carburante”, le cellule lavorano male e muoiono precocemente, con gravi conseguenze per diversi tessuti e organi, soprattutto per quelli con maggiore richiesta energetica – come muscoli, cuore e cervello. Arianna studierà nuovi approcci farmacologici a queste gravi malattie – per cui al momento non esistono terapie risolutive. Studi preliminari hanno individuato in una piccola porzione dell’enzima mtLeuRS, la capacità di migliorare il funzionamento dei mitocondri difettosi. Ora, basandosi su complesse analisi biochimiche e di genetica molecolare, Arianna studierà come migliorare l’efficacia di questi farmaci e il loro assorbimento da parte dei mitocondri malati.

Montemiglio  Linda Celeste

Ricerca: Taming HIPK2 kinase activity to tackle cellular diseases and cancer

La proteina HIPK2: un potenziale bersaglio terapeutico da modulare per contrastare lo sviluppo di tumori e di patologie fibrotiche. (01-01-2015 - 31-12-2018)
Con questo progetto Linda è alla ricerca di strategie farmacologiche innovative per contrastare l’insorgenza di tumori e di altre patologie cellulari. In particolare, si prefigge di “colpire” un bersaglio molecolare preciso: HIPK2 – una proteina multifunzionale, fondamentale durante lo sviluppo embrionale e nella risposta cellulare in seguito al danno del materiale genetico. L’attivazione della proteina HIPK2 porta alla morte di cellule neoplastiche mentre la sua inattivazione può avere effetti benefici in patologie quali la fibrosi renale. Per questo motivo, HIPK2 rappresenta un potenziale bersaglio da colpire per sviluppare terapie sempre più efficaci e dai minimi effetti collaterali. Con il suo lavoro, Linda, partendo dallo studio della struttura della proteina, analizzerà farmaci e composti naturali di derivazione batterica, saggiandone la capacità di modulare l’attività di HIPK2 – potenziandola o inibendola a seconda delle necessità terapeutiche. Il progetto prevede anche di realizzare nuovi “proiettili” (farmaci) anti-HIPK2 utilizzando sofisticati metodi di biologia computazionale.

Musarò Antonio

Characterization of the muscle-nerve crosstalk in a neuromuscular disease (01-01-2014 - 31-12-2017)
Stem cell therapy represents a promising tool to treat genetic diseases. However, this approach is not definitive yet and several hurdles limit the immediate translation of this strategy into clinic. Our working hypothesis is that the hostile microenvironment that characterize several pathologic conditions, including muscular dystrophy, might interfere with and limit the efficacy of stem cell-mediated therapy. In this project we aim to test the hypothesis that modifying/perturbing it we may improve the effectiveness of stem cell-mediated therapy. Preliminary evidences demonstrated that the local form of Insulin-like Growth Factor–1 (mIGF-1) sustains muscle hypertrophy and regeneration in senescent skeletal muscle, enhances the recruitment of circulating stem cells in injured muscle and counteracts muscle wasting in mdx dystrophic mice, reducing the inflammatory response and improving muscle mass and strength and elevating pathways associated with muscle survival and regeneration. One of the molecular target of mIGF-1 that was significantly modulated in mdx muscle is the pro-inflammatory cytokine IL-6. Thus, the aims of this project are to prove the hypothesis that IL−6 is a pivotal mediator of muscle damage and to verify whether inhibition of IL-6 expression and accumulation in dystrophic mice is a potential therapeutic strategy to counteract muscle wasting and to improve the restoration of functional dystrophin synthesis, by stem cell transplantation. The design of a combination of stem cell and tissue niche therapies might become a potential cornerstone approach for future therapeutic approaches in which a chronic inflammatory response limit the ability of stem cells to function properly.

Negri Rodolfo

Role of the COP9 signalosome in transcription modulation and chromatin organization in yeast and plants (01-01-2014 - 31-12-2017)
Histone demethylases have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. We recently discovered in S.cerevisiae an interesting conditional negative genetic interaction between the unique JARID histone demethylase Jhd2, responsible for H3K4 demethylation, and Not4, a protein which is involved in several different regulatory processes, including transcriptional regulation, RNA stability and Jhd2 degradation. The double deletion mutant jhd2/not4 is hypersensitive to rapamycin and its sensitivity is promptly suppressed by episomal Jhd2 expression in the double deletion strain. While this genetic interaction could be useful for the understanding of the transcriptional role of Jhd2 in yeast, which is still elusive, it is also an ideal system for in vivo screening of inhibitors specific for JARID demethylases. In a pilot screening on 45 candidate small molecules, we identified a compound which specifically inhibits Jhd2 in vivo, leading to a consistent increase in trimethyl-H3K4. The compound inhibits human JARID 1D in vitro and shows a strong cytostatic effect, a mild cytotoxicity and a selective increase of trimethyl-H3K4 in HeLa cells. We propose to better characterize the inhibitor’s effects in yeast and mammalian cells (in particular for what concerns the cytostatic effect); to extend the screening to new candidate molecules and to investigate on the meaning of the genetic interaction between Jhd2 and Not4 which could enlighten some aspects of the role of Jhd2 in transcription.

Palamara Anna Teresa

Selective inhibition of Hemagglutinin maturation by cellular oxidoreductases: a potential target for novel anti-influenza strategies (01-01-2013 - 31-12-2017)
Influenza virus (IV) infections are a big problem for public health because of lack of effective treatments. In fact, the efficacy of anti-influenza agents is limited by the rapid occurrence of drug resistance. Thus, many efforts have been devoted to identify new therapeutic strategies targeting the virus-host cell interactions. IV uses several intracellular pathways for its replication. In particular, redox-sensitive cellular oxidoreductases, like PDI and ERp57, normally promoting cellular glycoprotein folding in the Endoplasmic Reticulum (ER), are exploited for folding and oligomerization of viral surface glycoprotein hemagglutinin (HA). We recently showed that GSH-C4, a glutathione (GSH) derivative, blocked IV replication by impairing HA maturation. This effect was related to the correction of virus-induced depletion of intracellular GSH that, in turn, affected the redox state of PDI. Interestingly, GSH-C4 did not affect the glycoprotein secretion in uninfected cells, suggesting that cytosolic redox conditions play a pivotal role in ER folding activities. The main goal of this project is to determine whether HA is a specific substrate for one or more cellular oxidoreductases and whether virus-induced alterations in intracellular redox state play a role in this specificity. We will also investigate the possibility to selectively inhibit the activity of oxidoreductase/s involved in HA folding by using molecules able to modulate intracellular redox state. Since the degradation of unfolded HA in small peptides is a key event for their recognition by MHC1, we will test the hypothesis that the selective inhibition of HA folding could promotes the formation of MHC 1-HA complex.

Paolini Rossella

Molecular mechanisms involved in attenuating FceRI expression and signaling (01-01-2013 - 31-12-2016)
In addition to being major effector cells in the elicitation of allergic response, mast cells play a significant role in host defense against invading parasites. Indeed, mast cells can produce, store and release a broad spectrum of pro-inflammatory mediators and deliver exosomes throughout the body after activation by several stimuli, being the ligation of the high affinity receptor for IgE (FcRI) the most efficient and the best characterized. FcRI belongs to a multisubunit immunoreceptor family characterized by the presence of subunits, distinct for each receptor, used for ligand binding and others that share conserved cytoplasmic motifs responsible for cell activation. The receptor complex lacks intrinsic enzymatic activity but transduces intracellular signals through association with cytoplasmic protein tyrosine kinases. Regulation of the signals generated as a consequence of antigen interaction with specific IgE bound to FcRI, causes a shift in the resting state equilibrium of phosphorylation and dephosphorylation that serves to maintain homeostasis. The outcome of this activated state is the release of preformed mediators and the de novo synthesis of eicosanoids, cytokines and chemokines that result in both immediate and late-phase reactions. The complex and highly regulated apparatus leading to mast cell activation is counterbalanced by an equally sophisticated series of inhibitory mechanisms. Notably, a negative-feedback regulation of FcRI-mediated signals is provided by the internalization and delivery of activated receptor complexes to lysosomes for degradation, mainly occurring via clathrin-dependent endocytosis. More recently, the implication of a lipid raft-dependent FcRI ubiquitination in controlling the endocytosis of activated receptors has been also envisaged. In such context, the outcome of our project is to characterize in depth the molecular mechanisms that ensure a proper delivery of internalized FcRI complexes to lysosomes for degradation, thus providing a negative-feedback regulation of receptor activity and mast cell functions.

Piconese  Silvia

Ricerca: Immunometabolic checkpoints of Treg performance in HCV-related metabolic inflammation and cancer

Immunità e Metabolismo. (01-01-2015 - 31-12-2018)
Immunità e metabolismo sono strettamente correlati e intervengono l’uno nella regolazione dell’altro. Per esempio, un sottotipo di cellule del sangue, le cellule T regolatorie (Treg), svolge un ruolo essenziale nel proteggere il nostro organismo dall’intensa infiammazione che si sviluppa non solo in seguito a infezione, ma anche a causa di disordini di tipo metabolico. Con il suo progetto Silvia intende analizzare come il metabolismo dei grassi possa influenzare l’attività delle cellule Treg. Per questi suoi studi Silvia utilizzerà modelli sperimentali dell’obesità e di malattie del fegato (come il fegato grasso e il cancro) correlate all’infezione da virus dell’epatite C.

Raffa  Grazia Daniela

Ricerca: A Drosophila model for Spinal Muscular Atrophy (SMA)

Il moscerino della frutta: un modello sperimentale per la ricerca contro la SMA. (01-01-2015 - 31-12-2018)
L’atrofia muscolare spinale (SMA) è una malattia caratterizzata dalla progressiva morte dei motoneuroni, le cellule nervose che impartiscono ai muscoli il comando di movimento. Questa grave patologia (è la più comune causa genetica di morte infantile) è causata da un difetto nel gene che codifica per una proteina chiamata SMN. Con Il suo progetto Grazia Daniela si pone l’obiettivo di identificare e caratterizzare fattori che interagiscono e modulano SMN. A tale scopo la giovane ricercatrice utilizzerà, come modello sperimentale della SMA, il moscerino della frutta (Drosophila melanogaster).

Riminucci Mara

Role of Gs? signalling in the bone marrow hematolymphopoietic microenvironment as revealed by novel transgenic models (01-01-2015 - 31-12-2017)
This project is inscribed in our group’s long standing interest in the BM niche (biology and disease). We will refine and fully characterize functionally our system for heterotopic transplantation of skeletal progenitors, which results in the establishment of a BM niche in immunocompromised mice. This will involve in situ characterization and functional probing of the capacity of the niche to host blood borne human hematopoietic progenitor/stem cells and cancer cells as a probe of niche promiscuity. We will then attempt and develop innovative models of the interaction between malaria gametocytes and human BM. To this end, we will use a) immunocompromised mice, to analyze the bulk biodistribution of infused human RBCs infected with immature and mature gametocytes; b) mice carrying human bone marrow stroma in heterrotopic organoids; c) mice carrying human blood vessels in heterotopic organoids; c) humanized immunocompromised mice with human erythropoiesis, BM stroma, and/or blood vessels. This studies will address, in addition to the biology of the human HSC niche in vivo, its emerging potential key role as a relais in the human-to-mosquito parasite transmission and survival of in a broad ecosystem.

Sabatini Sabrina

Size matters: the molecular mechanisms maintaining meristem size during root growth (01-01-2013 - 31-12-2015)
In Arabidopsis roots, stem cells in the apical region of the meristem self-renew and produce daughter cells that differentiate in the distal meristem transition zone. We have shown that maintenance of the Arabidopsis root meristem size - and consequently root growth - is ensured by the interaction between two hormones in the vascular tissue of the meristem transition zone: cytokinin, which promotes cell differentiation, and auxin, which promotes cell division. To investigate the relationship between developmental events and gene expression, cell-specific resolution of gene activity is critical. Unfortunately, the vascular tissue is a very heterogeneous tissue where cell of different types and at different developmental stages are difficult to distinguish, thus severely limiting investigations at the single cell resolution. However, we recently discovered that cytokinin controls cell differentiation also from another root tissue , the lateral root cup (LCR), composed of a single cell layer. Aim of this project is to identify components of the molecular mechanism through which cytokinin controls root meristem size from the LRC. To achieve this, we will utilize state-of-the-art techniques such as fluorescence-activated cell sorting (FACS) coupled to expression profiling, to examine the transcriptional profiles of individual cell types, the LCR, in the Arabidopsis root meristem.

Santoni Angela

Characterization of functional properties and migration capacity of different NK cell subsets in haematological malignancies. ( - )
NK cells are innate lymphocytes capable of killing malignant or infected cells and to produce a wide array of cytokines and chemokines following activation. Chemokines play critical roles in the regulation of NK cell tissue distribution in normal conditions as well as in their rapid recruitment to the parenchyma of injured organs during inflammation. In this regard, differences in chemokine receptor expression have been reported on specialized NK cell subsets with distinct effector functions and tissue distribution. Haematopoietic tumors are life-threatening diseases characterized by clonal growth and accumulation of progenitor cells. It is becoming increasingly clear that NK cells are the major accountable of the therapeutic effect of haematopoietic stem cell transplantation (HSCT) from an HLA-disparate relative in patients with of acute myeloid leukemia (AML), being able to directly kill tumor cells. In multiple myeloma patients, a number of evidence strongly support the anti-tumor potential of NK cells in response to immunomodulatory drugs, or following allogeneic stem cell transplantation. Although NK cells have been widely used as tools for adoptive immunotherapy in haematological diseases, the mechanisms governing their tissue homing and accumulation capacity and responsible for their anti-tumor activity in situ, are poorly investigated. Bone marrow (BM) is the main place of NK cell differentiation and represents the site where most hematologic tumors grow. This proposal is aimed at characterizing the NK cell populations that are endowed with anti-tumor activity and exhibit a preferential BM homing, by analyzing the effector capacity of NK cells expressing selected chemokine receptors, both in the mouse and humans. We also plan to identify the mechanisms that regulate the mobilization of different NK cell subsets from BM into blood, the influence of tumor growth on NK cell homing/trafficking to the BM and how this could affect NK cell-based anti-tumor response. A better understanding of such mechanisms has high relevance for clinical settings in which NK cells are mobilized and recruited to the sites of inflammation, injury or tumor growth for host defense and tissue repair. More importantly, the results obtained might be considered to enrich anti-tumor effectors in human peripheral blood during BM cell mobilizing protocols. These are relevant issues in order to optimize NK cell infusion-based clinical trials in patients with haematological diseases.

Scagnolari  Carolina

Ricerca: Interleukin 32:molecular expression, epigenetic regulation and biological activities in human HIV-1 infection

Interleuchina 32: espressione molecolare, regolazione epigenetica e attività biologica nel corso dell’infezione da HIV (01-01-2013 - 31-12-2016)
 Con questo progetto, Carolina si propone di analizzare il ruolo dell’interleuchina 32 (IL-32) nel corso dell’infezione da HIV-1. L’IL-32 è una citochina che riveste un ruolo importante in numerose malattie autoimmuni e infiammatorie e studi recenti ne hanno evidenziato il coinvolgimento durante le infezioni virali. In particolare, la ricerca condotta da Carolina è volta a caratterizzare – prima in vitro e poi in pazienti HIV-positivi – l’attività biologica e i principali meccanismi che contribuiscono alla regolazione dell’espressione dell’IL-32. Questa ricerca può contribuire ad accrescere le nostre conoscenze sulle strategie utilizzate da HIV per evadere la difese immunitarie dell’ospite, aiutando i ricercatori a sviluppare approcci terapeutici alternativi.

Screpanti Isabella

Dissection of Notch signaling-dependent pathways involved in the progression of T cell leukemia (01-01-2014 - 31-12-2017)
Deregulated Notch signaling is considered the major cause of T cell acute lymphoblastic leukemia (T-ALL), suggesting Notch pathway as an attractive therapeutic target. However, therapeutic use of the available Notch signaling-inhibiting gamma-secretase inhibitors (GSI), revealed severe side effects. The molecular dissection of Notch pathway may reveal novel opportunities to develop less toxic targeted therapies. We demonstrated Notch3 capacity to drive the development of T-ALL, by the generation of a unique mouse model of T-ALL, driven by the enforced expression of constitutively active Notch3. Moreover, we and others reported that the vast majority of T-ALL patients display increased expression of Notch3. In the present proposal we plan to dissect the molecular mechanisms regulating the interplay of Notch3 with other oncogenic pathways. Hedgehog signaling pathway has been recently related to T cell development. Although Hedgehog role is widely studied in several cancer, including myeloid leukemias, its involvement in T-ALL has however not been elucidated. We will address Hedgehog and Notch pathways interplay in the purpose to demonstrate their integration in a regulatory circuitry coordinating T cell differentiation and leukemogenesis. The importance of microRNA pathway has been recently established in various human cancer including T-ALL. However, the mechanisms by which deregulated miRNA expression occurs in T-ALL, the target genes, as well as miRNA relationships with Notch signaling are not fully understood. Thus, the idea that Notch3 function is required to instruct and maintain a specific miRNA signature in T-ALL may add insights to the role of Notch signaling in T-ALL.

Silvestri Romano

New non-nucleoside antiviral agents targeting HIV-1 reverse transciptase (01-01-2013 - 31-12-2017)
Aim of the research project is the development of new non-nucleoside agents as potent, selective and safe inhibitors of HIV-1 reverse transcriptase (NNRTIs). NNRTIs became standard components of highly active antiretroviral therapy (HAART) of AIDS/HIV-1 infection as they showed effective inhibition of drug resistant mutant strains and better tolerance than protease inhibitors. Despite this consistent progress, current NNRTI drugs still show side effects, drug resistance and pharmacokinetic problems. Indolylarylsulfone (IAS) proved to be a robust scaffold for potent HIV-1 NNRTIs. In this proposal we aim at the develepment of new HIV-1 NNRTIs by chemical modification of regions A-C of the IAS scaffold using modern environment-preserving synthetic procedures. We have designed new IAS NNRTIs that share structural characteristics common to second generation ETV and RPV showing broad spectrum of activity against drug resistant HIV-1 mutant strains. By using an OBCM protocol developed in 2012, we are able simulate a high number of compounds for an expedite selection of putative mutation-resistant inhibitors. Goal is the achievement of new NNRTIs overcoming drug-resitance limitation, toxicity and pharmacokinetic problems of currently approved HIV-1 drugs.

Torrisi Maria Rosaria

Molecular mechanisms integrating endocytosis and signalling of fibroblast growth factor receptors (01-01-2014 - 31-12-2016)
The E5 oncoprotein encoded by the human papillomavirus (HPV) type 16 transforms epithelial cells by deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Out-of-context and deregulated oncogenic signaling of the FGFs and their receptors have been shown to play a role in the pathogenesis of different types of cancer and in tumor progression. Dysregulated expression and activity of TGF TGFRI/IIand SMADs have been also frequently described in human cancer in association with tumor progression. Therefore, it is possible that, in early infection and in the context of low-grade and high-grade lesions, HPV16 E5 might exert its oncogenic activity through modulation of FGF and TGF signaling. At the light of our recent demonstration of a functional crosstalk among 16E5 protein and KGFR/FGFR2b, aim of our research project will be to investigate the effects of 16E5 expression and expression/signaling of growth factor receptors, focusing in particular on FGFR2 epithelial and mesenchymal isoforms and on TGFRII. We plan also to identify the molecular mechanisms and pathways linking dysregulated growth factor signaling and altered receptor expression to viral infection. Specific aims of the project will be: a) to analyze the modulated expression in vitro of FGFR2b/FGFR2c and TGFRII in 16E5-expressing cellular models as well as in lesional tissues; b) to identify the molecular mechanisms and signaling pathways linking 16E5 with the altered receptor expression and cellular response; c) to evaluate the possible role of 16E5 in the induction of epithelial-mesenchymal transition through FGFR isoform switching and dysregulated TGF signaling.

Tramontano Anna

Ricerca: Computational design of peptides binding to a given protein surface

Tripodi Marco

Development of molecular tools to prevent EMT in liver fibrosis ( - )
This project is focused on the molecular networks controlling the epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) in hepatocyte differentiation and transformation. We previously characterized the hepatocyte EMT/MET dynamics in both differentiation and maintenance of epithelial identity. In particular, we unveiled a simple cross-regulatory circuit between the EMT master gene Snail and the hepatocyte differentiation master gene HNF4α in which these transcriptional factors repress each other with a consequent mutually exclusive expression. This minicircuitry was found to control the hepatocyte cellular identity and its unbalance the transition toward a mesenchymal/malignant phenotype. Recently, we integrated this molecular network with microRNA-200 family members and microRNA-34a, demonstrating that transcription of these stemness inhibiting miRs is controlled by Snail and HNF4 in opposite manner. Overall, Snail/HNF4α dynamics define the outcome of the transition between the mesenchymal/undifferentiated and the epithelial/differentiated phenotype in both physiological and pathological situations. Microenvironmental cytokines influence these dynamics. In particular, we and others demonstrated that the soluble factor TGFβ induces Snail upregulation/stabilization and HNF4 downregulation/inactivation triggering EMT in normal hepatocytes and tumor progression in transformed cells. Aims of this project are: 1) to develop molecular tools for the prevention/reversion of the TGFβ-induced EMT, final goal of this approach being the control of pathological hepatocyte transdifferentiation. 2) the dissection of the hepatocyte machinery controlling EMT/MET molecular mechanisms. In particular, we will focus on the role of the H19 non-coding RNA and of specific microRNAs we hypothesize to influence the Snail/ HNF4 balance.

Tuosto Loretta

CD28 co-stimulatory molecule as a key regulator of NF-?B signalling pathway: role of cytoskeleton in coupling CD28 to NF-?B activation (01-01-2014 - 31-12-2017)
CD28 is one of the most relevant costimulatory receptor that delivers both TCR-dependent and TCR-independent signals regulating a wide range of signalling pathways crucial for cytokine and chemokine gene expressions, T cell survival and proliferation. Most of the CD28-dependent signalling functions are initiated by the recruitment and activation of class IA phosphatidylinositol 3-kinase (PI3K), which catalyzes the conversion of phosphatidylinositol 4,5-biphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3), thus generating the docking sites for key signalling proteins. Hence, PIP2 turnover is an essential regulatory step to ensure the activation of PI3K following CD28 engagement. We have recently found that CD28 regulates PIP2 turnover by recruiting and activating phosphatidylinositol 4-phosphate 5-kinases  (PIP5K) in human primary CD4+ T lymphocytes. This event leads to the neo-synthesis of PIP2 and to its consumption by CD28-activated PI3K. We also evidenced that PIP5K activation is required for both CD28 unique signals regulating IL-8 gene expression as well as for CD28/TCR-induced Ca2+ mobilization, NF-AT nuclear translocation and IL-2 gene transcription. Our findings elucidate a novel mechanism that involves PIP5K as a key modulator of CD28 costimulatory signals. In searching for specific molecules involved in coupling CD28 to both the recruitment and activation of PIP5K, we identified Vav-1. Vav-1 is a GDP-GTP exchange factor for Rac-1 and Cdc42 small G proteins that is strongly tyrosine phosphorylated and activated by CD28 and is a key upstream regulator of both actin reorganization and CD28 regulated signalling pathways. Starting from these findings, the present project will focus on the characterization of PIP5K/Vav-1 molecular interaction and its functional relevance in CD28 signalling.

Vallone Beatrice

Ricerca: Bacterial P450 Cytochromes as tools for designing novel antimicrobial agents

Verga Falzacappa  Cecilia

Ricerca: Study of the thyroid hormone T3 in skeletal muscle homeostasis: a possible role in muscle wasting

L’ormone tiroideo T3 e un suo possibile ruolo nell’atrofia muscolare. (01-01-2015 - 31-12-2018)
Con questo progetto Cecilia si propone di indagare il ruolo svolto dall’ormone tiroideo T3 nel preservare il trofismo muscolare compromesso in seguito ad anoressia, immobilità o malattie neurodegenerative. Il muscolo è un noto bersaglio dell’azione ormonale tiroidea, ma gli effetti degli ormoni tiroidei nell’ atrofia muscolare (riduzione o perdita della massa e forza muscolare) non sono noti. Tali effetti sono oggetto dello studio di Cecilia. I risultati preliminari ottenuti in vitro e in vivo mostrano che la somministrazione dell’ormone T3 è in grado di proteggere la massa muscolare dall’atrofia indotta dal digiuno. Si analizzeranno ora i meccanismi molecolari alla base degli effetti osservati, e si valuterà poi se il trattamento ormonale possa essere benefico anche in altri modelli di atrofia muscolare, quali quella neurogenica o quella dovuta ad inutilizzo. La prospettiva futura è quella di considerare il trattamento ormonale tiroideo come terapia efficace in pazienti con atrofia muscolare.

Vicini Elena

Biological characterization and in vitro culture of human spermatogonial stem cells (01-01-2013 - 31-12-2015)
In the male gonads of mammals, the germline stem cells belong to a subpopulation of undifferentiated spermatogonia localized in the basal layer of the seminiferous epithelium. While, spermatogenesis in rodents and primates, including humans, has been considered dissimilar with important biological differences in the identity and behavior of stem cells, recent studies from our laboratory and others recently challenged this concept. Expect for the existence of type Apale and Adark spermatogonia, and that Adark spermatogonia are quiescient not much is known about the spermatogonial compartment in humans. Clearly, there is need of new data in order to obtain insight in the regulation of human germline compartment. In this direction, a new re-assesment of the stages of the seminiferous epithelium (cellular associations), the analysis of spermatogonial behaviors in whole mounted seminiferous tubules, as well as development of spermatogonial stem cells lines are highly warranted. Feasibility of this proposal strongly relay on the availability of unfixed human testicular cells. To this end we are collaborating with the group of Prof. Berloco at the Department of General and Special Surgery of Sapienza University and we have already obtained human testicular cells taken from healthy heart beating organ donors. Data obtained will be highly relevant to the study of molecular mechanisms regulating early step of spermatogenesis, for future applications on male infertility, and to unveil molecular mechanisms underlying pluripotency.

Vittorioso Paola

Study of the role of DAG1 and GAI in embryogenesis and seed germination in Arabidopsis thaliana (01-01-2013 - 31-12-2015)
The transcription factor DAG1 plays a central role in light-mediated seed germination. DAG1 acts downstream of PIL5, the master negative regulator of this process, and negatively regulates gibberellin (GA) biosynthesis by directly repressing the AtGA3ox1 gene. In darkness, PIL5 directly induces the expression of GAI and RGA, encoding two DELLA proteins, known as functionally redundant repressors of the GA-mediated processes. We are interested in studying the functional relationship between DAG1, GAI and RGA, by using a combined genetic and molecular approach. Recently we have found compelling evidence suggesting that GAI and RGA are not functionally redundant, as previously reported, and that GAI only seems to cooperate with DAG1 in repressing seed germination. Interestingly, genetic analyses indicate that DAG1 and GAI could also have an essential function during embryogenesis. We propose to study the molecular and functional interactions between DAG1 and GAI, during embryogenesis, seed development and germination. In additions we propose to identify their putative common targets.