New Insights into the Role of Metals in Host-Pathogen Interactions

: Almost eighty years have passed since the publication of the studies by Arthur Schade and Leona Caroline, which we can consider as the first investigations that began to disclose the importance of metals in host-pathogen interactions [...]

Broad band acoustic spectroscopy in disordered systems

2013/2014The present work of thesis is situated within the framework of the study of disordered systems as liquids and glasses. A liquid is a system characterized by long range translational invariance and by a short range ordered structure. In the liquid state, contrarily to the crystalline one, there is not structural periodicity and all we learnt from solid state physics (Block’s theorem, phonons, Brillouin’s zones, eigen-states of plane waves, etc.) must be fully revised. The macroscopic collective properties of condensed matter are the result of inter and intra-molecular interactions that are typified by characteristic time and space scales. A longstanding and powerful tool to investigate the collective nature of the microscopic processes inside the system is the acoustic spectroscopy. While in the case of crystalline phase we could take advantage of the periodical structure of the system, limiting our investigation to the so called first Brillouin zone, in order to characterize a disordered system we need to explore a widest as possible spectral range to access the all time and space scales in which the dynamical phenomena occur. This approach can be called Broad Band Acoustic Spectroscopy . My PhD activity was devoted to the development of new experimental methods and techniques allowing the exploration with continuity of dynamics evolving with timescales from tenth’s of ns’s to ps’s. I could test such Broad Band Acoustic Spectroscopy on a prototypical sample: acetonitrile, the liquid with highest dipole moment, known for its many different inter- and intra-molecular dynamics. Using several experimental and computational approaches I could characterize the main dynamical processes for such compound in its whole liquid phase. Thanks to the crossing of the all acquired information, it was possible to identify a mutual influence between different relaxations whose behaviour otherwise was not possible to correctly understand. After a brief introduction to contextualize the Broad Band Acoustic Spectroscopy in disordered systems, in Chapter 1 is presented an overview of the experimental techniques used to perform the measurements within this work. In this chapter, the first my original contribution to the extension of acoustic spectral range finds the place. Thanks to the design and realization of particular spatial filters it is now possible to perform Brillouin Light Scattering experiments with angle tunabil- ity without incurring in annoying spurious contributions issues. In the chapter are also recalled the main physical principles at the ground of every presented tech- nique, in particular stressing the complementarity of the energy and time resolved spectroscopies. Because all the information from the inner dynamics of the sample are mediated by the acoustic modes interaction, Chapter 2 is dedicated to the formalism of the density fluctuations, highlighting the differences between the hydrodynamic model, valid at macroscopic length-scales, and the memory functions approach, necessary to describe the mesoscopic region where the characteristic length-scales of the acoustic modes are comparable to the inter-particles distances. In Chapter 3 are shown the experimental results obtained thanks to the Broad Band Spectroscopy in the whole temperature range of the liquid phase of acetoni- trile. We could first measure some thermodynamical quantities by a non linear spectroscopy named Transient Grating, shedding light on the literature debate on them. Among the obtained results, we appreciated a temperature dependency of the heat capacity ratio which is usually considered a constant. Starting from these results and using the filtering approach introduced in Chapter 1, we could profitably study the vibrational relaxation of acetonitrile discovering some relevant discrepan- cies with previous studies. In this way we demonstrated the importance to avoid eventual spurious contributions and to have complementary information from dif- ferent spectral domains. Then there will be shown the results for the so called structural relaxation, obtained in the high energy domain by Inelastic X-ray Scat- tering. A really interesting correlation has been found with the result obtained by the ultrafast response of the Transient Grating method. Once again, to obtain such comparison was necessary to combine the information arising from many different spectral ranges, fact that highlights the utility and endorses the multi-techniques broad band acoustic spectroscopy method. The temperature dependence of the aforementioned relaxation processes sug- gested to evaluate if any coupling there exist between the two. This is exactly what we were able to appreciate in the deep-UV domain and we proposed a phenomeno- logical model to give a picture of the occurring interaction. To further investigate such dynamics coupling effect, could be extremely useful to explore the soft-UV spectral range but so far any technique was able to access such energy domain. In Chapter 4 we thus present our innovative solution to the problem: a novel table top Fabry-Perot interferometer conceived to operate with UV laser source and with only reflecting optics. The last chapters of the thesis describe my contribution to the forthcoming Free Electron Laser (FEL) based experiment to further increase the accessible spectral range to the acoustic spectroscopy: the TIMER project. In particular I will show the result obtained with the pilot experiment named “mini-TIMER” in which we could demonstrate the feasibility of a Transient Grating experiment in the Extreme Ultra Violet (EUV) domain. When TIMER will be operative it will be the first experimental setup able to probe the EUV mesoscopic region of crucial importance for the study of glasses and nano-structures. This result paves the way to a new class of intriguing experiments only matter of theoretical considerations so far: the so called four wave mixing experiments with elemental selectivity.Il presente lavoro di tesi si colloca nell’ambito dello studio dei sistemi disordi- nati quali liquidi e vetri. Un liquido ´e un sistema caratterizzato da invarianza traslazionale a lungo raggio e da una struttura ordinata a corto raggio. Lo stato liquido, a differenza di quello cristallino, non gode di una periodicit`a strutturale e tutto ci`o che avevamo imparato dalla fisica dello stato solido (teorema di Block, fononi, zone di Brillouin, autostati di onde piane, etc.) deve essere completamente rivisto. Le propriet`a collettive della materia sono il risultato di interazioni a livello inter- e intra-molecolare identificate da caratteristiche scale temporali e spaziali. Uno strumento ormai affermato e utile per lo studio della natura collettiva dei processi microscopici attivi all’interno del sistema ´e la spettroscopia acustica. Mentre nel caso della fase cristallina si poteva sfruttare la natura periodica della struttura del sistema, limitando lo studio alla cosiddetta prima zona di Brillouin, per caratteriz- zare un sistema disordinato ´e necessario esplorare un intervallo spettrale il pi`u ampio possibile cosí da poter aver accesso alle scale spazio-temporali in cui le dinamiche avvengo. Questo tipo di approccio pu`o essere chiamato Spettroscopia Acustica ad Ampia Banda Spettrale . Il mio dottorato ´e stato dedicato allo sviluppo di nuovi metodi e tecniche speri- mentali per esplorare con continuit`a i processi dinamici la cui evoluzione avviene su scale di tempi tra le decine di ns e i ps. Tale Spettroscopia Acustica ad Ampia Banda Spettrale ´e stata testata su un campione prototipo di acetonitrile, il liquido con il pi´u alto momento di dipolo esistente, noto per le sue molteplici dinamiche di orgine inter- e intra-molecolare. Usando diversi approcci sperimentali e computazionali ´e stato possibile caratterizzare i principali processi di rilassamento per tale composto in tutta la sua fase liquida. Combinando poi tutte le informazioni acquisite, ´e stato possibile indentificare una mutua influenza tra i diversi processi di rilassamento il cui comportamento altrimenti sarebbe rimasto incompreso. Dopo una breve introduzione per contestualizzare la Spettroscopia Acustica ad Ampia Banda Spettrale nell’ambito dei sistemi disordinati, nel Capitolo 1 viene offerta una panoramica delle tecniche sperimentali usate per effettuare le misure nel corso di questo lavoro. Trova posto in questo capitolo la descrizione del mio primo originale contributo all’estensione dell’intervallo spettrale acustico. Grazie alla progettazione e realizzazione di particolari filtri spaziali ´e ora possibile effet- tuare esperimenti di diffusione di luce Brillouin con angolo di scattering variabile senza dover incorrere in fastidiosi problemi di contributi spuri. Nel capitolo vengono anche richiamati i principali elementi di fisica alla base di ogni tecnica, marcando in particolare la caratteristica di complementariet`a tra esperimenti risolti in tempo e in energia. Siccome tutte le informazioni a proposito delle dinamiche interne del campione sono mediate dall’interazione coi modi acustici, il Capitolo 2´e dedicato al formalismo delle fluttuazioni di densit`a, evidenziando le differenze tra il modello idrodinamico, valido per scale spaziali macroscopiche, e l’approccio delle funzioni memoria, nec- essario per descrivere la regione mesoscopica dove le dimensioni caratteristiche dei modi acustici diventano confrontabili con le distanze tra le particelle. Nel Capitolo 3 sono riportati i risultati sperimentali ottenuti mediante la Spet- troscopia Acustica ad Ampio Intervallo Spettrale nell’intero range di temperature in cui l’acetonitrile permane allo stato liquido. Per prima cosa, attraverso un tecnica fotonica di spettroscopia non lineare (Transient Grating) ´e stato possibile misurare alcune variabili termodinamiche, potendo fare chiarezza tra i vari contributi presenti in letteratura. Tra i risultati ottenuti, la dipendenza in temperatura del rapporto tra i calori specifici laddove usualmente viene considerata costante. Partendo da questi risultati e usando l’approccio di filtraggio introdotto nel Capitolo 1, ´e stato possibile studiare il rilassamento vibrazionale dell’acetonitrile scoprendo alcune ril- evanti discrepanze con i precedenti lavori riportati in letteratura. Abbiamo cosí dimostrato l’importanza di eliminare eventuali contributi spuri e di poter attingere a informazioni complementari da diversi domini spettrali. Verr`a inoltre mostrato il risultato dello studio del cosiddetto rilassamento strutturale effettuato nel range di alte energie grazie allo Scattering di raggi X. Una interessante correlazione si ´e po- tuta riscontrare tra i risultati di questo esperimento e quelli ottenuti con la tecnica Transient Grating nella sua risposta ultraveloce. Ancora una volta, per ottenere tale risultato ´e stato necessario combinare informazioni provenienti da molti inter- valli spettrali diversi, confermando la validit`a di un approccio multi-tecnica come quello della spettroscopia acustica a larga banda spettrale. L’andamento in tem- peratura dei suddetti processi di rilassamento suggerivano di valutare la presenza di un eventuale accoppiamento tra i due fenomeni. ´E esattamente ci`o che abbiamo osservato esplorando il dominio del profondo ultravioletto e per il quale abbiamo proposto un modello fenomenologico in grado di fornire una rappresentazione delle interazioni in gioco. Per poter approfondire tale fenomeno di accoppiamento sarebbe estremamente utile poter esplorare l’intervallo spettrale degli UV soffici, ma fino ad oggi nessuna tecnica era in grado accedere a tale range. Nel Capitolo 4 mostriamo la nostra innovativa soluzione al problema: un nuovo interferometro Farby-Perot “table-top”, concepito per lavorare con una sorgente UV laser e con sole ottiche riflettive. Gli ultimi capitoli della tesi descrivono il mio contributo al prossimo esperi- mento avente come sorgente di luce un laser ad elettroni liberi: il progetto TIMER, destinato ad aumentare ulteriormente il range spettrale sperimentalmente acces- sibile con la spettroscopia acustica. In particolare, mostrer`o i risultati ottenuti nell’esperimentopilotachiamato“mini-TIMER”graziealqualedimostratolapossibilit`a di effettuare un esperimento Transient Grating anche nel range spettrale dell’estremo UV (EUV). Quando TIMER sar`a operativo sar`a il primo setup sperimentale in grado di sondare la regione mesoscopica nell’EUV, zona di cruciale importanza per lo studio dei vetri e delle nanostrutture. Questo risultato apre inoltre la strada ad una nuova classe di interessanti esperimenti ad oggi oggetto solo di considerazioni teoriche: i cosiddetti esperimenti di “four wave mixing” combinati con la selettivit`a elementale che la radiazione EUV pu`o fornire.XXVI Ciclo198

Exploiting the Potential Value of Over-the-Counter Drugs through Brand Equity: An Analytic Network Process Approach

We propose a network model to identify the main drivers of consumer-based brand equity. We apply our research to assess the value of three over-the-counter drug brands. Our aim is to help manufacturers to improve their position in the market of self-medication. This market has very peculiar characteristics: consumers buy products in response to their specific health needs; nonetheless, the market is not strictly regulated in the same way that the prescription market, which allows firms to choose their pricing and communication strategies. Moreover, consumers are not forced by physicians to buy one specific drug, but they can choose the one they prefer. To develop our model we use the Analytic Network Process methodology, which allows integrating qualitative and quantitative judgments from many decision makers and deals with non-regular preference structures. The output of the model is a ranking of the brand value drivers, according to their importance in influencing the consumers' purchase intentions. We find that advertising plays a major role in this setting. To test our model and validate our results, we analyse three Italian brands that produce over-the-counter (OTC) Diclofenac-based drugs. In addition, we compare our results with their market share

Impairment of the Zn/Cd detoxification systems affects the ability of Salmonella to colonize Arabidopsis thaliana

Salmonella capacity to colonize different environments depends on its ability to respond efficiently to fluctuations in micronutrient availability. Among micronutrients, Zn, besides playing an essential role in bacterial physiology, is a key element whose concentration can influence bacterial survival in a particular niche. Plant colonization by Salmonella enterica was described for several years, and some molecular determinants involved in this host-pathogen interaction have started to be characterized. However, it is still unclear if Zn plays a role in the outcome of this interaction, as well established for animal hosts that employ nutritional immunity strategies to counteract pathogens infections. In this study, we have investigated the involvement of Salmonella Typhimurium main effectors of zinc homeostasis in plant colonization, using Arabidopsis thaliana as a model host. The results show that to colonize plant tissues, Salmonella takes advantage of its ability to export excess metal through the efflux pumps ZntA and ZitB. In fact, the deletion of these Zn/Cd detoxification systems can affect bacterial persistence in the shoots, depending on metal availability in the plant tissues. The importance of Salmonella ability to export excess metal was enhanced in the colonization of plants grown in high Zn conditions. On the contrary, the bacterial disadvantage related to Zn detoxification impairment can be abrogated if the plant cannot efficiently translocate Zn to the shoots. Overall, our work highlights the role of Zn in Salmonella-plant interaction and suggests that modulation of plant metal content through biofortification may be an efficient strategy to control pathogen colonization

Analytic Hierarchy Process for New Product Development

The success of a New Product Development (NPD) process strongly depends on the deep comprehension of market needs and Analytic Hierarchy Process (AHP) has been commonly used to find weights for customers’ preferences. AHP best practices suggest that low‐consistency respondents should be considered untrustworthy; however, in some NPD cases – such as the one presented here – this stake can be extremely big. This paper deals with the usage of AHP methodology to define the weights of customer needs connected to the NPD process of a typical impulse buying good, a snack. The aim of the paper is to analyse in a critical way the opportunity to exclude or include non‐consistent respondents in market analysis, addressing the following question: should a non‐consistent potential customer be excluded from the analysis due to his inconsistency or should he be included because, after all, he is still a potential consumer? The chosen methodological approach focuses on evaluating the compatibility of weight vectors among different subsets of respondents, filtered according to their consistency level. Results surprisingly show that weights do not significantly change when non‐consistent respondents are excluded

An Analysis of the Effect of Operations Management Practices on Performance

In this paper we investigate the possible relationships among some optimization techniques used in Operations Management and the performance of SMEs that operate in the manufacturing sector. A model based on the Structural Equation Modelling (SEM) approach is used to analyse a dataset of small and medium-sized Italian enterprises. The model is expressed by a system of simultaneous equations and is solved through regression analysis. Taking advantage of the contributions presented previously, we focus our research on the Italian economy, highlighting the importance of Operations Management practices, which are relevant drivers of these firms’ performances

Crystallographic Study of Mutant Lysl20Leu Xenopus laevis Cu,Zn Superoxide Dismutase

Theoretical calculations and experimental measurements on the Xenopus laevis Cu,Zn superoxide dismutase (XSODB) wild-type protein and on some of its engineered mutants showed that the electrostatic arrangement around the active site channel plays a fundamental role in determining the catalytic properties of the en-zyme. Lysl20, which lies on the lip of the active site channel, about 11 Å from the catalytic copper ion, influences the enzyme electrostatic environment and binding selectivity. Neutralization of this residue has the effect of decreasing the activity of the enzyme versus the negatively charged substrate. In order to get precise information about the mutated residue and its effects on the structure of the engineered protein, the crystal structure of single site Lysl20Leu mutant XSODB was determined at 2.0 Å resolution, and refined to an R-factor value of 0.181. The structure of Lysl20Leu mutant XSODB is little affected by the amino-acid substitution, suggesting that the main effect of the mutation is perturbation of the electrostatic properties of the SOD catalytic center

Pichia pastoris Fep1 is a [2Fe-2S] protein with a Zn finger that displays an unusual oxygen-dependent role in cluster binding

Fep1, the iron-responsive GATA factor from the methylotrophic yeast Pichia pastoris, has been characterised both in vivo and in vitro. This protein has two Cys(2)-Cys(2) type zinc fingers and a set of four conserved cysteines arranged in a Cys-X-5-Cys-X-8-Cys-X-2-Cys motif located between the two zinc fingers. Electronic absorption and resonance Raman spectroscopic analyses in anaerobic and aerobic conditions indicate that Fep1 binds iron in the form of a [2Fe-2S] cluster. Site-directed mutagenesis shows that replacement of the four cysteines with serine inactivates this transcriptional repressor. Unexpectedly, the inactive mutant is still able to bind a [2Fe-2S] cluster, employing two cysteine residues belonging to the first zinc finger. These two cysteine residues can act as alternative cluster ligands selectively in aerobically purified Fep1 wild type, suggesting that oxygen could play a role in Fep1 function by causing differential localization of the [Fe-S] cluster

Role of ZnuABC and ZinT in Escherichia coli O157:H7 zinc acquisition and interaction with epithelial cells

BACKGROUND: Zinc is an essential element for all living cells. Recent studies have shown that the ZnuABC zinc uptake system significantly contributes to the ability of several pathogens to multiply in the infected host and cause disease, suggesting that zinc is scarcely available within different tissues of the host. To better understand the role of zinc in bacterial pathogenicity, we have undertaken a functional characterization of the role of the ZnuABC-mediated zinc uptake pathway in enterohemorrhagic Escherichia coli O157:H7. RESULTS: In this work we have analyzed the expression and the role in metal uptake of ZnuA, the periplasmic component of the ZnuABC transporter, and of ZinT, another periplasmic protein which has been shown to contribute to zinc recruitment. We report that the expression of zinT and znuA, regulated by Zur, is induced in zinc-poor media, and that inactivation of either of the genes significantly decreases E. coli O157:H7 ability to grow in zinc depleted media. We also demonstrate that ZinT and ZnuA have not a redundant function in zinc homeostasis, as the role of ZinT is subordinated to the presence of ZnuA. Moreover, we have found that znuA and zinT are strongly induced in bacteria adhering to cultured epithelial cells and that lack of ZnuA affects the adhesion ability. In addition we have found that a fraction of apo-ZinT can be secreted outside the cell where the protein might sequester environmental zinc, inducing a condition of metal starvation in surrounding cells. CONCLUSIONS: The here reported results demonstrate that ZnuABC plays a critical role in zinc uptake also in E. coli O157:H7 and that ZinT contributes to the ZnuA-mediated recruitment of zinc in the periplasmic space. Full functionality of the zinc import apparatus is required to facilitate bacterial adhesion to epithelial cells, indicating that the microbial ability to compete with the host cells for zinc binding is critical to establish successful infections. The observation that ZinT can be secreted when it is in the apo-form suggests that its presence in the extracellular environment may somehow contribute to metal uptake or facilitate bacterial colonization of the intestinal epithelia

Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn Superoxide Dismutases

<p>Abstract</p> <p>Background</p> <p>Highly virulent enterohemorrhagic <it>Escherichia coli </it>O157:H7 strains possess three <it>sodC </it>genes encoding for periplasmic Cu, Zn superoxide dismutases: <it>sodC</it>, which is identical to the gene present in non-pathogenic <it>E. coli </it>strains, and <it>sodC</it>-F1 and <it>sodC</it>-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent <it>sodC </it>redundancy in <it>E. coli </it>O157:H7 has not yet been investigated.</p> <p>Results</p> <p>We report that strains deleted of one or more <it>sodC </it>genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different <it>sodC </it>genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various <it>E. coli </it>O157:H7 SodC enzymes. We have found that the chromosomal and prophagic <it>sodC </it>genes are differentially regulated <it>in vitro</it>. <it>sodC </it>is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, <it>sodC</it>-F1 and <it>sodC</it>-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.</p> <p>Conclusion</p> <p>Our data show that the chromosomal and bacteriophage-associated <it>E. coli </it>O157:H7 <it>sodC </it>genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.</p