Proteolysis of the receptor for advanced glycation end products by matrix metalloproteinases

RAGE mediates diverse physiological and pathological effects by binding a variety of ligands. Despite incomplete understanding of RAGE-mediated disorders soluble RAGE (sRAGE) has been identified as a potential biomarker for RAGE-related diseases and possibly represents a hopeful pharmaceutical against RAGE-mediated disorders. Nevertheless, the source of sRAGE remains poorly investigated. Currently sRAGE is thought to be derived exclusively from alternative splicing of mRNA. In this thesis it was investigated whether sRAGE can also be released as a result of ectodomain shedding of full-length RAGE. Using cells overexpressing RAGE as a model system, it was demonstrated clearly that RAGE undergoes ectodomain shedding in both constitutive and regulated manner. Several stimuli including PMA, AMPA, calcium and chelerythrine stimulated the release of sRAGE into cell culture medium. Moreover, possible mechanisms that regulate ectodomain shedding of RAGE were investigated and it was found that shedding of RAGE is likely independent from PKC and MAPK pathways. By using gain of function and loss of function approaches MMP9 but not ADAM10, ADAM17 or MT1-MMP was characterized as the metalloproteinase that mediates shedding of RAGE. Furthermore, it was shown that cytoplasmic domain of RAGE is not essential for shedding of RAGE. In addition, the potential cleavage site of RAGE by MMP9 was investigated and a lack of sequence specificity for the RAGE processing proteinase was demonstrated by mutation analysis. Finally the physiopathological significance of shedding of RAGE is discussed. In conclusion, for the first time ectodomain shedding of human RAGE and the underlying regulatory mechanisms were investigated. The data open a new field for modulation of RAGE shedding as a novel intervention approach against RAGE-mediated diseases.

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

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

Dynamic resolution of alpha-substituted dihydrocinnamic aldehydes. A new asymmetric synthesis of pharmaceutically important amine building blocks.

Crystallization-induced diastereoisomer transformation (CIDT) was successfully employed in the enantioselective synthesis of 2-alkyl-3-aryl-propan-1-amines. These products are seen as potentially useful building blocks in the field of asymmetric organic chemistry, notably for pharmaceutically relevant compounds. The procedure was based on a recently reported protocol for deracemization of dihydrocinnamic aldehydes in which enantiomerically enriched 1-(amino(phenyl)methyl)naphthalen-2-ol (Betti base) is employed as a resolving agent. Additionally, fenpropimorph, a biologically active substance which contains the 2-alkyl-3-aryl-propan-1-amine moiety was synthetized, as an attempt to assess the usefulness of the enantiomerically enriched amines.

New catalytic processes for the upgrading of furfural and 5-hydroxymethylfurfural to chemicals and fuels

The demand of energy, fuels and chemicals is increasing due to the strong growth of some countries in the developing world and the development of the world economy. Unfortunately, the general picture derived sparked an exponential increase in crude oil prices with a consequent increase of the chemical, by-products and energy, depleting the global market. Nowadays biomass are the most promising alternative to fossil fuels for the production of chemicals and fuels. In this work, the development of three different catalytic processes for the valorization of biomass-derived has been investigated. 5-hydroxymethylfurfural oxidation was studied under mild reaction condition using gold and gold/copper based catalysts synthetized from pre-formed nanoparticles and supported onto TiO2 and CeO2. The analysis conducted on catalysts showed the formation of alloys gold/copper and a strong synergistic effect between the two metals. For this reason the bimetallic catalysts supported on titania showed a higher catalytic activity respect to the monometallic catalysts. The process for the production of 2,5-bishydroxymethyl furan (BHMF) was also optimized by means the 5-hydroxymethylfurfural hydrogenation using the Shvo complex. Complete conversion of HMF was achieved working at 90 °C and 10 bar of hydrogen. The complex was found to be re-usable for at least three catalytic cycles without suffering any type of deactivation. Finally, the hydrogenation of furfural and HMF was carried out, developing the process of hydrogen transfer by using MgO as a catalyst and methanol as a hydrogen donor. Quantitative yields to alcohols have been achieved in a few hours working in mild condition: 160 °C and at autogenous pressure. The only by-products formed were light products such as CO, CO2 and CH4 (products derived from methanol transformation), easily separable from the reaction solution depressurizing the reactor.

Lipid quality and oxidative stability in food products from animal origin, as affected by breeding factor

Nowadays it is requested more investigations on alternative rearing systems that are able to improve poultry welfare and to warrant high-quality and safe meat products. This thesis work was focused on the evaluation of the oxidative stability of poultry meats, obtained with different rearing systems, diets (supplemented with bioactive compounds), and packaging conditions. The thesis work was divided into the following parts: - Evaluation of the effects of different rearing systems on the quality, fatty acid composition and oxidative stability of poultry thigh and breast meat belonging to different product categories (“rotisserie” and “cut-up” carcasses); - Evaluation of the effects of different rearing systems and packaging conditions on the shelf-life of poultry thigh meat stored at 4°C for 14 days, and the effects of feed supplementation with thymol (control diet and diet with 2 different concentration of thymol) and packaging conditions on lipid oxidation of poultry thigh meat shelf-life (stored at 4°C for 14 days). The oxidative stability of poultry meat was studied by means of the spectrophotometric determinations of peroxide value and thiobarbituric acid reactive substances. - Evaluation of anti-inflammatory effects of different flavonoids (thymol, luteolin, tangeretin, sulforaphane, polymethoxyflavones, curcumin derivates) to detect their biological activity in LPS-stimulated RAW 264.7 macrophage cells in vitro, in order to study more in depth their action mechanisms. It was evaluated the cell vitality (MTT assay), nitrite concentration and protein profile. The study was focused on the identification of potential dietary bioactive compounds in order to investigate their biological activity and possible synergic effects, and to develop new suitable strategies for long-term promotion of human health, in particular against cancer.

Milk and dairy products: evaluation of bioactive components by analytical techniques

Milk and dairy products are important source of bioactive compounds useful to satisfy the nutritional and physiological needs of any newborns of mammalian species and useful to guarantee adequate growth and development of infants as well as provide a complete nourishment of adults. Physico-chemical, nutritional and organoleptic properties of the main constituents and the “minor” components have a crucial role in the quality of milk and milk products. Although in the past decades dietary milk fat was often regarded as harmful for the human health, recent researches suggest that milk contains specific fatty acids with nutritional and physiological health benefits. For these reasons, a major attention is given to the quantity and quality of total fat intake. In the recent years, as a result of the new concept of multifunctional agriculture and the changing behaviours about diet, consumer demands in favor of high-quality, security and safety dairy products are increased. Moreover, milk proteins and milk-derived bioactive peptides are recognized to have a high nutritive value, several health-promoting functional activities and excellent technological properties. Accordingly, growing interest in the development of functional dairy products and preparation of infant formulae for babies who cannot be breast-fed, has been give in order to meet the specific consumer’s requests. This manuscript presents the main results obtained during my PhD research aimed to evaluate the main bioactive lipids and proteins in milk and dairy products using innovative analytical techniques. The experimental section of this manuscript is divided in two sections where are reported the main results obtained during my research activities on dairy products and human milks in order to characterize their bioactive compounds for functional food applications.

Possibilities for the healthy and nutritional improvement of confectionery and sweet products.

The overall objective of this PhD was to investigate the possibility to increase the nutritional value of confectionary products by the use of natural ingredients with healthy functions. The first part of the thesis focused on the possible substitution of the most characteristic component of confectionary products, i.e. refined sugar. Many natural whole sweetening alternatives are available, though not widely used; the use of molasses, the byproduct of sugar beet and cane production, still rich in healthy components as minerals and phytochemicals is hereby discussed; after having verified molasses effectiveness in oxidative stress counteraction on liver cultured cells, the higher antioxidant capacity of a sweet food prepared with molasses instead of refined sugar was confirmed. A second step of the project dealt with another main ingredient of various sweet products, namely wheat. Particularly, the exploitation of soft and durum wheat byproducts could be another sustainable strategy to improve the healthy value of confectionery. The isolation of oligosaccharides with bioactive functions form different fractions of the wheat milling stream was studied and the new ingredients were shown to have a high dietary fiber and antioxidants content. As valid alternative, product developers should consider the appealing and healthy addition of ancient grains flour to sweet baked goods. The possibility of substituting the modern whole durum wheat with the ancient Kamut® khorasan was considered, and the antioxidant and anti-inflammatory effects of these grains were evaluated and compared both in vitro and in vivo on rats. Finally, since high consumption of confectionery is a risk factor for obesity, a possible strategy for the counteraction of this disease was investigated. The ability of three bioactives in inhibiting adipocytes differentiation was investigated. In fact, theoretically, compounds able to influence adipogenesis could be used in the formulation of functional sweet products and contribute to prevent obesity.

A study of new and more sustainable catalytic routes for the synthesis of adipic acid

The aim of my PhD research project was to investigate new and more sustainable routes, compared to those currently used, for the production of adipic acid (AA). AA is a very important chemical intermediate. The main use of AA is the production of Nylon-6,6 fibers, resins, polyesters, plasticizers. My project was divided into two parts: 1. The two-step oxidation of cyclohexene, where the latter is first oxidized into trans-1,2-cyclohexanediol (CHD) with aqueous hydrogen peroxide, and then the glycol is transformed into AA by reaction with molecular oxygen. Various catalysts were investigated in this process, both heterogeneous (alumina-supported Ru(OH)x and Au nanoparticles supported on TiO2, MgO and Mg(OH)2) and homogeneous (polyoxometalates). We also studied the mechanism of CHD oxidation with oxygen in the presence of these catalysts. 2. Baeyer-Villiger oxidation of cyclohexanone with aqueous hydrogen peroxide into ɛ-caprolactone, as a first step on the way to produce AA. Study on the mechanism of the uncatalyzed (thermal) oxidation of cyclohexanone were also carried out. Investigation on how the different heterogeneous catalysts affect the formation of the reaction products and their distribution was done.

Cannabinoid system combined to classic targets for a new MTDL strategy: design and synthesis of natural inspired molecules for Alzheimer's disease

In this thesis is described the design and synthesis of potential agents for the treatment of the multifactorial Alzheimer’s disease (AD). Our multi-target approach was to consider cannabinoid system involved in AD, together with classic targets. In the first project, designed modifications were performed on lead molecule in order to increase potency and obtain balanced activities on fatty acid amide hydrolase and cholinesterases. A small library of compounds was synthesized and biological results showed increased inhibitory activity (nanomolar range) related to selected target. The second project was focused on the benzofuran framework, a privileged structure being a common moiety found in many biologically active natural products and therapeutics. Hybrid molecules were designed and synthesized, focusing on the inhibition of cholinesterases, Aβ aggregation, FAAH and on the interaction with CB receptors. Preliminary results showed that several compounds are potent CB ligands, in particular the high affinity for CB2 receptors, could open new opportunities to modulate neuroinflammation. The third and the fourth project were carried out at the IMS, Aberdeen, under the supervision of Prof. Matteo Zanda. The role of the cannabinoid system in the brain is still largely unexplored and the relationship between the CB1 receptors functional modification, density and distribution and the onset of a pathological state is not well understood. For this reasons, Rimonabant analogues suitable as radioligands were synthesized. The latter, through PET, could provide reliable measurements of density and distribution of CB1 receptors in the brain. In the fifth project, in collaboration with CHyM of York, the goal was to develop arginine analogues that are target specific due to their exclusively location into NOS enzymes and could work as MRI contrasting agents. Synthesized analogues could be suitable substrate for the transfer of polarization by p-H2 molecules through SABRE technique transforming MRI a more sensitive and faster technique.

NMR Based Foodomics to Investigate the Digestibility of Protein-Rich Food Products

Nowadays, in developed countries, the excessive food intake, in conjunction with a decreased physical activity, has led to an increase in lifestyle-related diseases, such as obesity, cardiovascular diseases, type -2 diabetes, a range of cancer types and arthritis. The socio-economic importance of such lifestyle-related diseases has encouraged countries to increase their efforts in research, and many projects have been initiated recently in research that focuses on the relationship between food and health. Thanks to these efforts and to the growing availability of technologies, the food companies are beginning to develop healthier food. The necessity of rapid and affordable methods, helping the food industries in the ingredient selection has stimulated the development of in vitro systems that simulate the physiological functions to which the food components are submitted when administrated in vivo. One of the most promising tool now available appears the in vitro digestion, which aims at predicting, in a comparative way among analogue food products, the bioaccessibility of the nutrients of interest.. The adoption of the foodomics approach has been chosen in this work to evaluate the modifications occurring during the in vitro digestion of selected protein-rich food products. The measure of the proteins breakdown was performed via NMR spectroscopy, the only techniques capable of observing, directly in the simulated gastric and duodenal fluids, the soluble oligo- and polypeptides released during the in vitro digestion process. The overall approach pioneered along this PhD work, has been discussed and promoted in a large scientific community, with specialists networked under the INFOGEST COST Action, which recently released a harmonized protocol for the in vitro digestion. NMR spectroscopy, when used in tandem with the in vitro digestion, generates a new concept, which provides an additional attribute to describe the food quality: the comparative digestibility, which measures the improvement of the nutrients bioaccessibility.

Study of a new method for high energy top tagging at the ATLAS experiment

Since its discovery, top quark has represented one of the most investigated field in particle physics. The aim of this thesis is the reconstruction of hadronic top with high transverse momentum (boosted) with the Template Overlap Method (TOM). Because of the high energy, the decay products of boosted tops are partially or totally overlapped and thus they are contained in a single large radius jet (fat-jet). TOM compares the internal energy distributions of the candidate fat-jet to a sample of tops obtained by a MC simulation (template). The algorithm is based on the definition of an overlap function, which quantifies the level of agreement between the fat-jet and the template, allowing an efficient discrimination of signal from the background contributions. A working point has been decided in order to obtain a signal efficiency close to 90% and a corresponding background rejection at 70%. TOM performances have been tested on MC samples in the muon channel and compared with the previous methods present in literature. All the methods will be merged in a multivariate analysis to give a global top tagging which will be included in ttbar production differential cross section performed on the data acquired in 2012 at sqrt(s)=8 TeV in high phase space region, where new physics processes could be possible. Due to its peculiarity to increase the pT, the Template Overlap Method will play a crucial role in the next data taking at sqrt(s)=13 TeV, where the almost totality of the tops will be produced at high energy, making the standard reconstruction methods inefficient.

New synthetic strategies towards indolizines and pyrroles

Indolizines and pyrroles are considered as “privileged” structures since their skeletons were found in many biologically active natural products and they possess a wide range of pharmaceutical properties. Syntheses of these small drug-like molecules are very important in medicinal chemistry. However, most existent methodologies are usually limited to specific substitution patterns or require impractical starting materials or expensive catalysts. Therefore, developing new methodologies for the synthesis of indolizines and pyrroles from commercially available or readily accessible sources is highly desirable.rnIn this PhD thesis, several methods has been described for the synthesis of indolizines and pyrroles. In the first part, indolizines carrying substituents in positions 1-3 were synthesized via a formal [3+2]-cycloaddition of pyridinium ylides and nitroalkenes. Pyridinium salts were prepared by N-alkylation of pyridines with cyanohydrin triflates which could be prepared from corresponding aldehydes via a Strecker reaction followed by O-triflylation. Nitroalkenes were simply prepared from the corresponding aldehydes and nitroalkanes in a nitroaldol condensation. Overall, this modular approach allows to construct the indolizine framework with various substitution patterns starting from a pyridine, two different aldehydes and a nitroalkane. In contrast to reported methods, the produced indolizines do not have to contain an electron-withdrawing group.rnIt has also been found that nitrile-stabilized 2-alkylpyridinium ylides cyclize to unstable 2-aminoindolizines via an intramolecular 5-exo-dig cyclization. Using an in situ acetylation of the amino group, N-protected 2-aminoindolizines could be synthesized. As a less common substitution pattern, indolizines carrying substituents in positions 5–8 were synthesized from enones and 2-(1H-pyrrol-1-yl)nitriles obtained from α-aminonitriles using a modified Paal-Knorr pyrrole synthesis. The decoration of the pyridine unit in the indolizine skeleton has been achieved by a one-pot conjugate addition/cycloaromatization sequence.rnIn the second part of the thesis, the diversity-oriented synthesis of pyrroles from 3,5-diaryl substituted 2H-pyrrole-2-carbonitriles (cyanopyrrolines) obtained in a cyclocondensation of enones with aminoacetonitrile hydrochloride is being discussed. 2,4-Di-, 2,3,5-trisubstituted pyrroles, pyrrole-2-carbonitriles and 2,2’-bipyrroles were synthesized in a one- or two-step protocol. While the microwave-assisted thermal elimination of HCN from cyanopyrrolines gave 2,4-disubstituted pyrroles, DDQ-oxidation of the same intermediates furnished pyrrole-2-carbonitriles. Furthermore, 2,3,5-trisubstituted pyrroles were obtained via a C-2-alkylation of the deprotonated cyanopyrrolines followed by the elimination of HCN. Finally, it has also been found that tetraaryl substituted 2,2’-bipyrroles could be synthesized by the oxidative dimerization of cyanopyrrolines using copper (II) acetate at 100 °C.rn

New markov chain based methods for single and cross-domain sentiment classification

Nowadays communication is switching from a centralized scenario, where communication media like newspapers, radio, TV programs produce information and people are just consumers, to a completely different decentralized scenario, where everyone is potentially an information producer through the use of social networks, blogs, forums that allow a real-time worldwide information exchange. These new instruments, as a result of their widespread diffusion, have started playing an important socio-economic role. They are the most used communication media and, as a consequence, they constitute the main source of information enterprises, political parties and other organizations can rely on. Analyzing data stored in servers all over the world is feasible by means of Text Mining techniques like Sentiment Analysis, which aims to extract opinions from huge amount of unstructured texts. This could lead to determine, for instance, the user satisfaction degree about products, services, politicians and so on. In this context, this dissertation presents new Document Sentiment Classification methods based on the mathematical theory of Markov Chains. All these approaches bank on a Markov Chain based model, which is language independent and whose killing features are simplicity and generality, which make it interesting with respect to previous sophisticated techniques. Every discussed technique has been tested in both Single-Domain and Cross-Domain Sentiment Classification areas, comparing performance with those of other two previous works. The performed analysis shows that some of the examined algorithms produce results comparable with the best methods in literature, with reference to both single-domain and cross-domain tasks, in $2$-classes (i.e. positive and negative) Document Sentiment Classification. However, there is still room for improvement, because this work also shows the way to walk in order to enhance performance, that is, a good novel feature selection process would be enough to outperform the state of the art. Furthermore, since some of the proposed approaches show promising results in $2$-classes Single-Domain Sentiment Classification, another future work will regard validating these results also in tasks with more than $2$ classes.

A New Reactor Concept for Efficient Solar-Thermochemical Fuel Production

We describe and analyze the efficiency of a new solar-thermochemical reactor concept, which employs a moving packed bed of reactive particles produce of H2 or CO from solar energy and H2O or CO2. The packed bed reactor incorporates several features essential to achieving high efficiency: spatial separation of pressures, temperature, and reaction products in the reactor; solid–solid sensible heat recovery between reaction steps; continuous on-sun operation; and direct solar illumination of the working material. Our efficiency analysis includes material thermodynamics and a detailed accounting of energy losses, and demonstrates that vacuum pumping, made possible by the innovative pressure separation approach in our reactor, has a decisive efficiency advantage over inert gas sweeping. We show that in a fully developed system, using CeO2 as a reactive material, the conversion efficiency of solar energy into H2 and CO at the design point can exceed 30%. The reactor operational flexibility makes it suitable for a wide range of operating conditions, allowing for high efficiency on an annual average basis. The mixture of H2 and CO, known as synthesis gas, is not only usable as a fuel but is also a universal starting point for the production of synthetic fuels compatible with the existing energy infrastructure. This would make it possible to replace petroleum derivatives used in transportation in the U.S., by using less than 0.7% of the U.S. land area, a roughly two orders of magnitude improvement over mature biofuel approaches. In addition, the packed bed reactor design is flexible and can be adapted to new, better performing reactive materials.

A New Reactor Concept for Efficient Solar-Thermochemical Fuel Production

We describe and analyze the efficiency of a new solar-thermochemical reactor concept, which employs a moving packed bed of reactive particles produce of H-2 or CO from solar energy and H2O or CO2. The packed bed reactor incorporates several features essential to achieving high efficiency: spatial separation of pressures, temperature, and reaction products in the reactor; solid-solid sensible heat recovery between reaction steps; continuous on-sun operation; and direct solar illumination of the working material. Our efficiency analysis includes material thermodynamics and a detailed accounting of energy losses, and demonstrates that vacuum pumping, made possible by the innovative pressure separation approach in our reactor, has a decisive efficiency advantage over inert gas sweeping. We show that in a fully developed system, using CeO2 as a reactive material, the conversion efficiency of solar energy into H-2 and CO at the design point can exceed 30%. The reactor operational flexibility makes it suitable for a wide range of operating conditions, allowing for high efficiency on an annual average basis. The mixture of H-2 and CO, known as synthesis gas, is not only usable as a fuel but is also a universal starting point for the production of synthetic fuels compatible with the existing energy infrastructure. This would make it possible to replace petroleum derivatives used in transportation in the U. S., by using less than 0.7% of the U. S. land area, a roughly two orders of magnitude improvement over mature biofuel approaches. In addition, the packed bed reactor design is flexible and can be adapted to new, better performing reactive materials.