Thesis (Ph. D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Joseph D. Seymour. Includes bibliographical references (leaves 189-201).

This thesis describes an investigation into the folding behaviour of hen lysozyme by characterisation of the structure and the dynamics of different conformational states of the protein. The native state, a partially structured state generated by the addition of a cosolvent, trifluoroethanol (TFE), and a highly denatured state of the protein in presence of urea at low pH, have been studied at equilibrium by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The principal methods utilised in-fchis thesis are the measurement and interpretation of amide hydrogen exchange and ¹⁵ N relaxation data. Amide hydrogen exchange rates from a highly denatured state of hen lysozyme in 8 M urea, at pH 2.0, are well approximated by recent estimates for intrinsic exchange rates from unstructured polypeptides, implying that residual .structures cannot be persistent in this state. In the native protein, by contrast, protection factors are measured to be of the order of 10⁴-10⁸ and can be rationalised by the involvement of amides in secondary structure and burial from the protein surface. Surface accessibility has also been found to be an important determinant of the dynamics of main and sidechain groups monitored by ¹⁵N relaxation. For this purpose ¹⁵N resonances of native lysozyme were assigned and order parameters derived. The dynamic behaviour was considered in the light of features of the crystal and NMR structures, suggesting that a lack of van der Waals contacts is a major determinant for mobility. A denatured state of hen lysozyme, formed by addition of TFE cosolvent, has been developed as a model for a partially ordered conformation of the protein. Amide hydrogen exchange measurements show that sites with the highest protection factors, up to 250, are located in stable native-like helices. Near complete assignment of ¹⁵N-edited NMR spectra allowed a detailed description of secondary structure in the TFE denatured state. Non-native α-helical structure is present as extensions to the native helices and in a region of the polypeptide which forms part of the β-sheet in the native state. These structures have been shown to be in accord with the helical propensities of the primary sequence. Preliminary structure calculations suggest that, despite the absence of extensive and persistent tertiary interactions, topological restraints exist in parts of the TFE denatured state, resulting in considerable propensities for native-like arrangements of the secondary structure. Mainchain dynamics determined by ¹⁵N relaxation, although increased in magnitude, appear to be related to that of the native state and are dominated by the position of disulphide bonds and by chain hydrophobicity. Thus the structural and the dynamic behaviour of the polypeptide chain in the TFE denatured state could be rationalised, at least in part, by consideration of features of the primary sequence.

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: Sarah L. Codd. Includes bibliographical references (leaves 141-154).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Joseph Seymour. Includes bibliographical references (leaves 69-70).

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 106-111).

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: Sarah L. Codd. Includes bibliographical references (leaves 119-132).

L’imagerie par résonnance magnétique (IRM) est devenue un outil incontournable en recherche biomédicale. Le développement d’outils de neuroimagerie pour le petit animal est primordial pour valider des hypothèses biologiques et tester l’efficacité de nouvelles thérapies. Dans le contexte de la maladie d’Alzheimer (MA), l’utilisation d’IRM à haut champ magnétique permet l’imagerie des plaques amyloïdes, signature moléculaire de cette pathologie. Cependant, seule une faible proportion de ces lésions sont détectées et aucune méthode de neuroimagerie ne permet actuellement d’imager les dégénérescences neurofibrillaires, deuxième signature microscopique de la MA. Dans cette thèse, deux méthodes complémentaires, utilisant différents agents de contraste à base de Gadolinium (Gd), ont été étudiées pour améliorer la détection des plaques amyloïdes par IRM. La première partie de cette thèse s’est intéressée à la méthode du « Gd- staining ». Une étude comparative réalisée sur différents modèles murins d’amyloïdose et sur des primates non-humains, a permis de mieux comprendre les mécanismes impliqués dans la détection des plaques amyloïdes grâce à cette technique. Également, nous avons montré pour la première fois qu’il est possible d’imager les lésions Aβ humaines, en post mortem, par Gadolinium-staining. La deuxième partie de cette thèse s’est intéressée au développement d’agents de contraste vectorisés, ciblant spécifiquement les lésions de la MA. Ces molécules seraient capables de traverser spontanément la barrière hémato-encéphalique, principale limite au développement de molécules à visée cérébrale, et permettraient de cibler spécifiquement les deux lésions caractéristiques de la MA. Ces propriétés font de ces molécules de outils potentiels intéressants pour l’imagerie cérébrale de la MA. / Magnetic resonance imaging (MRI) has become a key tool for both clinical and preclinical research. Developing innovative neuroimaging techniques specifically designed for small animal models is therefore crucial to validate biological hypotheses and screen new drugs. Amyloid plaques and neurofibrillary tangles are the major lesions characterizing Alzheimer’s disease (AD) and intensive research has been carried out in order to enable the accurate detection of amyloid plaques using high-field MRI. However, only a small portion of plaques are spontaneously detected by MRI and no method is so far available for neurofibrillary tangles imaging. Here, we develop two contrast-enhanced MRI techniques to improve amyloid plaques detection using high-field MRI. The first part of this work focuses on Gadolinium-Staining for amyloid plaques detection by MRI. We performed a comparative study between mice model of amyloi-dosis and non-human primate models, which al-lowed us to gain a better understanding of the origin of the contrast induced by amyloid plaques when performing contrast-enhanced MRI. We also showed for the first time that Gd-stained MRI is able to detect amyloid plaques in human-AD brain tis-sues. The second part of this manuscript describes two novel single-domain antibodies (VHHs or nano-bodies) that are able to specifically recognize and bind amyloid plaques or neurofibrillary tangles. The detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of the blood-brain barrier. Interestingly, VHH exhibiting a basic isoelectric point are able to transmigrate across the blood-brain barrier, thus making them promising tools for in vivo imaging of AD’s lesion at high-field MR.

Recent progress in magnetic resonance imaging (MRI) has opened the way for micron-scale resolution, and thus for imaging biological cells. In this thesis work, we performed magnetic resonance microscopy (MRM) on the nervous system of Aplysia californica, a model particularly suited due to its simplicity and to its very large neuronal cell bodies, in the aim of studying cellular-scale processes with various MR contrasts. Experiments were performed on a 17.2 Tesla horizontal magnet, at resolutions down to 25 µm isotropic. Initial work consisted in conceiving and building radiofrequency microcoils adapted to the size of single neurons and ganglia. The first major part of the project consisted in using the manganese ion (Mn2+) as neural tract tracer in the buccal ganglia of Aplysia. Manganese is an MR contrast agent that enters neurons via voltage-gated calcium channels. We performed the mapping of axonal projections from motor neurons into the peripheral nerves of the buccal ganglia. We also confirmed the existence of active Mn2+ transport inside the neural network upon activation with the neurotransmitter dopamine. In the second major part of the project, we tested the potential of two diffusion MRI sequences for microscopy. On the one hand, we explored a very original mechanism for diffusion weighting, DESIRE (Diffusion Enhancement of SIgnal and REsolution), particularly suited for small samples. The two-dimensional DESIRE sequence was implemented and successfully tested on phantoms. The measured enhancement was consistent with theoretical predictions. Using this sequence to produce diffusion weighted images with an unprecedented contrast in biological tissue remains a challenge. On the other hand, a more "standard" sequence was implemented to measure the apparent diffusion coefficient (ADC) in nervous tissue with MRM. This sequence was a three-dimensional DP-FISP (Diffusion Prepared Fast Imaging with Steady-state free Precession), which met criteria for high resolution in a short acquisition time, with minimal artifacts. Using this sequence, we studied the changes in water ADC at different scales in the nervous system, triggered by cellular challenges. The challenges were hypotonic shock or exposure to ouabain. ADC measurements were performed on single isolated neuronal bodies and on ganglia tissue, before and after challenge. Both types of stress produced an ADC increase inside the cell and an ADC decrease at tissue level. The results favor the hypothesis that the increase in membrane surface area associated with cell swelling is responsible for the decrease of water ADC in tissue, typically measured in ischemia or other conditions associated with cell swelling.