Proton nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the interaction of yeast phosphoglycerate kinase (PGK) with its phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The application of one-dimensional and, for the first time, two-dimensional proton NMR techniques to this large protein has enabled specific resonance assignments to be made. Assignment has been aided by the investigation of specifically deuterated protein and site-specific mutant forms of the protein, including the isolated N- and C-domains. The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast PGK have been characterised and the assigned resonances used as local probes of structural and dynamic changes. Two binding sites have been determined for the nucleotide substrate, ATP, the occupancies of which are dependent on Mg²⁺ concentration. One site corresponds to the catalytic site determined crystallographically. A single binding site was found for 3-PG. This binding was shown to cause highly specific conformational changes throughout the N-domain and the interdomain region, which involve the relative movement of at least three α-helices. Investigation of 3-PG binding to several site-specific mutant forms of yeast PGK revealed a critical role for arginine 168 in the propagation of these changes. The general binding of anions to yeast PGK was investigated using the paramagnetic probes [Cr(CN)₆]^3- and [Fe(CN)₆]^3-, and the diamagnetic anion [Co(CN)₆]^3-. The primary anion binding site was determined from [Cr(CN)₆]^3- broadening data and found to share some side-chains involved in 3-PG binding, namely histidine 62 and arginine 168. Evidence for a secondary anion site was found. The anion binding data is discussed in view of the complex activation/inhibition effects of anions on the catalytic activity. Investigation of the isolated N- and C-domains showed that both can fold independently and confirmed that the C-domain is a nucleotide binding domain. It appears that the presence of the interdomain residues and/or the C-terminal peptide are necessary for 3-PG binding to the N-domain. This work shows that the specificity of the substrates is in binding, as expected, but also in the motions induced in the protein as a whole.

No description available.

Problems with the low back resulting from diseases of the lumbar disc account for much human suffering and medical expense. Through early identification of changes in the disc, Magnetic Resonance Imaging offers a potential method for recognition of those at risk of low back trouble. An understanding of the chemical and structural basis of the MR image will therefore be of great help in understanding the degenerative mechanism itself. With this in mind, NMR studies of human intervertebral discs were performed to extract data that could be correlated with different stages of degeneration. In vitro samples of anulus fibrosus and nucleus pulposus of grades II and IV discs were examined. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used to obtain T₂ relaxation data, which were analysed with a non-negative least squares algorithm and presented as a continuous spectrum of exponentials. An inversion-recovery sequence was used to obtain T₁ data which were analysed with the same algorithm, then presented as a discrete exponential spectrum. The same procedures were used to produce relaxation times from samples of isolated disc collagen and proteoglycan. Also, T₂ relaxation data were obtained with a twelve-echo CPMG imaging sequence from healthy volunteers, and these data analysed with the same procedure used for in vitro data. In vivo and in vitro results were compared and found to agree in terms of the T₂ relaxation values. Most change between grades occurred in the nucleus, where both T₁ and T₂ values decreased from grade II to grade IV. The results of the proteoglycan and collagen studies support the theory that structure, not chemical composition, is responsible for the observed changes in relaxation times. Based on estimates of the percentages of protons in the nucleus due to water, proteoglycan and collagen, tentative assignments were made to the components of the T₂ spectra for grades II and IV nucleus, and these were supported by the results of all parts of the study. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

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In this thesis work, a system for proton beam alignment was studied and optimized in many of its functional areas. The resulting system was named Positioning Alignment Control System (PACS). The PACS system is an integrated and efficient system as a result of the work done on it in the course of this thesis work.

There is a dogma in molecular biology that biological functions of peptides are determined by their structure ("function" code), coded in their primary structure ("structure" code). This work describes a new approach that attempts to elucidate these relationships by peptide topology design based on intriguing conformational properties of pipecolic acid based amino acids--like 1,2,3,4 tetrahydroisoquinoline (Tic). Opioid peptides, owing to the heterogeneity of opioid receptors, display a wide variety of physiological actions. The mu opioid receptor selective octapeptide I (D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂) is a model compound for topographical modifications induced by sequential substitutions by Tic residue. Thus, the closely related peptides I and II (Gly-D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂, obtained by coupling Gly residue to I) have contrasting affinities for the mu opioid receptor (IC₅₀ = 1.2 and 278 nM, respectively). Conformational analysis of I and II by means of 1D and 2D ¹H NMR spectroscopy allowed to determine dramatic differences in the side chain orientation of D-Tic in both peptides and to propose features of the bioactive conformation. The extended conformation of I (due to g(-) side chain conformation of D-Tic) is well recognized by the mu receptor in contrast to the folded conformation of II (due to a g(+) side chain conformation of D-Tic¹, that places the aromatic ring on the opposite side of the molecule), which is not. Peptide III (D-Phe-Cys-Tic-D-Trp-Orn-Thr-Pen-Thr-NH₂), featuring replacement of Tyr³ by Tic³, binds very weakly to the mu opioid receptor, due to rotation of the Tic aromatic side chain to the opposite side of the molecule (Tic side chain is in a g(+) conformation again). As these substitutions conserve the conformation of the backbone, constrained cyclic amino acids (picolic acid derivatives) can modify the topography of the peptide in a predictable manner, and (in conjunction with biological data) disclose structural elements of bioactive conformations. The mechanisms of pipecolic acid side chain rotamer selection, will be discussed in the context of design principles.

Proton magnetic resonance spectroscopy has been widely applied to the diagnosis of brain diseases. In the meanwhile, the classification of brain abscesses plays an important role on the accurate prognosis in clinics. Recently, the interest in using proton MRS to classify pyogenic brain abscesses has been arising because of its non-invasive property and good accuracy in detecting metabolites. The brain abscess can be classified by means of the metabolites observed in the MR spectra, which may thus benefit the accuracy of the brain abscess diagnosis clinically. However, the interpretation of MR spectra by experienced radiologists can be also very subjective and therefore results in the variation of diagnosis. In this study, we investigate the potential possibility of using Principal Component Analysis (PCA) to classify the short TE MR spectra in more objective way.

Magnetic Resonance Imaging (MRI) can obtain the tissues of in vivo non-invasively. Proton MR Spectroscopy uses the resonance principle to collect the signals of proton and transforms them to spectrums. It provides information of metabolites in patient¡¦s brain for doctors to observe the change of pathology. Observing the metabolites of brain abscess patients is most important process in clinical diagnosis and treatment. Then, doctors use different spectrums of echo time (TE) to enhance the accuracy in the diagnosis. In our study, we use independent component analysis (ICA) to analyze MR spectroscopy. After analyzing, the independent components represent the elements which compose the input data. Then, we use the projection which is mentioned by Ssu-Ying Lu¡¦s Thesis to help us observe the relationship between independent components and spectrums of patients. We also discuss the result of spectrums with using ICA and PCA and discover some questions (whether it need to do scale normalization before inputting data or not, the result of scale normalization doesn¡¦t expect, and the peak in some independent components confuse us by locating in indistinct place) to discuss and to find possible reason after experiments.

No description available.

Traumatic brain injury (TBI) continues to be a widespread problem in the United States with approximately 1.7 million occurrences annually [1]. Current automotive crash test standards use the Head Injury Criterion (HIC) [2] to assess head injury potential, but this metric does not relate an impact to underlying damage. For an injury metric to effectively predict TBI, it is crucial to relate level of impact to resulting injury. The research presented in this dissertation explains the development and repeatability of two novel injury devices, impact response characterization over the course of 24 hours in the Gottingen minipig and the relationships between metabolite changes, underlying disruption, and impact kinematics, and the characterization of impact response over the course of 72 hours. The translation-input and combined translation and rotation-input injury devices were shown to be repeatable, minimizing the number of animals needed for testing. Impact response over the course of 24 hours showed axonal disruption through immunostaining and proton magnetic resonance spectroscopy. The translation-input injury group metabolite analyses revealed the initial stages of glutamate excitotoxicity while the combined-input injury group showed a clear pathway for glutamate excitotoxicity. Numerous correlative relationships and potential underlying disruption predictors were found between metabolites, immunostaining, and kinematics. The most promising predictor combination for the translation-input injury device was N-acetylaspartylglutamate/Scyllo at 24 hours compared to 1 hour and linear speed for predicting underlying light neurofilament disruption. For the combined-input injury device, the strongest predictor combination was Glutamine/N-acetylaspartylglutamate at 24 hours compared to baseline and angular acceleration for predicting underlying light neurofilament disruption. Statistically significant predictors were found between Glutamate+Glutamine/Total Creatine at 24 hours compared to baseline and all kinematics and injury metrics with an angular component for predicting heavy neurofilament disruption. Analyses over the course of 72 hours revealed persistent axonal disruption and metabolite perturbations. Overall, this dissertation and the complementary parts of this project have many societal implications. Due to the high incidence of traumatic brain injury, there is a need for prevention, mitigation, and treatment strategies. Developing a new injury metric will help improve prevention strategies, especially in the automotive, sporting, and military environments. 1 Faul, M., Xu, L., Wald, M. M., and Coronado, V. G. (2010). Traumatic Brain Injury in the United States. Atlanta, GA: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control. 2 Versace, J. (1971). A Review of the Severity Index. SAE Technical Paper. No. 710881 / Ph. D.