published_or_final_version / abstract / toc / Physics / Master / Master of Philosophy

published_or_final_version / Physics / Master / Master of Philosophy

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Although neurotrophins are conventionally associated with the proper growth and survival of developing neurons, there is increasing evidence that they play an equally significant role in the functions of adult neurons. Specifically, brain derived neurotrophic factor activation of its preferred receptor TrkB is essential in the regulation of motoneuronal activity. Neurotrophin‐dependent and independent activation of TrkB regulates the motoneuronal dendritic integrity, and maintains unique classes of synapses. In addition, it regulates the expression and function of ion channels as well as the strength of inhibitory and excitatory synapses via different intracellular pathways. The recent physiological findings in the regulatory roles of TrkB are implicative of its presence on motoneuronal dendrites. Although, the expression of TrkB in the soma has long been confirmed, its distribution on the dendritic tree of motoneurons remains unknown. We aimed to examine the distribution of TrkB in the cytoplasm and membrane‐associated regions of the dendritic tree of adult neck motoneurons. We have determined, via confocal microscopy, that TrkB is present and abundant throughout the cytoplasm and the membrane‐associated regions of motoneuronal dendrites as well as the soma. TrkB is organized in clusters and its distribution is best described as punctated. We then developed a technique to separately extract and quantify the TrkB immunoreactivity associated with the membrane and the cytoplasm as function of distance from the soma and dendritic tree. We have demonstrated that there is no bias in TrkB immunoreactivity to a specific region of the dendritic tree in five trapezius motoneurons. These observations were confirmed for both cytoplasmic and membrane‐associated TrkB. There is compelling evidence that both mature full‐length and immature hypoglycosylated TrkB isoforms are active in strengthening the response to excitatory synapses in motoneurons. We identified the full length TrkB as well as 3 hypoglycosylated isoforms in cervical spinal segments that contain trapezius motoneurons and phrenic motoneurons. Taken together, these data indicate that TrkB is likely involved in regulating and maintaining different classes of ion channels and synapses on the dendrites as well as the soma. Various isoforms of TrkB may also be involved in regulating motoneuronal activity. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2014-01-14 12:48:21.357

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Cellulose is the main load-bearing component in plant fibre due to its covalent β-1→4- link that bonds glucose molecules into a flat ribbon and tight network of intra- and intermolecular hydrogen bonds. It is possible to manipulate the intra- and intermolecular hydrogen bonds in order to embed highly crystalline cellulose in a matrix of non-crystalline cellulose, thereby creating self-reinforced cellulose composites. Cellulose is an excellent choice of raw material for the production of sustainable and high-strength composites by selfconsolidation of cellulose since it is readily biodegradable and widely available. Nowadays, the cellulose industry makes extensive use of solvents. A multitude of solvents for cellulose is available but only a few have been explored up to the semi-industrial scale and can qualify as "sustainable" processes. An effective solvent for cellulose is a mixture of the LiCl salt and organic solvent N,N-Dimethylacetamide (DMAc). Once cellulose has been dissolved, the cellulose/LiCl/DMAc mixture can be precipitated in water. Preliminary results showed that a solution of 1 wt.% kraft cellulose in 8 wt.% LiCl/DMAc that was precipitated in water formed an hydrogel where cellulose chains were held in their amorphous state and in which no crystalline phase was detected by wide angle X-ray diffraction (WAXD). The initially amorphous cellulose started crystallizing by cross-linking of hydrogen bonds between the hydroxyl groups of the cellulose chains when the cellulose gel was dried and the water to cellulose ratio reached 7 g/g. The final form was poorly crystalline but distinct from amorphous cellulose. In order to study all-cellulose composites at a fundamental level, model all-cellulose composite films were prepared by partly dissolving microcrystalline cellulose (MCC) powder in an 8% LiCl/DMAc solution. Cellulose solutions were precipitated and the resulting gels were dried by vacuum-bagging to produce films approximately 0.2-0.3 mm thick. Wide-angle X-ray scattering (WAXS) and solid-state ¹³C NMR spectra were used to characterize molecular packing. The MCC was transformed to relatively slender crystallites of cellulose I in a matrix of paracrystalline and amorphous cellulose. Paracrystalline cellulose was distinguished from amorphous cellulose by a displaced and relatively narrow WAXS peak, by a 4 ppm displacement of the C-4 ¹³C NMR peak, and by values of T₂(H) closer to those for crystalline cellulose than disordered cellulose. Cellulose II was not formed in any of the composites studied. The ratio of cellulose to solvent was varied, with greatest transformation observed for c < 15%, where c is the weight of cellulose expressed as percentage of the total weight of cellulose, LiCl and DMAc. The dissolution time was varied between 1 and 48 h, with only slight changes occuring beyond 4 h. Transmission electron microscopy (TEM) was employed to assess the morphology of the composites. During dissolution, MCC in the form of fibrous fragments were split into thinner cellulose fibrils. The composites were tested in tension and fracture surfaces were inspected by scanning electron microscopy (SEM). It was found that the mechanical properties and final morphology of all-cellulose composites is primarily controlled by the rate of precipitation, initial cellulose concentration and dissolution time. All-cellulose composites were produced with a tensile strength of up to 106 MPa, modulus up to 7.6 GPa and strain-to-failure around 6%. The precipitation conditions were found to play a large role in the optimisation of the mechanical properties by limiting the amount of defects induced by differential shrinkage. Dynamic mechanical analysis was used to study the viscoelasticity of all-cellulose composites over temperatures ranging from -150℃ to 370℃. A β relaxation was found between -72 and -45℃ and was characterized by an activation energy of ~77.5±9.9 kJ/mol, which is consistent with the relaxation of the main chain through co-operative inter- and intramolecular motion. The damping at the β peak generally decreases with an increase in the crystallinity due to enhanced restriction of the molecular motion. For c≤15%, the crystallinity index and damping generally decreased with an increasing dissolution time, whereas the size distribution of the mobile entities increases. A simple model of crystallinity-controlled relaxation does not explain this phenomenon. It is proposed that the enhanced swelling of the cellulose in solution after higher dissolution times provides a more uniform distribution of the crystallites within the matrix resulting in enhanced molecular constriction of the matrix material. For c = 20%, however, the trend was the opposite when the dissolution time was increased. In this case, a slight increase in crystallinity and an increasing damping were observed along with a decrease in the size distribution of the mobile entities. This phenomenon corresponds to a re-crystallisation accompanied with a poor consolidation of the composite. A relaxation ɑ₂ at ~200℃ is attributed to the micro-Brownian motion of cellulose chains and is believed to be the most important glass transition for cellulose. The temperature of ɑ₂ decreased with an increase in crystallinity supposedly due to enhanced restriction of the mobile molecular phase. A high temperature relaxation which exhibited two distinct peaks, ɑ₁﹐₂ at ~300℃ and ɑ₁﹐₁ at ~320℃, were observed. ɑ₁﹐₂ is prevalent in the cellulose with a low crystallinity. A DMA scan performed at a slow heating rate enabled the determination of the activation energy for this peak as being negative. Consequently, ɑ₁﹐₂ was attributed to the thermal degradation onset of the surface exposed cellulose chains. ɑ₁﹐₁ was prevalent in higher crystallinity cellulose and accordingly corresponds to the relaxation of the crystalline chains once the amorphous portion starts degrading, probably due to slippage between crystallites. The relative ɑ₁﹐₁/ɑ₁﹐₂ peak intensity ratio was highly correlated to the amount of exposed chains on the surface of the cellulose crystallites. Novel aerogels (or aerocellulose) based on all-cellulose composites were also prepared by partially dissolving microcrystalline cellulose (MCC) in an 8 wt.% LiCl/DMAc solution. Cellulose gels were precipitated and then processed by freeze-drying to maintain the openness of the structure. The density of aerocellulose increased with the initial cellulose concentration and ranged from 116 to 350 kg.m⁻³. Aerocellulose with relatively high mechanical properties were successfully produced. The flexural strength and modulus of the aerocellulose was measured up to 8.1 MPa and 280 MPa, respectively.

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.