Browsing by Subject "Biophysical Sciences and Medical Physics"
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Item Metabolic alterations associated with hemorrhagic shock and traumatic injury as measured in the urine.(2011-12) Lusczek, Elizabeth RoseIntroduction: This dissertation focuses on profiling the changes in metabolism that occur as a result of hemorrhagic shock and traumatic injury as observed in urine obtained from a porcine model. Hemorrhagic shock and traumatic injury are responsible for the majority of deaths under the age of 44. A porcine model of hemorrhagic shock and traumatic injury was used to examine changes in metabolism due to hemorrhagic shock and traumatic injury. Changes in metabolism were hypothesized to be dependent upon the metabolic state the animal occupies upon injury (fed or fasted). It was also hypothesized that a metabolite or metabolites could serve as a marker of mortality. Materials and Methods: Nuclear magnetic resonance (NMR) spectroscopy and Chenomx software were used to generate individual profiles of metabolite concentrations (the metabolome) for each urine sample obtained. Because hemorrhagic shock produces drastic changes in urine concentration and large fluctuations in the concentrations of endogenous metabolites in the urine, urine must be normalized to urine output to allow for accurate comparisons between urine samples. The urinary metabolome was analyzed with partial least squares discriminant analysis (PLS-DA) and with a scale-free network. Results: PLS-DA models showed that metabolites associated with respiration, ischemia/reperfusion injury, and cell membrane damage were observed in the urine two hours after the initiation of resuscitation. Other metabolites which may be associated with injury sustained from surgical preparation were observed at 20 hours after the initiation of resuscitation. The metabolites 1,6-Anhydro β-D-glucose and mannose were associated with the metabolome of fed animals. The network analysis of the metabolome is reflective of the scale-invariant nature of metabolism. Respiratory metabolites were separated by whether they are involved in aerobic or anaerobic respiration. Anaerobic metabolites were also associated with markers of cell membrane rupture. Lactate, pyruvate, 2-oxoglutarate, succinate, taurine, glycine, dimethylamine, and creatinine were identified as well-connected hub metabolites in the network. No metabolites were associated with survival alone. Conclusions: The pathophysiology associated with hemorrhagic shock can be treated as a scale invariant process particularly with respect to metabolism. Results from a traditional metabolomics treatment of the data with PLS-DA models were congruent with results from a scale-invariant, weakly modular network of the urinary metabolome. Other samples obtained from these experiments (muscle, liver, and serum) could benefit from a similar treatment. Additional work in (a) integrating the metabolomes of all four biological samples and (b) creating a physics-based theory of hemorrhagic shock may provide important information about mortality and the propagation of injury.Item Structural transitions of myosin associated with force generation in spin-labeled muscle fibers.(2012-06) Mello, Ryan NicholasMuscle contraction is driven by the actin-activated hydrolysis of ATP by myosin, resulting in the relative sliding of actin and myosin filaments. Current models propose that filament sliding is driven by a structural transition of myosin’s catalytic domain (CD) and light chain domain (LCD). The goal of this research is to measure structural transitions of myosin II (muscle and nonmuscle) that are associated for force generation. Structural measurements were made using electron paramagnetic resonance (EPR) spectroscopy. This work is comprised of two separate, but related, projects. In the first project (Chapter 3), thiol crosslinking and EPR were used to resolve structural transitions of myosin’s LCD and CD that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain (RLC) for endogenous RLC, with full retention of function. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), post-hydrolysis intermediate (A.M.D.P), and rigor (A.M.D). To trap myosin in a structural state analogous to the elusive post-hydrolysis ternary complex A.M.D.P, we used pPDM to crosslink SH1 (Cys707) to SH2 (Cys697) on the CD. EPR showed that the LCD of crosslinked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to crosslink SH1 to SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 crosslinking traps a state in which both the LCD and CD are in a structural state intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the crosslinked state is an A.M.D.P analog on the force generation pathway. In the second project, we present a method for obtaining high-resolution structural information of proteins using EPR of a bifunctional spin label (BSL). Two complimentary EPR techniques were employed to measure dynamics and orientation (conventional EPR) and intraprotein distances (dipolar electron-electron resonance). The exploitation of BSL is a key feature of this work. BSL attaches at residue positions i and i+4, which drastically restricts probe motion compared to monofunctional probes. For comparison, measurements were also made with the monofunctional spin label MSL. Subfragment 1 of Dictyostelium myosin II (S1dC) was used to exemplify the increased resolution provided by BSL. Using this approach, we demonstrate with experiments that BSL significantly increases resolution when measuring distance and orientation compared to MSL. And while this work does focus on the methodology, there is significant biological insight into myosin’s nucleotide-dependent structural transitions.