Hyperpolarization via the Dynamic Nuclear Polarization (DNP) technique has revolutionized our ability to study metabolic changes in real time. The aim of this thesis was to build upon previous work centered around the use of DNP within the isolated perfused rat heart, a well established model system for the study of cardiac metabolism, to enhance the information that can be obtained through the combination of DNP with perfused organs. Initially this was done by using the widely studied DNP probe, [1-¹³C]pyruvate, to generate images of metabolism within the isolated perfused rat heart. The developed technique was then successfully demonstrated in two models of myocardial infarction. The thesis then proceeds to develop an understanding of how the supra-physiological concentrations of [1-¹³C]pyruvate commonly used in DNP experiments can affect metabolism in the isolated perfused rat heart, and the way in which the myocardium responds to those changes if it is not adequately supplied with substrates ordinarily present in vivo, namely fatty acids. New methods of providing the heart with these required substrates were developed, without significant interference to the biochemical information acquired from DNP experiments. As [1-¹³C]pyruvate only provides information on a small subset of carbohydrate metabolism, the next chapter develops new compounds to be used with DNP, which would allow the exploration of short chain fatty acid metabolism (butyrate) as well as ketone body metabolism (β-hydroxybutyrate and acetoacetate), and other aspects of carbohydrate metabolism (lactate and alanine). These compounds were developed and then tested for their potential usefulness in the isolated perfused heart. Finally, as the isolated perfused rat heart lacks the diversity of genetic disease models available in the mouse, the final chapter expanded the use of DNP to the isolated perfused mouse heart with all the size challenges that this entails, and makes the transition from the heart to the liver, in order to provide an alternative metabolic viewpoint on the biochemistry of disease models. This thesis thereby permits studies involving isolated perfused organs to be carried out whilst exploiting all the tools that DNP has to offer and consequentially, allows for a vast array of physiologically derived information to help us better understand metabolic diseases.

Signal Amplification by Reversible Exchange (SABRE) is a hyperpolarization technique that utilizes parahydrogen for the NMR signal enhancement of nuclear spins. SABRE is related to Parahydrogen Induced Polarization (PHIP), another means of hyperpolarization using parahydrogen; PHIP achieves hyperpolarization via chemical reduction. Although PHIP and SABRE share many similarities in experimentation, PHIP ultimately requires the presence of an unsaturated chemical bond as well as pairwise-addition of parahydrogen. No permanent chemical change occurs during SABRE, and instead may be considered as a merely physical exchange between molecules with sites on a catalyst. PHIP and SABRE may be compared to Dynamic Nuclear Polarization (DNP), arguably the most well-known and researched method for hyperpolarization; despite all that has been achieved with DNP, PHIP and SABRE offer vastly more-rapid, less-expensive, and more-simplified approaches for achieving hyperpolarization. The focus of this work is experimentation with SABRE processes and methods designed to overcome certain experimental challenges associated with this technique.

<p>Recent work has shown that hyperpolarized magnetic resonance spectroscopy (HP-MRS) can trace in vivo metabolism of biomolecules and is therefore extremely promising for diagnostic imaging. The most severe challenge this technique faces is the short signal lifetime for hyperpolarization, which is dictated by the spin-lattice (T1) relaxation. In this thesis we show with theory, simulation and experiment that the long-lived nuclear spin states in chemically equivalent or near equivalent spin systems offer a solution to this problem. Spin polarization that has lifetime much longer than T1 (up to 70-fold) has been demonstrated with pulse sequence techniques that are compatible with clinical imaging settings. Multiple classes of molecules have been demonstrated to sustain such long-lived hyperpolarization.</p> / Dissertation

In the recent years, Dynamic Nuclear Polarization (DNP) has emerged as a very promising technique for enhancing the sensitivity of the magnetic resonance spectroscopy and imaging (MRSI). A number of nuclei, namely 13C, 15N, 29Si, 89Y, and 129Xe, have been successfully polarized and a few of them have been employed in the in-vivo studies for functional imaging and metabolism. Hyperpolarized 13C-labeled compounds have wide applications in the metabolic and perfusion studies and can be used for early stage disease diagnosis, response to treatment, prognosis etc. DNP has been demonstrated in the 31P nucleus in nucleotides triphosphates as an application for the structural analysis and identification techniques. In this work, 31P DNP has been successfully applied, optimized and demonstrated in Dimethyl Methyl Phosphonate (DMMP) for the first time. DMMP is a freely diffusible tracer and hyperpolarized DMMP can potentially be used in the perfusion studies using MR imaging and spectroscopic techniques. The polarization buildup and signal enhancements were optimized for two different radicals, a nitroxyl radical TEMPO and a trityl radical OX063. Microwave frequency sweeps were done for both the radicals to find out the optimum frequencies for maximum polarization, Maximum signal enhancement (¡Ö2300 folds) and maximum percent polarization buildup (2.15%) were achieved by polarizing DMMP with the radical OX063 at the microwave frequency of 94.080 GHz with a glassing matrix containing D2O and glycerol and by using D2O in the dissolution step. DMMP was hyperpolarized at the optimum conditions and injected in a mouse for in-vivo spectroscopy and imaging. The results show that hyperpolarized DMMP is a potential candidate for functional imaging and metabolism.

<p>The purpose of this thesis is to report technological developments in contrast mechanisms for MRI. The search for new forms of contrast is on-going, with the hope that new contrast mechanisms and new contrast agents will provide unique insights into various molecular processes and disease states. In this thesis, we will describe new contrast mechanisms developed by manipulating the inherent physics of the system, as well as the development of exogenous contrast agents. More specifically, we will describe the application of iMQCs (intermolecular multiple quantum coherences) to thermometry and structural imaging, and the unique information provided from these studies. We will also describe methods for migrating iMQC-based pulse sequences from a Bruker research console onto a clinical GE console, thus enabling the application of iMQCs to humans. We will describe the development of hyperpolarized contrast agents which have the potential to provide an unprecedented level of molecular contrast to MRI and the development of techniques to enhance the lifetime of these hyperpolarized contrast agents. Finally, we will discuss a new type of T2 -weighted imaging which significantly improves the refocusing of CPMG-type sequences.</p> / Dissertation

<p>There are two fundamental limitations in magnetic resonance: the poor signal amplitude and the short duration before the system return to equilibrium. Hyperpolarization methods solve the problem of signal amplitude, however, the duration of the hyperpolarized signal is still limited by the spin-lattice relaxation time, T1. Disconnected eigenstates provide a mechanism by which hyperpolarization can be stored for several times T1. This thesis contributes to the knowledge of these states in four important ways. First, the decay of hyperpolarized magnetization of gas is simulated in lung tissue with a contrast agent, yielding insights about the optimal field strength for imaging. Second, I show that it is possible to rapidly discover and characterize disconnected eigenstates by showing that they can be measured without synthesizing the isotopically labeled compound. Third, I extend the spin systems that can support disconnected eigenstates by expanding the theory to include spin-1 nuclei. Finally, I show that disconnected states with long lifetimes can be populated in conjunction with hyperpolarization techniques to simultaneously yield large signal amplitudes for long durations. </p><p>Applications of hyperpolarized spin order are likely to be in complex geophysical or biological structures. Understanding the effect of the inhomogeneous fields created when such structures are placed in a magnetic field on hyperpolarized spin order is a necessity to characterize the experimental signal. An example case of hyperpolarized 3He and 129Xe diffusing through lung tissue is examined. In particular a Monte Carlo simulation tool, combined with a magnetic field map of the inhomogeneous field created by mouse lung tissue, is used to determine the dephasing rate of hyperpolarized 3He and 129Xe in the presence of SuperParamagnetic Iron Oxide Nanoparticles (SPION). Contributions to the dephasing rate include the inhomogeneous field, the SPION magnetic field, and dephasing caused by collisions with the confining geometry. The sensitivity of either gas to SPION increases with increasing SPION concentration and decreasing field strength.</p><p>There are some general rules about what makes for a disconnected eigenstate (or singlet state) with a long lifetime. However, no systematic experimental study has been undertaken due to the cost and time-constraints of synthesizing the labeled species for study. I show that synthesis is not a barrier for characterizing the long-lived states. Instead the lifetimes may be determined by using the naturally occurring doubly-labeled isotopomer. I verified this method with two compounds, diphenyl acetylene (DPA) and diethyl oxylate (DEO). The former was determined to have a singlet lifetime TS = 251.40 ±3.16 s from the synthesized species, while the naturally occurring isotopomer yielded a lifetime TS = 202 ±55.30 s, both substantially longer than the spin-lattice relaxation time, T1 = 1.63 ±0.01s. In DEO, the lifetime from the disconnected eigenstate was determined to be TS = 14.62 ±0.76 s (synthesized), TS = 19.32 ±3.16 s (naturally occurring). This method is applied to a range of compounds ranging from simple four-spin systems, such as diacetylene (TS = 48.80 ±22.74 s, T1 = 18.66 ±1.16 s) to eight spin systems in dimethylmaleic anhydride (TS = 27.25 ±3.39 s, T1 = 9.38 ±0.43 s). Additionally, a family of compounds including naphthalene (TS = 4.37 ±0.34 s, T1 = 11.33 ±4.89 s), biphenyl (TS = 3.09 ±0.66 s, T1 = 4.69 ±0.10 s), and DPA show that the rotation of the phenyl rings and intermolecular dipole-dipole relaxation can be critical to the relaxation dynamics.</p><p>One particular method of accessing the disconnected eigenstate involves coupling a chemically equivalent spin-1/2 pair asymmetrically to an auxiliary spin-1/2 pair. I demonstrate that the disconnected state may still be accessed when the auxiliary nuclei are spin-1. This has two distinct advantages. When the auxiliary nuclei change from proton to deuterium, the couplings are reduced by a factor of ~6.5 which prevents the disconnected state from relaxing as rapidly back to equilibrium. This is demonstrated in diacetylene-d2 and DPA-d10, where the singlet lifetime was extended by a factor of ~1.7 via deuteration (TS,1H = 49 ±23 s, TS,2H = 83 ±30 s for diacetylene and TS,1H = 274 ±6.1 s, TS,2H = 479 ±83 s for DPA). Additionally, by reducing the coupling strength, deuteration allows additional structural moieties to be explored, such as RDC=CDR. One such structure is explored in trans-ethylene-d2, where the singlet character of the protons can be accessed by the reduced coupling to the deuterium. Additionally, this allows for a relatively strong deuterium-deuterium scalar coupling, requiring modification to the theory. This is carried out analytically, and implications for the relaxation properties are performed using a spin-dynamics numerical simulation. The lifetime of the disconnected state was determined to be TS = 30.2 ±12.3 s, compared to the T1 = 1.1 ±0.2 s at high concentration (270 mM), and increasing to TS = 117. ±9.80 s at low concentration (52 mM). The variation in long lifetime is attributed to intermolecular dipole-dipole relaxation.</p><p>Ultimately, the gains in lifetime from using disconnected eigenstates provide a means to the practical implementation of hyperpolarization in a wider range of experiments. A recent hyperpolarization method, Signal Amplification By Reversible Exchange in Shield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH) is shown to directly hyperpolarize long lived spin order in a diazirine containing molecule. Diazirine rings are three member N=N-C groups that can replace a methylene group and serve as a versatile MR and optical molecular tag. Hyperpolarization is accomplished by bubbling parahydrogen through a solution containing the diazirine and an iridium catalyst. Due to the chemical inequivalence of the 15N of the diazirine, hyperpolarization of longitudinal magnetization and singlet character could be observed by transfer to the high field spectrometer. Signal enhancements of over 14,000 were observed. The magnetic field strength required for buildup of magnetization and singlet character was derived and is in agreement with the experiment. The magnetization lifetime was observed to be T1 = 5.75 ±0.18 minutes and independent of field strength, while the lifetime of the singlet character was observed to be as long as TS = 30.1 ±13.4 minutes at low field (3 Gauss).</p><p>The combination of these experiments – understanding lifetimes in inhomogeneous magnetic fields that will be encountered in experiment, identification of disconnected eigenstates with long lifetimes via the naturally occurring isotopomer and extending these lifetimes even further with deuteration, and finally, the direct generation of long-lived hyperpolarized spin order – allows a measurement that required hyperpolarized spin order for the enhanced signal amplitude, to be carried out.</p> / Dissertation

Novel Ca²⁺ signalling pathways in both endothelial cells and smooth muscle cells of rat small resistance arteries were investigated using a combination of confocal imaging, isometric tension recordings, and electrophysiology to study freshly isolated arteries and cells. We first examined the hypothesis that hyperpolarization could alter endothelial cell Ca²⁺ events. Hyperpolarization evoked by direct opening of K_ATP channels in the smooth muscle with levcromakalim triggered an increase in the frequency of Ca²⁺ events in the endothelium of rat cremaster arterioles. These Ca²⁺ events were discrete in nature, requiring subcellular regions of interest to reliably identify them. Opening of K_ATP channels indirectly through &beta;-adrenoceptor stimulation with isoprenaline, caused a similar increase in the frequency of endothelial cell Ca²⁺ events in rat mesenteric third order arteries. These events also had a similar, focal profile. Pharmacological investigation suggested that the response to isoprenaline was receptor-mediated, and dependent on Ca²⁺ influx and opening of K_ATP channels. The presence of &beta;-adrenoceptors on endothelial cells was confirmed using fluorescently-tagged &beta;-adrenoceptor ligands, which showed punctate labelling in smooth muscle and endothelial cells of rat mesenteric arteries. Freshly isolated endothelial cells also showed Ca²⁺ increases to isoprenaline, although this was not consistently observed. Following on from the observed endothelial cell Ca²⁺ response to hyperpolarization, we tested the hypothesized involvement of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels using the channel inhibitor, ZD7288. Pre-treatment with ZD7288 (1 &mu;M) reduced both the endothelial cell Ca²⁺ response to isoprenaline (in mesenteric arteries) and levcromakalim (in cremaster arterioles). HCN channel subtypes were identified in cremaster arterioles through immunolabelling. We also observed an interesting effect of higher concentrations of ZD7288 to potentially inhibit K⁺ channels, including endothelial cell KCa channels, since hyperpolarization to isoprenaline, levcromakalim or acetylcholine (ACh) was reduced by 10 &mu;M ZD7288, and relaxation to ACh was partially inhibited. ACh-mediated relaxation was also partially inhibited by the clinically used HCN channel blocker, ivabradine (0.3-30 &mu;M). Finally, we identified an interaction of the Ca²⁺-releasing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) with BKCa channels in the smooth muscle. NAADP-mobilised Ca²⁺ has been reported to interact with ryanodine receptors hence we hypothesized an interaction with BK_Ca channels via Ca²⁺ sparks. We found that NAADP-AM relaxed and hyperpolarized rat mesenteric arteries, which was blocked by iberiotoxin (BK_Ca channel inhibitor) and high extracellular [K⁺] (45 mM). Furthermore, NAADP increased paxilline-sensitive K⁺ currents and the frequency and amplitude of spontaneous transient outward currents (STOCs) in freshly isolated vascular smooth muscle cells patched in the whole-cell configuration, further supporting an action at BK_Ca channels. All together these data identify novel Ca²⁺ signalling pathways in resistance arteries that are both activated by and promote hyperpolarization, which is a key determinant of vascular tone.

<p>N-Heterocycles are ubiquitous in biologically active natural products and pharmaceuticals. Yet, new syntheses and modifications of N-heterocycles are continually of interest for the purposes of expanding chemical space, finding quicker synthetic routes, better pharmaceuticals, and even new handles for molecular labeling. There are several iterations of molecular labeling; the decision of where to place the label is as important as of which visualization technique to emphasize. </p><p>Piperidine and indole are two of the most widely distributed N-heterocycles and thus were targeted for synthesis, functionalization, and labeling. The major functionalization of these scaffolds should include a nitrogen atom, while the inclusion of other groups will expand the utility of the method. Towards this goal, ease of synthesis and elimination of step-wise transformations are of the utmost concern. Here, the concept of electrophilic amination can be utilized as a way of introducing complex secondary and tertiary amines with minimal operations.</p><p>Molecular tags should be on or adjacent to an N-heterocycle as they are normally the motifs implicated at the binding site of enzymes and receptors. The labeling techniques should be useful to a chemical biologist, but should also in theory be useful to the medical community. The two types of labeling that are of interest to a chemist and a physician would be positron emission tomography (PET) and magnetic resonance imaging (MRI). </p><p>Coincidentally, the 3-positions of both piperidine and indole are historically difficult to access and modify. However, using electrophilic amination techniques, 3-functionalized piperidines can be synthesized in good yields from unsaturated amines. In the same manner, 3-labeled piperidines can be obtained; the piperidines can either be labeled with an azide for biochemical research or an 18F for PET imaging research. The novel electrophiles, N-benzenesulfonyloxyamides, can be reacted with indole in one of two ways: 3-amidation or 1-amidomethylation, depending on the exact reaction conditions. Lastly, a novel, hyperpolarizable 15N2-labeled diazirine has been developed as an exogenous and versatile tag for use in magnetic resonance imaging.</p> / Dissertation

Introdução: Em pacientes deprimidos, a mortalidade cardíaca devido à doença cardíaca coronária (CHD) é duas vezes maior do que em pacientes coronarianos nãodeprimidos, possivelmente devido à presença de disfunção endotelial. Pode haver uma associação entre a utilização de drogas antipsicóticas e doenças cardiovasculares, que são associadas à disfunção endotelial vasotônica. Os estudos relacionados aos efeitos das drogas antipsicóticas sobre a função endotelial vascular são praticamente inexistentes. Objetivo: Esse estudo foi delineado para avaliar os mecanismos envolvidos na reatividade vascular dependente do endotélio sob ação da droga antipsicótica atípica clozapina. Materiais e método: As curvas concentraçãoresposta em anéis pré-contraídos com PE (1 ?M), foram registradas na presença e na ausência de inibidores dos inibidores L-NAME e indometacina, os quais foram incubados, isolados e/ou em associação, por 30 minutos. Para estudar a participação dos canais para K+ foram realizados experimentos com os bloqueadores Tetraetilamonio e Cloreto de potássio (Kcl). Para finalizar o protocolo foram realizados experimentos com bloqueadores da guanilato ciclase (ODQ e azul de metileno) e os inibidores dos canais de K+ (apamina, paxilina e glibenclamida). Resultados: A clozapina causou relaxamento dependente do endotélio envolvendo as três vias de relaxamento (GMPc/NO, AMPc/PGI2 e hiperpolarização). Essa observação foi embasada na ineficiência dos bloqueadores, L-NAME, Indometacina e Tetraetilamônio (TEA), respectivamente. O relaxamento dependente do endotélio foi atenuado pela associação L-NAME/Indometacina e abolido em vasos contraídos por cloreto de potássio e incubados com glibenclamida. Conclusão: Estes resultados sugerem que há participação sinérgica, provavelmente através de um mecanismo de cross-talk, dos sistemas cAMP, cGMP e hiperpolarização. Portanto, mais estudos sobre possíveis funções endoteliais relacionadas aos efeitos colaterais dos antipsicóticos cardiovasculares seriam uma direção de pesquisa adequada. / Objective: In depressed patients, cardiac mortality due to coronary heart disease (CHD) is twice as high as in non-depressed coronary patients, possibly due to the presence of endothelial dysfunction. There may be an association between the use of antipsychotic drugs and cardiovascular diseases, which are associated with vasotonic endothelial dysfunction. Studies related to the effects of antipsychotic drugs on vascular endothelial function are practically non-existent. Aims: This study was designed to evaluate the mechanisms involved in endothelium-dependent vascular reactivity under the action of the atypical antipsychotic drug clozapine. Method: As concentration-response curves in pre-contracted rings with PE (1 ?M), they were recorded in the presence and absence of inhibitors of the L-NAME and indomethacin inhibitors, which were incubated, isolated and / or in association, for 30 minutes. To study the participation of the K + channels, experiments were performed with the tetraethylammonium and potassium chloride (Kcl) blockers. In order to obtain the protocol they were performed with guanylase cyclase blockers (ODQ and methylene blue) and K + channel inhibitors (apamina, paxilin and glibenclamide). Results: Clozapine caused endothelium-dependent relaxation as the three relaxation pathways (cGMP / NO, cAMP / PGI2 and hyperpolarization). This observation was based on the inefficiency of the blockers, L-NAME, Indomethacin and Tetraethylammonium (TEA), respectively. Endothelium-dependent relaxation was attenuated by the association LNAME / Indomethacin and abolished in vessels contracted by potassium chloride and incubated with glibenclamide. Conclusion: These results suggest that there is synergistic participation, probably through a cross-talk mechanism, of the cAMP, cGMP and hyperpolarization systems. Therefore, more studies of possible endothelium function related to cardiovascular antipsychotics side effects would be a proper research direction.

Introdução: Em pacientes deprimidos, a mortalidade cardíaca devido à doença cardíaca coronária (CHD) é duas vezes maior do que em pacientes coronarianos nãodeprimidos, possivelmente devido à presença de disfunção endotelial. Pode haver uma associação entre a utilização de drogas antipsicóticas e doenças cardiovasculares, que são associadas à disfunção endotelial vasotônica. Os estudos relacionados aos efeitos das drogas antipsicóticas sobre a função endotelial vascular são praticamente inexistentes. Objetivo: Esse estudo foi delineado para avaliar os mecanismos envolvidos na reatividade vascular dependente do endotélio sob ação da droga antipsicótica atípica clozapina. Materiais e método: As curvas concentraçãoresposta em anéis pré-contraídos com PE (1 ?M), foram registradas na presença e na ausência de inibidores dos inibidores L-NAME e indometacina, os quais foram incubados, isolados e/ou em associação, por 30 minutos. Para estudar a participação dos canais para K+ foram realizados experimentos com os bloqueadores Tetraetilamonio e Cloreto de potássio (Kcl). Para finalizar o protocolo foram realizados experimentos com bloqueadores da guanilato ciclase (ODQ e azul de metileno) e os inibidores dos canais de K+ (apamina, paxilina e glibenclamida). Resultados: A clozapina causou relaxamento dependente do endotélio envolvendo as três vias de relaxamento (GMPc/NO, AMPc/PGI2 e hiperpolarização). Essa observação foi embasada na ineficiência dos bloqueadores, L-NAME, Indometacina e Tetraetilamônio (TEA), respectivamente. O relaxamento dependente do endotélio foi atenuado pela associação L-NAME/Indometacina e abolido em vasos contraídos por cloreto de potássio e incubados com glibenclamida. Conclusão: Estes resultados sugerem que há participação sinérgica, provavelmente através de um mecanismo de cross-talk, dos sistemas cAMP, cGMP e hiperpolarização. Portanto, mais estudos sobre possíveis funções endoteliais relacionadas aos efeitos colaterais dos antipsicóticos cardiovasculares seriam uma direção de pesquisa adequada. / Objective: In depressed patients, cardiac mortality due to coronary heart disease (CHD) is twice as high as in non-depressed coronary patients, possibly due to the presence of endothelial dysfunction. There may be an association between the use of antipsychotic drugs and cardiovascular diseases, which are associated with vasotonic endothelial dysfunction. Studies related to the effects of antipsychotic drugs on vascular endothelial function are practically non-existent. Aims: This study was designed to evaluate the mechanisms involved in endothelium-dependent vascular reactivity under the action of the atypical antipsychotic drug clozapine. Method: As concentration-response curves in pre-contracted rings with PE (1 ?M), they were recorded in the presence and absence of inhibitors of the L-NAME and indomethacin inhibitors, which were incubated, isolated and / or in association, for 30 minutes. To study the participation of the K + channels, experiments were performed with the tetraethylammonium and potassium chloride (Kcl) blockers. In order to obtain the protocol they were performed with guanylase cyclase blockers (ODQ and methylene blue) and K + channel inhibitors (apamina, paxilin and glibenclamide). Results: Clozapine caused endothelium-dependent relaxation as the three relaxation pathways (cGMP / NO, cAMP / PGI2 and hyperpolarization). This observation was based on the inefficiency of the blockers, L-NAME, Indomethacin and Tetraethylammonium (TEA), respectively. Endothelium-dependent relaxation was attenuated by the association LNAME / Indomethacin and abolished in vessels contracted by potassium chloride and incubated with glibenclamide. Conclusion: These results suggest that there is synergistic participation, probably through a cross-talk mechanism, of the cAMP, cGMP and hyperpolarization systems. Therefore, more studies of possible endothelium function related to cardiovascular antipsychotics side effects would be a proper research direction.