No description available.

Background: Delirium is defined as an acute disorder of attention and cognition. Delirium is common in hospitalized elderly patient and is associated with increased morbidity, length of stay and patient care costs. Although Delirium can develop at any time during hospitalization, it typically presents early in the post-operative period (Post-Operative Delirium, POD) in the surgery context. The molecular mechanism and possible genetics basis of POD onset are not known, as well as all the risk factors are not completely defined. Our hypothesis is that genetic risk factor involving the inflammatory response could have possible effects on the immunoneuroendocrine system. Moreover, our previous data (inflamm-aging) suggest that aging is associated with an increase of inflammatory status, favouring age-related diseases such as neurodegenerative diseases, frailty, depression among other. Some pro-inflammatory or anti-inflammatory cytokines, seem to play a crucial role in increasing the inflammatory status and in the communication and regulation of immunoneuroendocrine system. Objective: this study evaluated the incidence of POD in elderly patients undergoing general surgery, clinical/physical and psychological risk factors of POD insurgency and investigated inflammatory and genetic risk factors. Moreover, this study evaluated the consequence of POD in terms of institutionalization, development of permanent cognitive dysfunction or dementia and mortality Methods: patients aged over 65 admitted for surgery at the Urgency Unit of S.Orsola-Malpighi Hospital were eligible for this case–control study. Risk factors significantly associated with POD in univariate analysis were entered into multivariate analysis to establish those independently associated with POD. Preoperative plasma level of 9 inflammatory markers were measured in 42 control subjects and 43 subjects who developed POD. Functional polymorphisms of IL-1 α , IL-2, IL-6, IL-8, IL-10 and TNF-alpha cytokine genes were determined in 176 control subjects and 27 POD subjects. Results: A total of 351 patients were enrolled in the study. The incidence of POD was 13•2 %. Independent variables associated with POD were: age, co-morbidity, preoperative cognitive impairment, glucose abnormalities. Median length of hospital stay was 21 days for patients with POD versus 8 days for control patients (P < 0•001). The hospital mortality rate was 19 and 8•4 % respectively (P = 0•021) and mortality rate after 1 year was also higher in POD (P= 0.0001). The baseline of IL-6 concentration was higher in POD patients than patients without POD, whereas IL-2 was lower in POD patients compared to patients without POD. In a multivariate analysis only IL-6 remained associated with POD. Moreover IL-6, IL-8 and IL-2 are associated with co-morbidity, intra-hospital mortality, compromised functional status and emergency admission. No significant differences in genotype distribution were found between POD subjects and controls for any SNP analyzed in this study. Conclusion: In this study we found older age, comorbidity, cognitive impairment, glucose abnormalities and baseline of IL-6 as independent risk factors for the development of POD. IL-6 could be proposed as marker of a trait that is associated with an increased risk of delirium; i.e. raised premorbid IL-6 level predict for the development of delirium.

No description available.

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

Translational control has a direct impact on cancer development and progression. Quantitative and qualitative changes of cap-dependent translation initiation contribute to neoplastic transformation and progression. However, the idea that “alternative” mechanisms of translation initiation, such as IRES-dependent translation, can be involved in the tumorigenesis is emerging. Because the relevance of this kind of translation initiation in cancer progression is not so well clarified, the purpose of my work was to study the impact of IRES-dependent mRNA translation on tumourigenesis and cancer progression with particular regard for breast cancer. The data obtained clarify the function of cap-independent translation in cancer. Particularly they suggested that the deregulation of IRES-dependent translation can be considered a sort of pro-oncogenic stimulus characterized by the inhibition of the expression of some proteins that block cell growth and proliferation and by the over expression of other proteins that contributed to cell survival. In addition, under stress condition, such as a hypoxia, in immortalized epithelial cell lines, changes in cap-independent translation are associated with an induction of expression of stem cell markers, and with the selection of a sub group of cells that have an increased ability to self-renewing and therefore in the acquisition of a more aggressive phenotype.

Il lavoro svolto nel corso del mio dottorato ha avuto per oggetto lo studio dell’ inibizione della glicolisi aerobia (il principale processo metabolico utilizzato dalle cellule neoplastiche per produrre energia) ottenuta mediante il blocco dell’enzima lattato deidrogenasi (LDH). La mia attività si è concentrata sulla possibilità di utilizzare questo approccio allo scopo di migliorare l’efficacia della terapia antitumorale, valutandone gli effetti su colture di carcinoma epatocellulare umano Inizialmente, per valutare gli effetti della inibizione della LDH, è stato usato l’acido ossamico ( OXA). Questo composto è l’unico inibitore noto specifico per LDH ; è una molecola non tossica in vivo, ma attiva a concentrazioni troppo elevate per consentirne un uso terapeutico. Un importante risultato ottenuto è stata la dimostrazione che l’ inibizione della LDH ottenuta con OXA non è solo in grado di innescare una risposta di morte nelle cellule trattate, ma, associata alla somministrazione di sorafenib, aumenta fortemente l’efficacia di questo farmaco, determinando un effetto di sinergismo. Questo forte effetto di potenziamento dell’azione del farmaco è stato spiegato con la dimostrazione che il sorafenib ha la capacità di inibire il consumo di ossigeno delle cellule trattate, rendendole più dipendenti dalla glicolisi. Grazie alla collaborazione con il Dipartimento di Scienze Farmaceutiche il nostro gruppo di ricerca è arrivato alla identificazione di un composto (galloflavina) che inibisce la LDH con una efficienza molto maggiore di OXA. I risultati preliminari ottenuti sulle cellule di epatocarcinoma suggeriscono che la galloflavina potrebbe essere un composto promettente nel campo degli inibitori metabolici tumorali e inducono a una sua valutazione più approfondita come potenziale farmaco antineoplastico. / The aim of this work was to study the inhibition of aerobic glycolysis (the main metabolic pathway used by cancer cells to produce energy) achieved by blocking the enzyme lactate dehydrogenase (LDH). My activity has focused on the possibility of using this approach in order to improve the efficacy of anticancer therapy, evaluating its effects on cultured human hepatocellular carcinoma. In order to evaluate the effects of LDH inhibition, in a first set of experiments, we used oxamic acid (OXA). This compound is the only known specific inhibitor of LDH; it is a non-toxic molecule in vivo, but it is active at too high concentrations to allow its therapeutic use. An important result was the demonstration that LDH inhibition achieved by OXA not only triggers cell death signals but, when combined with the administration of sorafenib, also greatly increases the efficacy of this drug, leading to a synergistic effect. This strong potentiating effect of the drug action has been explained with the demonstration that sorafenib has the ability to inhibit the oxygen consumption of the treated cells, making them more dependent on glycolysis for ATP generation. In collaboration with the Department of Pharmaceutical Sciences our research group has identified a compound (galloflavin) that inhibits LDH with much higher efficacy than OXA. To our knowledge, inhibition of LDH is the only biochemical effect described for galloflavin. According to the results obtained on hepatocarcinoma cultured cells, galloflavin might be a promising lead candidate in the field of tumor metabolic inhibitors, deserving a more exhaustive evaluation as a potential anticancer agent.

Antimicrobial peptides (AMPs) are an important component of the innate immune system of the plants. Plant defensins are a large family of antimicrobial peptides with several interesting features, such as small dimension, high stability and broad spectrum of action. The discovery of new molecules and the study of their mechanism of action allow to consider them attractive for biotechnological applications. In this PhD thesis a defensin from Prunus persica (PpDFN1) and four novel DEFensin Like (DEFL) peptides from Vitis vinifera have been studied. In order to characterize the antimicrobial activity of these molecules, the recombinant mature peptides have been expressed in Escherichia coli and purified to homogeneity by chromatography techniques. PpDFN1 is able to inhibit the growth of B. cinerea, P. expansum and M. laxa with different intensity. The recombinant peptide is capable of membrane permeabilization as demonstrated by SYTOX green fluorescence uptake in treated mycelia. Its interaction with membranes containing sphingolipid species has been shown by artificial lipid monolayers. Furthermore, PpDFN1 displays stronger interaction with monolayers composed by lipids extracted from sensitive fungi with the highest interaction against P. expansum, the most sensitive fungi to PpDFN1 action. DEFL 13, a defensin from grapevine, resulted the strongest antibotrytis peptides. It is electrostatically attracted to the fungal membranes as shown by the antagonist effect of the cations and is able to membrane permeabilization in B. cinerea hyphae. DEFL 13 is internalized in fungal cells and leads to fungal death by activation of some signaling pathways as demonstrated by screening of a mutant collection of B. cinerea

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.