The characteristics of crystallized fats depend on their solid fraction (SF) and fractal structures, which are affected by shear during crystallization. Binary mixtures of trilaurin (LLL) and trimyristin (MMM) diluted in triolein were used as samples. Pure diluted LLL and MMM were also studied. Samples were examined at different crystallization temperatures either statically or at shear rates of 800, 80, and 8 1/s. The sample cell combined a rheometer with a nuclear magnetic resonance (NMR) device to measure SF value and apparent viscosity. The measurements were compared to equations that describe the dependency of viscosity on solid volume fraction, to understand the effect of crystallites orientation at higher shear rates. Phase transitions during crystallization were observed by time-resolved synchrotron X-ray diffraction under similar conditions. Shear induced a strong reduction in phase onset and transition time and variations in phase distributions and the crystal size.

Cholesterol is critical to ensure proper functioning of a membrane. Despite this, the movement of cholesterol within the cell is not fully understood. The molecular order of binary and ternary mixtures of polyunsaturated fatty acids with varying degrees of hy- drocarbon chain unsaturation with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and/or cholesterol was studied using 2H NMR. The introduction of cholesterol into sam- ples of 18:1PC, 18:2PC (unsaturated lipid/DMPC-d54/CHOL, 75:5:20mol%) increased the C-2H bond order by ∼30%. Similar bond ordering was found for 20:4PC and 22:6PC samples, however, they were temperature dependent. A two-phase region (lo-ld) was found for 22:6PC:DMPC-d54/CHOL (75:5:20mol%) for temperatures below 286.7 K. The reorientation axis formed an angle of 78±4◦ with respect to the C3-2H bond vector re- gardless of the lipid. The order parameter of cholesterol was temperature independent, and ranged from 0.69±0.04 to 0.78±0.04 depending on the lipid unsaturation. The re- orientation axis of cholesterol was oriented at ∼25◦ to the bilayer normal.

Soy whey protein isolate (SWPI) – Fenugreek gum conjugates were developed and their molecular characteristics and emulsifying properties were investigated. SWPI was extracted from soy whey of tofu processing. SWPI exhibited excellent emulsifying properties comparable to soy protein isolate. However, to improve the emulsifying properties of SWPI for some applications, it was conjugated to fenugreek gum. The extent of conjugation was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared (FTIR) and High performance size exclusion chromatography (HPSEC). The SDS-PAGE of the conjugates showed polydispersed bands at the top of the separating gel in the conjugates suggesting the formation of high molecular weight products. Refractive index spectrum of HPSEC profiles showed a reduction of protein peak of unconjugated mixture and shifted a peak to higher molecular weight of the conjugates. Ultraviolet spectrum of HPSEC showed an increase of protein peak intensity at polysaccharide region. FTIR spectrum showed an amide band I and II were still observed in the conjugates after the unreacted proteins were removed. 1D NMR spectra showed that fenugreek gum was covalently bound to proteins through interaction between the reducing end of mannose residue and lysine. The protein solubility of SWPI – Fenugreek gum conjugates improved as compared to SWPI and SWPI – Fenugreek gum mixture when assessed in the pH range 3 to 8 at 22oC, especially at isoelectric point of protein (pl). A 1:3 and 1:5 ratio of SWPI – Fenugreek gum gave rise to better emulsion stabilization compared to 1:1 ratio. Particle size analysis revealed that conjugation of SWPI – Fenugreek gum at 60oC for 3 days was enough to produce relatively small droplet sizes in oil-in-water emulsions. SWPI – Unhydrolyzed fenugreek gum conjugates exhibited better emulsifying properties compared to SWPI – Partially hydrolyzed fenugreek gum conjugates. The conjugates improved emulsifying properties of SWPI, particularly around the pl of protein. The emulsifying properties were greatly increased by heating the conjugates before emulsification. The conjugates also improved emulsion stability at high salt concentration compared to SWPI. In summary, incorporation of SWPI into fenugreek gum improved emulsifying properties of SWPI near the pl of protein and at high salt concentration. / No / No

The E2A proteins are transcription factors critical for B-lymphopoiesis. A chromosomal translocation involving the E2A gene promotes acute lymphoblastic leukemia (ALL) through expression of the oncoprotein E2A-PBX1. Two activation domains of E2A-PBX1, AD1 and AD2, have been implicated in transcription mediated by recruitment of the transcriptional co-activator CBP/p300. A motif has been identified within AD1 that is important for recruiting CBP/p300, known as PCET. This recruitment requires an interaction between the activation domains of E2A-PBX1 and the KIX domain of CBP/p300. The KIX domain recognizes a generic ΦXXΦΦ sequence (Φ corresponds to a hydrophobic residue) found in the activation domains of numerous transcription factors. Mutation of leucine 20 in PCET has been shown to abrogate ex vivo immortalization of murine bone marrow and oncogenesis in a murine bone marrow transplantation model. A similar sequence is also found in AD2 and is implicated in E2A transcriptional activity and recruitment of CBP/p300. The structural details of these interactions remain largely unknown. NMR spectroscopy was used to determine the solution structure of the PCET:KIX complex, and the functional consequences of the Leu20Ala mutation were structurally rationalized. Other PCET mutations informed by this structure were tested and correlations were found between in vitro binding affinities and both transcriptional activation and immortalization. The binding site of the ΦXXΦΦ-containing E2A AD2 peptide was mapped to the same site on the KIX domain used by the PCET motif. A model of this complex was generated and mutations were tested using a similar approach as was used for PCET. E2A AD2 binds the KIX domain with lower affinity than the PCET motif and is not required for immortalizing bone marrow. A mutation that increases the affinity of E2A AD2 for the KIX domain to levels approaching that seen for the PCET:KIX interaction restores transcriptional activation and immortalization, demonstrating that immortalization by E2A-PBX1 is an affinity dependent process involving the KIX domain of CBP/p300. These studies indicate that the activation domains of E2A-PBX1 serve to support the in vivo function of the oncoprotein and that the PCET:KIX complex is a potential target for novel therapeutics in E2A-PBX1+ leukemia. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-09-13 13:30:48.848

Tat1-72, is an intrinsically disordered protein at pH 4.1 as previously indicated by NMR chemical shifts and coupling constants, and confirmed by 15N-relaxation parameters. The presence of SDS elicits a conformational change to α-helicity in Tat1-72. In the presence of the non-ionic DDM detergent and zinc, Tat was found to be soluble at pH 4 when bound to TAR RNA; TAR binding also elicits a conformational shift to α-helicity in Tat1-72. The β-sheet content of Tat1-72 is increased in the presence of NaCl. In similar conditions, Tat1-72 aggregates stained with Congo Red displayed a yellow-green birefringence and a red-shift in the Congo Red absorbance that is typical of β-amyloid fibril. The web-based algorithm “WALTZ” identifies the majority of the Tat1-72 hydrophobic core region as amyloidogenic. The helical propensity of Tat1-72 in TFE was determined by two-dimensional NMR spectroscopy.

The interaction of deoxyribonucleic acid (DNA) and cellular proteins is absolutely central to any biological understanding of DNA replication, transcription, and even gene regulation. Because an incumbent protein latches not onto the four bases but onto the backbone phosphate groups of the nucleic acid, backbone dynamics directly pertain to an understanding of basic cell processes. Studies have unambiguously proven that DNA exists in a balance of two conformations, BI and BII, defined by the difference in their backbone torsion angles. A given DNA sequence expresses a preference for either BI or BII, though both exist in most samples (and are presented as a ratio). Factors affecting that ratio include flanking sequence and methylation. When a DNA sample is methylated, which occurs at a cytosine, backbone dynamics at that site and perhaps even its neighbors are theoretically quenched due to the steric strain of a large attached group. DNA methylation is implicated in cancer diagnosis by new studies focusing on hypermethylation in CpG islands, This thesis uses solid-state deuterium NMR to study the backbone dynamics of the Dickerson dodecamer, [d(CGCGAATTCGCG)]2, which was the first synthetic BII conformer successfully crystallized (allowing for analysis in the solid state) and which contains the EcoRI binding site, GAATTC. This molecule is a good model system because a massive amount of information has been gathered on it not only using NMR, both high-resolution and solid-state, but also using x-ray diffraction, electron paramagnetic resonance, and all-atom molecular dynamics simulation. This thesis research shows the quenching of backbone dynamics due to C9 methylation.

Chemical species carry information in two dimensions, in their concentrations and their isotopic signatures. The concentrations of metabolites or synthetic compounds describe the composition of a chemical or biological system, while isotopic signatures describe processes in the system by their reaction pathways, regulation, and responses to external stimuli. Stable isotopes are unique tracers of these processes because their natural abundances are modulated by isotope effects occurring in physical processes as well as in chemical reactions. Nuclear magnetic resonance (NMR) spectroscopy is a prime technique not only for identification and quantification of small molecules in complex systems but also for measuring intramolecular distribution of stable isotopes in metabolites and other small molecules. In this thesis, we use quantitative NMR in three fields: in food science, environmental pollutant tracing, and plant-climate science. The phospholipid (PL) composition of food samples is of high interest because of their nutritional value and technological properties. However, the analysis of PLs is difficult as they constitute only a small fraction of the total lipid contents in foods. Here, we developed a method to identify PLs and determine their composition in food samples, by combining a liquid-liquid extraction approach for enriching PLs, with specialized 31P,1H-COSY NMR experiments to identify and quantify PLs. Wide-spread pollution with synthetic compounds threatens the environment and human health. However, the fate of pollutants in the environment is often poorly understood. Using quantitative deuterium NMR spectroscopy, we showed for the nitrosamine NDMA and the pesticide DDT how intramolecular distributions (isotopomer patterns) of the heavy hydrogen isotope deuterium reveal mechanistic insight into transformation pathways of pollutants and organic compounds in general. Intramolecular isotope distributions can be used to trace a pollutant’s origin, to understand its environmental transformation pathways and to evaluate remediation approaches. The atmospheric CO2 concentration ([CO2]) is currently rising at an unprecedented rate and plant responses to this increase in [CO2] influence the global carbon cycle and will determine future plant productivity. To investigate long-term plant responses, we developed a method to elucidate metabolic fluxes from intramolecular deuterium distributions of metabolites that can be extracted from historic plant material. We show that the intramolecular deuterium distribution of plant glucose depends on growth [CO2] and reflects the magnitude of photorespiration, an important side reaction of photosynthesis. In historic plant samples, we observe that photorespiration decreased in annual crop plants and natural vegetation over the past century, with no observable acclimation, implying that photosynthesis increased. In tree-ring samples from all continents covering the past 60 – 700 years, we detected a significantly smaller decrease in photorespiration than expected. We conclude that the expected “CO2 fertilization” has occurred but was significantly less pronounced in trees, due to opposing effects. The presented applications show that intramolecular isotope distributions not only provide information about the origin and turnover of compounds but also about metabolic regulation. By extracting isotope distributions from archives of plant material, metabolic information can be obtained retrospectively, which allows studies over decades to millennia, timescales that are inaccessible with manipulation experiments.

Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the 15N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that the 15N chemical shift tensors of flavins engaged in different hydrogen bonds will differ from one another. Tetraphenylacetyl riboflavin (TPARF) is soluble in benzene to over 250 mM, so, this flavin alone and in complexes with binding partners provides a system for studying the effects of formation of specific hydrogen bonds. For N5, the redoxactive N atom, one of the chemical shift principle values (CSPVs) changed 10 ppm upon formation of a hydrogen bonded complex, and the results could be replicated computationally. Thus our DFT-derived frontier orbitals are validated by spectroscopy and can be used to understand reactivity. Indeed, our calculations indicate that the electron density in the diazabutadiene system diminishes upon H-bond complex formation, consistent with the observed 100 mV increase in reduction midpoint potential. Thus, the current studies of TPARF and its complexes provide a useful baseline for further SSNMR studies aimed at understanding flavin reactivity in enzymes.

A model for studying magnetisation during FC-PEDRI experiments has been developed and verified experimentally. The optimum EPR irradiation frequency, which maximises the Overhauser enhancement while minimising the RF power deposition in sample, was investigated using the FC-DNP technique. Experiments to verify the model were carried out in the EPR frequency range between 20 and 180 MHz and with different applied ERP RF power for free radicals with different EPR linewidths. The optimum EPR irradiation frequency was found to be dependent on the applied power level and on the EPR linewidths. Methods to obtain NMR images with T₁-weighted were also investigated. The most successful method was found to be a combination of Inversion Recovery and FC-PEDRI. This technique yields both high <I>T</I>₁ contrast between samples and good contrast between images with different <I>T</I>₁ values. The free radical distribution is recorded with the same SNR as those collected with the conventional FC-PEDRI techniques. Rapid imaging of FC-PEDRI has also been studied. The number of EPR irradiation periods can be optimised to give a good free radical distribution image while reducing the power deposition in the sample. With 4 EPR irradiation periods, the difference image displays the free radical distribution comparable with the conventional technique (64 EPR irradiation periods). The power deposition in the sample is reduced by the factor of 16 and acquisition time is reduced by a factor of 4. Finally, inversion recovery spin preparation has been added to the hybrid fast sample. It was found that an IR hybrid FISP FC-PEDRI pulse sequence could be used to generate <I>T</I>₁ contrast in images which show similar detail and intensity of the free radical distribution as those obtained using hybrid FISP-PEDRI.

Anion exchange membrane fuel cells (AEMFCs) are an alternative to proton exchange membrane fuel cells (PEMFCs) with potential benefits that include low cost (i.e., platinum-free), facile electro-kinetics, low fuel crossover, and use of CO-resistant metal catalysts. Despite these advantages, AEMFCs have not been widely used because they require more highly conductive anion exchange membranes (AEMs) that do not exhibit impaired physical properties. Therefore, the issues that this research is dealing with are to maximize conductivity and to improve chemical stability. As model materials for these studies, I synthesize a series of multiblock copolymers with which polymer structures and morphologies can be easily controlled. Chapter 2 presents the synthesis and the chemical structure determination of the multiblock copolymers. With the objective of maximizing conductivity, an understanding of the impact of structural features such as organization, size, polarity and connectivity of ionic domains and channels within AEMs on ion/water transporting properties is necessary for the targeted and predictable design of an enhanced material. Chapters 3 to 5 describe three characterization techniques that reveal the role of these structural features in the transport process. Specifically, Chapter 3 demonstrates the possibility that the NMR relaxation times of water could be an indicator of the efficiency of ion channels. Low-temperature DSC measurements differentiate the state of water (i.e., bound water and free water) inside the membranes by measuring freezing temperature drop and enthalpy. Chapter 4 demonstrates that the number of water molecules in each state correlates with conductivity and suggests a major anion-conducting mechanism for the multiblock AEM systems. In Chapter 5, the measurement of the activation energy of diffusion characterizes ion transporting behavior that occurs on the sub-nanometer scale. For the characterization of the chemical stability of the AEMs under high pH conditions, I employ automated 1H NMR measurements as a function of time as well as diffusion-ordered NMR spectroscopy (DOSY) as shown in Chapter 6. Finally, I demonstrate that new multiblock copolymers are successfully utilized as an ionomer for a hybrid cell in Chapter 7. The properties of the polymer strongly influence overall cell performance. I believe that the combination of the techniques presented in this thesis will provide insight into the ion/water transporting mechanism in a polymer ion conductor and guidance for improving conductivity and the chemical stability of the AEMs.