Several cell lineages derived from the mesectoderm occupy and contact axons in the midline of the developing Drosophila CNS. Which of these midline cell lineages contribute to commissural axon morphogenesis? In the absence of the midline cells as in mutant embryos of the single-minded gene, the longitudinal axons collapse at the midline and commissural axons are absent. Despite the similarity in axon tract phenotype, the midline cells in slit mutant embryos survive but are displaced. Correct cytoarchitecture of the midline cells is therefore dependent on the activity of Sli protein which is in turn necessary for commissure formation. In mutant embryos displaying a fused commissure phenotype (rhomboid and Star), the anterior and middle midline glia cells failed to migrate and died by apoptosis after commissure development. In these mutants the number of cells in midline neuronal lineages was reduced before defects in midline glia were apparent. In wildtype embryos approximately 50% of cells in three midline glia lineages died by apoptosis after commissure separation as shown by ultrastructural and enhancer trap analysis. Midline glia lineages died by apoptosis as shown morphologically and by their survival in embryos deficient in the cell death gene reaper. Quantitative analysis revealed variable survival of cells in the anterior, middle and posterior midline glial lineages during embryogenesis suggesting heterogeneity among these cells. The presence of extra anterior, middle and posterior midline glial lineages relative to wildtype numbers in reaper mutant embryos suggested that cell death regulates either midline glial proliferation or cell fate determination during wildtype embryogenesis. Alterations in axon-glia contact correlated with changes in midline glia survival. What happens to apoptotic cells in the Drosophila embryonic central nervous system? A variety of glia in the nervous system were capable of phagocytic activity including midline glia, longitudinal tract glia, nerve root glia and subperineurial glia, revealed by electron microscopy. However, the majority of apoptotic cells in the central nervous system were engulfed by subperineurial glia. In the absence of phagocytic haemocytes in embryos mutant for the Bicaudal-d gene, most apoptotic cells were retained in subperineurial glia at the outer edges of the central nervous system. Apoptotic cells were expelled from the central nervous system of Bicaudal-d mutant embryos suggesting that phagocytic haemocytes participate in the removal of apoptotic cells from the central nervous system but are not essential for this process. / Thesis / Doctor of Philosophy (PhD)

<p> Osteoarthritis (OA) is the most common arthritis, and the second most common diagnosis leading to disability. While loss of joint function is disabling, patients report that the greatest disabler of OA is the pain. Unfortunately, OA pain remains an unmet medical need. Numerous mechanisms have been proposed for the pathogenesis of OA pain. However, none of these mechanisms has led to satisfactory evidence-based treatment for OA pain. There is a critical need to address the mechanisms for OA pain due to the aging demographics and the prevalence of OA in older adults. This thesis project was aimed to study neural mechanisms for OA pain. The general hypothesis was that the pain of OA arises as a result of phenotypic changes in primary sensory neurons, especially in larger diameter A-fiber neurons. In vivo intracellular recordings were used to determine changes in specific populations of DRG neuron in a surgical knee derangement model of OA in the rat. It was found that AB-fiber low threshold mechanoreceptors, particularly muscle spindle afferents underwent significant changes (including changes in action potential configurations and in responses to repetitive stimulation) one month following the model induction when histopathological changes of the knee joint and the nocifensive behaviors of the affected lower limb favor OA. Nociceptors, including C-, As- and AB-fiber neurons remained largely unchanged at one month OA. AB-fiber high threshold mechanoreceptors exhibited significant changes at two month OA, a later phase during the progression of OA. The data demonstrate that distinct populations of dorsal root ganglia neuron are altered during the progression of OA, which might be the neuronal basis for clinical presentations of sensory deficit in OA including pain and loss of proprioception. The data also suggest that the pain in OA might be a form of neuropathic pain. </p> / Thesis / Doctor of Philosophy (PhD)

This thesis focuses on an adaptive quadratic spiking model of a motoneuron that is both versatile in its ability to represent a range of experimentally observed neuronal firing patterns as well as computationally efficient for large network simulation. The objective of research is to fit membrane voltage data to the model using a parameter estimation approach involving simulated annealing. By manipulating the system dynamics of the model, a realizable model with linear parameterization (LP) can be obtained to simplify the estimation process. With a persistently excited current input applied to the model, simulated annealing is used to efficiently determine the best model parameters that minimize the square error function between the membrane voltage reference data and data generated by the LP model. Results obtained through simulation of this approach show feasibility to predict a range of different neuron firing patterns.

Spinal cord injury (SCI) often leads to irreversible damage, and permanent paralysis inferior to the injury is common (Leibinger et al., 2013). Injury to the spinal cord occurs in two phases. In the first phase, components of the spinal cord are subject to mechanical trauma causing direct damage. In the second phase, damage spreads from the area of injury through molecular processes. Several studies have linked M1 "pro-inflammatory" macrophages to exacerbation of damage by inducing dieback of dystrophic axons, but not healthy axons, through direct cellular contact. Several studies have identified the presence of macrophage subtypes at specific time. A literature review was conducted in order to summarize these findings (Busch, Horn, Silver, & Silver, 2009; Evans et al., 2014; Horn, Busch, Hawthorne, van Rooijen, & Silver, 2008; Kigerl et al., 2009; Shechter et al., 2013). Although the full mechanism behind the process of M1 macrophage-mediated dieback of dystrophic axons is unclear, matrix metalloproteinase-9 (MMP-9) produced by these macrophages has been shown to play a role. However, the specific interaction between MMP-9 and neurons is under investigation. The research described explores the relationship between MMP-9 and fractalkine (CX3CL1), a surface protein expressed by CNS neurons. SDS-PAGE and western blot were used to determine whether the presence of MMP-9 increases the cleavage of fractalkine at several time intervals. At a concentration of 300ng/ml, MMP-9 was not found to demonstrate cleavage of fractalkine.

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Lhx genes are a sub-family of Hox genes that play important roles in animal development. In Caenorhabditis elegans there are seven Lhx genes, including the founding family member lin-11. The lin-11 gene is necessary for the specification of neuronal and reproductive tissues. My thesis work has involved understanding the mechanism of lin-11 regulation and its function in these tissues. To this end, I addressed two distinct but complementary questions, one of which focused on how transcriptional regulation of lin-11 occurs and the second on the role of LIN-11 protein domains/regions. My work on the transcriptional regulation has uncovered important roles of two of the largest lin-11 introns, intron 3 and intron 7. These introns promote lin-11 expression in non-overlapping sets of amphid neurons. Based on gene expression patterns and behavioural assays, intron 3 is capable of restoring lin-11 function in lin-11(n389 ) null mutant allele. Comparison of intron 3-driven reporter expression in the neuronal cell types between C. elegans and C. briggsae has revealed cis and trans evolutionary changes in lin-11 regulation between the two species. Functional dissection of the introns in C. elegans has led to the identification of three distinct non-overlapping enhancers, each specific for a single amphid neuron, i.e., RIC, AIZ, and AVG. I have also identified four transcription factors, SKN-1, CEH-6, CRH-1, and CES-1, that act through these enhancers to regulate neuronal expression of lin-11. Furthermore, I have characterized the function of the LIM domains and a proline-rich (PRR) C-terminus region of LIN-11 in the specification of neuronal and reproductive tissues. My work shows that while the LIM domains are required for LIN-11 function in these tissues, the PRR region is dispensable. I have also examined the functional conservation of lin-11 domains using two other Lhx genes, Drosophila melanogaster (dLim1) and Mus musculus (Lhx1 ), and found that both of these genes were able to rescue lin-11 defects. Together, my work has significantly advanced our understanding of transcriptional regulation of lin-11, the importance of LIM domains in tissue formation, and functional conservation of Lhx genes across phyla. / Thesis / Doctor of Philosophy (PhD)