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.

Indigestible cellulose and lignin in determining feces production and apparent digestibility in horses

Four crossbred geldings were used in a randomized blocks experimental design. The objective was to study the use of the internal markers indigestible cellulose (iCEL) and indigestible lignin (iLIG), obtained in situ (cattle) or in vivo (equine) to predict nutrient apparent digestibility in horses. Treatments consisted of different methodologies to determine digestibility: direct method with total feces collection (TC), and indirect method using internal markers iCEL and iLIG obtained either by in situ incubation in bovine rumen or in vivo (IV) using the mobile nylon bag (MNB) technique in horses. Feces production was 2.80 kg in DM, and average recovery rate (p > 0.05) was 101%. Nutrient digestibility coefficient (p > 0.05) estimates were adequately predicted by iCEL and iLIG, obtained in situ or in vivo, with average values of 52.63, 54.17, 64.90, 43.73 and 98.28% for dry matter, organic matter, crude protein, neutral detergent fiber and starch, respectively. It can be concluded that iCEL and iLIG may be obtained in vivo by MNB in horses consuming a forage-concentrate diet, to predict nutrient digestibility coefficients.

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.

Longterm peripheral baroreflex and chemoreflex function after bilateral eversion carotid endarterectomy

Introduction The “eversion” technique for carotid endarterectomy (e-CEA), that involves the transection of the internal carotid artery at the carotid bulb and its eversion over the atherosclerotic plaque, has been associated with an increased risk of postoperative hypertension possibly due to a direct iatrogenic damage to the carotid sinus fibers. The aim of this study is to assess the long-term effect of the e-CEA on arterial baroreflex and peripheral chemoreflex function in humans. Methods A retrospective review was conducted on a prospectively compiled computerized database of 3128 CEAs performed on 2617 patients at our Center between January 2001 and March 2006. During this period, a total of 292 patients who had bilateral carotid stenosis ≥70% at the time of the first admission underwent staged bilateral CEAs. Of these, 93 patients had staged bilateral e-CEAs, 126 staged bilateral s- CEAs and 73 had different procedures on each carotid. CEAs were performed with either the eversion or the standard technique with routine Dacron patching in all cases. The study inclusion criteria were bilateral CEA with the same technique on both sides and an uneventful postoperative course after both procedures. We decided to enroll patients submitted to bilateral e-CEA to eliminate the background noise from contralateral carotid sinus fibers. Exclusion criteria were: age >70 years, diabetes mellitus, chronic pulmonary disease, symptomatic ischemic cardiac disease or medical therapy with b-blockers, cardiac arrhythmia, permanent neurologic deficits or an abnormal preoperative cerebral CT scan, carotid restenosis and previous neck or chest surgery or irradiation. Young and aged-matched healthy subjects were also recruited as controls. Patients were assessed by the 4 standard cardiovascular reflex tests, including Lying-to-standing, Orthostatic hypotension, Deep breathing, and Valsalva Maneuver. Indirect autonomic parameters were assessed with a non-invasive approach based on spectral analysis of EKG RR interval, systolic arterial pressure, and respiration variability, performed with an ad hoc software. From the analysis of these parameters the software provides the estimates of spontaneous baroreflex sensitivity (BRS). The ventilatory response to hypoxia was assessed in patients and controls by means of classic rebreathing tests. Results A total of 29 patients (16 males, age 62.4±8.0 years) were enrolled. Overall, 13 patients had undergone bilateral e-CEA (44.8%) and 16 bilateral s-CEA (55.2%) with a mean interval between the procedures of 62±56 days. No patient showed signs or symptoms of autonomic dysfunction, including labile hypertension, tachycardia, palpitations, headache, inappropriate diaphoresis, pallor or flushing. The results of standard cardiovascular autonomic tests showed no evidence of autonomic dysfunction in any of the enrolled patients. At spectral analysis, a residual baroreflex performance was shown in both patient groups, though reduced, as expected, compared to young controls. Notably, baroreflex function was better maintained in e-CEA, compared to standard CEA. (BRS at rest: young controls 19.93 ± 2.45 msec/mmHg; age-matched controls 7.75 ± 1.24; e-CEA 13.85 ± 5.14; s-CEA 4.93 ± 1.15; ANOVA P=0.001; BRS at stand: young controls 7.83 ± 0.66; age-matched controls 3.71 ± 0.35; e-CEA 7.04 ± 1.99; s-CEA 3.57 ± 1.20; ANOVA P=0.001). In all subjects ventilation (VÝ E) and oximetry data fitted a linear regression model with r values > 0.8. Oneway analysis of variance showed a significantly higher slope both for ΔVE/ΔSaO2 in controls compared with both patient groups which were not different from each other (-1.37 ± 0.33 compared with -0.33±0.08 and -0.29 ±0.13 l/min/%SaO2, p<0.05, Fig.). Similar results were observed for and ΔVE/ΔPetO2 (-0.20 ± 0.1 versus -0.01 ± 0.0 and -0.07 ± 0.02 l/min/mmHg, p<0.05). A regression model using treatment, age, baseline FiCO2 and minimum SaO2 achieved showed only treatment as a significant factor in explaining the variance in minute ventilation (R2= 25%). Conclusions Overall, we demonstrated that bilateral e-CEA does not imply a carotid sinus denervation. As a result of some expected degree of iatrogenic damage, such performance was lower than that of controls. Interestingly though, baroreflex performance appeared better maintained in e-CEA than in s-CEA. This may be related to the changes in the elastic properties of the carotid sinus vascular wall, as the patch is more rigid than the endarterectomized carotid wall that remains in the e-CEA. These data confirmed the safety of CEA irrespective of the surgical technique and have relevant clinical implication in the assessment of the frequent hemodynamic disturbances associated with carotid angioplasty stenting.

Catalytic liquid- and gas-phase oxidations for the synthesis of intermediates and specialty chemicals: some examples of industrial relevance

Nowadays, it is clear that the target of creating a sustainable future for the next generations requires to re-think the industrial application of chemistry. It is also evident that more sustainable chemical processes may be economically convenient, in comparison with the conventional ones, because fewer by-products means lower costs for raw materials, for separation and for disposal treatments; but also it implies an increase of productivity and, as a consequence, smaller reactors can be used. In addition, an indirect gain could derive from the better public image of the company, marketing sustainable products or processes. In this context, oxidation reactions play a major role, being the tool for the production of huge quantities of chemical intermediates and specialties. Potentially, the impact of these productions on the environment could have been much worse than it is, if a continuous efforts hadn’t been spent to improve the technologies employed. Substantial technological innovations have driven the development of new catalytic systems, the improvement of reactions and process technologies, contributing to move the chemical industry in the direction of a more sustainable and ecological approach. The roadmap for the application of these concepts includes new synthetic strategies, alternative reactants, catalysts heterogenisation and innovative reactor configurations and process design. Actually, in order to implement all these ideas into real projects, the development of more efficient reactions is one primary target. Yield, selectivity and space-time yield are the right metrics for evaluating the reaction efficiency. In the case of catalytic selective oxidation, the control of selectivity has always been the principal issue, because the formation of total oxidation products (carbon oxides) is thermodynamically more favoured than the formation of the desired, partially oxidized compound. As a matter of fact, only in few oxidation reactions a total, or close to total, conversion is achieved, and usually the selectivity is limited by the formation of by-products or co-products, that often implies unfavourable process economics; moreover, sometimes the cost of the oxidant further penalizes the process. During my PhD work, I have investigated four reactions that are emblematic of the new approaches used in the chemical industry. In the Part A of my thesis, a new process aimed at a more sustainable production of menadione (vitamin K3) is described. The “greener” approach includes the use of hydrogen peroxide in place of chromate (from a stoichiometric oxidation to a catalytic oxidation), also avoiding the production of dangerous waste. Moreover, I have studied the possibility of using an heterogeneous catalytic system, able to efficiently activate hydrogen peroxide. Indeed, the overall process would be carried out in two different steps: the first is the methylation of 1-naphthol with methanol to yield 2-methyl-1-naphthol, the second one is the oxidation of the latter compound to menadione. The catalyst for this latter step, the reaction object of my investigation, consists of Nb2O5-SiO2 prepared with the sol-gel technique. The catalytic tests were first carried out under conditions that simulate the in-situ generation of hydrogen peroxide, that means using a low concentration of the oxidant. Then, experiments were carried out using higher hydrogen peroxide concentration. The study of the reaction mechanism was fundamental to get indications about the best operative conditions, and improve the selectivity to menadione. In the Part B, I explored the direct oxidation of benzene to phenol with hydrogen peroxide. The industrial process for phenol is the oxidation of cumene with oxygen, that also co-produces acetone. This can be considered a case of how economics could drive the sustainability issue; in fact, the new process allowing to obtain directly phenol, besides avoiding the co-production of acetone (a burden for phenol, because the market requirements for the two products are quite different), might be economically convenient with respect to the conventional process, if a high selectivity to phenol were obtained. Titanium silicalite-1 (TS-1) is the catalyst chosen for this reaction. Comparing the reactivity results obtained with some TS-1 samples having different chemical-physical properties, and analyzing in detail the effect of the more important reaction parameters, we could formulate some hypothesis concerning the reaction network and mechanism. Part C of my thesis deals with the hydroxylation of phenol to hydroquinone and catechol. This reaction is already industrially applied but, for economical reason, an improvement of the selectivity to the para di-hydroxilated compound and a decrease of the selectivity to the ortho isomer would be desirable. Also in this case, the catalyst used was the TS-1. The aim of my research was to find out a method to control the selectivity ratio between the two isomers, and finally to make the industrial process more flexible, in order to adapt the process performance in function of fluctuations of the market requirements. The reaction was carried out in both a batch stirred reactor and in a re-circulating fixed-bed reactor. In the first system, the effect of various reaction parameters on catalytic behaviour was investigated: type of solvent or co-solvent, and particle size. With the second reactor type, I investigated the possibility to use a continuous system, and the catalyst shaped in extrudates (instead of powder), in order to avoid the catalyst filtration step. Finally, part D deals with the study of a new process for the valorisation of glycerol, by means of transformation into valuable chemicals. This molecule is nowadays produced in big amount, being a co-product in biodiesel synthesis; therefore, it is considered a raw material from renewable resources (a bio-platform molecule). Initially, we tested the oxidation of glycerol in the liquid-phase, with hydrogen peroxide and TS-1. However, results achieved were not satisfactory. Then we investigated the gas-phase transformation of glycerol into acrylic acid, with the intermediate formation of acrolein; the latter can be obtained by dehydration of glycerol, and then can be oxidized into acrylic acid. Actually, the oxidation step from acrolein to acrylic acid is already optimized at an industrial level; therefore, we decided to investigate in depth the first step of the process. I studied the reactivity of heterogeneous acid catalysts based on sulphated zirconia. Tests were carried out both in aerobic and anaerobic conditions, in order to investigate the effect of oxygen on the catalyst deactivation rate (one main problem usually met in glycerol dehydration). Finally, I studied the reactivity of bifunctional systems, made of Keggin-type polyoxometalates, either alone or supported over sulphated zirconia, in this way combining the acid functionality (necessary for the dehydrative step) with the redox one (necessary for the oxidative step). In conclusion, during my PhD work I investigated reactions that apply the “green chemistry” rules and strategies; in particular, I studied new greener approaches for the synthesis of chemicals (Part A and Part B), the optimisation of reaction parameters to make the oxidation process more flexible (Part C), and the use of a bioplatform molecule for the synthesis of a chemical intermediate (Part D).

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.

Study of Candidate Genes for production and carcass traits in Italian Heavy Pigs: identification of a new DNA Markers, Expression Studies and Association Analysis using Different Experimental Design

Heavy pig breeding in Italy is mainly oriented for the production of high quality processed products. Of particular importance is the dry cured ham production, which is strictly regulated and requires specific carcass characteristics correlated with green leg characteristics. Furthermore, as pigs are slaughtered at about 160 kg live weight, the Italian pig breeding sector faces severe problems of production efficiency that are related to all biological aspects linked to growth, feed conversion, fat deposition and so on. It is well known that production and carcass traits are in part genetically determined. Therefore, as a first step to understand genetic basis of traits that could have a direct or indirect impact on dry cured ham production, a candidate gene approach can be used to identify DNA markers associated with parameters of economic importance. In this thesis, we investigated three candidate genes for carcass and production traits (TRIB3, PCSK1, MUC4) in pig breeds used for dry cured ham production, using different experimental approaches in order to find molecular markers associated with these parameters.

Funktionsanalyse von Gamma2-Adaptin: ein zellulärer Adaptor der Hepatitis-B-Virus-Morphogenese

Im Replikationszyklus umhüllter Viren entstehen neue Viruspartikel durch die Knospung an Membranen der Wirtszelle. An diesem Prozess sind verschiedene zelluläre Faktoren und Mechanismen beteiligt, speziell die ESCRT-Proteinkomplexe, welche die Vesikelbildung an den MVBs steuern. Auch bei HBV ist davon auszugehen, dass Komponenten der Wirtszelle an der Umhüllung und Freisetzung der Virionen beteiligt sind, allerdings sind diese noch weitgehend unbekannt. Ziel dieser Arbeit war es daher, die zellulären Faktoren genauer zu charakterisieren und ihre Funktion bei der Virusumhüllung aufzuklären. Den Ausgangspunkt für die hier durchgeführten Untersuchungen bildeten vorangegangene Arbeiten, in denen die spezifische Interaktion des L-Hüllproteins von HBV mit g2-Adaptin nachgewiesen werden konnte. Diese ist für die Morphogenese von HBV essentiell, allerdings ist die zelluläre ebenso wie die virusspezifische Funktion von g2-Adaptin bislang unbekannt. Im Rahmen dieser Arbeit sollte daher untersucht werden, wo und wie g2-Adaptin in der Zelle funktionell ist, um daraus Rückschlüsse auf die Vorgänge bei der Morphogenese von HBV ziehen zu können. Die Grundlage für die Charakterisierung von g2-Adaptin bildete seine Ähnlichkeit zu zellulären Clathrin-Adaptorproteinen. So konnte hier gezeigt werden, dass auch g2-Adaptin ein Clathrin-Bindungsmotiv besitzt, welches eine Interaktion mit Clathrin ermöglicht. Außerdem konnte ein Ubiquitin-Interaktions-Motiv (UIM) identifiziert werden, das die Bindung an ubiquitinierte Proteine vermittelt. Diese Beobachtung deutet darauf hin, dass g2-Adaptin zu einer Gruppe monomerer Adaptorproteine zählen könnte, welche als Ubiquitin-Rezeptoren in der Zelle funktionell sind. Die folgenden Analysen zeigten eine weitere Gemeinsamkeit, da auch g2-Adaptin selbst durch Ubiquitin modifiziert wird, wobei die Ubiquitinierung von einem intakten UIM abhängt. Dieser als Coupled Monoubiquitination bezeichnete Prozess wird hierbei durch die Ubiquitin-Ligase Nedd4 vermittelt, die direkt mit g2-Adaptin interagiert. Dabei konnte nachgewiesen werden, dass die C2-Domäne von Nedd4 ebenfalls mit Ubiquitin modifiziert ist, wodurch der Kontakt zum UIM von g2-Adaptin erfolgt. Die meisten der bislang bekannten Ubiquitin-bindenden Adaptorproteine, spielen bei der Vesikelentstehung an verschiedenen zellulären Membranen eine Rolle, wo sie an der Sortierung der vorwiegend ubiquitinierten Membranproteine beteiligt sind und zelluläre Komponenten rekrutieren, welche die Vesikelabschnürung vermitteln. Die Adaptorproteine sind dabei meist mit der jeweiligen Membran assoziiert, was auch für g2-Adaptin nachgewiesen werden konnte. Diese Membranbindung wird durch den N-terminalen Proteinbereich von g2-Adaptin vermittelt und erfolgt unabhängig von den Ubiquitin-bindenden Eigenschaften und von Nedd4. Allerdings scheint die Ubiquitin-Modifikation von g2-Adaptin ausschließlich in membrangebundener Form zu erfolgen. An welchen Membranen g2-Adaptin lokalisiert ist, wurde in Immunfluoreszenzstudien untersucht, wobei eine enge Assoziation von g2-Adaptin mit späten Endosomen bzw. MVBs zu beobachten war. Bei weiteren Analysen konnte auch ein funktioneller Einfluss auf die Vesikelentstehung an den MVBs nachgewiesen werden, da durch die Depletion von g2-Adaptin stark vergrößerte, defekte MVBs induziert wurden. Dies deutet darauf hin, dass g2-Adaptin als Ubiquitin-Rezeptor an diesen Prozessen beteiligt sein könnte. Ebenso wie andere Adaptorproteine könnte es hier an die Cargo-Proteine binden, diese durch den Kontakt zu Clathrin lokal konzentrieren und die Vesikelabschnürung durch die Rekrutierung der MVB-Maschinerie vermitteln. Möglicherweise stellt g2-Adaptin hierbei den bislang nicht identifizierten Adaptor dar, der die Verbindung zwischen Nedd4 und der MVB-Kaskade herstellt. Eine ähnliche Funktion für g2-Adaptin ist auch bei der Morphogenese von HBV denkbar. Aufgrund der durchgeführten Lokalisationsstudien ist anzunehmen, dass die Umhüllung der HBV-Partikel direkt an den MVBs erfolgt. Vermutlich bindet g2-Adaptin hier an das L-Hüllprotein, wobei es durch die Rekrutierung von Clathrin zu einer lokalen Anreicherung der Hüllproteine kommt. g2-Adaptin interagiert zudem in UIM-abhängiger Weise mit dem Nukleokapsid, wobei der Kontakt direkt erfolgen könnte oder durch die Ubiquitin-Ligase Nedd4 vermittelt wird, welche über eine Late-Domäne ebenfalls mit dem Nukleokapsid verbunden ist. Anscheinend gelangt das Nukleokapsid durch den Einfluss von g2-Adaptin und Nedd4 zum Ort der Virusmorphogenese, wo die eigentliche Umhüllung und die Abschnürung der Viruspartikel erfolgen. Vermutlich sind auch hier Komponenten der MVB-Maschinerie beteiligt, die womöglich durch g2-Adaptin rekrutiert werden.