Fattore Luigi

Ricerca 2018 - 2019 (Finanziamento Ricercatori Under 35 - call 2018 Progetti "Teresa Ariaudo"): Novel miRNAs as therapeutic tools for intervention in melanoma drug resistance

Novel miRNAs as therapeutic tools for intervention in melanoma drug resistance (01-01-2018 - 31-12-2019)
Melanoma is the most aggressive and lethal type of skin cancer. Metastatic disease is poorly influenced by standard chemotherapy. The recent introduction of target therapy with BRAF and MEK kinase inhibitors in BRAF V600 mutated patients and of immunotherapy with checkpoint inhibitors has dramatically improved clinical outcome. Unfortunately, both types of therapies are plagued by drug resistance. In the case of kinase inhibitors in the majority of cases the long term efficacy of these therapies is hampered by the acquisition of resistance during the course of therapy. It is generally accepted that resistance to MAPK inhibitors could be caused both by genetic and non-genetic mechanisms. Our group has recently focused its attention on this second type of mechanisms and in particular on the role of miRNAs, since they are powerful regulators of gene expression both in healthy and disease conditions. In particular, we have identified a panel of miRNA, which is strongly deregulated in melanoma cells upon acquisition of resistance to MAPKi. In the present project I will further investigate the involvement of these miRNAs in the establishment of resistance to targeted therapies. In particular, I plan to study miRNA capability to modulate stromal cells by reprogramming the tumor microenvironment through the deregulation of specific cytokines. In addition, I will further assess the possibility to use these miRNAs as novel therapeutic agents able to potentiate the efficacy of current therapies.

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 Giovanni

Ricerca 2018 - 2019 (Finanziamento Progetti call 2018 - Seed International Research Projects) - collaborazione con IP Sofia - Bulgaria NK cell-based regulation of mesenchymal stem cell differentiation in experimental models of bone formation and cartilage degeneration

NK cell-based regulation of mesenchymal stem cell differentiation in experimental models of bone formation and cartilage degeneration (01-01-2018 - 31-12-2019)
Unresolved inflammation often leads to chronic diseases, characterized by tissue dysfunction and failure in tissue repair in response to trauma or infection due to a delay in the initiation of the anabolic phase. Abnormal formation of new bone tissue has been observed in osteoarthritis (OA), a degenerative disease affecting joint, cartilage and bone. A series of events characterize OA, including degradation of cartilage matrix components associated with increased synthesis and activation of pro-inflammatory mediators and enhanced expression of bone erosion factors and upregulation of bone remodeling proteins. Although, much is known about the pro-inflammatory and anti-inflammatory cytokines milieu contributing to bone remodeling and repair, the immune cell source of these factors and their reciprocal interaction are poorly understood. This is an important topic of investigation as several immune cell lineages were shown to regulate mesenchymal stem cell (MSC) migration and osteogenic capacity on one side and cartilage degradation on the other. We have documented that NK cells and neutrophils have a disease-promoting role in experimental OA. Since our preliminary observations indicate a role of NK cells in the repair phase of the disease, we hypothesized that NK cell interaction with other immune cells could affect the local growth factor environment, regulating osteogenesis and chondrogenesis. Indeed, the reciprocal crosstalk of immune cells (i.e. neutrophil and NK cells) in the OA joint is important for the perpetuation of the disease since in vivo removal of neutrophils reduced the accumulation of NK cells into synovia. Thus, we hypothesized that interfering with the positioning of one cell lineage (i.e. NK cells) will influence the state of activation of counteracting cell types and shift the balance of catabolic and anabolic events to promote/limit bone repair and formation. This proposal is aimed to analyze the ability of NK cells to alter MSC chondrogenic/osteogenic potential in vitro and in vivo. Particular attention will be devoted to the factors that govern NK cell migration as well as to the immune cell-produced factors in the joint environment responsible for the impairment of MSC chondrogenic/osteogenic capacity. We will study in vivo the MSCsmediated bone formation after depletion of NK cells or of neutrophils using monoclonal antibody. We will study in vitro how NK cells will modulate the osteogenic vs chondrogenic differentiation of MSCs in normal conditions or in conditions guiding (BMP-2, GDF-5 or histone deacetylase inhibitors, HDACi) MSC differentiation toward a specific cell lineage. We will investigate the contribution of CXCR3 in the migration of NK cells at the damage/remodeling phase of collagenase-induced OA and in MSC regenerative capacity. The study will provide novel facets on the NK cell-based mechanisms controlling bone/cartilage repair via inflammation that undoubtedly will be of interest for regenerative medicine relying on boosting the individual recovery.

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.

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.

Canettieri Gianluca

Ricerca 2018 - 2010 (Fianziamento Rciercatori Under 60 - Call 2018 Progetti "Anna Tramontano"): Targeting MYC translation in Colorectal Cancer

Targeting MYC translation in Colorectal Cancer (01-01-2018 - 31-12-2018)
Colorectal cancer (CRC) is a major cause of death from cancer worldwide. Despite the progresses made with early diagnosis and improvement of therapeutic protocols, the prognosis of the advanced stages is still poor. For this reason, understanding the molecular determinants of CRC tumorigenesis represents an indispensable step to find novel therapeutic opportunities. Several genes and pathways have been found mutated in CRC and most of them converge on the activation of MYC, thus making this oncogene an attractive therapeutic target. However, attempts to find direct MYC inhibitors have been disappointing, suggesting that alternative strategies, aimed at reducing MYC expression or activity are preferable options. One avenue is the inhibition of MYC translation, although inhibitors of general translation, such as PI3K/mTor inhibitors, have shown the paradox effect to increase MYC translation via non-canonical, compensatory mechanisms. Our preliminary work has led to the identification of MTR (MYC translational regulator), a regulator of MYC expression that is essential for the growth of CRC. Our working model is that MTR promotes MYC translation by binding a G-rich element in the coding sequence of MYC, thereby resolving stable mRNA structures and relieving ribosome stalling. Moreover, we have found that this protein is required for the translational upregulation of MYC in response to PI3K/mTOR inhibitors, likely via a phosphorylation-dependent stabilization mechanism. The proposed project aims at elucidating these issues by addressing the following three main tasks: 1) We will study the effect of MTR ablation in colorectal cancer mouse models and human cells 2) We will elucidate how MTR promotes MYC translation and how PI3K signaling affects this mechanism 3) We will study the MTR/MYC axis in human samples of CRCs and its correlation with different stages of the disease Results of this project will inform us about an unprecedented mechanism of regulation of MYC translation and the consequences of its targeting in CRC.

Carnevale  Daniela

Ricerca (2018 - 2019 - Finanziamento Ricercatori Under 45): Search for immune-related pathogenetic mechanisms of aortic aneurysm, induced by disrupted TGF-beta signaling in smooth muscle cells, to find novel therapeutic targets.Ricerca (2014 - 2016 - Finanziamento Ricercatori Under 40): Characterization of immune reservoirs involved in the adaptative cardiac remodeling to pressure overload

Search for immune-related pathogenetic mechanisms of aortic aneurysm, induced by disrupted TGF-beta signaling in smooth muscle cells, to find novel therapeutic targets. (01-01-2018 - 31-12-2019)
The project proposes to study the immune response involved in the pathophysiology of the aortic aneurysms associated to disrupted transforming growth factor beta (TGFβ) signaling in smooth muscle cells (SMCs), with the last aim to find novel mechanisms to be targeted for translational purposes. Thus far, genetic approaches to aortic pathology described a pivotal role of TGFβ signaling in aneurysm development, mainly related to mutations in the genes involved in to this pathway. However, despite several hypotheses have been proposed, we still miss a clear framework of how a disruption of TGFβ signaling lastly determines a complex pathology like aneurysm’ formation, which also depends on the infiltration of immune cells in vascular walls. The TGFβ pathway is mediated through the phosphorylation and interaction of SMAD2 and SMAD3 with the common interactor SMAD4, forming a complex that regulates gene transcription in the nucleus. Interestingly, syndromes with SMAD4 mutations in human also include aortic aneurysms at high frequency. Considering that SMAD4 is an obligatory component of the canonical TGFβ pathway, targeting this gene has been considered an appealing strategy to unravel the role of TGFβ signaling in aortic pathology. Since Smad4 null mice are embryonic lethal, we will pursue the strategy to study a model of selective Smad4 deletion in SMCs of adult mice, in the attempt to recapitulate the complexity of aortic pathology due to disrupted TGFβ signaling, which usually develops in adults. Our previous results obtained in this model, highlighted that Smad4 deletion in SMCs releases the brake on IL-1β production as a danger signal mediating the interaction between vascular and immune compartments in the aortic walls. The research design of the actual proposal will rely on a multidisciplinary approach addressing manifold issues relevant to the pathology. Basically, we will investigate how Smad4 in SMCs could affect: i) non-cell autonomous mechanisms leading to interaction with immune system and ii) design of novel pharmacological therapies based on the molecular mechanisms characterized. In particular we will focus on molecular mechanisms relevant to preserve the vascular-immune interface in aortic aneurysms. Worth noticing, the actual therapies for aortic aneurysms are limited to tight monitoring of pathology progression and surgical intervention to prevent rupture. Thus the identification of novel molecular targets is needed to propose pharmacological approaches aimed at limiting pathology progression, striving for avoiding surgical intervention.

Cenci Giovanni

Ricerca (2018 -2020 - Finanziamento Ricercatori Under 60 - Progetti Call 2018 "Anna Tramontano") : Characterization of the role of Separase in the regulation of Lamins and Rad50 Progetto PTR (collaboration with IP Paris): Role of HP1/Cbx protein ubiquitination in chromatin organizationRicerca 2014- 2016 : Identification of new factors required for telomere capping in Drosophila

Characterization of the role of Separase in the regulation of Lamins and Rad50 (01-01-2018 - 31-12-2021)
Defective DNA repair and perturbation of nuclear architecture can threaten genome integrity. The comprehension of molecular mechanisms that underlie these effects can help to understand how cells prevent genome instability, which is a prevalent feature of several human genetic diseases including cancer. Our preliminary genetic and molecular results point towards an unforeseen function of Separase, a conserved endo-peptidase that resolves sister chromatid cohesion through cleavage of cohesins, in maintaining nuclear architecture and chromosome integrity. We will exploit a combination of genetic and molecular approaches in both Drosophila and human cells to study the involvement of Separase in these processes. We believe that our results, which highlight new and conserved functions of Separase beyond cohesion cleavage, will reveal new insights on the regulation of genome maintenance.

Cirone Mara

Ricerca 2018 - 2020 (Finanziamento Under 60 - Call 2018 progetti "Anna Tramontano"): Autophagy manipulation as a strategy to counteract EBV- and KSHV-drivenmalignancies.

Autophagy manipulation as a strategy to counteract EBV- and KSHV-driven malignancies. (01-01-2018 - 31-12-2020)
The object of this study is to evaluate whether autophagy dysregulation is involved in the malignant transformation induced by EBV and KSHV, two gammaherpesviruses strongly associated to human cancer. Autophagy plays an important role in the different phases of cancerogenesis or in cancer maintenance and progression. Preliminary findings indicate that both viruses inhibit autophagy, encoding for proteins that directly or indirectly impair the several autophagic steps. Interestingly, autophagy inhibition has been reported to promote the initial steps of the malignant transformation, impairing the elimination of misfolded proteins and damaged organelles, whose accumulation promotes cancerogenesis. Base on this knowledge, we propose to evaluate whether counteracting gammaherpesvirus-mediated autophagy inhibition could help to prevent their-associated malignancies.

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.

Contestabile Roberto

Ricerca 2018 - 2020 (Finanziamento Ricercatori Under 60 - Call 2018 Progetti "Anna Tramontano"): Regulation of vitamin B6 metabolism and bioavailability in Eubacteria.

Regulation of vitamin B6 metabolism and bioavailability in Eubacteria. (01-01-2018 - 31-12-2020)
Pyridoxine, pyridoxal, pyridoxamine and their phosphorylated forms, collectively referred to as vitamin B6, play several fundamental biological functions, the best-known example being the catalytic role of pyridoxal 5’-phosphate (PLP), which is an essential cofactor for dozens of enzymes involved in essential metabolisms. Vitamin B6 also plays important roles in biological processes such as response to oxidative stress, gene regulation and bacterial virulence. Because of its aldehyde group, PLP is a very reactive molecule that readily combines with thiols and amines; therefore, it is potentially toxic and its cellular concentration must be kept at a low level. At the same time, large amounts of the cofactor are needed to saturate PLP-dependent enzymes and satisfy cell needs. Such considerations raise the question of how all these requirements are met and how PLP, once made available from biosynthesis, is delivered to the apoenzymes that require it as cofactor. As a matter of fact, in spite of the pivotal importance of vitamin B6 in all organisms, very little is known on the regulation of its metabolism at the level of gene transcription, enzyme activity and membrane transport. Our research project will rely on an integrated computational and experimental approach to investigate several understudied aspects of vitamin B6 metabolism in Eubacteria, focusing on model organisms of biotechnological and medical relevance such as Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa. We will explore several aspects of the regulation of vitamin B6 metabolism and bioavailability, such as the distribution of different biosynthetic pathways in Eubacteria, the regulation of the expression of PLP biosynthetic genes, the transport of B6 vitamers across the membrane and the role of proteins that might serve as PLP carriers involved in PLP homeostasis and delivery to apoenzymes.

Cruciani Fulvio

Ricerca 2018 - 2020 (Finanziamento Ricercatori Under 60 - Call 2018  Progetti "Anna Tramontano"):  Dynamics of intra-chromosomal gene conversion between palindrome arms of the human Y chromosome.Ricerca 2013 - 2015: Sequence diversity and evolution of human endogenous retroviral LTRs: the role of ectopicgene conversion

Dynamics of intra-chromosomal gene conversion between palindrome arms of the human Y chromosome. (01-01-2018 - 31-12-2020)
One of the most striking feature of the male-specific region of the human Y chromosome (MSY) is the presence of eight massive palindromes (P1-P8), composed of two inverted repeats (arms) separated by a spacer sequence. These elements, which in total span 5.7 Mb of the MSY, contain many genes important for sperm production and exhibit > 99.9% arm-to-arm sequence identity, due to abundant gene conversion. The independent appearance of large palindromes in sex chromosomes of different species suggests they can have a profound biological significance that has yet to be fully clarified. Despite thousands of human Y chromosomes have been sequenced in their non-repetitive regions, little is known about within-population sequence diversity of palindromes, which could illuminate about Y-Y gene conversion features, dynamics and evolutionary meaning. In a recent next generation sequencing (NGS) analysis of the P7 palindrome performed in our lab, we obtained new unbiased data suggesting that gene conversion may strongly affect the evolutionary patterns of palindromes. To shed light into the dynamics and the evolutionary meaning of this process, we will undertake a comparative NGS (50×) analysis of different human Y palindromes in 104 samples, whose phylogenetic relationships were assessed in a previous study. The availability of a stable and reliable Y chromosome phylogeny should allow us to identify peculiar mutational patterns within palindrome arms and to test if gene conversion is biased toward the elimination of new variants and the conservation of old ones. Moreover, by mapping conversion events across the Y phylogeny, we could be able to calculate a precise Y-Y gene conversion rate for each palindrome here analysed.

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

Ricerca 2018 - 2019: Finanziamento Seed research International Projetcs (in collaborazione con IP del Brasile - Fondazione Oswaldo Cruz): Whole transcriptome (RNA-Seq) combined with biochemical studies to evaluate the contribution of the metabolism of glutamate and GABA in Mycobacterium tuberculosis strains circulating in Salvador, Bahia, Brazil.

Ricerca 2013 -2015:  Involvement of Escherichia coli acid resistance genes in virulence expression and in the adaptation to changing environmental conditions

Whole transcriptome (RNA-Seq) combined with biochemical studies to evaluate the contribution of the metabolism of glutamate and GABA in Mycobacterium tuberculosis strains circulating in Salvador, Bahia, Brazil. (01-01-2018 - 31-12-2019)
Mycobacterium tuberculosis is the World most deadly bacterial pathogen. In 2015, the World Health Organization reported that 10.4 million people were newly diagnosed with Tuberculosis, and that 1.8 million people died from it. The emergence of M. tuberculosis strains multi(MDR), extensively(XDR) and totally (TDR) drug resistant is disappointingly accompanied by little knowledge about i) the bacterial mechanisms of resistance and ii) the host factors that provide a selective pressure driving the evolution of these strains. What is known is that the stress caused by the antibiotics (oxidative stress) or in the macrophage (oxidative and acid stress) can trigger a bacterial response that leads to the “stepwise” building-up of mutations that eventually lead to MDR, XDR and TDR. A better knowledge on the points above is highly desirable. As part of this “Seed International Research Project”, we will evaluate the correlation between specific metabolic pathways and stress resistance in M. tuberculosis strains representatives of two contemporaneous bacterial collections obtained in the city of Salvador (Bahia, Brazil) and available at the Fiocruz Institute. We will investigate by RNA-Seq the whole transcriptome of MDR strains isolated from patients for which a history and follow-up are available. This study will therefore contribute to the understanding of how changes in gene expression correlate to drug resistance. In addition, we will look at changes in the expression levels of glutamate decarboxylase (Gad) and of the GABA-catabolizing enzymes. M. tuberculosis Gad will be expressed in a recombinant form for inhibition studies by selected compounds and for the production of anti-Gad antibodies that will be employed for immunoblot analysis on cellular lysates of specific strains.

Della Torre Alessandra

Ricerca 2018 - 2020: Finanziamento Ricercatori Under 60 - Call 2018 Progetti "Anna Tramontano": EXPLOITATION of NOVEL GENOMIC RESOURCES to DEVELOP MOLECULAR TOOLS for GENOTYPING AFRO-TROPICAL MALARIA VECTORS and STUDY ECOLOGICAL SPECIATIONRicerca 2014 - 2016: Genetic and phenotypic characterization of species and “molecular forms” of the Anopheles gambiaecomplex (Diptera: Culicidae), afrotropical malaria vectors

The present project builds on the existing expertize of the research group on genomics, population genetics and ecology of Afrotropical malaria vectors and on our participation to the international “Anopheles gambiae 1.000 Genome” Consortium and to the ExGenMal project (awarded by the 2017 Actions Concertées InterPasteuriennes), with the aim to develop novel molecular tools for a straightforward and large scale analysis of contemporary introgression between the two main malaria vectors species in sub-Saharan Africa, Anopheles gambiae and A. coluzzii. The newly developed tools will be applied to two case studies to analyse more in detail the adaptive introgression on a trajectory towards hybrid speciation recently revealed at the edges of the species distribution, which has the potential to affect the dynamics of malaria transmission and the effectiveness of vector control interventions. Results are expected to provide innovative valuable tools for studying and monitoring these two major malaria vector species, as well as to contribute to evolutionary biology by providing new details on ecological speciation of these organisms at the forefront of research on novel species formation.

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 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.

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.

Fazi Francesco

Ricerca 2018 - 2019 (Fianziamento Ricercatori Under 45): Development of a combination strategy based on ER and oxidative stress in Acute Myeloid Leukemia

Development of a combination strategy based on ER and oxidative stress in Acute Myeloid Leukemia (01-01-2018 - 31-12-2019)
AML is a very heterogeneous disease caused by different genetic aberrations. These result in the expression of fusion or mutant proteins that in all cases cause impaired differentiation and enhanced proliferation and survival of hematopoietic progenitors. At present, the only highly effective molecular targeted therapy for AML is based on all-trans retinoic acid (RA) and chemotherapy and/or arsenic trioxide (ATO) in acute promyelocytic leukemia (APL), which express the promyelocytic leukemia (PML)–retinoic acid receptor α (RARα) fusion protein. We have previously shown that APL cell lines and primary leukemic blasts induced to differentiate by RA become highly sensitive to small amounts of ER stress inducing drugs, not detrimental for the same cells in the absence of RA. Furthermore the same cells resulted sensitive to a combination of ER stress inducers and ATO because of generation of oxidative stress. Importantly the same treatments resulted not toxic on hematopoietic progenitors obtained from healthy bone marrows. We observed that ER stress in combination with RA caused increased amounts of disulphide-bound high molecular weight aggregates of PML-RARα and PML, exacerbating the alteration of cellular proteostasis already generated by induction of ER stress. This observation provides the rationale to translate the findings we obtained in APL to other types of AML characterized by fusion or mutant proteins. The presence of mutant proteins that are easily prone to aggregation or mis-folding, because of their mutant structure or because of mis-localization, could render the cells sensitive to levels of ER and oxidative stress that could be recovered in their absence. In the present project we propose to evaluate the impact of ER and oxidative stress inducers, used at low doses in combination with RA, in AML cell lines and primary human blasts as synergistic strategy to promote AML cell death. We obtained preliminary data that support our hypothesis in ML-2 and MV-4-11 AML cell lines. We plan to identify different AML sub-types that will result sensitive to the combination of ER and oxidative stress in the presence or not of RA and to clarify the underlying molecular mechanisms. The 5-year relative survival rate for AML is about 40% with the exception of APL where it reaches more than 90%. Except APL other types of AML do not respond as well to RA because of the molecular mechanisms underlying carcinogenesis. Identification of new molecular targets like the ER stress response would allow the design of new sub-type related therapeutic strategies.

Giannini Giuseppe

Ricerca 2018 - 2020: Finanziamento Ricercatori Under 60 - Call 2018 Progetti "Anna Tramontano" : Harnessing replication stress to understand and tackle MYCN-dependent tumors.Ricerca 2015 - 2017: Functional interactions between the MRN complex and N-Myc in neuronal development and carcinogenesis

Harnessing replication stress to understand and tackle MYCN-dependent tumors. (01-01-2018 - 31-12-2020)
Preservation of genome integrity is a priority issue in mammalian tissues, especially in the nervous system, where a defective DNA damage response (DDR) is associated with developmental and neoplastic disorders. Recently, our work focused on the functional interactions between MYCN and the replication stress (RS)-response in neuronal cells. We showed that MYCN regulates the expression of a number of proteins, including the MRN complex and PARP, to control RS, prevent DNA damage and cell death. Moreover, inhibition of either MRN or PARP+CHK1 very effectively kills MYCN-dependent neuroblastoma (NB) and medulloblastoma (MB), tumors that are still largely incurable. Based on our own and other’s work we generated some testable hypotheses: i) the MRN complex is essential to control RS in neuronal progenitors; ii) subtle variations in MRN activity may uncover its oncosuppressive function in neuronal tumorigenesis; iii) MYCN-dependent neoplastic cells retain a strict requirement for controlling the otherwise deleterious effects of RS; iv) MYCN supports a coordinated pattern of replication-, DNA repair- and checkpoint-related genes whose pharmaceutical modulation might be of utmost importance for the therapy of MYCN-overexpressing tumors. We will test these hypothesis via pharmacological and genetic manipulations of designed targets in NB and MB mouse and cell models. Through dry and wet lab work, we will further screen for more effective combinations of “RS-response” inhibitors. We expect our efforts will provide: i) an increased understanding of the molecular mechanisms linking oncogene-induced RS and neuronal carcinogenesis; ii) a proof of concept that inhibiting the RS-R can be synthetic lethal with MYCN-overexpression; iii) a number of validated animal and cellular models for further studies. Finally, by showing that targeting the “RS response” is possible and effective, we will hopefully establish a novel approach for the therapy of deadly tumors, such as MYCN-amplified NB.

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.

Imperi Francesco

Ricerca 2018 - 2019 (Finanziamento Ricercatori Under 45): Functional characterization and pharmacological inhibition of colistin resistance inPseudomonas aeruginosaRicerca 2013 - 2015 (FInanziamento Ricercatori Under 40): Exploring the Pseudomonas aeruginosa cell envelope as a source of novel protein drug targets

Functional characterization and pharmacological inhibition of colistin resistance in Pseudomonas aeruginosa (01-01-2018 - 31-12-2019)
The old antibiotic colistin represents our last-line treatment option against many multi-drug resistant Gram-negative bacteria. However, reports of colistin resistance in clinical isolates are rising, calling for alternative therapeutic strategies and/or efforts to preserve the clinical efficacy of colistin. Aminoarabinosylation of LPS is a common colistin resistance mechanism in Gram-negative bacteria, and recent evidences indicate that this LPS modification is strictly required for the development of colistin resistance in Pseudomonas aeruginosa, one of the most dreaded opportunistic pathogens for which effective therapeutic options are very limited. By taking advantage of recently-published structural and functional information on the last enzymatic step of LPS aminoarabinosylation, we carried out a docking-based virtual screening of an in house natural compounds library, which led to the identification of a putative inhibitor of LPS aminoarabinosylation showing significant synergism with colistin against a reference P. aeruginosa colistin-resistant isolate. This project is aimed at further development and validation of this inhibitor, through structurebased design of derivatives with improved binding affinity, and evaluation of their colistin synergistic activity and cytotoxicity on eukaryotic cells in vitro. The range of activity of the most promising compounds will be then assessed on a collection of P. aeruginosa colistin resistant clinical isolates, as well as on other Gram-negative pathogens. Moreover, the inhibition of LPS aminoarabinosylation in inhibitor-treated bacteria will be confirmed by mass spectrometry. Finally, the colistin synergistic activity of selected compounds will be verified in vivo using a simple animal model of infection. In parallel, the cellular pathways involved in the development of high-level adaptive resistance to colistin in P. aeruginosa will be investigated by functional genomics, with the final goal to identify new potential targets for the design of additional colistin synergists with different mechanisms of action. The expected deliverables of this project, in terms of both antibacterial compounds and information, would provide new pharmacological opportunities to extend the clinical lifetime of colistin that, at present, represents our last resort for many recalcitrant Gram-negative infections.

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.

Mangoni Maria Luisa

Ricerca 2018 - 2020 (Finanziamento Ricercatori Under 60 - call 2018 Progetti "Anna Tramontano"): Development of novel peptide-based formulations and nano/bio-materials against pulmonary and ocular surface microbial infectionsRicerca 2014 - 2016: Fighting microbial infections: a multidisciplinary startegy to develop short-sized native peptide-based antimicrobials

Development of novel peptide-based formulations and nano/bio-materials against pulmonary and ocular surface microbial infections (01-01-2018 - 31-12-2020)
New antimicrobial agents are urgently needed, as the growing number of microorganisms resistant to the available drugs represents one of the most serious life threats. Antimicrobial peptides (AMPs) of innate immunity hold promise as novel anti-infective agents with alternative mode(s) of action and multiple functions. Our research group identified amphibian skin-derived AMPs (e.g. esculentins and temporins) which rapidly kill important human pathogens, e.g. Staphylococcus aureus and Pseudomonas aeruginosa with a membrane perturbing activity that limits the induction of resistance. Remarkably, one of these AMPs was found to cause significant bacterial clearance in mouse models of acute P. aeruginosa pulmonary infection and keratitis. Nevertheless, a relevant aim which needs to be achieved for the usage of AMPs in therapy includes a proper delivery system to the target site. To this goal, the production of peptide-loaded nanoparticles is an attractive approach to assist peptide release to the therapeutic target. Furthermore, a promising strategy to circumvent AMPs’ short half-life and cytotoxicity associated with high concentration of soluble peptide molecules is given by their immobilization on biomaterial’s surface. In line with the above, the major objectives of the present proposal are (i) to develop new antimicrobial formulations based on our frog skin AMPs/optimized analogs to apply in a suitable solution or delivered via nanoparticles, for treatment of lung/ocular surface microbial infections; and (ii) to develop peptide-coated contact lenses capable of preventing microbial colonization of lenses and therefore the emergence of keratitis.

Messina Giovanni

Ricerca 2018 - 2019 (Finanziamento Under 35 - Call 2018 Progetti "Teresa Ariaudo"): Unmasking the encrypted role of Tip60 chromatin remodelling complex in cell division

Unmasking the encrypted role of Tip60 chromatin remodelling complex in cell division (01-01-2018 - 31-12-2019)
This project aims to utilise a range of cutting-edge tools for dissecting dTip60 chromatin remodelling complex function during cell division, and unveiling mechanisms underpinning the mitotic targeting of dTip60 complex subunits and their functions in cell cycle control in Drosophila. In addition to its role in exchanging phospho-H2A.V with an unmodified H2A.V (Kusch et al, 2014) to regulate chromatin structure and gene expression, dTip60 complex has been shown to be involved in cell cycle regulation. Indeed, four subunits of the dTip60 complex, DOM-A, YETI, Tip60 and MRG15, are recruited to the mitotic apparatus in Drosophila S2 cells (Messina et al, 2017). Moreover, recent data demonstrate that: 1) subunits of two human chromatin remodelling complexes (SRCAP and P400/Tip60) related to Drosophila dTip60 complex, relocate from chromatin to the mitotic structures; and 2) RNAi-depletion of these proteins affect chromosome segregation, carrying out to polyploidy and multinucleation. Thus, the translocation of remodelling factors from interphase chromatin to the mitotic apparatus suggests highly regulated mechanism of localization possibly related to their function of poorly understood evolutionary conserved phenomenon which is worth elucidating. For this purpose, I will exploit CRISPR/Cas9 technology to specifically destroy the endogenous genes (dom, yeti, tip60 or mrg15), and repair them with the ‘Auxin inducible’-tagged versions for rapid protein-specific degradation. I will use Drosophila embryo as the experimental system because of its peculiar enability to undergo a series of rapid synchronous syncytial nuclear divisions that can be easily imaged by using in vivo time-lapse microscopy to depict chromosome segregation and mitotic defects.

Miranda Banos María Elena

Ricerca 2018 - 2019 (Finanziamento Ricercatori Under 45): Cellular pathways involved in the toxicity of neuroserpin polymers that cause dementia FENIBRicerca 2010 -2012: Handling and toxicity of mutant serpins underlying human disease

Cellular pathways involved in the toxicity of neuroserpin polymers that cause dementia FENIB (01-01-2018 - 31-12-2019)
The dementia FENIB is a rare but fatal neurodegenerative condition due to point mutations in the neuronal protein neuroserpin (NS). NS is one of the serpins (serin protease inhibitors), a conserved superfamily of proteins that inhibit serin proteases by a mechanism that requires high structural flexibility, rendering serpin proteins very sensitive to point mutations that alter their folding and promote their intracellular polymerisation and accumulation within the endoplasmic reticulum of the cell. This constitutes the molecular mechanism underlying the serpinopathies. Although the pathological manifestations of serpin polymerisation depend on the inhibitory target and place of action of each specific serpin, the molecular mechanism is common and several aspects remain obscure for all serpinopathies. Particularly, little is known about the cell toxicity due to polymer accumulation in the endoplasmic reticulum. We have recently created a neural expression system and performed a RNA sequencing comparison of control cells and cells expressing a severe pathological variant of NS. We have found that cells expressing polymerogenic NS upregulate several anti-oxidant genes and undergo apoptosis when the anti-oxidant defences are blocked. Our preliminary results also indicate an alteration of mitochondrial physiology in these cells. Here we propose to characterise the nature of the ER stress caused by accumulation of NS polymers in neurons and to investigate the mitochondrial alterations in cells expressing mutant NS, particularly in connexion with oxidative stress, ER-mitochondria interaction and Ca2+ signalling, and their role in the neuronal degeneration underlying the dementia FENIB.

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

Ricerca 2018 - 2020 (Finanziamento Ricercatori Under 60 - call 2018 Progetti "Anna Tramontano"): Understanding and combating sarcopenia: the role of metabolic disorders andcytokines-mediated inflamm-aging.Ricerca 2014 - 2016: Characterization of the muscle-nerve crosstalk in a neuromuscular disease

Understanding and combating sarcopenia: the role of metabolic disorders and cytokines-mediated inflamm-aging. (01-01-2018 - 31-12-2020)
Broad objectives and specific aims The aims of the project are to define the pathogenic mechanisms of sarcopenia and the metabolic disorders leading to sarcopenic obesity, to disclose muscle and systemic/serological factors that can be used as biomarkers of muscle frailty, and to verify potential epigenetic alterations that impinge muscle stem cells activity and muscle function in sarcopenic conditions. Background/Rationale Sarcopenia is the age-related loss of muscle mass and function. The causes of sarcopenia are unknown. Current hypotheses indicate that it may be the result of several factors, including hormonal changes, inflammatory pathway activation, fatty infiltration, altered mechanisms regulating the turnover of contractile proteins and organelles, neuro-muscular function as well as altered production and tissue responsiveness of trophic factors. Metabolic disorders, such as obesity, have been suggested as a risk factor for sarcopenia. Sarcopenic obesity, which describes the process of muscle loss combined with increased body fat as people age, is associated with loss of strength and function, reduced quality of life, and early death. Recent findings demonstrate an adverse confluence between sarcopenia and excessive adiposity, as the co-existence of such adverse alterations in body composition may exacerbate systemic inflammation and muscle wasting in the elderly. However, the underlying pathogenic concept of "sarcopenic obesity" is mainly based on phenotypical data derived from clinical observations. Description of the project Based on the previous and preliminary data, we will define the molecular interaction between sarcopenic obesity and inflammation and will verify whether altered inflammatory cytokines expression recapitulate the pathologic phenotype of sarcopenia. Anticipated output The identification of the molecular players of sarcopenia, sarcopenic obesity, and inflamm-aging will reveal if a common signature characterizes the three morbidity conditions and identify biomarkers to monitor the rate of decline in body functions during aging.

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.

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.

Riminucci Mara

Ricerca 2018 - 2019 (Finanziamento per "Seed International Research Projects - call 2018, in collaborazione con IP Paris: Cell mechanical properties of infected erythrocytes in the maturation of Plasmodium falciparum transmission stages in the bone marrow niche investigated with a humanized mouse model Ricerca 2015 - 2017: Role of Gs? signalling in the bone marrow hematolymphopoietic microenvironment as revealed by novel transgenic models

Cell mechanical properties of infected erythrocytes in the maturation of Plasmodium falciparum transmission stages in the bone marrow niche investigated with a humanized mouse model (01-01-2018 - 31-12-2019)
Recent studies revealed an unsuspected role of the human bone marrow and its extravascular compartment as a privileged niche for the maturation of the gametocytes of the human malaria parasite Plasmodium falciparum, the stages transmissible to the Anopheles mosquitoes, an evidence proposing a key role of the host-parasite interplay in this organ in the transmission of this disease. This project will combine a Bone Marrow Humanized Mouse model based on osteoprogenitor cell transplantation, able to reproduce a hematopoiesis microenvironment, with transgenic P. falciparum parasite lines. These parasites produce gametocytes expressing bioluminescent reporters specific for different maturation stages for a sensitive and reliable gametocyte quantification in the animal and/or are mutated in specific gene products and enzymes which regulate the cell deformability of the gametocyte-infected erythrocytes. A role of cell mechanical properties in the ability of P. falciparum gametocytes to circulate and to be transmitted to mosquitoes is emerging in recent studies conducted by the Institut Pasteur laboratory participating as a partner in this project. This collaboration will provide specific transgenic lines, compounds and inhibitors enabling the project to evaluate how changes in gametocyte-infected red blood cell deformability affect the ability of immature and mature gametocytes to home, colonize and be released from the bone marrow microenvironment in our humanized mouse model. The project is expected to reveal molecular mechanisms and players of the host parasite interactions involved in the maturation of P. falciparum transmission stages in the human bone marrow microenvironment.

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.

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.

Toto Angelo

 Ricerca 2018 - 2019 (Finanziamento Under 35 - Call 2018 Progetti "Teresa Ariaudo"): Analysing the NTAIL interaction with the PXD domain to develop antiviral strategies against Paramyxoviridae

Analysing the NTAIL interaction with the PXD domain to develop antiviral strategies against Paramyxoviridae (01-01-2018 - 31-12-2019)
The interaction between the C-terminal, intrinsically disordered domain of the nucleoprotein (NTAIL) and the X domain of the phosphoprotein (PXD) has a key role in the replication and transcription of the genome of three human pathogens, Measles (MeV), Hendra (HeV) and Nipah (NiV) viruses. NTAIL represents an extremely attractive target for the perspective of developing pharmaceutical strategies able to inhibit its interaction with PXD as to interrupt the life cycle of the viruses. Aims of the project are: 1. Mechanistic characterization of NTAIL folding upon binding reaction. 2. Structural characterization on NTAIL key residues involved in the recognition and binding events 3. Pharmacophore design and in silico screening of drug libraries, to be validated in vitro

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.

Visentini Marcella

Ricerca 2018 - 2019 (Finanziamento Ricercatori Under 45): Molecular biomarkers predictive of hematological response to direct antiviral therapy in hepatitis C virus associated lymphoproliferative disorders 

Molecular biomarkers predictive of hematological response to direct antiviral therapy in hepatitis C virus associated lymphoproliferative disorders (b) (01-01-2018 - 31-12-2019)
Hepatitis C virus associated monoclonal B-cell lymphoproliferative disorders (LPDs) range from benign mixed cryoglobulinemia (MC) to indolent or aggressive B-cell lymphomas (BNHL). Dependence on HCV infection is suggested by the expression of stereotyped crossidiotypes (XId) putatively recognizing HCV in B cells from these LPDs and by the regression of these disorders after clearance of the virus. The new direct acting antivirals (DAA), unlike interferon that yields ~50% sustained virologic responses (SVR), allow HCV clearance in 90-100% of patients. A recent study of DAA therapy in 46 HCV+ indolent NHL yielded 98% SVR but only 67% hematologic responses, leaving ~1/3 of patients in the need of alternative treatments. The factors most likely associated with hematologic nonresponse to DAA are: 1. lack of expression of stereotyped XId (putatively HCV-unrelated NHL); 2. specific mutations associated with B-cell neoplasia. This projects aims to investigate the structural and functional characteristics of the B cell receptor (BCR) expressed in B cells from patients with HCV+ LPDs, the mutational profile of these cells and to correlate the findings with hematological response to DAA treatment. These analyses will be performed in the premalignant B cells from patients with MC and in the more aggressive malignant B cells from patients with NHL. The different pattern of molecular response in these disorders, possibly related to a different grade of HCV-dependency, may allow the definition of molecular biomarkers predictive of hematological response and may shed light in the pathogenesis of these virus-associated LPDs.