Nutraceutical approach to the modulation of cholesterol metabolism: in vitro and in vivo studies

Negli ultimi anni, si sono diffusi nuove strategie per il trattamento delle malattie cardiovascolari, che possano supportare una terapia medica, o in alcuni casi, sostituirla. Infatti, l’abbandono delle terapie è il più importante problema di salute pubblica del mondo occidentale, soprattutto per le malattie croniche. Ciò è dovuto alla complessità delle terapie farmacologiche e ai numerosi e in alcuni casi gravi effetti collaterali dei farmaci somministrati. Di conseguenza, una riduzione di questi effetti migliorerebbe le condizioni di vita del paziente e quindi diminuirebbe il rischio di abbandono della terapia. Per ottenere ciò, è possibile affiancare al trattamento farmacologico una terapia nutraceutica, consistente nella somministrazione di complessi molecolari o microorganismi, provenienti da piante, latte o cibi funzionali. Lo scopo generale di questo studio è indagare le attività ipolipidemizzanti di un composto nutraceutico e di un ceppo batterio specifico nel modello animale che presenta elevati alti livelli plasmatici di colesterolo. Inoltre, sono stati analizzati gli effetti del trattamento nutraceutico sui meccanismi fisiologici che contrastano la creazione della placca aterosclerotica come l’efflusso di colesterolo dalle “foam cells” presenti nell’ateroma, o la riduzione dell’assorbimento intestinale di colesterolo. La presente tesi è divisa in due parti. Nella prima parte, abbiamo analizzato la capacità dei Bifidobacteria di ridurre i livelli di colesterolo nel medium di crescita. Dall’analisi, si è osservato che vari ceppi del genere Bifidobacteria presentano un’ampia capacità di assimilazione del colesterolo all’interno della cellula batterica, in particolare il Bifidobacterium bifidum PRL2010. Le analisi di trascrittomica del Bb PRL2010 incubato in presenza di colesterolo, hanno rivelato un significativo aumento dei livelli di trascrizione di geni codificanti trasportatori e riduttasi, responsabili del meccanismo di accumulo all’interno della cellula batterica e della conversione del colesterolo in coprostanolo. L’attività ipolipidemizzante del Bb PRL2010 è stata poi valutata nel modello murino, mostrando la modificazione del microbiota dei topi trattati dopo somministrazione del batterio in questione. Nella seconda parte del progetto di ricerca, abbiamo indagato sugli effetti di un composto coperto da brevetto, chiamato “Ola”, sull’efflusso di colesterolo di criceti trattati con questo composto nutraceutico. L’efflusso di colesterolo è il primo step del meccanismo fisiologico noto come Trasporto Inverso del Colesterolo, che consente l’eliminazione del colesterolo dalle placche aterosclerotiche, attraverso l’interazione fra le HDL, presenti nella circolazione sanguigna, e specifici trasportatori delle foam cells, come ABCA1/G1 e SR-BI. In seguito, le lipoproteine rilasciano il colesterolo alle cellule epatiche, dove è metabolizzato ed escreto attraverso le feci. Per valutare l’effetto dell’Ola sul profilo lipidico dei criceti, sono state condotte analisi in vitro. I risultati mostrano un aumento dell’efflusso di colesterolo in cellule che esprimono il trasportatore ABCA1, comparato con il gruppo controllo. Questi due studi mostrano come l’approccio nutraceutico può essere un importante modo per contrastare l’aterosclerosi. Come mostrato in letteratura, gli effetti dei composti nutraceutici sull’aterosclerosi e su altre malattie croniche, hanno portato a un ampio uso come supporto alle terapie farmacologiche, ed in alcuni casi hanno rimpiazzato la terapia farmacologica stessa.

Intermolecular and Intramolecular Charge Transfer in Functional Molecular Materials

This thesis is devoted to the investigation of inter and intramolecular charge transfer (CT) in molecular functional materials and specifically organic dyes and CT crystals. An integrated approach encompassing quantum-chemical calculations, semiempirical tools, theoretical models and spectroscopic measurements is applied to understand structure-property relationships governing the low-energy physics of these materials. Four main topics were addressed: 1) Spectral properties of organic dyes. Charge-transfer dyes are constituted by electron donor (D) and electron acceptor (A) units linked through bridge(s) to form molecules with different symmetry and dimensionality. Their low-energy physics is governed by the charge resonance between D and A groups and is effectively described by a family of parametric Hamiltonians known as essential-state models. These models account for few electronic states, corresponding to the main resonance structures of the relevant dye, leading to a simple picture that is completed introducing the coupling of the electronic system to molecular vibrations, treated in a non-adiabatic way, and an effective classical coordinate, describing polar solvation. In this work a specific essential-state model was proposed and parametrized for the dye Brilliant Green. The central issue in this work has been the definition of the diabatic states, a not trivial task for a multi-branched chromophore. In a second effort, we have used essential-state models for the description of the early-stage dynamics of excited states after ultrafast excitation. Crucial to this work is the fully non-adiabatic treatment of the coupled electronic and vibrational motion, allowing for a reliable description of the dynamics of systems showing a multistable, broken-symmetry excited state. 2) Mixed-stack CT salts. Mixed-stack (MS) CT crystals are an interesting class of multifunctional molecular materials, where D and A molecules arrange themselves to form stacks, leading to delocalized electrons in one dimension. The interplay between the intermolecular CT, electrostatic interactions, lattice phonons and molecular vibrations leads to intriguing physical properties that include (photoinduced) phase transitions, multistability, antiferromagnetism, ferroelectricity and potential multiferroicity. The standard microscopic model to describe this family of materials is the Modified Hubbard model accounting for electron-phonon coupling (Peierls coupling), electron-molecular vibrations coupling (Holstein coupling) and electrostatic interactions. We adopt and validate a method, based on DFT calculations on dimeric DA structures, to extract relevant model parameters. The approach offers a powerful tool to shed light on the complex physics of MS-CT salts. 3) Charge transfer in organic radical dipolar dyes. In collaboration with the group of Prof. Jaume Veciana (ICMAB- Barcellona), we have studied spectral properties of a special class of CT dyes with D-bridge-A structure where the acceptor group is a stable radical (of the perchlorotriphenylmethyl, PTM, family), leading to an open-shell CT dyes. These materials are of interest since they associate the electronic and optical properties of CT dyes with magnetic properties from the unpaired electron. The first effort was devoted to the parametrization of the relevant essential-state model. Two strategies were adopted, one based on the calculation of the low-energy spectral properties, the other based on the variation of ground state properties with an applied electric field. 4) The spectral properties of organic nanoparticles based on radical species are investigated in collaboration with Dr. I. Ratera (ICMAB- Barcellona). Intriguing spectroscopic behavior was observed pointing to the presence of excimer states. In an attempt to rationalize these findings, extensive calculations (TD-DFT and ZINDO) were performed. The results for the isolated dyes are validated against experimental spectra in solution. To address intermolecular interactions we studied dimeric structures in the gas phase, but the preliminary results obtained do not support excimer formation.

Synthesis and Characterisation of Functional Magnetic and Plasmonic Nanomaterials

The main aim of this thesis is the controlled and reproducible synthesis of functional materials at the nanoscale. In the first chapter, a tuning of morphology and magnetic properties of magnetite nanoparticles is presented. It was achieved by an innovative approach, which involves the use of an organic macrocycle (calixarene) to induce the oriented aggregation of NPs during the synthesis. This method is potentially applicable to the preparation of other metal oxide NPs by thermal decomposition of the respective precursors. Products obtained, in particular the multi-core nanoparticles, show remarkable magnetic and colloidal properties, making them very interesting for biomedical applications. The synthesis and functionalisation of plasmonic Au and Ag nanoparticles is presented in the second chapter. Here, a supramolecular approach was exploited to achieve a controlled and potentially reversible aggregation between Au and Ag NPs. This aggregation phenomena was followed by UV - visible spectroscopy and dynamic light scattering. In the final chapters, the conjugation of plasmonic and magnetic functionalities was tackled through the preparation of dimeric nanostructures. Au - Fe oxide heterodimeric nanoparticles were prepared and their magnetic properties thoroughly characterised. The results demonstrate the formation of FeO (wustite), together with magnetite, during the thermal decomposition of the iron precursor. By an oxidation process that preserves Au in the dimeric structures, wustite completely disappeared, with the formation of either magnetite and / or maghemite, much better from the magnetic point of view. The plasmon resonance of Au results damped by the presence of the iron oxide, a material with high refractive index, but it is still present if the Au domain of the nanoparticles is exposed towards the bulk. Finally, remarkable hyperthermia, also in vitro, was found for these structures.