Studio della trasduzione del Segnale Nucleare Inositide-Dipendente: identificazione di eEF1A2 come nuovo Fosfosubstrato di PKC βI

Introduction Phospholipase Cb1 (PLC-β1) is a key player in the regulation of nuclear inositol lipid signaling and of a wide range of cellular functions, such as proliferation and differentiation (1,2,3). PLCb1 signaling depends on the cleavage of phosphatidylinositol 4,5-bisphosphate and the formation of the second messengers diacylglycerol and Inositol tris-phosphate which activate canonical protein kinase C (cPKC) isoforms. Here we describe a proteomic approach to find out a potential effector of nuclear PLC-b1 dependent signaling during insulin stimulated myogenic differentiation. Methods Nuclear lysates obtained from insulin induced C2C12 myoblasts were immunoprecipitated with anti-phospho-substrate cPKC antibody. Proteins, stained with Comassie blue, were excised, digested and subsequently analysed in LC-MS/MS. For peptide sequence searching, the mass spectra were processed and analyzed using the Mascot MS/MS ion search program with the NCBI database. Western blotting, GST-pull down and co-immunoprecipitation were performed to study the interaction between eEF1A2 and cPKCs. Site direct mutagenesis was performed to confirm the phosphorylated motif recognized by the antibody. Immunofluorescence analysis, GFP-tagged eEF1A2 vector and subcellular fractionation were performed to study nuclear localization and relative distribution of eEF1A2. Results We have previously shown that PLC-β1 is greatly increased at the nuclear level during insulin-induced myoblasts differentiation and that this nuclear localization is essential for induction of differentiation. Thus, nuclear proteins of insulin stimulated C2C12 myoblasts, were immunoprecipitated with an anti-phospho-substrate cPKC antibody. After Electrophoretic gel separation of proteins immunoprecipitated, several molecules were identified by LC-MS/MS. Among these most relevant and unexpected was eukaryotic elongation factor 1 alpha 2 (eEF1A2). We found that eEF1A2 is phosphorylated by PKCb1 and that these two molecules coimmunolocalized at the nucleolar level. eEF1A2 could be phosphorylated in many sites among which both threonine and serine residues. By site direct mutagenesis we demonstrated that it is the serine residue of the motif recognized by the antibody that is specifically phosphorylated by PKCb1. The silencing of PLCb1 gives rise to a reduction of expression and phosphorylation levels of eEF1A2 indicating this molecule as a target of nuclear PLCb1 regulatory network during myoblasts differentiation.

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

New DAG-dependent mechanisms modulate cell cycle progression

Through the years, several studies reported the involvement of nuclear lipid signalling as highly connected with cell cycle progression. Indeed, nuclear Phosphatidylinositol-4,5-Biphosphate (PIP2) hydrolisis mediated by Phospholipases C (PLC), which leads to production of the second messengers Diacylglycerol (DAG) and Inositol-1,4,5-Triphosphate (IP3), is a fundamental event for both G1/S and G2/M checkpoints. In particular, we found that nuclear DAG production was mediated by PLCbeta1, enzyme mainly localized in the nucleus of K562 human erythroleukemia cells. This event triggered the activation and nuclear translocation of PKCalpha, which, in turn, resulted able to affect cell cycle via modulation of Cyclin D3 and Cyclin B1, two important enzymes for G1/S transition and G2/M progression respectively.

Il nucleotide extracellulare UTP: induzione della migrazione di cellule staminali emopoietiche CD34+

La letteratura scientifica degli ultimi anni si è arricchita di un numero sempre crescente di studi volti a chiarire i meccanismi che presiedono ai processi di homing di cellule staminali emopoietiche e del loro attecchimento a lungo termine nel midollo osseo. Tali fenomeni sembrano coinvolgere da un lato, l’interazione delle cellule staminali emopoietiche con la complessa architettura e componente cellulare midollare, e dall’altro la riposta ad un’ampia gamma di molecole regolatrici, tra le quali chemochine, citochine, molecole di adesione, enzimi proteolitici e mediatori non peptidici. Fanno parte di quest’ultimo gruppo anche i nucleotidi extracellulari, un gruppo di molecole-segnale recentemente caratterizzate come mediatori di numerose risposte biologiche, tra le quali l’allestimento di fenomeni flogistici e chemiotattici. Nel presente studio è stata investigata la capacità dei nucleotidi extracellulari ATP ed UTP di promuovere, in associazione alla chemochina CXCL12, la migrazione di cellule staminali umane CD34+. E’ così emerso che la stimolazione con UTP è in grado di incrementare significativamente la migrazione dei progenitori emopoietici in risposta al gradiente chemioattrattivo di CXCL12, nonché la loro capacità adesiva. Le analisi citofluorimetriche condotte su cellule migranti sembrano inoltre suggerire che l’UTP agisca interferendo con le dinamiche di internalizzazione del recettore CXCR4, rendendo così le cellule CD34+ maggiormente responsive, e per tempi più lunghi, al gradiente attrattivo del CXCL12. Saggi di homing competitivo in vivo hanno parallelamente mostrato, in topi NOD/SCID, che la stimolazione con UTP aumenta significativamente la capacità dei progenitori emopoeitci umani di localizzarsi a livello midollare. Sono state inoltre indagate alcune possibili vie di trasduzione del segnale attivate dalla stimolazione di recettori P2Y con UTP. Esperimenti di inibizione in presenza della tossina della Pertosse hanno evidenziato il coinvolgimento di proteine Gαi nella migrazione dipendente da CXCL12 ed UTP. Ulteriori indicazioni sono provenute dall’analisi del profilo trascrizionale di cellule staminali CD34+ stimolate con UTP, con CXCL12 o con entrambi i fattori contemporaneamente. Da questa analisi è emerso il ruolo di proteine della famiglia delle Rho GTPasi e di loro effettori a valle (ROCK 1 e ROCK 2) nel promuovere la migrazione UTP-dipendente. Questi dati sono stati confermati successivamente in vitro mediante esperimenti con Tossina B di C. Difficile (un inibitore delle Rho GTPasi) e con Y27632 (in grado di inibire specificatamente le cinasi ROCK). Nel complesso, i dati emersi in questo studio dimostrano la capacità del nucleotide extracellulare UTP di modulare la migrazione in vitro di progenitori emopoietici umani, nonché il loro homing midollare in vivo. L’effetto dell’UTP su questi fenomeni si esplica in concerto con la chemochina CXCL12, attraverso l’attivazione concertata di vie di trasduzione del segnale almeno parzialmente condivise da CXCR4 e recettori P2Y e attraverso il reclutamento comune di proteine ad attività GTPasica, tra le quali le proteine Gαi e i membri della famiglia delle Rho GTPasi.

Meccanismi evolutivi dei parapoxvirus: caratterizzazione genomica di pseudocowpoxvirus e messa a punto di sistemi per lo studio delle ricombinazioni

The Poxviruses are a family of double stranded DNA (dsDNA) viruses that cause disease in many species, both vertebrate and invertebrate. Their genomes range in size from 135 to 365 kbp and show conservation in both organization and content. In particular, the central genomic regions of the chordopoxvirus subfamily (those capable of infecting vertebrates) contain 88 genes which are present in all the virus species characterised to date and which mostly occur in the same order and orientation. In contrast, however, the terminal regions of the genomes frequently contain genes that are species or genera-specific and that are not essential for the growth of the virus in vitro but instead often encode factors with important roles in vivo including modulation of the host immune response to infection and determination of the host range of the virus. The Parapoxviruses (PPV), of which Orf virus is the prototypic species, represent a genus within the chordopoxvirus subfamily of Poxviridae and are characterised by their ability to infect ruminants and humans. The genus currently contains four recognised species of virus, bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV) both of which infect cattle, orf virus (OV) that infects sheep and goats, and parapoxvirus of red deer in New Zealand (PVNZ). The ORFV genome has been fully sequenced, as has that of BPSV, and is ~138 kb in length encoding ~132 genes. The vast majority of these genes allow the virus to replicate in the cytoplasm of the infected host cell and therefore encode proteins involved in replication, transcription and metabolism of nucleic acids. These genes are well conserved between all known genera of poxviruses. There is however another class of genes, located at either end of the linear dsDNA genome, that encode proteins which are non-essential for replication and generally dictate host range and virulence of the virus. The non-essential genes are often the most variable within and between species of virus and therefore are potentially useful for diagnostic purposes. Given their role in subverting the host-immune response to infection they are also targets for novel therapeutics. The function of only a relatively small number of these proteins has been elucidated and there are several genes whose function still remains obscure principally because there is little similarity between them and proteins of known function in current sequence databases. It is thought that by selectively removing some of the virulence genes, or at least neutralising the proteins in some way, current vaccines could be improved. The evolution of poxviruses has been proposed to be an adaptive process involving frequent events of gene gain and loss, such that the virus co-evolves with its specific host. Gene capture or horizontal gene transfer from the host to the virus is considered an important source of new viral genes including those likely to be involved in host range and those enabling the virus to interfere with the host immune response to infection. Given the low rate of nucleotide substitution, recombination can be seen as an essential evolutionary driving force although it is likely underestimated. Recombination in poxviruses is intimately linked to DNA replication with both viral and cellular proteins participate in this recombination-dependent replication. It has been shown, in other poxvirus genera, that recombination between isolates and perhaps even between species does occur, thereby providing another mechanism for the acquisition of new genes and for the rapid evolution of viruses. Such events may result in viruses that have a selective advantage over others, for example in re-infections (a characteristic of the PPV), or in viruses that are able to jump the species barrier and infect new hosts. Sequence data related to viral strains isolated from goats suggest that possible recombination events may have occurred between OV and PCPV (Ueda et al. 2003). The recombination events are frequent during poxvirus replication and comparative genomic analysis of several poxvirus species has revealed that recombinations occur frequently on the right terminal region. Intraspecific recombination can occur between strains of the same PPV species, but also interspecific recombination can happen depending on enough sequence similarity to enable recombination between distinct PPV species. The most important pre-requisite for a successful recombination is the coinfection of the individual host by different virus strains or species. Consequently, the following factors affecting the distribution of different viruses to shared target cells need to be considered: dose of inoculated virus, time interval between inoculation of the first and the second virus, distance between the marker mutations, genetic homology. At present there are no available data on the replication dynamics of PPV in permissive and non permissive hosts and reguarding co-infetions there are no information on the interference mechanisms occurring during the simultaneous replication of viruses of different species. This work has been carried out to set up permissive substrates allowing the replication of different PPV species, in particular keratinocytes monolayers and organotypic skin cultures. Furthermore a method to isolate and expand ovine skin stem cells was has been set up to indeep further aspects of viral cellular tropism during natural infection. The study produced important data to elucidate the replication dynamics of OV and PCPV virus in vitro as well as the mechanisms of interference that can arise during co-infection with different viral species. Moreover, the analysis carried on the genomic right terminal region of PCPV 1303/05 contributed to a better knowledge of the viral genes involved in host interaction and pathogenesis as well as to locate recombination breakpoints and genetic homologies between PPV species. Taken together these data filled several crucial gaps for the study of interspecific recombinations of PPVs which are thought to be important for a better understanding of the viral evolution and to improve the biosafety of antiviral therapy and PPV-based vectors.

Data and behavioral contracts for web services

The recent trend in Web services is fostering a computing scenario where loosely coupled parties interact in a distributed and dynamic environment. Such interactions are sequences of xml messages and in order to assemble parties – either statically or dynamically – it is important to verify that the “contracts” of the parties are “compatible”. The Web Service Description Language (wsdl) is a standard used for describing one-way (asynchronous) and request/response (synchronous) interactions. Web Service Conversation Language extends wscl contracts by allowing the description of arbitrary, possibly cyclic sequences of exchanged messages between communicating parties. Unfortunately, neither wsdl nor wscl can effectively define a notion of compatibility, for the very simple reason that they do not provide any formal characterization of their contract languages. We define two contract languages for Web services. The first one is a data contract language and allow us to describe a Web service in terms of messages (xml documents) that can be sent or received. The second one is a behavioral contract language and allow us to give an abstract definition of the Web service conversation protocol. Both these languages are equipped with a sort of “sub-typing” relation and, therefore, they are suitable to be used for querying Web services repositories. In particular a query for a service compatible with a given contract may safely return services with “greater” contract.

Progetto e realizzazione del sistema di gestione autonoma del volo e controllo in remoto per un velivolo UAV ad ala rotante

This PhD thesis presents the results, achieved at the Aerospace Engineering Department Laboratories of the University of Bologna, concerning the development of a small scale Rotary wing UAVs (RUAVs). In the first part of the work, a mission simulation environment for rotary wing UAVs was developed, as main outcome of the University of Bologna partnership in the CAPECON program (an EU funded research program aimed at studying the UAVs civil applications and economic effectiveness of the potential configuration solutions). The results achieved in cooperation with DLR (German Aerospace Centre) and with an helicopter industrial partners will be described. In the second part of the work, the set-up of a real small scale rotary wing platform was performed. The work was carried out following a series of subsequent logical steps from hardware selection and set-up to final autonomous flight tests. This thesis will focus mainly on the RUAV avionics package set-up, on the onboard software development and final experimental tests. The setup of the electronic package allowed recording of helicopter responses to pilot commands and provided deep insight into the small scale rotorcraft dynamics, facilitating the development of helicopter models and control systems in a Hardware In the Loop (HIL) simulator. A neested PI velocity controller1 was implemented on the onboard computer and autonomous flight tests were performed. Comparison between HIL simulation and experimental results showed good agreement.

Assimilation of Meteosat Second Generation (MSG) satellite data in a regional numerical weather prediction model using a one-dimensional variational approach

The quality of temperature and humidity retrievals from the infrared SEVIRI sensors on the geostationary Meteosat Second Generation (MSG) satellites is assessed by means of a one dimensional variational algorithm. The study is performed with the aim of improving the spatial and temporal resolution of available observations to feed analysis systems designed for high resolution regional scale numerical weather prediction (NWP) models. The non-hydrostatic forecast model COSMO (COnsortium for Small scale MOdelling) in the ARPA-SIM operational configuration is used to provide background fields. Only clear sky observations over sea are processed. An optimised 1D–VAR set-up comprising of the two water vapour and the three window channels is selected. It maximises the reduction of errors in the model backgrounds while ensuring ease of operational implementation through accurate bias correction procedures and correct radiative transfer simulations. The 1D–VAR retrieval quality is firstly quantified in relative terms employing statistics to estimate the reduction in the background model errors. Additionally the absolute retrieval accuracy is assessed comparing the analysis with independent radiosonde and satellite observations. The inclusion of satellite data brings a substantial reduction in the warm and dry biases present in the forecast model. Moreover it is shown that the retrieval profiles generated by the 1D–VAR are well correlated with the radiosonde measurements. Subsequently the 1D–VAR technique is applied to two three–dimensional case–studies: a false alarm case–study occurred in Friuli–Venezia–Giulia on the 8th of July 2004 and a heavy precipitation case occurred in Emilia–Romagna region between 9th and 12th of April 2005. The impact of satellite data for these two events is evaluated in terms of increments in the integrated water vapour and saturation water vapour over the column, in the 2 meters temperature and specific humidity and in the surface temperature. To improve the 1D–VAR technique a method to calculate flow–dependent model error covariance matrices is also assessed. The approach employs members from an ensemble forecast system generated by perturbing physical parameterisation schemes inside the model. The improved set–up applied to the case of 8th of July 2004 shows a substantial neutral impact.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Design and implementation of bioinformatics tools for large scale genome annotation

The continuous increase of genome sequencing projects produced a huge amount of data in the last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced and publically available. However the sole sequencing process of a genome is able to determine just raw nucleotide sequences. This is only the first step of the genome annotation process that will deal with the issue of assigning biological information to each sequence. The annotation process is done at each different level of the biological information processing mechanism, from DNA to protein, and cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time consuming when applied at a this large scale level. Thus, in silico methods need to be used to accomplish the task. The aim of this work was the implementation of predictive computational methods to allow a fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences. The first part of the work was focused on the implementation of a new machine learning based method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent from biases present in the training dataset, which causes the over‐prediction of the most represented examples in all the other available predictors developed so far. This important result was achieved by a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the currently available state‐of‐the‐art methods for this prediction task. BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with experimental and similarity‐based annotations. In the second part of the work a new, machine learning based, method was implemented for the prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of false positive prediction as low as 0.1%. GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in the different considered regions. Furthermore the hypothesis that compositional biases are present among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected. All the developed predictors and databases are freely available at: BaCelLo http://gpcr.biocomp.unibo.it/bacello eSLDB http://gpcr.biocomp.unibo.it/esldb GPIPE http://gpcr.biocomp.unibo.it/gpipe

Optically active photoresponsive multifunctional polymeric materials

In the last year [1], Angiolini and co-workers have synthesized and investigated methacrylic polymers bearing in the side chain the chiral cyclic (S)-3-hydroxypyrrolidine moiety interposed between the main chain and the trans-azoaromatic chromophore, substituted or not in the 4’ position by an electron-withdrawing group. In these materials, the presence of a rigid chiral moiety of one prevailing absolute configuration favours the establishment of a chiral conformation of one prevailing helical handedness, at least within chain segments of the macromolecules, which can be observed by circular dichroism (CD). The simultaneous presence of the azoaromatic and chiral functionalities allows the polymers to display both the properties typical of dissymmetric systems (optical activity, exciton splitting of dichroic absorptions), as well as the features typical of photochromic materials (photorefractivity, photoresponsiveness, NLO properties). The first part of this research was to synthesize analogue homopolymers and copolymers based on bisazoaromatic moiety and compare their properties to those of the above mentioned analogue derivatives bearing only one azoaromatic chromophore in the side chain. We focused also the attention on the effects induced on the thermal and chiroptical behaviours by the insertion of particulars achiral comonomers characterized by different side-chain mobility and grown hindrance (MMA, tert-BMA and TrMA). On the other hand carbazole containing polymers [2] have attracted much attention because of their unique features. The use of these materials in advanced micro- and nanotechnologies spreads in many different applications such as photoconductive and photorefractive polymers, electroluminescent devices, programmable optical interconnections, data storage, chemical photoreceptors, NLO, surface relief gratings, blue emitting materials and holographic memory. The second part of the work was focused on the synthesis and the characterization polymeric derivatives bearing in the side chain carbazole or phenylcarbazole moieties linked to the (S)- 2-hydroxy succinimide or the (S)-3-hydroxy pyrrolidinyl ring as chiral groups covalently linked to the main chain through ester bonds. The last objective of this research was to design, synthesize, and characterize multifunctional methacrylic homopolymers and copolymers bearing three distinct functional groups (i.e. azoaromatic, carbazole and chiral group of one single configuration) directly linked in the side chain. This polymeric derivatives could be of potential interest for several advanced application fields, such as optical storage, waveguides, chiroptical switches, chemical photoreceptors, NLO, surface relief gratings, photoconductive materials, etc.

Light energy management in peach: utilization, photoprotection , photodamage and recovery. Maximizing light absorption in orchard is not always the best solution

The relation between the intercepted light and orchard productivity was considered linear, although this dependence seems to be more subordinate to planting system rather than light intensity. At whole plant level not always the increase of irradiance determines productivity improvement. One of the reasons can be the plant intrinsic un-efficiency in using energy. Generally in full light only the 5 – 10% of the total incoming energy is allocated to net photosynthesis. Therefore preserving or improving this efficiency becomes pivotal for scientist and fruit growers. Even tough a conspicuous energy amount is reflected or transmitted, plants can not avoid to absorb photons in excess. The chlorophyll over-excitation promotes the reactive species production increasing the photoinhibition risks. The dangerous consequences of photoinhibition forced plants to evolve a complex and multilevel machine able to dissipate the energy excess quenching heat (Non Photochemical Quenching), moving electrons (water-water cycle , cyclic transport around PSI, glutathione-ascorbate cycle and photorespiration) and scavenging the generated reactive species. The price plants must pay for this equipment is the use of CO2 and reducing power with a consequent decrease of the photosynthetic efficiency, both because some photons are not used for carboxylation and an effective CO2 and reducing power loss occurs. Net photosynthesis increases with light until the saturation point, additional PPFD doesn’t improve carboxylation but it rises the efficiency of the alternative pathways in energy dissipation but also ROS production and photoinhibition risks. The wide photo-protective apparatus, although is not able to cope with the excessive incoming energy, therefore photodamage occurs. Each event increasing the photon pressure and/or decreasing the efficiency of the described photo-protective mechanisms (i.e. thermal stress, water and nutritional deficiency) can emphasize the photoinhibition. Likely in nature a small amount of not damaged photosystems is found because of the effective, efficient and energy consuming recovery system. Since the damaged PSII is quickly repaired with energy expense, it would be interesting to investigate how much PSII recovery costs to plant productivity. This PhD. dissertation purposes to improve the knowledge about the several strategies accomplished for managing the incoming energy and the light excess implication on photo-damage in peach. The thesis is organized in three scientific units. In the first section a new rapid, non-intrusive, whole tissue and universal technique for functional PSII determination was implemented and validated on different kinds of plants as C3 and C4 species, woody and herbaceous plants, wild type and Chlorophyll b-less mutant and monocot and dicot plants. In the second unit, using a “singular” experimental orchard named “Asymmetric orchard”, the relation between light environment and photosynthetic performance, water use and photoinhibition was investigated in peach at whole plant level, furthermore the effect of photon pressure variation on energy management was considered on single leaf. In the third section the quenching analysis method suggested by Kornyeyev and Hendrickson (2007) was validate on peach. Afterwards it was applied in the field where the influence of moderate light and water reduction on peach photosynthetic performances, water requirements, energy management and photoinhibition was studied. Using solar energy as fuel for life plant is intrinsically suicidal since the high constant photodamage risk. This dissertation would try to highlight the complex relation existing between plant, in particular peach, and light analysing the principal strategies plants developed to manage the incoming light for deriving the maximal benefits as possible minimizing the risks. In the first instance the new method proposed for functional PSII determination based on P700 redox kinetics seems to be a valid, non intrusive, universal and field-applicable technique, even because it is able to measure in deep the whole leaf tissue rather than the first leaf layers as fluorescence. Fluorescence Fv/Fm parameter gives a good estimate of functional PSII but only when data obtained by ad-axial and ab-axial leaf surface are averaged. In addition to this method the energy quenching analysis proposed by Kornyeyev and Hendrickson (2007), combined with the photosynthesis model proposed by von Caemmerer (2000) is a forceful tool to analyse and study, even in the field, the relation between plant and environmental factors such as water, temperature but first of all light. “Asymmetric” training system is a good way to study light energy, photosynthetic performance and water use relations in the field. At whole plant level net carboxylation increases with PPFD reaching a saturating point. Light excess rather than improve photosynthesis may emphasize water and thermal stress leading to stomatal limitation. Furthermore too much light does not promote net carboxylation improvement but PSII damage, in fact in the most light exposed plants about 50-60% of the total PSII is inactivated. At single leaf level, net carboxylation increases till saturation point (1000 – 1200 μmolm-2s-1) and light excess is dissipated by non photochemical quenching and non net carboxylative transports. The latter follows a quite similar pattern of Pn/PPFD curve reaching the saturation point at almost the same photon flux density. At middle-low irradiance NPQ seems to be lumen pH limited because the incoming photon pressure is not enough to generate the optimum lumen pH for violaxanthin de-epoxidase (VDE) full activation. Peach leaves try to cope with the light excess increasing the non net carboxylative transports. While PPFD rises the xanthophyll cycle is more and more activated and the rate of non net carboxylative transports is reduced. Some of these alternative transports, such as the water-water cycle, the cyclic transport around the PSI and the glutathione-ascorbate cycle are able to generate additional H+ in lumen in order to support the VDE activation when light can be limiting. Moreover the alternative transports seems to be involved as an important dissipative way when high temperature and sub-optimal conductance emphasize the photoinhibition risks. In peach, a moderate water and light reduction does not determine net carboxylation decrease but, diminishing the incoming light and the environmental evapo-transpiration request, stomatal conductance decreases, improving water use efficiency. Therefore lowering light intensity till not limiting levels, water could be saved not compromising net photosynthesis. The quenching analysis is able to partition absorbed energy in the several utilization, photoprotection and photo-oxidation pathways. When recovery is permitted only few PSII remained un-repaired, although more net PSII damage is recorded in plants placed in full light. Even in this experiment, in over saturating light the main dissipation pathway is the non photochemical quenching; at middle-low irradiance it seems to be pH limited and other transports, such as photorespiration and alternative transports, are used to support photoprotection and to contribute for creating the optimal trans-thylakoidal ΔpH for violaxanthin de-epoxidase. These alternative pathways become the main quenching mechanisms at very low light environment. Another aspect pointed out by this study is the role of NPQ as dissipative pathway when conductance becomes severely limiting. The evidence that in nature a small amount of damaged PSII is seen indicates the presence of an effective and efficient recovery mechanism that masks the real photodamage occurring during the day. At single leaf level, when repair is not allowed leaves in full light are two fold more photoinhibited than the shaded ones. Therefore light in excess of the photosynthetic optima does not promote net carboxylation but increases water loss and PSII damage. The more is photoinhibition the more must be the photosystems to be repaired and consequently the energy and dry matter to allocate in this essential activity. Since above the saturation point net photosynthesis is constant while photoinhibition increases it would be interesting to investigate how photodamage costs in terms of tree productivity. An other aspect of pivotal importance to be further widened is the combined influence of light and other environmental parameters, like water status, temperature and nutrition on peach light, water and phtosyntate management.

Importanza prognostica delle citochine e loro ruolo nell'immunomodulazione: risultati di studi sperimentali in vivo e in vitro

Citokines are proteins produced by several cell types and secreted in response to various stimuli. These molecules are able to modify the behaviour of other cells inducing activities like growth, differentiation and apoptosis. In the last years, veterinary scientists have investigated the role played by these factors; in fact, cytokines can act as intercellular communicative signals in immune response, cell damage repair and hematopoiesis. Up to date, various cytokines have been identified and in depth comprehension of their effects in physiology, pathology and therapy is an interesting field of research. This thesis aims to understand the role played by these mediators during natural or experimentally induced pathologies. In particular, it has been evaluated the genic and protein expressions of a large number of cytokines during several diseases and starting from different matrix. Considering the heterogeneity of materials used in experimentations, multiple methods and protocols of nucleic acids and proteins extractions have been standardized. Results on cytokines expression obtained from various in vitro and in vivo experimental studies have shown how important these mediators are in regulation and modulation of the host immune response also in veterinary medicine. In particular, the analysis of inflammatory and septic markers, like cytokines, has allowed a better understanding in the pathogenesis during horse Recurrent Airway Obstruction, foal sepsis, Bovine Viral Diarrhea Virus infection and dog Parvovirus infection and the effects of these agents on the host immune system. As experimentations with mice have shown, some pathologies of the respiratory and nervous system can be reduced or even erased by blocking cytokines inflammatory production. The in vitro cytokines expression evaluation in cells which are in vivo involved in the response to exogenous (like pathogens) or endogenous (as it happens during autoimmune diseases) inflammatory stimuli could represent a model for studying citokines effects during the host immune response. This has been analyzed using lymphocytes cultured with several St. aureus strains isolated from bovine mastitic milk and different colostrum products. In the first experiment different cytokines were expressed depending on enterotoxins produced, justifying a different behaviour of the microrganism in the mammal gland. In the second one, bone marrow cells derived incubated with murine lymphocytes with colostrum products have shown various cluster of differentiation expression , different proliferation and a modified cytokines profile. A better understanding of cytokine expression mechanisms will increase the know-how on immune response activated by several pathogen agents. In particular, blocking the cytokine production, the inhibition or catalyzation of the receptor binding mechanism and the modulation of signal transduction mechanism will represent a novel therapeutic strategy in veterinary medicine.