Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: School of Nursing. Bibliography: leaves 165-183. Also available online via the Internet.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

1] Spinal cords from mouse embryos were successfully prepared and maintained in primary dissociated cell culture, for periods in excess of 10 weeks. 2] Stable intracellular recordings were made from spinal neurones which had been sustained in these cultures. 3] Experiments were made on these spinal neurones using various amino acids and peptides. Solutions of these compounds were discretely applied by pressure ejection. 4] L-Glutamate, GABA and glycine evoked responses which appeared the same as those documented previously. 5] Ethylene-diamine did not evoke a response from the spinal neurones tested. 6] Only a small percentage of the spinal neurones responded to met5- and leu5 - enkephalin, FMRFamide, neurotensin and glycyl L-glutamine. Supplementing the cultures with tissue from other organs did not increase the percentage of spinal neurones which were capable of responding to peptide. 7] Met5 -enkephalin and leu5 -enkephalin each evoked responses from the spinal neurones. 8] The enkephalin-evoked depolarizations accompanied by an increased input resistance were apparently voltage dependent. These responses were abolished at potentials more negative than -90mV and did not invert under normal recording conditions. 9] The enkephalin-evoked depolarizations associated with a decreased input resistance had extrapolated inversion potentials of -20mV. No voltage dependence was seen. 10] Enkephalins also evoked responses which had an inversion potential close to the resting membrane potential. These were accompanied by a decreased input resistance and were not desensitized by prolonged application of peptide. 11] None of these responses showed obvious desensitization with prolonged application, however, they were all attenuated by naloxone. 12] Met5 -enkephalin was apparently more potent than leu5 -enkephalin on a small number of neurones. Furthermore, met5 -enkephalin application, during the weaker response from those neurones to leu5 -enkephalin, evoked a attenuated response. 13] FMRFamide evoked two responses from these spinal neurones. These responses were seen separately and mixed. In the latter case they were referred to as biphasic responses. 14] The depolarizing response to FMRFamide was accompanied by an increase in input resistance. Potassium had some involvement in these responses. 15] The FMRFamide responses which were accompanied by a decreased input resistance showed a great variety of inversion potentials between neurones. These actions were dependent upon sodium and chloride ions. 16] Enkephalin and FMRFamide, when applied separately to the same spinal neurone, did not evoke the same response. 17] Responses evoked by neurotensin were hyperpolarizations associated with a decreased input resistance. These responses were dependent upon potassium and independent of chloride ions. 18] Glycyl L-glutamine evoked two types of hyperpolarizing response from the spinal neurones. These could appear separately or combined. 19] The faster responses to glycyl L-glutamine were apparently dependent on potassium ions. 20] The slower responses to glycyl L-glutamine were apparently insensitive to changes in extracellular potassium or chloride. However, these responses were sensitive to intracellular injection of chloride ions. 21] At concentrations of peptide which evoked a response from other spinal neurones, none of the peptides produced any measurable modulation of amino acid-evoked responses.

The question of how a single cell can grow, divide, and ultimately acquire a distinct function within an adult animal is central to the field of developmental biology. An elegant way to address this question is by studying the specification of a specific cell type, for example, vertebrate motoneurons. For an animal to be able to move and behave appropriately, individual motoneurons (MNs) must correctly innervate specific muscles. For this to happen, MNs must first be specified and then must differentiate into distinct subtypes, each of which is classified in part by the muscle it innervates. MN subtype specification is dependent on both the acquisition of MN-specific characteristics as well as the failure to acquire characteristics specific to interneurons, cells that only innervate other neurons. The entire process of specification is initiated in progenitor cells and relies on the correct spatial and temporal expression of specific genes. Previous work in various vertebrate models has identified some of the key genes involved in MN specification, most notably transcription factors such as olig2, nkx6s, lhxs, mnxs, and islet1. In this dissertation, I use the zebrafish model to demonstrate novel roles in MN specification for two of these families of transcription factors - the lhxs and the mnxs. I provide evidence that both lhx3 and lhx4 are necessary for normal MN and ventral interneuron (IN) development and work by preventing MNs from expressing IN-specific characteristics. I also show that mnx1, mnx2a, and mnx2b are necessary in MNs both to promote the acquisition of some MN subtype-specific characteristics and to prevent the acquisition of some IN-specific characteristics and appear to be working in part through interactions with islet1. Finally, I identify an intermediate filament gene, inab, as being expressed in a subset of zebrafish MNs and a ventral IN and as having a potential role in the axon outgrowth of a specific MN subtype. Together, this work provides evidence for a mechanism of MN specification dependent on the expression of genes that both promote aspects of MN fate and inhibit aspects of IN fate. This dissertation includes previously unpublished co-authored material.

No description available.

M.A. / The power of positive thinking to promote and maintain well-being has enjoyed widespread popular appeal. Spurred largely by the development of the Life Orientation Test (LOT) (Scheier & Carver, 1985), mounting evidence now attests to the apparent benefits conferred by an optimistic life outlook. Optimism, the inclination to expect favourable outcomes, has been linked to both psychological and physical well-being (Taylor, et al., 1992; Cheng & Hamid, 1997; Marshall, Wortman, Kusalas, Hervig, & Vickers, 1992; Scheier, Carver & Bridges, 1994; Scheier et al., 1989; Segerstrom, Taylor, Kemeny, & Fahey, 1998). Optimists and pessimists have been shown to differ in the manner in which they cope with the challenges in their lives. They differ in their stable coping tendencies and in the kinds of coping responses that they spontaneously generate when given hypothetical coping situations (Scheier, Weintraub, & Carver, 1986). Optimists also differ from pessimists in the manner in which they cope with serious disease and with concerns about specific health threats (Friedman, et al., 1992), and these coping mechanisms in optimists in turn have been linked to improved physical and psychological functioning. There is also a clear biological link between optimism and physical well-being and this has been noted in such areas as immune functioning (Peterson & Bossio, 2002). The current study examines these apparent benefits of optimism on health and explores the medical bases for this connection. The study also examines whether these benefits may be of use in the treatment and recovery from the unique secondary medical complications experienced by those who are spinal cord injured.

Spinal Cord Injury (SCI) often causes cell death, demyelination, axonal degeneration and cavitation, resulting in functional motor and sensory loss below the site of injury. In an attempt to overcome SCI, the regenerating neurons require a permissive environment to promote their ability to reconnect. We report a novel thiolated hyaluronic acid (HA) hydrogel scaffold that can be used to repair the injured spinal cord. More specifically, thiolated hyaluronic acid hydrogels with varying thiol concentrations were successfully synthesized. The amount of thiol groups was measured spectrophotometrically using Ellman’s test. HA gels with different crosslinking densities were synthesized and the water content of the hydrogels was determined. The thermal behavior of the HA gels were studied by DSC. The strength of the hydrogels with varying thiol group content was evaluated by a rheometer. In addition, in vitro enzymatic degradation was performed through submerge the hydrogels in 200U/ml of hyaluronidase solution and incubate at 37°C. According to the result of the present study, this novel hydrogel shows great potential to serve as a 3D cell-patterning scaffold which can be inserted into a hollow fiber channel that could be used to promote regeneration after the SCI.

Maturation of motor control, including movements that can be autonomously generated by spinal circuits, relies on the development of key inputs to spinal circuitry. In particular, the development of supraspinal, sensory and motor fibers come together to form organized spinal circuits capable of producing skilled movements that are volitionally controlled. Primitive reflexes such as the palmar grasp reflex (PGR) are known to disappear during development; presumably giving way to more volitional control of hand grasping. However, the underlying changes to the spinal circuitry responsible for this transition remain to be determined. dI3 INs, a class of dorsal spinal interneurons, have positioned themselves as key mediators of reflexive grasping in early development and grasping in adult mice. The first aim of the study focused on determining the developmental time point at which the PGR disappeared. Our studies demonstrated that the PGR was lost by the third week of development. The second aim of this study focused on identifying changes in sensory innervation, presynaptic inhibition and supraspinal excitation to dI3 INs that might account for the loss of this reflex. Our studies demonstrated that while sensory innervation remained constant during development, presynaptic inhibitory terminals onto sensory afferents were found to increase during development. In addition, we report that dI3 INs receive decreasing corticospinal (CST) input during development. While these developmental changes do not fully account for the disappearance of the PGR, they provide valuable insights into how a reflex centered on a particular population develops.

Successful engraftment is highly dependent on the quality and quantity of stem cells and nucleated cells in cord blood. Storage of umbilical cord blood is expensive and it will be very useful if factors that influence cell count and viability could be identified to aid in the decision to process and store cord blood collections for the ultimate aim of successful engraftment. This study determined the standards for laboratory parameters of haematopoietic potential, such as collection volume, post processing volume, CD34+/45+ cell counts and viability of stem cells and leukocytes and cell ratios for the cord blood bank. In this research we determined whether maternal age and infant gender have an effect on laboratory parameters. We studied the effect of 4°C and room temperature (RT) as well as the period of storage on the same laboratory parameters. The quality and recovery of stem cells and leukocytes after laboratory manipulation was determined. Established standards for leukocyte count, stem cell count and collection volume compare well with other international UCB banks. Maternal age and infant gender have no influence on laboratory parameters and could therefore not be used as a measure of cell quantity and quality prior to processing. Cell count and cell viability of stem cells is not significantly influenced by 4°C or RT temperature or 24h and 48h storage. Leukocyte cell count and viability is not affected by storage temperature, but increased storage periods showed high levels of leukocyte cell count deterioration and decreased leukocyte cell viability. Processing of UCB causes significant cell loss in stem cells and leukocytes. Processing or no processing of UCB has showed no affect on the viability of stem cells stored at different storage periods and temperature. Temperature has no affect on leukocyte cell counts and viability of either processed or unprocessed leukocytes but increased storage periods dramatically decrease leukocyte count and viability. The information generated by this study will assist in the process of optimizing the storage of UCB. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Immunology / unrestricted

This work presents an alternative approach to the production of cellulose-based biomaterials. Instead of extracting, processing and regenerating plant and or bacteria-derived cellulose into a biomaterial, my work established a decellularization protocol to remove cellular plant content from plant tissue resulting in a scaffold composed of cellulose with the evolved architecture of the plant cell wall. Tracheophyte plants, including clubmosses, horsetails, and ferns, gymnosperms and angiosperms, have evolved distinct vascular structures that support the transport of water and nutrients in xylem and phloem that form the vascular bundles (VBs)1. This thesis took it’s inspiration from the dense, linearly arranged, parallel microchannels which include (VBs) in the stalks of Asparagus officinalis possess an architecture with striking similarities to biomaterial scaffolds intended to repair damaged tissue. My work demonstrated that the plant cell wall contains many of the ideal characteristics of a medical biomaterial. The scaffold is biocompatible with mammalian cells and maintains high viability even with cell densities comparable to commercially available scaffolds. The cellulose scaffold could be biochemically functionalized or cross-linked to control the scaffolds' surface biochemistry and mechanical properties. My in vivo model demonstrated that the lignocellulose scaffold did not elicit a foreign body response. The scaffold was permissive to host cell invasion, including active host fibroblast, leading to the deposition of host collagen extracellular matrix. Importantly, active blood vessels formed within the scaffold to support the population of host cells. The scaffold retained much of its original shape and provided an inert, pro-vascular long-term environment for host cells to invade. Taken together, this led to the hypothesis that the innate plant cell wall architecture could restore the function of injured tissue, specifically that the vascular bundles could be used to promote axonal regeneration in spinal cord injuries. Rats with complete spinal cord transection were implanted with cellulose scaffolds with vascular bundles. Animals that received plant-derived scaffolds demonstrated a significant improvement in motor function. This thesis defines a novel and parallel route for exploiting naturally occurring plant microarchitectures of the underlying crystalline cellulose scaffold.