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Il presente documento restituisce una sintesi delle attività del progetto IMPACT ed è diviso in due parti. La prima parte evidenzia gli strumenti completati durante la durata del progetto, in particolare la rete di radar costieri per il monitoraggio delle correnti marine e la piattaforma webGIS per la consultazione dei dati raccolti ed elaborati dai partner di progetto. La seconda parte capitalizza sui suddetti strumenti per fornire sia indicazioni metodologiche che di governance. Nello specifico, la rete di radar costieri viene utilizzata per creare mappe di potenziale contaminazione e indicare in che condizioni le attività portuali possano essere più sostenibili. Le misure demografiche e i calcoli di ritenzione indicano il livello d'efficacia delle dimensioni attuali delle AMP mentre le misure di contaminazione suggeriscono di considerare un numero maggiore di stazioni nell'ottica di migliorare i piani di monitoraggio esistenti. In quest'ottica, le indicazioni rappresentano il piano d'azione e le linee guida del progetto. L'ulteriore espansione della rete di radar costieri prevista nei progetti SICOMAR plus e SINAPSI è la prova che la conclusione delle attività del progetto IMPACT sia in realtà solo un importante punto di partenza.

BACKGROUND: Novel anti-schistosomal multi-stage drugs are needed because only a single drug, praziquantel, is available for the treatment of schistosomiasis and is poorly effective on larval and juvenile stages of the parasite. Schistosomes have a complex life-cycle and multiple developmental stages in the intermediate and definitive hosts. Acetylation and deacetylation of histones play pivotal roles in chromatin structure and in the regulation of transcription in eukaryotic cells. Histone deacetylase (HDAC) inhibitors modulate acetylation of several other proteins localized both in the nucleus and in the cytoplasm and therefore impact on many signaling networks and biological processes. Histone post-translational modifications may provide parasites with the ability to readily adapt to changes in gene expression required for their development and adaptation to the host environment. The aim of the present study was to screen a HDAC class I inhibitor library in order to identify and characterize novel multi-stage hit compounds. METHODS: We used a high-throughput assay based on the quantitation of ATP in the Schistosoma mansoni larval stage (schistosomula) and screened a library of 1500 class I HDAC inhibitors. Subsequently, a few hits were selected and further characterized by viability assays and phenotypic analyses on adult parasites by carmine red and confocal microscopy. RESULTS: Three compounds (SmI-124, SmI-148 and SmI-558) that had an effect on the viability of both the schistosomula larval stage and the adult worm were identified. Treatment with sub-lethal doses of SmI-148 and SmI-558 also decreased egg production. Moreover, treatment of adult parasites with SmI-148, and to a lesser extent Sm-124, was associated with histone hyperacetylation. Finally, SmI-148 and SmI-558 treatments of worm pairs caused a phenotype characterized by defects in the parasite reproductive system, with peculiar features in the ovary. In addition, SmI-558 induced oocyte- and vitelline cell-engulfment and signs of degeneration in the uterus and/or oviduct. CONCLUSIONS: We report the screening of a small HDAC inhibitor library and the identification of three novel compounds which impair viability of the S. mansoni larval stage and adult pairs. These compounds are useful tools for studying deacetylase activity during parasite development and for interfering with egg production. Characterization of their specificity for selected S. mansoni versus human HDAC could provide insights that can be used in optimization and compound design.

Motivation: Tyrosine Kinase inhibitors (TKIs) constitute the most promising frontier of the last decade in the field of cancer treatment. However, the development of resistance mechanisms often makes the tumour insensitive to TKI-targeted therapy. Identification of the factors responsible for the emergence of resistance remains a major clinical challenge for cancer treatment. Non Small Cell Lung Cancer (NSCLC) with mutations activating the Epidermal Growth Factor Receptor (EGFR) provides a suitable model for studying TKI resistance mechanisms. The majority of patients, after an initial positive response to EGFR- TKI drugs (e.g. Erlotinib), develops resistance to treatment for the onset of EGFR secondary mutations and/or the activation of alternative signaling pathways. The present work aims to identify genes and signaling pathways involved in the development of TKI-resistance. To this end, we developed cellular models of NSCLC and designed cellular, molecular and bioinformatics analyses to correlate genotypes to phenotypes. Methods: We generated six cell lines resistant to EGFR-TKI treatment by exposing NSCLC cell lines, harboring EGFR activating mutations in Exon 19, to Erlotinib (ERL) for 5 months. The selected resistant lines were then characterized at the cellular and molecular level to identify phenotypes associated with ERL-resistance. Briefly, immunofluorescence and confocal microscopy analysis as well as cell viability, wound healing and anchorage- independent cell growth assays were used to study cell morphology, cell growth, cell migration, and tumorigenic features. Sequence analysis, quantitative PCR and Western blot were used to characterize mutations, gene copy number variations (CNVs) and gene expression profiles of selected receptor tyrosine kinases (namely EGFR, KRAS, MET, AXL, AKT) that have been previously reported to be mutated or dysregulated in NSCLC. Next, CNVs and gene expression data were assayed for both parental and resistant derivative cell lines at genome-scale level by using microarray technology. We used R (cran.r-project.org/) and Bioconductor (www.bioconductor.org/) for data processing and analysis. The IGV genome browser (www.broadinstitute.org/igv/) and Gorilla web tool (cbl- gorilla.cs.technion.ac.il/) will be used, respectively, for interactive exploration and integrative analysis of the different data levels and for gene functional enrichment analysis. Results: Preliminary cellular and molecular characterization of the NSCLC cell lines highlighted several features associated to an ERL-resistant phenotype, including altered cell migration and cell adhesion, Epithelial-to-Mesenchimal-Transition (EMT) features and metabolic dysregulation. In-depth analysis is still on-going. Moreover, comparison of array data between resistant and sensitive cells array data identified several chromosomal regions that undergone amplification or deletion, as well as lists of thousands of genes aberrantly expressed in selected drug-resistant cell lines, and hundreds of genes aberrantly expressed in all resistant cell lines. Interestingly, preliminary functional enrichment analysis of genes, found consistently altered in all resistant cell lines, indicates their involvement in key biological processes. Further bioinformatics studies are in progress.

Title: IDDM12 LOCUS ANALYSIS IN T1DM: IDENTIFICATION AND CHARACTERIZATION OF FUNCTIONAL POLYMORPHISMS Keywords: T1D, CTLA4, ICOS Disease: Type 1 diabetes Research type: Basic science Authors: Ruberti G, Belfiore F, Malquori L, Carsetti L and Cascino I. Institute of Cell Biology Via E. Ramarini, 00016 Monterotondo scalo (RM) Tel. 06-90091201, Fax 06-90091260, Abstract: Type1 Diabetes (T1D) is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Increasing evidence both in humans and in animal models has shown that defect(s) in immunoregulation underlie T1D, similarly to other immune-mediated disorders. T cell activation results from the integration of signals generated through the T cell antigen receptor-CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. Genetic studies of human T1D indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in IDDM12 locus (1-3). Data obtained by our group in nuclear families and in a case-control study show an association with T1D of a series of markers in the IDDM12 region. These appear to belong to two separate association groups, one centered on the CTLA4 locus and the other on the ICOS locus. The specific aims of this project are to: 1. Identify and functionally characterize the disease variation in the ICOS region; 2. Study the functional role of the T1D-associated CTLA4 3'UTR (AT)n repeat and the CT60 polymorphism on mRNA stability and targeting. Aim 1. For the analysis of the region comprised between ICOS gene and d2s2189 microsatellite (150 kb) we have selected 12 SNPs, already described in the NCBI SNP database. The SNPs were selected for being located in the sequences that were at least 80% identical to the orthologous mouse region over a 100 bp. The underlying rationale is that essential regulatory elements are conserved among mammals, suggesting that cross-species sequence comparisons should identify them (4). We have set up the conditions for multiple PCRs and multiple microsequencing on 10 DNA control samples.Then, we have tested for association the 12 SNP in a case-control study. Three SNP who showed a trand of association with the disease, are now under TDT study on our T1D family panel with at least one affected child. Aim 2. CTLA-4 plays a critical role in downregulating T cell responses. T1D and other autoimmune diseases have shown genetic linkage to the CTLA4 locus. Here, we report that the 3'UTR of CTLA4 regulates Luciferase reporter gene expression, and CTLA4 mRNA decay and translation efficiency, providing the cells with tools to rapidly respond to changes in intracellular and extracellular stimuli. Intriguingly, we also observed that the length of the (AT)n repeat contributed to gene expression regulation. The 3'UTR T1D-associated (AT)n repeat medium (16AT) and long (24AT) alleles decreased the luciferase activity of 80% and 70% respectively in Jurkat and Hek 293T cells; while the short (6AT) allele decreased the activity of 70% and 60% in the hematopoietic and epithelial cell lines. The difference observed was statistically significant in both cell types (p

Project Abstract - T1DM is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Increasing evidence both in humans and in animal models has shown that defect(s) in immunoregulation underlie autoimmune diabetes, similarly to other immune-mediated disorders. T cell activation results from the integration of signals generated through the T cell receptor with those from additional positive and negative regulatory pathways. Disruption of this balance leads to a defective immune response or alternative over-activation of the system as observed in several human diseases. Genetic studies of human T1DM indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in IDDM12 locus. Data obtained by our group in nuclear families and in a case-control study show an association with T1DM of a series of markers in the IDDM12 region. These appear to belong to two separate association groups, one centered on the CTLA4 locus and the other on the ICOS locus. The specific aims of this project are to: 1. Identify and functionally characterize the disease variation in the ICOS region (Task A); 2. Study the functional role of the T1DM-associated CTLA4 3' UTR (AT)n repeat and the CT60 polymorphis on mRNA stability and targeting (Task B).

LAY ABSTRACT - Objective Alterations in the immune response contribute to the pathogenesis of type 1 diabetes (T1DM). CTLA-4 has an important role in the activation of T lymphocytes: Interestingly some variations of this gene are more frequent in diabetic patients as demonstrated by several genetic studies. The main goal of this project is to study the role of these variations on the funcion of the CTLA-4 product. In other words we are interested in evaluating whether modification in the gene may have an effect on the immune response of T lymphocytes. Background/Rationale T1DM is a polygenic disease caused by the immune-mediated destruction of islet insulin-secreting-cells. Genetic predisposition, enviromental factors and immune (dys) regulation participate in the development of autoimmune diseases. These complex diseases are not caused by a defect in a single gene, as in cystic fibrosis, rather they arise from the interaction of multiple genes and enviromental factors. Apart from HLA that regulates the specificity of the immune response, genetic studies indicate the presence of a disease region on chromosome 2q33 associated with T1DM and other autoimmune diseases in several populations. CTLA-4 gene located in this region has been intensively studied and evidence of its association with T1DM as well with other autoimmune diseases is sufficiently established. It is a challenge to study the effect of T1DM-associated CTLA-4 variations on its biological function and on the pathogenesis of diabetes. T cell activation results from the integration of signals generated through the receptor of T lymphocytes with those from additional positive and negative regulatory pathways such as CTLA4. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system as observed in several human diseases. Increasing evidence both in humans and in animal models have shown that defect(s) in immunoregulatory mechanisms underlie autoimmune diabetes, however the molecular basis and defects of this impaired immunoregulation have not been elucidated. Description of Project By using several molecular and cellular approaches we will study the variations of the CTLA-4 gene that are associated with diabetes. Several mutants will be produced to study the effect of these variations on the stability of the transcribed messenger RNA, the localization in the cell of both the RNA and the CTLA-4 gene product. CTLA-4 expression on the cell surface of T cells should be tightly regulated because, together with other molecules, CTLA-4 modulates the immune response. Thus modifications of CTLA-4 expression or sequestration into the cell may have important effects on T cell activation and function. Anticipated Outcome We should be able to evaluate the effect of disease associated CTLA-4 gene variations on the function of this important immunoregulatory molecule. These studies should contribute to the understanding of diabetes pathogenesis. Relevance to Type I Diabetes Genetic studies strongly point to an association of the CTLA-4 gene with T1DM and other autoimmune diseases. Defects in immunoregulatory mechanisms play an important role in the development of autoimmune diseases. A crucial goal for future efforts in the genetics of T1DM will be the transition from association studies to the identification of disease genes and functional characterization of disease pathways. For these reasons we strongly believe that studies of the effect of CTLA-4 gene variations on its biological function are going in the right direction and should contribute to the understanding of immunoregulation and disease development.

BACKGROUND: The T-cell lymphoma cell line HuT78B1, selected for resistance to Fas-mediated apoptosis, resulted unexpectedly resistant to the apoptotic and cytotoxic effects of gemcitabine (dFdC). We investigated whether this resistance was due to the impairment of the Fas/Fas-ligand (FasL) system. MATERIALS AND METHODS: dFdC effects were studied in HuT78B1 and in the parental Fas-sensitive HuT78 cells exposed to inhibitors of the Fas/FasL system. RESULTS: FasL- and Fas-blocking antibodies did not interfere with dFdC-induced apoptosis in HuT78 cells, whereas inhibitors of caspase-8, -9, -1 or -3 had partial inhibitory effects. Notably, in HuT78B1 cells there was a markedly reduced dFdC accumulation notwithstanding a high activity of the activating enzyme deoxycytidine kinase. dFdC accumulation in HuT78 cells was unaffected by a Fas-blocking antibody. CONCLUSION: This is the first time that the selection of a Fas-resistant cell line led to the isolation of a cell clone unable to accumulate the deoxycytidine analog dFdC. Our results show that this alteration is independent from the impairment of the Fas/FasL system.

FAS/Apo-1 (CD95) is an apoptosis-signaling cell surface receptor belonging to the TNF receptor family. Tumor cells resistant to Fas-mediated apoptosis have been described, but to date, the mechanisms responsible for this resistance are not well understood. We found that a series of apoptosis-resistant clones from human HUT78 lymphoma cells express a splicing variant coding for a truncated Fas molecule that lacks the intracellular death-signaling domain. The mutation responsible for the FasExo8Del expression was identified as a deletion-insertion in the intron 7/exon 8 region of the Fas gene. Moreover this mutation affects the phenotype in a dominant negative fashion, i.e., even in the presence of the normal receptor.

Fas/Apo-1 molecule is an apoptosis-signaling cell surface Ag belonging to the TNFR family. To investigate the possibility that soluble forms of the Fas receptor are expressed in human cells, we analyzed Fas mRNA transcripts obtained from activated peripheral mononuclear cells of healthy donors and from human tumor cell lines. We identified and characterized three human mRNA Fas variants: FasTMDel, FasDel2, and FasDel3. To determine whether the three transcripts were derived by alternative splicing, the Fas genomic intron/exon organization of the regions surrounding the deleted sequences was analyzed in Fas clones isolated from a human genomic library. Expression of the transcripts was studied in COS cells transiently transfected with the FasTMDel, FasDel2, and FasDel3 cDNAs. Immunocytochemical and in vitro apoptosis inhibition studies suggest that the transcripts are expressed as soluble Fas proteins that may play a functional role in the regulation of apoptosis.

Anti-hepatitis B surface (HBs) antibodies have been induced in vitro by human peripheral blood mononuclear cells (PBMC) from individuals immunized with hepatitis B surface antigen (HBsAg). Anti-HBs antibody production is antigen-specific, T-cell dependent, class II MHC-restricted and requires de novo synthesis. Furthermore, HBsAg-specific CD8 suppressor cells can also be induced in vitro after challenge with high antigen doses. Antigen presenting cells (APC) are required for the induction of suppression. These suppressor cells are antigen-specific since they do not suppress the antibody response to tetanus toxoid. Antigen-specific suppression is inhibited by cytotoxic treatment of CD8 CD11 cells with OKM1 monoclonal antibody (MoAb) and complement, suggesting that these suppressor CD8 cells may represent granular lymphocytes. Addition to cultures of high concentrations of a recombinant human IL-2 dose not affect suppression, ruling out the adsorption of IL-2 by suppressor cells as a possible mechanism for suppression.