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.

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The ongoing effort to improve the sensitivity and information content of NMR spectroscopy and MRI has important implications in scientific research and medical diagnostics. In this dissertation, a variety of approaches have been investigated and expanded on in an effort to contribute to this field. First, cryptophanes are cage-shaped molecules that have previously been used to encapsulate molecules of interest for a number of potential applications--including gas sensing and biosensing. In one set of studies, encapsulation of molecular hydrogen gas (H2) has shown different behavior compared to other small organic molecules in C111 (up until now, the smallest cryptophane). The transient, non-covalent binding was studied by variable-temperature NMR at different fields up to 950 MHz. A mathematical model that considers multiple-H2 binding was developed to better understand the physics and binding process, with predictions compared to experimental data (and rationalized in light of quantum chemical calculations on possible H2@C111 complexes). To our knowledge, C111 is the only system to reversibly trap multiple H2 gas molecules non-covalently under mild conditions. In a second series of studies, the interaction of laser-polarized xenon and a water-soluble cryptophane was studied. Despite the low concentration of xenon in aqueous solution, it was possible to achieve polarization transfer from xenon to cryptophane spins via the SPINOE (spin-polarization induced nuclear Overhauser effect). The SPINOE enhancements, along with the 129Xe NMR spectra, provide information about the interaction of the Xe-cryptophane complex (variants of which are now used in so-called xenon biosensors). This was our first in-house successful application of hyperpolarized xenon as a signal source for the spins of other molecules, leading the way to a number of ongoing studies. Although the absolute NMR enhancements obtained via the SPINOE were small, much larger enhancements were studied in a technique that uses para-hydrogen (pH2)--a spin isomer of normal molecular hydrogen)--as the source of spin order. As with the xenon experiments (and the H2 binding experiments), pH2 must be delivered as a gas to a sealed sample prior to performing the NMR experiments. Parahydrogen-induced polarization (PHIP) is an emerging field in enhancing the sensitivity in NMR experiments and may play an important role in MRI studies. Within this field a very recent phenomena of signal amplification by reversible exchange (SABRE) was investigated. The reproducibility of this recent discovery has been examined and new conclusions about the mechanism of this technique are delineated. NMR signal enhancements of nearly ~400-fold are reported. Moreover, a new water soluble NHC-Iridium catalyst was synthesized and investigated in SABRE related studies. We also report the first studies of SABRE-enhancement in biologically tolerable solvents--opening a door to the development of SABRE-hyperpolarized metabolic contrast agents for subsecond molecular imaging in the body. Although much of the above work was motivated by the desire to improve NMR/MRI sensitivity enhancement, other efforts concerned the other side of the equation--improving NMR/MRI information content. The next section concerns our efforts to investigate use of Variable-Angle (VA) NMR to study composite liquid crystal (LC) media comprised of stretched polyacrylamide gels (SAG) and embedded bacteriophage Pf1 particles. This in situ combination exploited the apparent interference between the different solute-aligning properties of the two LC components--yielding composite media with alignment properties that can differ in a tunable manner from those obtained with each medium alone. Characterization of alignment of both large and small molecules provides more insight into the nature of solute alignment that those composite phases introduce--with the goal of developing this approach as a new technique for studying molecular structure and dynamics via the dipolar and quadrupolar couplings that are restored in liquid-crystalline media. Finally the use of SPIONS--superparamagnetic iron oxide nanoparticles--as contrast agents is a relatively new approach to enhance information content in MRI studies; this is particularly true for SPIONs that have been surface-functionalized to achieve an environment-sensitive MR response. Novel surface-functionalized SPIONs were investigated by examining their effect on nuclear spin relaxation in aqueous environments simulating bodily tissues. More specifically, the pH and ionic strength dependent properties of selected dendron-functionalized and polymer-functionalized SPIONs have been examined. Of particular interest to this dissertation is how environment-mediated transient clustering of the SPIONs gives rise to changes in so-called transverse (homogeneous) spin relaxation rates as measured by following the decay of MR signals detected after the application of a series of radio-frequency (RF) pulses. In order to better understand these effects in the context of the SPIONs' behavior, a mathematical model is under development whose predictions are compared with experimental data. Aspects of the model are also compared to transmission electron micrography (TEM) and dynamic light scattering (DLS).

Signal Amplification by Reversible Exchange, or SABRE, is a type of PHIP (ParaHydrogen Induced Polarization) pioneered by Duckett, Green, and co-workers where an organometallic catalyst is used to co-locate parahydrogen (pH2) and a molecular substrate to be hyperpolarized. Like traditional PHIP, SABRE is of interest because it is cost-effective, potentially continuous, scalable, and rapid (achieving polarization enhancement in seconds). However unlike traditional PHIP, SABRE does not require permanent alteration of the substrate to hyperpolarize it. In addition to achieving 1H polarizations of several percent, SABRE in microTesla fields has enabled the creation of ~10% polarization for heteronuclear (15N) spins. I will discuss on a series of novel catalysts that I developed in my Ph.D program. Firstly of all, a heterogeneous SABRE ("HET-SABRE") catalyst where catalytic moieties were tethered to solid supports. Although NMR enhancements were modest (5), this initial work showed the feasibility of the approach. Next, two types of nanoscale catalysts were created to explore SABRE at the interface between heterogeneous and homogeneous conditions. Nanoparticle and polymer comb variants were synthesized by covalently tethering Ir-based catalysts to support materials comprised of TiO2/PMAA (poly methacrylic acid) and PVP (polyvinyl pyridine), respectively, and characterized by AAS, NMR, and DLS. Following pH2 delivery to mixtures containing one type of "nano-SABRE" catalyst, a target substrate, and ethanol, up to ~(-)40-fold and ~(-)7-fold 1H NMR signal enhancements were observed for pyridine using the nanoparticle and polymer comb catalysts, respectively, following transfer to high field (9.4 T). These enhancements appear to result from intact particles and not from any catalyst molecules leaching from their supports. Unlike the case with homogeneous SABRE catalysts, high-field (in situ) SABRE effects were generally not observed with the nanoscale catalysts. The potential for separation and reuse of such catalyst particles is also demonstrated. Besides the effort on green chemistry of SABRE catalyst, I have been investigating the preparation of different variants of the "standard" SABRE catalyst--[IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD=cyclooctadiene)]--for performing SABRE in otherwise "pure" aqueous environments. Because of the poor aqueous solubility of SABRE catalysts, previous promising efforts have used co-solvents to achieve SABRE in aqueous/organic mixtures. However, I have found that the chemical changes that accompany this catalyst's activation also endow it with water solubility. Complete removal of the organic solvent following activation and subsequent re-constitution of the activated structure in deuterated water allowed up to ~(-)33-fold 1H signal enhancements to be obtained for nicotinamide. Additionally, I have investigated chemical alteration of the structure of the pre-activated catalyst to endow greater water solubility. PEGylation of the aromatic carbine moiety provided much greater aqueous solubility, but while SABRE-active in organic solutions, the catalyst lost activity in >50% water (an effect under ongoing study). As an alternative approach, synthesis of a di-Ir complex precursor where the COD rings have been replaced by CODDA (1,2-dihydroxy-3,7-cyclooctadiene) permits creation of a water-soluble catalyst [IrCl(CODDA)IMes] that enables aqueous SABRE in a single step without need for any organic co-solvent; the potential utility of the catalyst is demonstrated with the ~(-)32-fold enhancement of 1H signals of pyridine in water with only 1 atm of pH2. Taken together, these results support the utility of rational design for improving SABRE and HET-SABRE for applications varying from fundamental studies of catalysis to biomedical imaging. In the following, I also investigate different aspects of how catalyst structure can affect resulting SABRE enhancements, including the interplay of catalyst structure and temperature for optimal SABRE, as well as the confounding effects on catalyst activation. Results from the "standard" Ir SABRE catalyst (1)--[IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD=cyclooctadiene)]--are compared with those obtained with variants respectively created by synthetically replacing the -Cl moiety with 4-amino-pyridine (4AP, 2), (diphenylphosphino)ethylamine (DPPA, 3), triphenyl phosphine (TPP, 4), and tribenzyl phosphine (TBP, 5); a sixth variant (6) was serendipitously created by an alternate synthetic route for (1) that appears to result in a polymorph according to x-ray crystallography. Studies of activation rate found that (4) and (5) activated the fastest under pH2 exposure (~20 s, an order of magnitude faster than (1)); activation rate was inversely correlated with SABRE enhancement, with peak 1H polarization enhancement ( ranging from only ~(-)44 for (4) to nearly ~(-)1900 for (1) (or PH~6%) for pyridine at 9.4 T, and ~(-)240 for nicotinamide. Although (1) gave the overall highest  values as expected, other catalysts gave rise to better SABRE performance in other temperature regimes: Optimal temperatures varied significantly, e.g. ~273 K for (2) to ~310-320 K for (1); the optimal temperature for (6) was considerably lower (<273 K) than that for (1), despite the apparent structural similarity. Taken together, these results show that full optimization of SABRE enhancement for a given experiment (with respect to substrate, target nucleus, etc.) may require systematic variation of parameters including catalyst ligand choice and temperature (to modulate binding affinities and off rates with respect to relevant spin-spin couplings), in addition to pH2 partial pressure, flow rate, and magnetic field. Finally, some research on an ssNMR will be represented, to show the potential application of ssNMR on the coating detection.

Coordinated polarity and differentiation of cells in the plane of a tissue layer are essential to the development of multicellular organisms. Arabidopsis thaliana root hairs and trichomes provide model systems to study the pathways that control planar polarity and cell fate specification in plants. A concentration gradient of the plant hormone auxin provides an instructive cue that coordinates polar assembly of signalling complexes at plasma membranes of root epidermal cells; however, knowledge about additional players and cytoskeletal effectors driving cell polarization prior to hair emergence remains limited. On the other hand, epidermal cell fate specification is controlled by a well-characterized gene network of transcription factors that translate positional signals and cell-to-cell communication into tissue-wide patterning. Yet, new components are continuously found to interact with the patterning pathway, shedding light on its connections with diverse developmental processes. This thesis presents the SABRE (SAB) gene as a novel player in planar polarity establishment and root epidermal patterning. SAB is a large protein with sequence similarity to proteins present in all eukaryotes and affects planar polarity as well as orientation of cell divisions and cortical microtubules. Genetic interaction with the microtubule-associated protein gene CLASP further supports involvement of SAB in microtubule arrangement, suggesting a role for this gene in cytoskeletal organisation. Strikingly, SAB also interacts genetically with ACTIN7 (ACT7), and both ACT7 and its modulator ACTIN INTERACTING PROTEIN 1-2 (AIP1-2) contribute to planar polarity of root hair positioning. Cell-file specific expression of AIP1-2 depends on the epidermal-patterning regulator WEREWOLF (WER), revealing a connection between actin organization, planar polarity and cell fate specification. Consistent with this finding, SAB also functions in patterning of the root epidermis by stabilizing cell fate acquisition upstream of the core patterning pathway. These results unveil new roles for SAB in planar polarity and epidermal patterning and suggest that organization of the microtubule and the actin cytoskeleton are important to both planar polarity establishment and cell fate specification. / Samordning av polaritet och differentiering av celler inom ett vävnadslager är avgörande för utvecklingen av multicellulära organismer. Rothår och bladhår hos Arabidopsis thaliana utgör modellsystem för att studera signalvägar som kontrollerar planpolaritet och specifikation av cellers öde hos växter. En koncentrationsgradient av växthormonet auxin ger en instruktiv signal som koordinerar polär hopsättning av signalkomplex vid plasmamembranet i rotepidermisceller; dock är kunskapen om ytterligare aktörer och hur cytoskelettets aktörer påverkar cellpolaritet innan rothår bildas begränsad. Vad gäller differentieringen av epidermala cellers öde kontrolleras dessa genom ett väl karakteriserat nätverk av transkriptionsfaktorer som överför positionssignaler och cell-till-cell kommunikation till vävnadsomfattande mönsterbildning. Fortfarande hittas dock nya komponenter som interagerar med signalvägarna för mönsterbildning, vilket ger nya insikter om dess förbindelser med diverse utvecklingsprocesser. Denna avhandling presenterar genen SABRE (SAB) som en ny aktör i etableringen av planpolaritet och mönsterbildning av rotepidermis. SAB är ett stort protein som har sekvenslikhet med proteiner som finns i alla eukaryoter och det påverkar planpolaritet, orientering av celldelning och kortikala mikrotubler. Genetisk interaktion med genen för det mikrotubuli-associerade proteinet CLASP stärker ytterligare inblandningen av SAB i organiserandet av mikrotubler och antyder att denna gen har en roll i organiserandet av cytoskelettet. Slående är att SAB även interagerar genetiskt med ACTIN7 (ACT7) och att både ACT7 och dess modulator ACTIN-INTERACTING PROTEIN1-2 (AIP1-2) bidrar till planpolaritet vid positionering av rothår. Cellfils-specifikt uttryck av AIP1-2 beror på den epidermala mönsterbildande genen WEREWOLF (WER), vilket påvisar ett samband mellan organisationen av aktin, planpolaritet och specifikationen av cellers öde. SAB fungerar även i mönsterbildning av rotens epidermis och stabiliserar förvärvet av cellöde uppströms av den centrala signalvägen för mönsterbildning. Dessa resultat visar på nya roller för SAB i planpolaritet och mönsterbildning av epidermis och indikerar att organiseringen av mikrotubler och aktin-cytoskelettet är viktiga både för etablerandet av planpolaritet och för specificeringen av cellers öde.

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