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.

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.

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.

The use of mobile single-sided NMR for the diagnosis of osteoporosis: a preliminary study

Una nuova ed originale tecnica è stata messa a punto, finalizzata alla realizzazione di una procedura per la diagnosi dell’osteoporosi, mediante l’utilizzo di scanner low field single-sided NMR. Tre differenti scanner (NMR MOLE, MOUSE PM 10 e MOUSE PM5) sono stati usati per determinare il Bone Volume-to-Total Volume ratio (BV/TV), parametro che fornisce indicazioni sulla microstruttura dell’osso. I risultati sono stati confrontati con le analisi micro-CT. Gli esperimenti sono stati condotti nel Lab. NMR del dipartimento DIFA di UNIBO e nel Lab. NMR della Victoria University di Wellington (NZ), durante un periodo di visita di cinque mesi, supportato da una borsa di studio della “Facoltà di Scienze” di UNIBO. Le analisi micro-CT sono state condotte presso il Lab. di Tecnologie Mediche dell’Istituto Ortopedico Rizzoli, Bologna. La ricerca è stata parzialmente finanziata dalla “Fondazione del Monte di Bologna e Ravenna”. La caratterizzazione dell’osso trabecolare di campioni animali e dei tessuti che lo circondano (come cartilagine e muscolo) è stata condotta tramite mappe di correlazione T1-T2 e D-T2 , dove T1 e T2 sono, rispettivamente, il tempo di rilassamento longitudinale e trasversale del nucleo 1H, e D è il coefficiente di autodiffusione molecolare. E’ stata sviluppata una sequenza di impulsi (Diffusion-Weighted T1-T2) per ottenere mappe T1-T2 pesate in diffusione. I risultati hanno consentito di mettere a punto una procedura che elimina il segnale NMR proveniente da cartilagine e muscolo, rendendo più realistico lo scenario di applicazione in-vivo. I tre diversi dispositivi NMR hanno dato risultati consistenti tra loro e con le immagini micro-CT. L’intera catena di esperimenti condotti ha mostrato che dispositivi NMR single-sided possono essere usati per valutare il BV/TV di ossa trabecolari, con il vantaggio di essere portatili, a basso costo e non invasivi, permettendo campagne di screening della popolazione a rischio osteoporosi.

Two-dimensional relaxation-diffusion analysis to study water compartmentalization in cells by a single-sided NMR device

In questo lavoro di tesi è presentato un metodo per lo studio della compartimentalizzazione dell’acqua in cellule biologiche, mediante lo studio dell’autodiffusione delle molecole d’acqua tramite uno strumento NMR single-sided. Le misure sono state eseguite nel laboratorio NMR all’interno del DIFA di Bologna. Sono stati misurati i coefficienti di autodiffusione di tre campioni in condizione bulk, ottenendo risultati consistenti con la letteratura. È stato poi analizzato un sistema cellulare modello, Saccharomyces cerevisiae, allo stato solido, ottimizzando le procedure per l’ottenimento di mappe di correlazione 2D, aventi come assi il coefficiente di autodiffusione D e il tempo di rilassamento trasversale T2. In questo sistema l’acqua è confinata e l’autodiffusione è ristretta dalle pareti cellulari, si parla quindi di coefficiente di autodiffusione apparente, Dapp. Mediante le mappe sono state individuate due famiglie di nuclei 1H. Il campione è stato poi analizzato in diluizione in acqua distillata, confermando la separazione del segnale in due distinte famiglie. L’utilizzo di un composto chelato, il CuEDTA, ha permesso di affermare che la famiglia con il Dapp maggiore corrisponde all’acqua esterna alle cellule. L’analisi dei dati ottenuti sulle due famiglie al variare del tempo lasciato alle molecole d’acqua per la diffusione hanno portato alla stima del raggio dei due compartimenti: r=2.3±0.2µm per l’acqua extracellulare, r=0.9±0.1µm per quella intracellulare, che è probabilmente acqua scambiata tra gli organelli e il citoplasma. L’incertezza associata a tali stime tiene conto soltanto dell’errore nel calcolo dei parametri liberi del fit dei dati, è pertanto una sottostima, dovuta alle approssimazioni connesse all’utilizzo di equazioni valide per un sistema poroso costituito da pori sferici connessi non permeabili. Gli ordini di grandezza dei raggi calcolati sono invece consistenti con quelli osservabili dalle immagini ottenute con il microscopio ottico.

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.

Investigation of different apple cultivars by high resolution magic angle spinning NMR. A feasibility study

(1)H HR-MAS NMR spectroscopy was applied to apple tissue samples deriving from 3 different cultivars. The NMR data were statistically evaluated by analysis of variance (ANOVA), principal component analysis (PCA), and partial least-squares-discriminant analysis (PLS-DA). The intra-apple variability of the compounds was found to be significantly lower than the inter-apple variability within one cultivar. A clear separation of the three different apple cultivars could be obtained by multivariate analysis. Direct comparison of the NMR spectra obtained from apple tissue (with HR-MAS) and juice (with liquid-state HR NMR) showed distinct differences in some metabolites, which are probably due to changes induced by juice preparation. This preliminary study demonstrates the feasibility of (1)H HR-MAS NMR in combination with multivariate analysis as a tool for future chemometric studies applied to intact fruit tissues, e.g. for investigating compositional changes due to physiological disorders, specific growth or storage conditions.

Enhanced Sensitivity by Nonuniform Sampling Enables Multidimensional MAS NMR Spectroscopy of Protein Assemblies

We report dramatic sensitivity enhancements in multidimensional MAS NMR spectra by the use of nonuniform sampling (NUS) and introduce maximum entropy interpolation (MINT) processing that assures the linearity between the time and frequency domains of the NUS acquired data sets. A systematic analysis of sensitivity and resolution in 2D and 3D NUS spectra reveals that with NUS, at least 1.5- to 2-fold sensitivity enhancement can be attained in each indirect dimension without compromising the spectral resolution. These enhancements are similar to or higher than those attained by the newest-generation commercial cryogenic probes. We explore the benefits of this NUS/MaxEnt approach in proteins and protein assemblies using 1-73-(U-C-13,N-15)/74-108-(U-N-15) Escherichia coil thioredoxin reassembly. We demonstrate that in thioredoxin reassembly, NUS permits acquisition of high-quality 3D-NCACX spectra, which are inaccessible with conventional sampling due to prohibitively long experiment times. Of critical importance, issues that hinder NUS-based SNR enhancement in 3D-NMR of liquids are mitigated in the study of solid samples in which theoretical enhancements on the order of 3-4 fold are accessible by compounding the NUS-based SNR enhancement of each indirect dimension. NUS/MINT is anticipated to be widely applicable and advantageous for multidimensional heteronuclear MAS NMR spectroscopy of proteins, protein assemblies, and other biological systems.

Characterization of HgCl2 Tridentate Amine Complexes by X-ray Crystallography, NMR and ESI-MS

Two new HgCl2 complexes of tridentate nitrogen ligands were characterized by X-ray crystallography, proton NMR spectroscopy and ESI-MS. The five-coordinate complex [Hg(BMPA)Cl-2] (1) (BMPA = bis(2-pyridylmethyl)amine) crystallized from acetonitrile/m-xylene by slow evaporation in the monoclinic space group P2(1)/n with a = 8.3896(8) , b = 12.8020(13) , c = 13.3526(13) , alpha = 90A degrees, beta A = 90.480(2)A degrees, gamma A = 90A degrees and z = 4. The square pyramidal structure (tau = 0.009) has approximate C (s) symmetry. Despite comparable Hg-N bond lengths in 1, inversion of the central nitrogen was rapid on the chemical shift time scale in dilute solution except at very low temperatures. The related complex [Hg(BEPA)Cl-2] (2) (BEPA = bis(2-{pyrid-2-yl}ethyl)amine) crystallized from acetonitrile/ethyl acetate/hexanes by slow diffusion in the orthorhombic space group Pnma with a = 13.424(3) , b = 14.854(3) , c = 8.118(2) , alpha = 90A degrees, beta A = 90A degrees, gamma A = 90A degrees and z = 4. The mixed geometry structure (tau = 0.56) also has crystallographic mirror symmetry as well as C (s) point group symmetry. In dilute acetonitrile solution, 1 was stable while 2 slowly converted to a more thermodynamically stable complex.

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC (1)H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS. (C) 2011 Elsevier B.V. All rights reserved.

Exploring High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy for Metabonomic Analysis of Apples

Classical liquid-state high-resolution (HR) NMR spectroscopy has proved a powerful tool in the metabonomic analysis of liquid food samples like fruit juices. In this paper the application of (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to apple tissue is presented probing its potential for metabonomic studies. The (1)H HR-MAS NMR spectra are discussed in terms of the chemical composition of apple tissue and compared to liquid-state NMR spectra of apple juice. Differences indicate that specific metabolic changes are induced by juice preparation. The feasibility of HR-MAS NMR-based multivariate analysis is demonstrated by a study distinguishing three different apple cultivars by principal component analysis (PCA). Preliminary results are shown from subsequent studies comparing three different cultivation methods by means of PCA and partial least squares discriminant analysis (PLS-DA) of the HR-MAS NMR data. The compounds responsible for discriminating organically grown apples are discussed. Finally, an outlook of our ongoing work is given including a longitudinal study on apples.