Hundertneunundzwanzig Xe NMR for investigations of complex and dynamic systems

In this work, the remarkable versatility and usefulness of applications of Xe-129 NMR experiments is further extended. The application of Xe-129 NMR spectroscopy to very different system is studied, including dynamic and static, solid and liquid, porous and non-porous systems. Using the large non-equilibrium polarization created by hyperpolarization of Xe-129, time-resolved NMR measurements can be used for the online-monitoring of dynamic systems. In the first part of this work, several improvements for medical applications of hyperpolarized Xe-129 are achieved and their feasibility shown experimentally. A large gain in speed and reproducibility of the accumulation process of Xe-129 as ice and an enhancement of the usable polarization in any experiment requiring prior accumulation are achieved. An enhancement of the longitudinal relaxation time of Xe-129 is realized by admixture of a buffer gas during the storage of hyperpolarized Xe-129. Pursuing the efforts of simplifying the accumulation process and enhancing the storage time of hyperpolarized Xe-129 will allow for a wider use of the hyperpolarized gas in (medical) MRI experiments. Concerning the use of hyperpolarized Xe-129 in MRI, the influence of the diffusion coefficient of the gas on parameters of the image contrast is experimentally demonstrated here by admixture of a buffer gas and thus changing the diffusion coefficient. In the second part of this work, a polymer system with unique features is probed by Xe-129 NMR spectroscopy, proving the method to be a valuable tool for the characterization of the anisotropic properties of semicrystalline, syndiotactic polystyrene films. The polymer films contain hollow cavities or channels with sizes in the sub-nanometer range, allowing for adsorption of Xe-129 and subsequent NMR measurements. Despite the use of a ’real-world’ system, the transfer of the anisotropic properties from the material to adsorbed Xe-129 atoms is shown, which was previously only known for fully crystalline materials. The anisotropic behavior towards atomar guests inside the polymer films is proven here for the first time for one of the phases. For the polymer phase containing nanochannels, the dominance of interactions between Xe-129 atoms in the channels compared to interactions between Xe atoms and the channel walls are proven by measurements of a powder sample of the polymer material and experiments including the rotation of the films in the external magnetic field as well as temperature-dependent measurements. The characterization of ’real-world’ systems showing very high degrees of anisotropy by Xe-129 are deemed to be very valuable in future applications. In the last part of this work, a new method for the online monitoring of chemical reactions has been proposed and its feasibility and validity are experimentally proven. The chemical shift dependence of dissolved Xe-129 on the composition of a reaction mixture is used for the online monitoring of free-radical miniemulsion polymerization reactions. Xe-129 NMR spectroscopy provides an excellent method for the online monitoring of polymerization reactions, due to the simplicity of the Xe-129 NMR spectra and the simple relationship between the Xe-129 chemical shift and the reaction conversion. The results of the time-resolved Xe-129 NMR measurements are compared to those from calorimetric measurements, showing a good qualitative agreement. The applicability of the new method to reactions other than polymerization reactions is investigated by the online monitoring of an enzymatic reaction in a miniemulsion. The successful combination of the large sensitivity of Xe-129, the NMR signal enhancements due to hyperpolarization, and the solubility of Xe-129 gives access to the large new field of investigations of chemical reaction kinetics in dynamic and complex systems like miniemulsions.

Festkörper-NMR an polymeren Kompositmaterialien

In dieser Arbeit wurden polymere Kompositmaterialien mit Hilfe von Festkörper-NMR-Techniken untersucht, um den Einfluß von Polymer-Festkörper-Kontakten auf molekulare Materialeigenschaften zu betrachten. Dabei wurden sowohl Analysen am Polymer als auch am Füllmaterial durchgeführt.rnrnIm ersten Teil der Arbeit wurde die Dynamik von Poly(ethylmethacrylat) (PEMA) in sphärischen Bürstenpartikeln gemessen. Diese Bürsten bestanden aus einem Poly(silsesquioxan)-Kern und verpfropften PEMA-Ketten, die über ATRP (atom transfer radical polymerization) an verschiedenen Kettensequenzen mit 13C an der Carboxylgruppe markiert wurden. Statische 13C-NMR-Messungen konnten zeigen, dass die Dynamik dieser Sequenzen unabhängig vom Abstand zur Oberfläche verlangsamt ist, was auf eine eingeschränkte Reptation zurückgeführt wurde.rnrnDer zweite Teil der Arbeit beschäftigt sich mit den molekularen Unterschieden von Silika-Naturkautschuk-Kompositen, die über mechanisches Mischen bzw. über eine Sol-Gel-Reaktion hergestellt wurden. Durch kinetische 1H-NMR-Messungen wurde der Umsatz der Sol-Gel-Reaktion bestimmt. Mittels heteronuklearen 29Si{1H}-NMR-Korrelationsexperimenten wurde ein direkter räumlicher Kontakt zwischen dem Inneren der Partikel und dem Polymer nachgewiesen. Dies belegt experimentell, dass im Kompositmaterial die Polymerketten in den durch Sol-Gel-Reaktion hergestellten Silikapartikeln eingeschlossen sind.

Synthesis of constrained peptidomimetics for therapeutic, diagnostic and theranostic applications

This thesis work deals, principally, with the development of different chemical protocols ranging from environmental sustainability peptide synthesis to asymmetric synthesis of modified tryptophans to a series of straightforward procedures for constraining peptide backbones without the need for a pre-formed scaffold. Much efforts have been dedicated to the structural analysis in a biomimetic environment, fundamental for predicting the in vivo conformation of compounds, as well as for giving a rationale to the experimentally determined bioactivity. The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, titration experiments, molecular dynamics, etc.), FT-IR and ECD spectroscopy. As a practical application, 3D rigid scaffolds have been employed for the synthesis of biological active compounds based on peptidomimetic and retro-mimetic structures. These mimics have been investigated for their potential as antiflammatory agents and actually the results obtained are very promising. Moreover, the synthesis of Amo ring permitted the development of an alternative high effective synthetic pathway for obtaining Linezolid antibiotic. The final section is, instead, dedicated to the construction of a new biosensor based on zeolite L SAMs functionalized with the integrin ligand c[RGDfK], that has showed high efficiency for the selective detection of tumor cells. Such kind of sensor could, in fact, enable the convenient, non-invasive detection and diagnosis of cancer in early stages, from a few drops of a patient's blood or other biological fluids. In conclusion, the researches described herein demonstrate that the peptidomimetic approach to 3D definite structures, allows unambiguous investigation of the structure-activity relationships, giving an access to a wide range bioactive compounds of pharmaceutical interest to use not only as potential drugs but also for diagnostic and theranostic applications.

Integrazione di misure NMR e microscopiche per la descrizione quantitativa degli effetti di stress esterni su colture cellulari

La tesi si occupa dell’integrazione di misure NMR e microscopiche per la descrizione quantitativa degli effetti di stress esterni su colture cellulari. Una maggiore comprensione dei dati ricavati tramite NMR potrebbe consentire di interpretare la vitalità e funzionalità cellulare attraverso la localizzazione spaziale degli idrogeni. L'ipotesi di partenza è che la compartimentazione degli idrogeni (associati a molecole di acqua) nel citoplasma possa fornire una stima del numero di cellule vitali (e quindi in grado di mantenere l'acqua al proprio interno mediante processi attivi). I risultati NMR, prodotti dal gruppo di ricerca del Dipartimento di Fisica e Astronomia dell’Università di Bologna della Prof.ssa Fantazzini, sono stati comparati con le informazioni acquisite dal candidato tramite metodologie di conteggio cellulare (sia vive che morte) presenti in un campione sottoposto a forte e prolungato stress (assenza di terreno di coltura e di bilanciamento del sistema tampone tramite CO2 per 400 ore). Cellule da una linea di glioblastoma multiforme (T98G) mantenute in condizioni di coltura standard sono state preparate secondo lo stesso protocollo per entrambe le tecniche di indagine, e monitorate per intervalli di tempo paragonabili. Le immagini delle cellule ottenute al microscopio ottico in modalità “contrasto di fase” sono state acquisite e utilizzate per l’analisi. Una volta definito un metodo di conteggio sperimentale, è stato possibile dedurre il comportamento delle concentrazioni di cellule sopravvissute in funzione del tempo di campionamento. Da una serie ripetuta di esperimenti è stato caratterizzato un andamento nel tempo di tipo esponenziale decrescente per il numero di cellule vive, permettendo la stima della costante di decadimento in buon accordo tra i vari esperimenti. Un analogo modello esponenziale è stato utilizzato per la descrizione dell'aumento del numero di cellule morte. In questo caso, la difficoltà nell'individuare cellule morte integre (per la frammentazione delle membrane cellulari dopo la morte) ha contribuito a determinare una sistematica sottostima nei conteggi. Il confronto dei risultati NMR e microscopici indica che la diminuzione del numero di cellule vive corrisponde ad una analoga diminuzione degli H1 compartimentalizzati fino ad un tempo specifico di circa 50-60 ore in tutti gli esperimenti. Oltre questo tempo, i dati di NMR mostrano invece un incremento di H1 compartimentalizzati, quando invece il numero di cellule vive continua a diminuire in modo monotono. Per interpretare i risultati ottenuti, è stato quindi ipotizzato che, a partire da cellule morte frammentate, strutture micellari e liposomiali formate dai lipidi di membrana in soluzione possano intrappolare gli H1 liberi, aumentando il segnale di risposta della componente compartimentalizzata come evidenziato in NMR.

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.

Hydrogels for DNA isolation from blood

This dissertation describes the synthesis of surface attached hydrogel biomaterials, characterization of their properties, evaluation of structuring concepts and the investigation of these materials in the isolation of DNA from human whole blood. Photosensitive hydrogel precursor materials on the basis of hydroxyethylmethacrylate (HEMA) were synthesized by free radical polymerization. In order to obtain surface bound hydrogel films, the precursors were deposited on a suitable substrate and subsequently irradatiated with UV - light to accomplish the formation of crosslinks in the film and create surface attachment. The composition of the polymerization precursor materials was determined by comprehensive NMR and GPC studies, revealing the copolymerizationrnbehaviour of the used monomers - HEMA derivatives and the photocrosslinkerrnMABP - and their respective distribution in the hydrogel precursors. The degree of crosslinking of the hydrogels was characterized with UV/vis spectroscopy. Stress-strain measurements were conducted in order to investigate the mechanical properties of the biomaterials. Moreover, the swelling process and biomolecule adsorption properties of the hydrogels were investigated with SPR/OW spectroscopy. For this, the deposition and binding of the hydrogels on gold or SiO2 surfaces was facilitated with photocrosslinkable adhesion promotors. The produced hydrogels were mechanically rigid and stablernunder the conditions of PCR and blood lysis. Furthermore, strategies towards the increase of hydrogel surface structure and porosity with porosigens, 2D laser interference lithography and photocleavable blockcopolymers were investigated. At last, a combinatorial strategy was used for the determination of the usefulness of hydrogels for the isolation from DNA from blood. A series of functionalized hydrogel precursors were synthesized, transferred to the surface inside a PCR tube and subsequently screened in regard to DNA adsorption properties with Taqman quantitative PCR. This approach yielded a promising candidate for a functional PCR tube coating that would allow the entire DNA isolation procedure being carried out in a single reaction container.rnThereforce, the practical application of such macromolecular architectures can be envisioned to improve industrial DNA diagnostic processes.

2D and 3D numerical modelling of multilayer detachment folding and salt tectonics

Numerical modelling was performed to study the dynamics of multilayer detachment folding and salt tectonics. In the case of multilayer detachment folding, analytically derived diagrams show several folding modes, half of which are applicable to crustal scale folding. 3D numerical simulations are in agreement with 2D predictions, yet fold interactions result in complex fold patterns. Pre-existing salt diapirs change folding patterns as they localize the initial deformation. If diapir spacing is much smaller than the dominant folding wavelength, diapirs appear in fold synclines or limbs.rnNumerical models of 3D down-building diapirism show that sedimentation rate controls whether diapirs will form and influences the overall patterns of diapirism. Numerical codes were used to retrodeform modelled salt diapirs. Reverse modelling can retrieve the initial geometries of a 2D Rayleigh-Taylor instability with non-linear rheologies. Although intermediate geometries of down-built diapirs are retrieved, forward and reverse modelling solutions deviate. rnFinally, the dynamics of fold-and-thrusts belts formed over a tilted viscous detachment is studied and it is demonstrated that mechanical stratigraphy has an impact on the deformation style, switching from thrust- to folding-dominated. The basal angle of the detachment controls the deformation sequence of the fold-and-thrust belt and results are consistent with critical wedge theory.rn

Rilassometria NMR per lo studio degli ioni cobalto nel cobaltismo da artroprotesi

Il rilascio di detriti d’usura metallici è una grave problematicità connessa ai sistemi protesici, e principalmente riguarda le protesi d’anca ad accoppiamento metallo su metallo in lega CoCr. La presenza di un livello di ioni Co nel siero che supera la soglia di tossicità è correlata a metallosi periprotesica eal fallimento del l’impianto. Recentemente è emersa un’altra casistica, presumibilmente connessa alla distribuzione e accumulo di questi ioni in tessuti di organi anche lontani dall’impianto, che si manifesta con una sintomatologia sistemica analoga a casi noti di avvelenamento da Cobalto. Nel contesto di questa nuova patologia sarebbe di grande interesse la possibilità di monitorare in-vivo la distribuzione del Cobalto rilasciato da protesi articolari, in organi o tessuti di pazienti che manifestano alti ivelli ionici di Co nel siero utilizzando metodiche non invasive come l’NMR. L’ipotesi sperimentale di applicabilità prende spunto dalle proprietà magnetiche che alcuni composti del Cobalto possono presentare nell’organismo. In questo lavoro sperimentale, nato dalla collaborazione tra il laboratorio NMR del DIFA dell’Università di Bologna e l’Istituto Ortopedico Rizzoli (IOR) di Bolgna, si presentano i risultati relativi allo studio di fattibilità condotto con diverse metodiche di rilassometria NMR su campioni biologici in presenza di Co. L’obiettivo riguarda la caratterizzazione delle proprietà di rilassamento con elettromagnete a temperatura ambiente e fisiologica, e la valutazione delle dinamiche molecolari dai profili NMRD ottenuti alle basse frequenze con metodica Fast Field Cycling, dei nuclei 1H di tali sistemi in presenza di Co.

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

A comparison between 2D and 3D cephalometry on CBCT scans of human skulls

The purpose of this study was to evaluate whether measurements on conventional cephalometric radiographs are comparable with 3D measurements on 3D models of human skulls, derived from cone beam CT (CBCT) data. A CBCT scan and a conventional cephalometric radiograph were made of 40 dry skulls. Standard cephalometric software was used to identify landmarks on both the 2D images and the 3D models. The same operator identified 17 landmarks on the cephalometric radiographs and on the 3D models. All images and 3D models were traced five times with a time-interval of 1 week and the mean value of repeated measurements was used for further statistical analysis. Distances and angles were calculated. Intra-observer reliability was good for all measurements. The reproducibility of the measurements on the conventional cephalometric radiographs was higher compared with the reproducibility of measurements on the 3D models. For a few measurements a clinically relevant difference between measurements on conventional cephalometric radiographs and 3D models was found. Measurements on conventional cephalometric radiographs can differ significantly from measurements on 3D models of the same skull. The authors recommend that 3D tracings for longitudinal research are not used in cases were there are only 2D records from the past.