NMR characterization of interstitial myocardial sodium

Thesis (Ph. D.)--Massachusetts Institute of Technology, Whitaker College of Health Sciences and Technology, 1991.Includes bibliographical references (leaves 138-146).by Brent D. Foy.Ph.D

NMR, water and plants

This Thesis describes the application of a non-destructive pulsed proton NMR method mainly to measure water transport in the xylem vessels of plant stems and in some model systems. The results are equally well applicable to liquid flow in other biological objects than plants, e.g. flow of blood and other body fluids in human and animals (Chapter 8). The method is based on a pulse sequence of equidistant πpulses in combination with a linear magnetic field gradient G.Following a general introduction and a survey of the properties of water in plants (Chapters 1 and 2), the basic NMR theory as well as reviews on the application of pulsed NMR to the determination of flow, diffusion and water content are presented in chapter 3.A mathematical treatment has produced analytical expressions for the shape of the signal S(t), based on a model in which the flowing fluid is thought to receive a ½π-τ-(π-τ-) n pulse train: a ½πpulse upon entering the r.f. coil followed by a sequence of equidistant πpulses until the fluid leaves the coil; simultaneously, this movement. of the fluid along a magnetic field gradient applied in the direction of flow produces a phase shift of the nuclear magnetization with respect to the rotating frame of reference (Chapter 4). Although this model does not lead to perfect agreement between the experimental and theoretical signal shape S(t), it correctly predicts the effects of experimental parameters on S(t) via analytical expressions. The main results from this theoretical treatment in combination with computer simulations, which have been experimentally verified in glass capillary systems, are:- as long as T 2≥ ½T 1 , the mean linear flow velocity v can be found from the time t max at which a maximum appears in the signal shape: v=C/t max , where C is a calibration constant, depending on G, τand the flow profile. If T 2 <½T 1 v can only be reliably determined when both T 1 and T 2 of the flowing fluid are known.- T 2 and the amount of flowing water in the coil V, and consequently the volume flowrate Q, can be determined from the height of the maximum S(t max ) and t max . Depending on the value of T 2 and the value of the ratio T 1 /T 2 , T 2 and V are found from a semilog plot of either S(t max ) vs. t max (T 1 >>T 2 ) or ∂[S(t max ) . t max ]/∂t max vs. t max (T 1≈ T 2 ).Based on flow measurements in plant stem segments (Chapter 5) it has been suggested that T 2 strongly depends on the vessel diameter for the narrow xylem capillaries. This behaviour of T 2 can explain negative results in plant stems with small vessel diameter. Under the present experimental conditions the method has been successfully applied to Cucurbitaceae (cucumber, gherkin, pumpkin) and tomato plants.T 2 measurements in wheat leaves have been shown to be insensitive to the presence of cell-bound paramagnetic ions (Chapter 7). The magnitude of T 2 of two separate water fractions (covering -90% of the total water content) has been found to be inversely proportional to water content. Measurements of flow and water content have been combined for an intact gherkin plant (Chapter 5), demonstrating that the combination of both NMR methods results in a powerful non-invasive method to study important parts of the plant water balance simultaneously. The results strongly suggest that the method can be used as an early warning for development of stress phenomena in plants, due to drought and other factors. From the flow measurements it has been shown how in a plant system the values of T 2 and T 1 of the water in the xylem vessels can be determined and estimated, respectively.A comparison between the results obtained with NMR, heat pulse and weight balance flow measurements is presented in Chapter 6. A linear relationship between the linear flow velocity obtained by NMR and the volume flowrate determined by the balance method yields an effective cross-sectional area available for flow of ~50% of the cross-sectional area of the xylem vessels measured by using a microscope. NMR measurements alone yield a slightly lower value of the effective cross-sectional area. Compared with the NMR method, the heat pulse method monitors only relative changes in the flow velocity. A plot of the flow velocity obtained by the heat pulse method versus the volume flowrate obtained by the balance method exhibits some unwanted experimental scatter.Chapter 8 suggests some applications of the pulsed NMR flow method, also to other systems than plants, and defines important instrumental requirements for these applications

Classification of breast malignancy using optimised advanced diffusion-weighted imaging : and surgical planning for breast tumour resection using MR-guided focused ultrasound

Intravoxel Incoherent Motion Imaging (IVIM) is a non-invasive MR-imaging technique that enables the measurement of cellularity and vascularity using diffusion-weighted (DW)-imaging. IVIM has been applied to various cancer types including breast cancer, and is becoming more popular but lacks standardisation. The quantitative parameters; diffusion, D, perfusion fraction, f, and pseudo micro capillary diffusion, D* are thought to be correlated with tumour physiognomies such as proliferation, angiogenesis and heterogeneity.In Part 1 of this thesis, an optimised clinical b-value protocol is produced using a robust statistical method. This optimised protocol and various fitting methodologies are investigated in healthy volunteers, and then the most precise approach is applied in a clinical trial in patients following diagnosis of breast cancer, before treatment, to correlate IVIM parameters with breast cancer grade, histological type and molecular subtype with statistically significant results supporting IVIM’s potential as a non-invasive biomarker for malignancy. Monte Carlo simulations support this clinical application, where real data mean squared errors due to SNR limitations lie within simulated errors. A computed DW-imaging program is also presented to produce better quality images than acquired high b-value images as an adjunct to the optimised IVIM protocol.In Part 2 of this thesis, MR-guided Focused Ultrasound (MRgFUS) is explored as a means to create a pre-surgical template of thermally induced palpable markers to enable a surgeon to resect occult lesions and potentially reduce positive tumour margin status and local recurrence after breast conserving surgery. A surrogate animal model with pseudo lesion is presented, as well as a clinical tool to plan spot markers around a lesion as seen on MRI

Theoretical Investigation of Interactions and Relaxation in Biological Macromolecules

One of the major challenges posed to our quantitative understanding of structure, dynamics, and function of biological macromolecules has been the high level of complexity of biological structures. In the present work, we studied interactions between G protein-coupled receptors (GPCRs), and also introduced a theoretical model of relaxation in complex systems, in order to help understand interactions and relaxation in biological macromolecules. GPCRs are the largest and most diverse family of membrane receptors that play key roles in mediating signal transduction between outside and inside of a cell. Oligomerization of GPCRs and its possible role in function and signaling currently constitute an exciting area of research, with implications on development of therapeutic regimens. We performed molecular dynamics (MD) simulations of fluorescent proteins attached through short linkers to GPCRs, in order to obtain distances between them and orientation factors of their transition dipole moments. Used in conjunction with Förster resonance energy transfer (FRET) experiments, this information is used for determination of binding interfaces between GPCR protomers (i.e., single molecules) within an oligomer. We simulated, with coarse-grained resolution, several configurations of dimers and tetramers of the M2 muscarinic acetylcholine receptor fused to the green fluorescent protein (GFP, a donor of energy in FRET) and yellow fluorescent protein (YFP, an acceptor of energy). From simulated distances and orientation factors for oligomers with different relative orientations of the protomers, we computed apparent FRET efficiencies for mixtures of monomers, dimers and tetramers based on the simulated data, and then compared them to experimental FRET data. Comparing the fitting residuals obtained for all tested oligomer configurations, we were able to determine, for the first time, the most probable quaternary structure of the M2 muscarinic receptor in living cells. The study of relaxation processes is still insufficiently developed for the case of complex systems. Although it is currently firmly established that the dielectric behavior of systems of coupled dipoles or systems with complex biological structures deviates markedly from classical Debye (in the frequency domain) or pure exponential decay (in the time domain), the exact ways in which these deviations occur and their significance are still debated issues. In the second part of my thesis, we use a new approach to this problem for systems that present hierarchical relationships between their parts, also known as fractals. We formulated a set of differential equations of physical quantities in the hierarchical structure and developed a method of solving it. As a test case, for which there is experimental data to relate to, we applied this method to dielectric relaxation, and successfully reproduced the Debye, and non-Debye behaviors in the frequency domain, as well as corresponding non-exponential behaviors in the time domain. The proposed approach will likely provide an adequate mathematical framework for such disparate phenomena as recombination of photodissociable molecules, distribution of income in large populations of humans, and non-exponential decay of fluorescence in systems with multiple, hierarchically organized energetic levels. This in turn could help, develop correct approaches for analyzing FRET measurements in the time domain, which currently pose many challenges

42nd Rocky Mountain Conference on Analytical Chemistry

Abstracts from the 42nd annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Broomfield, Colorado, July 30 - August 3, 2000

Modulating Calcium Signaling by Protein Design and Analysis of Calcium Binding Proteins

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium stores, and gated by membrane calcium channels/pumps. To closely monitor calcium dynamics there is a pressing need to develop calcium sensors that are targeted to high calcium environment such as the ER/SR with relatively low binding affinity and fast kinetic properties to complement the current calcium indicator toolkits. In this dissertation, the development of fast red florescent calcium binding protein using the protein design is reported. The results show the calcium dependent fluorescence increase of mCherry mutant MCD1 (RapidER) and MCD15 (RapidER’) is able to monitor the ER calcium release in several cell lines responding to perturbations of extracellular calcium signaling. The specific targeting to the ER membrane was achieved by fusing the ryanodine receptor 1 transmembrane domains for the spatio-temporal calcium imaging. To understand the underlying mechanism of calcium binding induced fluorescence increase in the designed calcium sensor CatchER, the fluorescence lifetime of CatchER was determined in calcium free and bound forms using time resolved florescence spectroscopy. The results suggest that calcium binding inhibits the geminate quenching, resulting in a longer lifetime when the anionic form is indirectly excited at 395 nm. It is believed that such unique calcium-induced lifetime change can be applied to monitor calcium signaling in cell imaging. NMR spectroscopy was used to investigate the protein-protein/ligand interaction in this dissertation. The residual dipolar coupling and T1, T2, NOE dynamic study were carried out to understand the binding mode of CaM and the N-terminal intracellular loop of connexin 43. The results show that both N and C terminal domains of Ca2+-CaM contact with the peptide, leading to a partially unwound and bending central helix of CaM. The ligand binding induced conformational change was demonstrated by selectively labeled proteins including extracellular domain of calcium sensing receptor and the bacterial membrane protein SecA fragments C34 and N68