Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Crystal engineering of co-crystals and host-guest systems for environmentally friendly applications: sunscreens stabilization, pollutant sequestering and herbicide reformulation.

Solid state engineered materials have proven to be useful and suitable tools in the quest of new materials. In this thesis different crystalline compounds were synthesized to provide more sustainable products for different applications, as in cosmetics or in agrochemistry, to propose pollutants removal strategy or to obtain materials for electrocatalysis. Therefore, the research projects presented here can be divided into three main topics: (i) sustainable preparation of solid materials of widely used active ingredients aimed at the reduction of their occurrence in the natural environment. The systems studied in this section are cyclodextrins host-guest compounds, obtained via mechanochemical and slurry synthesis. The first chemicals studied are sunscreens inclusion complexes, that proved to have enhanced photostability and desired photoprotection. The same synthetic methods were applied to obtain inclusion complexes of bentazon, a herbicide often found to leach in groundwaters. The resulting products showed to have desired water solubility properties. The same herbicide was also adsorbed on amorphous calcium phosphate nanoparticles, to obtain a biocompatible formulation of this agrochemical. This herbicide could benefit by the adsorption on nanoparticles for what concerns its kinetic release in different media as well as its photostability. (ii) Sustainable synthesis of co-crystals based on polycyclic aromatic hydrocarbons, for the proposal of a sequestering method with a resulting material with enhanced properties. The co-crystallization via mechanochemical means proved that these pollutants can be sequestered via simple solvent-free synthesis and the obtained materials present better photochemical properties when compared to the starting co-formers. (iii) Crystallization from mild solvents of nanosized materials useful for the application in electrocatalysis. The study of compounds based on nickel and cobalt metal ions resulted in the obtainment of 2D and 1D coordination polymers. Moreover, solid solutions were obtained. These crystals showed layered structures and, according to preliminary results, they can be exfoliated.

Cell Host-Microbe Interactions: Turning Pathogen Mechanisms Into Cell's Advantages

Host-Pathogen Interaction is a very vast field of biological sciences, indeed every year many un- known pathogens are uncovered leading to an exponential growth of this field. The present work lyes between its boundaries, touching different aspects of host-pathogen interaction: We have evaluate the permissiveness of Mesenchimal Stem cell (FM-MSC from now on) to all known human affecting herpesvirus. Our study demonstrate that FM-MSC are full permissive to HSV1, HSV2, HCMV and VZV. On the other hand HHV6, HHV7, EBV and HHV8 are susceptible, but failed to activate a lytic infection program. FM-MSC are pluripotent stem cell and have been studied intensely in last decade. FM-MSC are employed in some clinical applications. For this reason it is important to known the degree of susceptibility to transmittable pathogens. Our atten- tion has then moved to bacterial pathogens: we have performed a proteome-wide in silico analy- sis of Chlamydiaceae family, searching for putative Nuclear localization Signal (NLS). Chlamy- diaceae are a family of obligate intracellular parasites. It’s reasonably to think that its members could delivered to nucleus effector proteins via NLS sequences: if that were the case the identifi- cation of NLS carrying proteins could open the way to therapeutic approaches. Our results strengthen this hypothesis: we have identified 72 protein bearing NLS, and verified their func- tionality with in vivo assays. Finally we have conceived a molecular scissor, creating a fusion protein between HIV-1 IN protein and FokI catalytic domain (a deoxyexonuclease domain). Our aim is to obtain chimeric enzyme (trojIN) which selectively identify IN naturally occurring target (HIV LTR sites) and cleaves subsequently LTR carrying DNA (for example integrated HIV1 DNA). Our preliminary results are promising since we have identified trojIN mutated version capable to selectively recognize LTR carrying DNA in an in vitro experiments.

Drosophila melanogaster as a model to study host-parasitoid interactions: the case of the polydnaviral protein TnBVANK1

Parasitic wasps attack a number of insect species on which they feed, either externally or internally. This requires very effective strategies for suppressing the immune response and a finely tuned interference with the host physiology that is co-opted for the developing parasitoid progeny. The wealth of physiological host alterations is mediated by virulence factors encoded by the wasp or, in some cases, by polydnaviruses (PDVs), unique viral symbionts injected into the host at oviposition along with the egg, venom and ovarian secretions. PDVs are among the most powerful immunosuppressors in nature, targeting insect defense barriers at different levels. During my PhD research program I have used Drosophila melanogaster as a model to expand the functional analysis of virulence factors encoded by PDV focusing on the molecular processes underlying the disruption of the host endocrine system. I focused my research on a member of the ankyrin (ank) gene family, an immunosuppressant found in bracovirus, which associates with the parasitic wasp Toxoneuron nigriceps. I found that ankyrin disrupts ecdysone biosynthesis by impairing the vesicular traffic of ecdysteroid precursors in the cells of the prothoracic gland and results in developmental arrest.

Water, sediment and soil physicochemical interactions in freshwater, brackish and saline systems

The physicochemical interactions between water, sediment and soil deeply influence the formation and development of the ecosystem. In this research, different freshwater, brackish and saline subaqueous environments of Northern Italy were chosen as study area to investigate the physicochemical processes which occur at the interface between water and sediments, as well as the effects of soil submergence on ecosystem development. In the freshwater system of the Reno river basin, the main purpose was to define the heavy metals hazard in water and sediments of natural and artificial water courses. Heavy metals partitioning and speciation allowed to assess the environmental risk linked to the critical action of dredging canal sediments, for the maintenance of the hydraulic safety of plain lands. In addition, some bioremediation techniques were experimented for protecting sediments from heavy metals contamination, and for giving an answer to the problem of sediments management. In the brackish system of S. Vitale park, the development of hydromorphic and subaqueous soils was investigated. The study of soil profiles highlighted the presence of a soil continuum among pedons subjected to different saturation degrees. This investigation allowed to the identification of both morphological and physicochemical indicators, which characterize the formation of subaqueous soils and describe the soil hydromorphism in transitional soil systems. In the saline system of Grado lagoon, an ecosystem approach was used to define the role of water oscillation in soil characterization and plants colonization. This study highlighted the close relationship and the mutual influence of soil submergence and aeration, tide oscillation and vegetation cover, on the soil development. In view of climate change, this study contribute to understand and suppose how soil and landscape could evolve. However, a complete evaluation of hydromorphic soil functionality will be achieved only involving physiological and biochemical expertise in these kind of studies.

Functional photoactive and electroactive nanodevices

Most of current ultra-miniaturized devices are obtained by the top-down approach, in which nanoscale components are fabricated by cutting down larger precursors. Since this physical-engineering method is reaching its limits, especially for components below 30 nm in size, alternative strategies are necessary. Of particular appeal to chemists is the supramolecular bottom-up approach to nanotechnology, a methodology that utilizes the principles of molecular recognition to build materials and devices from molecular components. The subject of this thesis is the photophysical and electrochemical investigation of nanodevices obtained harnessing the principles of supramolecular chemistry. These systems operate in solution-based environments and are investigated at the ensemble level. The majority of the chemical systems discussed here are based on pseudorotaxanes and catenanes. Such supramolecular systems represent prototypes of molecular machines since they are capable of performing simple controlled mechanical movements. Their properties and operation are strictly related to the supramolecular interactions between molecular components (generally photoactive or electroactive molecules) and to the possibility of modulating such interactions by means of external stimuli. The main issues addressed throughout the thesis are: (i) the analysis of the factors that can affect the architecture and perturb the stability of supramolecular systems; (ii) the possibility of controlling the direction of supramolecular motions exploiting the molecular information content; (iii) the development of switchable supramolecular polymers starting from simple host-guest complexes; (iv) the capability of some molecular machines to process information at molecular level, thus behaving as logic devices; (v) the behaviour of molecular machine components in a biological-type environment; (vi) the study of chemically functionalized metal nanoparticles by second harmonic generation spectroscopy.

Studio dei semiochimici coinvolti nelle interazioni intra- e inter-specifiche in Gonocerus acuteangulatus (Goeze) (Heteroptera: Coreidae) in vista di un loro impiego nella difesa del nocciolo.

Gonocerus acuteangulatus (Hemiptera: Coreidae) è considerato uno dei principali fitofagi del nocciolo, in grado di causare con l’attività trofica pesanti perdite quali-quantitative di produzione. Nel triennio sono state quindi condotte indagini sulla bioetologia di G. acuteangulatus volte a: I) studiare comportamento alimentare ed effetti sulla produzione corilicola, II) identificare i feromoni e valutarne l’attività mediante biosaggi fisiologici e comportamentali in laboratorio, semi-campo e campo, III) rilevare le piante ospiti alternative al nocciolo. Mediante isolamento di adulti del coreide su rami di nocciolo con frutti è stata confermata l’assenza di correlazione fra entità del danno e numerosità degli individui presenti in corileto. Dalle analisi sensoriali su nocciole sane e danneggiate è emerso che le alterazioni causate delle punture di nutrizione sono rese più evidenti da conservazione e tostatura. Variazioni di tempi e temperature di tostatura potrebbero mitigare gli effetti del cimiciato. Nello studio dei feromoni, G. acuteangulatus, molto mobile nell’ambiente, è risultato poco adatto ai biosaggi in condizioni artificiali, come quelle in olfattometro e semi-campo. Le femmine sono tuttavia apparse attrattive per adulti di entrambi i sessi, mentre la miscela feromonale sintetizzata ha mostrato un’azione attrattiva, seppure non costante. Pertanto, ulteriori ripetizioni sono necessarie per convalidare questi risultati preliminari, modificando le condizioni di saggio in relazione alle caratteristiche della specie. Infine è stata accertata la preferenza del fitofago per alcune specie vegetali rispetto al nocciolo. Nel corso del triennio, popolazioni molto consistenti di G. acuteangulatus sono state rilevate su bosso, ciliegio di Santa Lucia, rosa selvatica, sanguinello, spino cervino, in corrispondenza del periodo di comparsa e maturazione dei frutti. Nell’impostazione di una strategia di difesa a basso impatto ambientale, l’attrattività di queste piante, in sinergia con eventuali feromoni di aggregazione, potrebbe essere utilmente sfruttata, per mantenere il coreide lontano dalla coltura.

Supramolecular functional assemblies with photo or chemical addressability

Supramolecular self-assembly represents a key technology for the spontaneous construction of nanoarchitectures and for the fabrication of materials with enhanced physical and chemical properties. In addition, a significant asset of supramolecular self-assemblies rests on their reversible formation, thanks to the kinetic lability of their non-covalent interactions. This dynamic nature can be exploited for the development of “self-healing” and “smart” materials towards the tuning of their functional properties upon various external factors. One particular intriguing objective in the field is to reach a high level of control over the shape and size of the supramolecular architectures, in order to produce well-defined functional nanostructures by rational design. In this direction, many investigations have been pursued toward the construction of self-assembled objects from numerous low-molecular weight scaffolds, for instance by exploiting multiple directional hydrogen-bonding interactions. In particular, nucleobases have been used as supramolecular synthons as a result of their efficiency to code for non-covalent interaction motifs. Among nucleobases, guanine represents the most versatile one, because of its different H-bond donor and acceptor sites which display self-complementary patterns of interactions. Interestingly, and depending on the environmental conditions, guanosine derivatives can form various types of structures. Most of the supramolecular architectures reported in this Thesis from guanosine derivatives require the presence of a cation which stabilizes, via dipole-ion interactions, the macrocyclic G-quartet that can, in turn, stack in columnar G-quadruplex arrangements. In addition, in absence of cations, guanosine can polymerize via hydrogen bonding to give a variety of supramolecular networks including linear ribbons. This complex supramolecular behavior confers to the guanine-guanine interactions their upper interest among all the homonucleobases studied. They have been subjected to intense investigations in various areas ranging from structural biology and medicinal chemistry – guanine-rich sequences are abundant in telomeric ends of chromosomes and promoter regions of DNA, and are capable of forming G-quartet based structures– to material science and nanotechnology. This Thesis, organized into five Chapters, describes mainly some recent advances in the form and function provided by self-assembly of guanine based systems. More generally, Chapter 4 will focus on the construction of supramolecular self-assemblies whose self-assembling process and self-assembled architectures can be controlled by light as external stimulus. Chapter 1 will describe some of the many recent studies of G-quartets in the general area of nanoscience. Natural G- quadruplexes can be useful motifs to build new structures and biomaterials such as self-assembled nanomachines, biosensors, therapeutic aptamer and catalysts. In Chapters 2-4 it is pointed out the core concept held in this PhD Thesis, i.e. the supramolecular organization of lipophilic guanosine derivatives with photo or chemical addressability. Chapter 2 will mainly focus on the use of cation-templated guanosine derivatives as a potential scaffold for designing functional materials with tailored physical properties, showing a new way to control the bottom-up realization of well-defined nanoarchitectures. In section 2.6.7, the self-assembly properties of compound 28a may be considered an example of open-shell moieties ordered by a supramolecular guanosine architecture showing a new (magnetic) property. Chapter 3 will report on ribbon-like structures, supramolecular architectures formed by guanosine derivatives that may be of interest for the fabrication of molecular nanowires within the framework of future molecular electronic applications. In section 3.4 we investigate the supramolecular polymerizations of derivatives dG 1 and G 30 by light scattering technique and TEM experiments. The obtained data reveal the presence of several levels of organization due to the hierarchical self-assembly of the guanosine units in ribbons that in turn aggregate in fibrillar or lamellar soft structures. The elucidation of these structures furnishes an explanation to the physical behaviour of guanosine units which display organogelator properties. Chapter 4 will describe photoresponsive self-assembling systems. Numerous research examples have demonstrated that the use of photochromic molecules in supramolecular self-assemblies is the most reasonable method to noninvasively manipulate their degree of aggregation and supramolecular architectures. In section 4.4 we report on the photocontrolled self-assembly of modified guanosine nucleobase E-42: by the introduction of a photoactive moiety at C8 it is possible to operate a photocontrol over the self-assembly of the molecule, where the existence of G-quartets can be alternately switched on and off. In section 4.5 we focus on the use of cyclodextrins as photoresponsive host-guest assemblies: αCD–azobenzene conjugates 47-48 (section 4.5.3) are synthesized in order to obtain a photoresponsive system exhibiting a fine photocontrollable degree of aggregation and self-assembled architecture. Finally, Chapter 5 contains the experimental protocols used for the research described in Chapters 2-4.

Synthetic strategies of new molecular paramagnetic mechanically-interlocked complexes

Supramolecular chemistry is a multidisciplinary field which impinges on other disciplines, focusing on the systems made up of a discrete number of assembled molecular subunits. The forces responsible for the spatial organization are intermolecular reversible interactions. The supramolecular architectures I was interested in are Rotaxanes, mechanically-interlocked architectures consisting of a "dumbbell shaped molecule", threaded through a "macrocycle" where the stoppers at the end of the dumbbell prevent disassociation of components and catenanes, two or more interlocked macrocycles which cannot be separated without breaking the covalent bonds. The aim is to introduce one or more paramagnetic units to use the ESR spectroscopy to investigate complexation properties of these systems cause this technique works in the same time scale of supramolecular assemblies. Chapter 1 underlines the main concepts upon which supramolecular chemistry is based, clarifying the nature of supramolecular interactions and the principles of host-guest chemistry. In chapter 2 it is pointed out the use of ESR spectroscopy to investigate the properties of organic non-covalent assemblies in liquid solution by spin labels and spin probes. The chapter 3 deals with the synthesis of a new class of p-electron-deficient tetracationic cyclophane ring, carrying one or two paramagnetic side-arms based on 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) moiety. In the chapter 4, the Huisgen 1,3-dipolar cycloaddition is exploited to synthesize rotaxanes having paramagnetic cyclodextrins as wheels. In the chapter 5, the catalysis of Huisgen’s cycloaddition by CB[6] is exploited to synthesize paramagnetic CB[6]-based [3]-rotaxanes. In the chapter 6 I reported the first preliminary studies of Actinoid series as a new class of templates in catenanes’ synthesis. Being f-block elements, so having the property of expanding the valence state, they constitute promising candidates as chemical templates offering the possibility to create a complex with coordination number beyond 6.

Synthetic strategies of new spin-labelled supramolecular systems

Molecular recognition and self-assembly represent fundamental issues for the construction of supramolecular systems, structures in which the components are held together through non-covalent interactions. The study of host-guest complexes and mechanical interlocked molecules, important examples in this field, is necessary in order to characterize self-assembly processes, achieve more control over the molecular organization and develop sophisticated structures by using properly designed building blocks. The introduction of paramagnetic species, or spin labelling, represents an attractive opportunity that allows their detection and characterization by the Electron Spin Resonance spectroscopy, a valuable technique that provides additional information to those obtained by traditional methods. In this Thesis, recent progresses in the design and the synthesis of new paramagnetic host-guest complexes and rotaxanes characterized by the presence of nitroxide radicals and their investigation by ESR spectroscopy are reported. In Chapter 1 a brief overview of the principal concepts of supramolecular chemistry, the spin labelling approach and the development of ESR methods applied to paramagnetic systems are described. Chapter 2 and 3 are focused on the introduction of radicals in macrocycles as Cucurbiturils and Pillar[n]arenes, due to the interesting binding properties and the potential employment in rotaxanes, in order to investigate their structures and recognition properties. Chapter 4 deals with one of the most studied mechanical interlocked molecules, the bistable [2]rotaxane reported by Stoddart and Heath based on the ciclobis (paraquat-p-phenylene) CBPQT4+, that represents a well known example of molecular switch driven by external stimuli. The spin labelling of analogous architectures allows the monitoring by ESR spectroscopy of the switch mechanism involving the ring compound by tuning the spin exchange interaction. Finally, Chapter 5 contains the experimental procedures used for the synthesis of some of the compounds described in Chapter 2-4.

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.

Modelling the influence of diradical character and aggregation on conjugated molecular materials

Molecular materials are made by the assembly of specifically designed molecules to obtain bulk structures with desired solid-state properties, enabling the development of materials with tunable chemical and physical properties. These properties result from the interplay of intra-molecular constituents and weak intermolecular interactions. Thus, small changes in individual molecular and electronic structure can substantially change the properties of the material in bulk. The purpose of this dissertation is, thus, to discuss and to contribute to the structure-property relationships governing the electronic, optical and charge transport properties of organic molecular materials through theoretical and computational studies. In particular, the main focus is on the interplay of intra-molecular properties and inter-molecular interactions in organic molecular materials. In my three-years of research activity, I have focused on three major areas: 1) the investigation of isolated-molecule properties for the class of conjugated chromophores displaying diradical character which are building blocks for promising functional materials; 2) the determination of intra- and intermolecular parameters governing charge transport in molecular materials and, 3) the development and application of diabatization procedures for the analysis of exciton states in molecular aggregates. The properties of diradicaloids are extensively studied both regarding their ground state (diradical character, aromatic vs quinoidal structures, spin dynamics, etc.) and the low-lying singlet excited states including the elusive double-exciton state. The efficiency of charge transport, for specific classes of organic semiconductors (including diradicaloids), is investigated by combining the effects of intra-molecular reorganization energy, inter-molecular electronic coupling and crystal packing. Finally, protocols aimed at unravelling the nature of exciton states are introduced and applied to different molecular aggregates. The role of intermolecular interactions and charge transfer contributions in determining the exciton state character and in modulating the H- to J- aggregation is also highlighted.

Study of the components of quality in SO2-free wines obtained by innovative vinification protocols. Evaluation of the pre-fermentative addition of lysozyme and oenological tannins.

The research performed during the PhD candidature was intended to evaluate the quality of white wines, as a function of the reduction in SO2 use during the first steps of the winemaking process. In order to investigate the mechanism and intensity of interactions occurring between lysozyme and the principal macro-components of musts and wines, a series of experiments on model wine solutions were undertaken, focusing attention on the polyphenols, SO2, oenological tannins, pectines, ethanol, and sugar components. In the second part of this research program, a series of conventional sulphite added vinifications were compared to vinifications in which sulphur dioxide was replaced by lysozyme and consequently define potential winemaking protocols suitable for the production of SO2-free wines. To reach the final goal, the technological performance of two selected yeast strains with a low aptitude to produce SO2 during fermentation were also evaluated. The data obtained suggested that the addition of lysozyme and oenological tannins during the alcoholic fermentation could represent a promising alternative to the use of sulphur dioxide and a reliable starting point for the production of SO2-free wines. The different vinification protocols studied influenced the composition of the volatile profile in wines at the end of the alcoholic fermentation, especially with regards to alcohols and ethyl esters also a consequence of the yeast’s response to the presence or absence of sulphites during fermentation, contributing in different ways to the sensory profiles of wines. In fact, the aminoacids analysis showed that lysozyme can affect the consumption of nitrogen as a function of the yeast strain used in fermentation. During the bottle storage, the evolution of volatile compounds is affected by the presence of SO2 and oenological tannins, confirming their positive role in scaveging oxygen and maintaining the amounts of esters over certain levels, avoiding a decline in the wine’s quality. Even though a natural decrease was found on phenolic profiles due to oxidation effects caused by the presence of oxygen dissolved in the medium during the storage period, the presence of SO2 together with tannins contrasted the decay of phenolic content at the end of the fermentation. Tannins also showed a central role in preserving the polyphenolic profile of wines during the storage period, confirming their antioxidant property, acting as reductants. Our study focused on the fundamental chemistry relevant to the oxidative phenolic spoilage of white wines has demonstrated the suitability of glutathione to inhibit the production of yellow xanthylium cation pigments generated from flavanols and glyoxylic acid at the concentration that it typically exists in wine. The ability of glutathione to bind glyoxylic acid rather than acetaldehyde may enable glutathione to be used as a ‘switch’ for glyoxylic acid-induced polymerisation mechanisms, as opposed to the equivalent acetaldehyde polymerisation, in processes such as microoxidation. Further research is required to assess the ability of glutathione to prevent xanthylium cation production during the in-situ production of glyoxylic acid and in the presence of sulphur dioxide.

Seeing and feeling the body

Recognizing one’s body as separate from the external world plays a crucial role in detecting external events, and thus in planning adequate reactions to them. In addition, recognizing one’s body as distinct from others’ bodies allows remapping the experiences of others onto one’s sensory system, providing improved social understanding. In line with these assumptions, two well-known multisensory mechanisms demonstrated modulations of somatosensation when viewing both one’s own and someone else’s body: the Visual Enhancement of Touch (VET) and the Visual Remapping of Touch (VRT) effects. Vision of the body, in the former, and vision of the body being touched, in the latter, enhance tactile processing. The present dissertation investigated the multisensory nature of these mechanisms and their neural bases. Further experiments compared these effects for viewing one’s own body or viewing another person’s body. These experiments showed important differences in multisensory processing for one’s own body, and for other bodies, and also highlighted interactions between VET and VRT effects. The present experimental evidence demonstrated that a multisensory representation of one’s body – underlie by a high order fronto-parietal network - sends rapid modulatory feedback to primary somatosensory cortex, thus functionally enhancing tactile processing. These effects were highly spatially-specific, and depended on current body position. In contrast, vision of another person’s body can drive mental representations able to modulate tactile perception without any spatial constraint. Finally, these modulatory effects seem sometimes to interact with high order information, such as emotional content of a face. This allows one’s somatosensory system to adequately modulate perception of external events on the body surface, as a function of its interaction with the emotional state expressed by another individual.

Modeling and simulations of some anisotropic soft-matter systems: from biaxial to chiral materials

We have modeled various soft-matter systems with molecular dynamics (MD) simulations. The first topic concerns liquid crystal (LC) biaxial nematic (Nb) phases, that can be possibly used in fast displays. We have investigated the phase organization of biaxial Gay-Berne (GB) mesogens, considering the effects of the orientation, strength and position of a molecular dipole. We have observed that for systems with a central dipole, nematic biaxial phases disappear when increasing dipole strength, while for systems characterized by an offset dipole, the Nb phase is stabilized at very low temperatures. In a second project, in view of their increasing importance as nanomaterials in LC phases, we are developing a DNA coarse-grained (CG) model, in which sugar and phosphate groups are represented with Lennard-Jones spheres, while bases with GB ellipsoids. We have obtained shape, position and orientation parameters for each bead, to best reproduce the atomistic structure of a B-DNA helix. Starting from atomistic simulations results, we have completed a first parametrization of the force field terms, accounting for bonded (bonds, angles and dihedrals) and non-bonded interactions (H-bond and stacking). We are currently validating the model, by investigating stability and melting temperature of various sequences. Finally, in a third project, we aim to explain the mechanism of enantiomeric discrimination due to the presence of a chiral helix of poly(gamma-benzyl L-glutamate) (PBLG), in solution of dimethylformamide (DMF), interacting with chiral or pro-chiral molecules (in our case heptyl butyrate, HEP), after tuning properly an atomistic force field (AMBER). We have observed that DMF and HEP molecules solvate uniformly the PBLG helix, but the pro-chiral solute is on average found closer to the helix with respect to the DMF. The solvent presents a faster isotropic diffusion, twice as HEP, also indicating a stronger interaction of the solute with the helix.