Physics
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Item QUANTATITIVE STUDY OF WATER DYNAMICS IN BIOMIMETIC MODELS AND LIVING TISSUE BY NMR AND MRI: PERSPECTIVES ON DIRECT DETECTION NEURONAL ACTIVITY(2015) Bai, Ruiliang; Briber, Robert M; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Detection of neuronal activity noninvasively and in vivo is a desideratum in medicine and in neuroscience. Unfortunately, the widely used method of functional magnetic resonance imaging (fMRI) only indirectly assesses neuronal activity via its hemodynamic response; limiting its temporal and spatial accuracy. Recently, several new fMRI methods have been proposed to measure neuronal activity claiming to be more direct and accurate. However, these approaches have proved difficult to reproduce and are not widely applied mainly because of a dearth of “ground truth” experiments that convincingly establish the correlation between the magnetic resonance (MR) signals and the underlying neuronal activity. In addition, limited knowledge of water dynamics in living tissue restricts our understanding of the underlying biophysical sources of these candidate fMRI signals. To address the first problem, we developed a novel test system to assess and validate fMRI methods, in which real-time fluorescent intracellular calcium images and MR recording were simultaneously acquired on organotypic rat-cortex cultures without hemodynamic confounds. This experimental design enables direct correlation of the candidate functional MR signals with optical indicia of the underlying neuronal activity. Within this test bed, MR signals with contrasts from water relaxation times, diffusion, and proton density were tested. Diffusion MR was the only one shown to be sensitive to the pathological condition of hyperexcitability, e.g., such as those seen in epilepsy. However, these MR signals do not appear to be sensitive or specific enough to detect and follow normal neuronal activity. Efforts were made toward improving our understanding of the water dynamics in living tissue. First, water diffusivities and relaxation times in a biomimetic model were measured and quantitatively studied using different biophysical-based mathematical models. Second, we developed and applied a rapid 2D diffusion/relaxation spectral MR method, to better characterize the heterogeneous nature of tissue water. While the present study is still far from providing a complete picture of water dynamics in living tissues, it provides novel tools for advancing our understanding of the possibilities and limits of detecting neuronal activity via MR in the future, as well as providing a reproducible and reliable way to assess and validate fMRI methods.Item Molecular basis of the kinematics of the kinesin step(2012) Zhang, Zhechun; Thirumalai, Devarajan; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Kinesin is an ATP-dependent cellular transporter that ferries cargos towards the plus-end of a microtubule. Despite significant advances in experiments, which have provided deep insights into the motility of kinesin, the molecular events that occur in a single step have not been fully resolved. In order to provide these details, this thesis develops a structure of the complex between kinesin and microtubule, and devises new simulation methods to probe the stepping kinetics over a wide range of conditions. Hundreds of molecular movies of kinesin walking on the microtubule are generated using coarse-grained simulation methods. Analysis of these movies shows that there are three major stages in the stepping kinetics of kinesin. In addition, an allosteric network within kinesin, responsible for controlling nucleotide release, is identified using microsecond all-atom simulations. These simulations are used to answer two important questions. First, does kinesin move by a "power stroke" or by diffusion? During a single step, the trailing head of the kinesin detaches from the microtubule, passes the microtubule-bound leading head, and attaches to the target binding site 16 nm away. The target binding site, however, is one of eight accessible binding sites on the microtubule. Is it possible that the "power stroke" (a large conformational change) in the leading head, pulls the trailing head into the neighborhood of the target binding site? This remained unclear because the fraction of the 16 nm step associated with the power stroke and diffusion had never been quantified. Second, how does the microtubule accelerate ADP release from kinesin, which is a key step in completing a single step? The ADP binding site of kinesin is more than 1.5 nm away from the microtubule binding surface. Therefore, the microtubule must affect the ADP binding site through an allosteric mechanism. However, the structural basis for transmitting signals through the underlying allosteric network was previously unknown. Analysis of hundreds of kinesin steps generated using coarse-grained simulations showed that the power stroke associated with the docking of the neck linker to the leading head, is responsible for only 4 nm of the 16 nm step, and the remaining 12 nm is covered by diffusion. However, the power stroke in the leading head constrains the diffusion of the trailing head, decreases the probability of side steps, and therefore biases the trailing head, to the target binding site. Additional all-atom simulations of the ADP-kinesin-microtubule complex, revealed a surprisingly simple allosteric network within kinesin that explains the acceleration of ADP release upon microtubule binding. The allosteric network also explains two additional experimental observations on ADP release from kinesin.