The Effects of Palmitic Acid on Nitric Oxide Production by Rat Skeletal Muscle: Mechanism via Superoxide and iNOS Activation

Background: Increased plasma concentrations of free fatty acids (FFA) can lead to insulin resistance in skeletal muscle, impaired effects on mitochondrial function, including uncoupling of oxidative phosphorylation and decrease of endogenous antioxidant defenses. Nitric oxide (NO) is a highly diffusible gas that presents a half-life of 5-10 seconds and is involved in several physiological and pathological conditions. The effects of palmitic acid on nitric oxide (NO) production by rat skeletal muscle cells and the possible mechanism involved were investigated. Methods: Primary cultured rat skeletal muscle cells were treated with palmitic acid and NO production was assessed by nitrite measurement (Griess method) and 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Nuclear factor-kappa B (NF-kappa B) activation was evaluated by electrophoretic mobility shift assay and iNOS protein content by western blotting. Results: Palmitic acid treatment increased nitric oxide production. This effect was abolished by treatment with NOS inhibitors, L-nitro-arginine (LNA) and L-nitro-arginine methyl esther (L-NAME). NF-kappa B activation and iNOS content were increased due to palmitic acid treatment. The participation of superoxide on nitric oxide production was investigated by incubating the cells with DAF-2-DA in the presence or absence of palmitic acid, a superoxide generator system (X-XO), a mixture of NOS inhibitors and SOD-PEG (superoxide dismutase linked to polyethylene glycol). Palmitic acid and X-XO system increased NO production and this effect was abolished when cells were treated with NOS inhibitors and also with SOD-PEG. Conclusions: In summary, palmitic acid stimulates NO production in cultured skeletal muscle cells through production of superoxide, nuclear factor-kappa B activation and increase of iNOS protein content. Copyright (C) 2012 S. Karger AG, Basel

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCe15A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

The role of AT1 receptor-mediated reproductive function in renovascular hypertension in male rats

There is an association between hypertension and reproductive dysfunction. Angiotensin II (Ang II) is involved in the pathogenesis of hypertension and the regulation of reproduction. The present study aimed to determine whether the angiotensinergic system mediates the effects of hypertension on ieproductive function in male rats subjected to a two-kidney, one-clip (2K1C) model. Sexual behavior parameters, gametogenesis and plasma concentrations of Ang II, testosterone, prolactin and corticosterone were evaluated in male rats 28 days after 2K1C or sham surgery and losartan (Los) treatment (a type 1 angiotensin II (All) receptor antagonist) or vehicle (V) treatment. The animals were divided into Sham + V, 2K1C + V. Sham + Los and 2K1C + Los groups. The 2KiC + V group showed a hypertensive response, inhibition of sexual behavior, spermatogenesis dysfunction, and increases in plasma Ang II and prolactin. Conversely, plasma testosterone decreased, and plasma corticosterone remained constant. Losartan treatment normalized blood pressure and prevented the changes in plasma testosterone and prolactin, sexual behavior and spermatogenesis in the 2KiC + Los group. In addition, losartan treatment caused an additional increase in circulating Ang II in both groups (She m + Los arid 2K1C + Los). Together, these results suggest that Ang II, acting through the All receptor, modulates behavioral and endocrine parameters of reproductive function during renovascular hypertension. In addition, the effects of circulating Ang II on plasma testosterone and prolactin seem to contribute to the spermatogenic and sexual dysfunctions in hypertensive rats. (C) 2012 Els.evier Inc. All rights reserved.

Production of Muons from Heavy Flavor Decays at Forward Rapidity in pp and Pb-Pb Collisions at root s(NN)=2.76 TeV

The ALICE Collaboration has measured the inclusive production of muons from heavy-flavor decays at forward rapidity, 2.5 < y < 4, in pp and Pb-Pb collisions at root s(NN) = 2.76 TeV. The p(t)-differential inclusive cross section of muons from heavy-flavor decays in pp collisions is compared to perturbative QCD calculations. The nuclear modification factor is studied as a function of p(t) and collision centrality. A weak suppression is measured in peripheral collisions. In the most central collisions, a suppression of a factor of about 3-4 is observed in 6 < p(t) < 10 GeV/c. The suppression shows no significant p(t) dependence.

D-S(+) meson production at central rapidity in proton-proton collisions at root s=7 TeV

The P-T-differential inclusive production cross section of the prompt charm-strange meson D-s(+) in the rapidity range vertical bar y vertical bar < 0.5 was measured in proton-proton collisions at root s = 7 TeV at the LHC using the ALICE detector. The analysis was performed on a data sample of 2.98 x 10(8) events collected with a minimum-bias trigger. The corresponding integrated luminosity is L-int = 4.8 nb(-1). Reconstructing the decay D-s(+) -> phi pi(+) with phi -> K-K+, and its charge conjugate, about 480 D-s(+/-) mesons were counted, after selection cuts, in the transverse momentum range 2 < P-T < 12 GeV/c. The results are compared with predictions from models based on perturbative QCD. The ratios of the cross sections of four D meson species (namely D-0, D+, D*+ and D-s(+)) were determined both as a function of p(T) and integrated over p(T)after extrapolating to full p(T) range, together with the strangeness suppression factor in charm fragmentation. The obtained values are found to be compatible within uncertainties with those measured by other experiments in e(+)e(-), ep and pp interactions at various centre-of-mass energies. (C) 2012 CERN. Published by Elsevier By. All rights reserved.

Multi-strange baryon production in pp collisions at root s=7 TeV with ALICE

A measurement of the multi-strange Xi(-) and Omega(-) baryons and their antiparticles by the ALICE experiment at the CERN Large Hadron Collider (LHC) is presented for inelastic proton-proton collisions at a centre-of-mass energy of 7 TeV. The transverse momentum (p(T)) distributions were studied at mid-rapidity (vertical bar y vertical bar < 0.5) in the range of 0.6 < p(T) < 8.5 GeV/c Xi(-) for and Xi(+) baryons, and in the range of 0.8 < P-T < 5 GeV/c for Omega(-) and<(Omega)over bar>(+). Baryons and antibaryons were measured as separate particles and we find that the baryon to antibaryon ratio of both particle species is consistent with unity over the entire range of the measurement. The statistical precision of the current data has allowed us to measure a difference between the mean p(T) of Xi(-) ((Xi) over bar)(+) and Omega(-) ((Omega) over bar (+)). Particle yields, mean pi, and the spectra in the intermediate pi range are not well described by the PYTHIA Perugia 2011 tune Monte Carlo event generator, which has been tuned to reproduce the early LHC data. The discrepancy is largest for Omega(-)((Omega) over bar (+)). This PYTHIA tune approaches the pi spectra of Xi(-) and Xi(+) baryons below p(T) <0.85 GeV/c and describes the Xi(-) and Xi(+) spectra above p(T) > 6.0 GeV/c. We also illustrate the difference between the experimental data and model by comparing the corresponding ratios of (Omega(-) +(Omega) over bar (+))/(Xi(-) + Xi(+)) as a function of transverse mass. (C) 2012 CERN. Published by Elsevier B.V. All rights reserved.

Light vector meson production in pp collisions at root s=7 TeV ALICE Collaboration

The ALICE experiment has measured low-mass dimuon production in pp collisions at root s = 7 TeV in the dimuon rapidity region 2.5 < y < 4. The observed dimuon mass spectrum is described as a superposition of resonance decays (eta, rho, omega, eta', phi) into muons and semi-leptonic decays of charmed mesons. The measured production cross sections for omega and phi are sigma(omega)(1 < p(t) < 5 GeV/c. 2.5 < y < 4) = 5.28 +/- 0.54(stat) +/- 0.49(syst) mb and sigma(phi)(1 < p(t) < 5 GeV/c. 2.5 < y < 4) = 0.940 +/- 0.084(stat) +/- 0.076(syst) mb. The differential cross sections d(2)sigma/dy dp(t) are extracted as a function of p(t) for omega and phi. The ratio between the rho and omega cross section is obtained. Results for the phi are compared with other measurements at the same energy and with predictions by models. (C) 2012 CERN. Published by Elsevier B.V. All rights reserved.

Interferon-gamma and interleukin-10 production by mononuclear cells from patients with advanced head and neck cancer

OBJECTIVE: This study aims to evaluate the production of interferon-gamma and interleukin-10 by stimulated peripheral blood mononuclear cells isolated from patients with supraglottic laryngeal cancer before and after surgical treatment. METHODS: Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cultures of peripheral blood mononuclear cells isolated during the preoperative and late postoperative periods were stimulated with concanavalin A and Bacille Calmette-Guerin, and the supernatant concentrations of interferon-gamma and interleukin-10 were measured. RESULTS: For non-stimulated cultures, the interferon-gamma levels produced by the preoperative period and the late postoperative period cultures were lower than the levels produced by the control group cultures. The interferon-gamma levels after stimulation with concanavalin A were higher in the late postoperative period cultures than in the preoperative evaluation cultures. Stimulation with Bacille Calmette-Guerin led to the production of similar levels of interferon-gamma and interleukin-10 by all cultures; thus, stimulation increased the levels of interferon-gamma produced by both the preoperative and postoperative cultures relative to the levels produced by the corresponding unstimulated cultures. CONCLUSION: Patients with advanced supraglottic laryngeal cancer exhibit an in vitro deficiency in interferongamma secretion by mononuclear cells. Stimulated cells seem to recover this function during the postoperative period.

Mesenchymal stem cells (MSC) prevented the progression of renovascular hypertension, improved renal function and architecture

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×10(5) cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT1 were elevated, whereas AT2 levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.

Altered phenotype and function of dendritic cells in individuals with chronic periodontitis

OBJECTIVE: To investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). DESIGN: m-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. RESULTS: Expression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). CONCLUSIONS: Bacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis

The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production

Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1β, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Measurement of low pT D0 meson production cross section at CDF II

In this thesis we present a study of the D0 meson (through one of its two-body decay channel, D0 → Kπ) collected by the CDF II experiment at the Tevatron pp ̄ collider at Fermilab. In particular we measured the differential production cross section as a function of the transverse momentum down to pT = 1.5 GeV/c.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

Pathogenic role of IL-33-mediated eosinophil infiltration and function in experimental inflammatory bowel disease

IL-33/ST2 axis is known to promote Th2 immune responses and has been linked to several autoimmune and inflammatory disorders, including inflammatory bowel disease (IBD), and recent evidences show that it can regulate eosinophils (EOS) infiltration and function. Based also on the well documented relationship between EOS and IBD, we assessed the role of IL-33-mediated eosinophilia and ileal inflammation in SAMP1/YitFc (SAMP) murine model of Th1/Th2 chronic enteritis, and we found that IL-33 is related to inflammation progression and EOS infiltration as well as IL-5 and eotaxins increase. Administering IL-33 to SAMP and AKR mice augmented eosinophilia, eotaxins mRNA expression and Th2 molecules production, whereas blockade of ST2 and/or typical EOS molecules, such as IL-5 and CCR3, resulted in a marked decrease of inflammation, EOS infiltration, IL-5 and eotaxins mRNA expression and Th2 cytokines production. Human data supported mice’s showing an increased colocalization of IL-33 and EOS in the colon mucosa of UC patients, as well as an augmented IL-5 and eotaxins mRNA expression, when compared to non-UC. Lastly we analyzed SAMP raised in germ free (GF) condition to see the microbiota effect on IL-33 expression and Th2 responses leading to chronic intestinal inflammation. We found a remarkable decrease in ileal IL-33 and Th2 cytokines mRNA expression as well as EOS infiltration in GF versus normal SAMP with comparable inflammatory scores. Moreover, EOS depletion in normal SAMP didn’t affect IL-33 mRNA expression. These data demonstrate a pathogenic role of IL-33-mediated eosinophilia in chronic intestinal inflammation, and that blockade of IL-33 and/or downstream EOS activation may represent a novel therapeutic modality to treat patients with IBD. Also they highlight the gut microbiota role in IL-33 production, and the following EOS infiltration in the intestinal mucosa, confirming that the microbiota is essential in mounting potent Th2 response leading to chronic ileitis in SAMP.