Abstract The first part of this research was focused on the relationship between injury severity and cell death mechanisms after spinal cord ischemia-reperfusion injury. The major blood supply to the thoracolumbar spinal cord comes from the segmental arteries originating from the thoracoabdominal aorta. Paraplegia cause by spinal cord ischemia is a devastating complication of thoracoabdominal aortic surgery. Previous studies indicated that ischemia-reperfusion injury of the central nervous system causes two distinct types of cell death, necrosis and apoptosis. It was also implicated that the intensity of injury can somehow affect the cell death mechanisms. In the first series of our experiments, by occluding the descending thoracic aorta with or without simultaneously inducing hypovolemic hypotension in rats, we established a model of experimental spinal cord ischemia-reperfusion (SCIR) in which the injury severity can be controlled. Recordings of carotid blood pressure (CBP) and spinal cord blood flow (SCBF) showed that aortic occlusion induced dramatic CBP elevation but SCBF drop in both the normotensive (NT) and hypotensive (HT) groups. However, the HT group demonstrated significantly lower SCBF during aortic occlusion, and much slower elevation of SCBF after reperfusion, and extremely poor neurological performance. Spinal cord lesions were characterized by infarction associated with extensive necrotic cell death, but little apoptosis and caspase-3 activity. In contrast, in the NT group, SCIR resulted in minor tissue destruction associated with persistently abundant apoptosis, augmented caspase-3 activity, and favorable functional outcome. The relative sparing of motoneurons in the ventral horns from apoptosis might have accounted for the minor functional impairment in the NT group. The severity of ischemia-reperfusion (I/R) injury was found to have substantial impact on the histopathological changes and cell death mechanisms, which correlated with neurological performance. These findings implicate that injury severity and duration after injury are two critical factors to be considered in therapeutic intervention. Based on the knowledge that bPrevious studies have implicated both excitotoxicity and apoptosis are involved in the pathogenesis of SCIR injury, we proposedtested the possibility that the N-methyl-D-aspartate (NMDA) receptor antagonist (dizocilpine maleate: (MK801) and the protein synthesis inhibitor (cycloheximide) would produce a synergic effect in the treatment of SCIR injury. In the second series of experiments, I/R iSpinal cord ischemia-reperfusion injury was induced by a thoracic aortic occlusion and blood volume reduction, followed by reperfusion and volume restoration. ischemia-reperfusion Rats were treated with vehicle, MK801, cycloheximide, or combination of MK801 and cycloheximide in combination. The MK801 and combined therapy group got a better recovery of hHind limb motor function recovery was better in the MK801 and combined-therapy groups than in the control and cycloheximide groups. On the 7th day after ischemia-reperfusion injury, all three treated groups showed significantly higher neuronal survival rates (NSR) than that of the control group. Among the three treated groups, the combined-treatment group showed the highest NSR. In addition, the Ttherapeutic effect of the combined-treatment group (27.4% increase of NSR) iwas better than the anticipated by the addition of MK801 and cycloheximide based on NSR data group. The number of apoptotic cells of was significantly reduced in the cycloheximide group and the combined-treatment group, as compared to that of the control group. It was unchanged in the MK-801 group. These results suggest that combined treatments directed at blocking both NMDA receptor-mediated excitotoxic necrosis and caspase-mediated apoptosis might have synergic therapeutic potential in reducing SCIR injury. Mitogen-activated protein kinases (MAPKs) including c-Jun N-terminal kinases (JNK), p38, and extracellular signal-regulated kinases (ERK), play important roles in the transduction of stressful signals and the integration of cellular responses. Although it has been generally held that the JNK and p38 pathways are related to cell death and degeneration, while the ERK pathway, cell proliferation and survival, controversy still exists. The roles of the ERK pathway in I/R injury of the CNS, in particular, remain to be clarified, because contradictory data have been reported by different investigators. Given this controversy, in the third series of experiments, we examined in injured spinal cords the temporal and spatial profiles of ERK1/2 activation following SCIR, and the effects of inhibiting the kinase that phosphorylates ERK1/2, MEK. The results showed that I/R injury induced an immediate phosphorylation of ERK1/2 in the spinal cord, which was alleviated by a MEK inhibitor, U0126. The control group was characterized by poorer neurological outcome, more severe tissue destruction, pronounced apoptosis, and lower neuronal survival. In contrast, the U0126-treated group demonstrated more apparent improvement of hind limb motor function, less tissue destruction, lack of apoptosis, and higher neuronal survival. In addition, administration of U0126 also significantly increased the activation of nuclear factor-£eB (NF-£eB) and the expression of cellular inhibitor of apoptosis protein 2 (c-IAP2). These findings implicate that the mechanisms underlying the neuroprotection afforded by ERK1/2 inhibition may be through the NF-£eB-c-IAP2 axis. The activation of the MEK-ERK signaling pathway appeared to be harmful in SCIR injury. Strategies aimed at blocking this pathway may bear potential therapeutic benefits in the treatment of SCIR injury. The second part of the research was focused on the pathophysiology of surgery-associated paraspinal muscle injury and measures to protect surgically violated paraspinal muscles. The wide dissection and forceful retraction of paraspinal muscles which are often required for posterior spinal sugery may severely jeopardize the muscles structurally and functionally. Immediate posteoperative pathological changes in the surgically violated paraspinal muscles may cause severe pain and a delay of patient ambulation. Long-term sequelae of surgical injury of paraspinal muscles include chronic back pain and impaired back muscle strength. Ironically, being a common complication of posterior spinal surgery, paraspinal muscle injury is so often neglected. Limited previous data indicate that the underlying pathophysiology of muscle damage involve both mechanical and ischemic mechanisms. We hypothesized that surgical dissection and retraction may produce oxidative stress within the paraspinal muscles. Meanwhile, we also proposed that the oxidative stress may trigger certain protective mechanisms within the insulted muscles. The first part of our study was a human study conducted to assess the significance of oxidative stress, and the relationship between it and the stress response mediated by heat shock protein 70 (HSP70) induction within paraspinal muscles under intraoperative retraction. A group of patients with lumbar spondylolisthesis treated with posterolateral lumbar spinal fusion, pedicle fixation and laminectomy were enrolled. Multifidus muscle specimens were harvested intraoperatively before, at designated time points during, and after surgical retraction. Muscle samples were analyzed for HSP70 and malondialdehyde (MDA) levels. Both HSP70 expression and MDA production within multifidus muscle cells were increased significantly by retraction. HSP70 expression then dropped after a peak at 1.5 hr of retraction, whereas MDA levels remained elevated even after release of retractors for reperfusion of the muscles. Histopathological and immunohistochemical evidence indicated that the decline of HSP70 synthesis within muscle cells after prolonged retraction was the result of severe muscle damage. These results highlighted the noxious impact of intraoperative retraction on human paraspinal muscles, and the significance of oxidative stress at the cellular and molecular levels. It is also implicated that intraoperative maneuvers aimed at reducing the oxidative stress within the paraspinal muscles may help attenuating surgery-associated paraspinal muscle damage. Given the findings of the first part of our study, and the knowledge that inflammation is a major postoperative pathological finding in surgically injured paraspinal muscles, we proceeded to examine the roles of two important inflammatory mediators, cyclooxygenase (COX)-2 and nuclear factor (NF)-£eB, in the pathogenesis of retraction-associated paraspinal muscle injury. A rat model of paraspinal muscle dissection and retraction that mimicks the conditions in human posterior spinal surgery was established. In the control group, paraspinal muscles were dissected from the spine through a dorsal incision, and then laterally retracted. Paraspinal muscle specimens were harvested before, and at designated time points during and after persistent retraction. The time course of NF-£eB activation as well as the expression of COX-2 were examined. Severity of inflammation was evaluated based on histopathology and myeloperoxidase (MPO) activity. NF-£eB activation was inhibited by the administration of pyrrolidine dithiolcarbamate (PDTC) in the PDTC-treated group. In the control group, retraction induced an early increase of NF-£eB/DNA binding activity in paraspinal muscle cells, which persited throughout the whole course of retraction. COX-2 expression was not detectable until 1 day after surgery, and reached a peak at 3 days. The time course of COX-2 expression correlated with that of inflammatory pathology and MPO activity. Extensive muscle fiber loss and collagen fiber replacement were observed at 7 days after surgery. Pretreatment with PDTC inhibited intraoperative NF-£eB activation and greatly downregulated postoperative COX-2 expression and inflammation in the muscles. Fibrosis following inflammation was also significantly abolished by PDTC administration. These findings indicate that NF-£eB-regulated COX-2 expression and inflammation play an important role in the pathogenesis of surgery-associated paraspinal muscle injury. Therapeutic strategies involving NF-£eB inhibition may be applicable to the prevention of such injury.

Knowledge of the mechanics of the lens capsule is crucial for improving cataract surgery as well as understanding better the physiological role of the lens capsule in the process of accommodation. Previous research on the mechanical properties of the lens capsule contains many gaps and contradictions due to experimental limitations and inappropriate assumptions. Thus, the goal of this work is to quantify fully the regional, multiaxial mechanical behavior of the lens capsule and to calculate the change in stress and strain fields as a result of cataract surgery. Determining in situ the multiaxial mechanical behavior of the lens capsule required the design and construction of an experimental device capable of altering stresses in the capsule while measuring localized surface deformations. Tests performed on this device reveal that the meridional and circumferential strains align with the principal directions and are equivalent through most of the anterior lens capsule, except close to the equator where the meridional strain is greater. Furthermore, preconditioning effects were also found to be significant. Most importantly, however, these tests provide the data necessary for calculating material properties. This experimental system is advantageous in that it allows reconstruction of 3D geometry of the lens capsule and thereby quantification of curvature changes, as well as measurement of surface deformations that result from various surgical interventions. For instance, a continuous circular capsulorhexis (CCC) is commonly used during cataract surgery to create a hole in the anterior lens capsule (typically with a diameter of 5 mm). After the introduction of a CCC, strain was found to redistribute evenly from the meridional direction (retractional strain) to the circumferential direction (extensional strain), where both directional components of strain reached magnitudes up to 20% near the edge of the CCC. Furthermore, the curvature was found to increase at the edge of the CCC and remain the same near the equator, indicating that the mere introduction of a hole in the lens capsule will alter the focal characteristics of the lens and must therefore be considered in the design of an accommodative intraocular lens.

This dissertation describes the work done on two distinct projects. In the first part I sought to unravel the mechanisms that underlie the activity-dependent modulation of response in the excitation-secretion coupling of the neurohypophysis. In the second part, I optically monitored and analyzed the secondary structure changes accompanying amyloid fibril formation along multiple pathways, under both denaturing and near-physiological conditions. Neuronal plasticity plays an important role in regulating various biological systems by modulating release of hormones or neurotransmitters. The changing response to the same stimulus, depending on the context and previous stimulation events, is also the basis of learning and all higher order brain functions. The mechanisms behind this modulation are widely varied, and are often poorly understood in specific tissues. In this work, we examined excitation-secretion coupling in the neurohypophysis, a tissue composed of densely packed axons that secretes the hormones arginine vasopressin and oxytocin. The release of hormones depends not only on the overall level of activity in the gland, but also upon the specifics of the temporal pattern of stimulation. By optically monitoring the electrical activity using voltage sensitive dyes, we were able to investigate this plasticity in the intact gland. Varying extracellular potassium concentration in the bath, increasing interstitial space via hypertonic saline, and retarding potassium reuptake with ouabain all showed that extracellular potassium accumulation drives the depression of excitability. This effect is hidden from glass micro-electrode recordings because of the inevitable damage sustained by the surrounding tissue. Furthermore, no calcium mediated release mechanism played any significant role in the depression. Numerical simulations confirmed the findings and give more insight to the details of the mechanism. Deposits of amyloid fibrils, long, unbranched polymeric protein aggregates, are the molecular hallmark for a variety of human diseases, including Alzheimer's disease, Parkinson's disease, and type II diabetes. While the amyloid fibrils all share a characteristic cross-beta sheet structure, the proteins that make up the aggregates have no unifying theme in either native structure or function. In this research, I characterized the structural reordering that accompanies this aggregation using Fourier transform infrared spectroscopy (FTIR). Hen egg white lysozyme forms fibrillar aggregates with two distinct morphologies, depending on the growth conditions. At acidic pH with low ionic concentrations, lysozyme forms the fibrils with standard amyloid morphology. These aggregates are long and stiff but with the cross sectional area of a single monomer. At higher salt concentrations, the aggregation follows another pathway, under which oligomers initially form and later assemble into protofibrils. The oligomeric protofibrils are thicker than the monomeric filaments, but are much more curvilinear. These fibrils are not universally recognized as amyloidogenic aggregates. Using FTIR, I showed that both this aggregates are indeed amyloid structures, but that they are structurally distinct. While it is generally accepted that partial unfolding of the protein is a prerequisite for amyloid fibril formation, we found that native protein can be the substrate for amyloid growth when seeded with preformed oligomeric or protofibrillar aggregates. These seeded fibrils grown under near-physiological conditions are structurally indistinguishable from those grown from partially unfolded protein under denaturing conditions. This incorporation and restructuring of native monomers is characteristic of prion-like assembly.

Factorial designs have been widely used in many scientific and industrial settings, where it is important to distinguish "active'' or real factorial effects from "inactive" or noise factorial effects used to estimate residual or "error" terms. We propose a new approach to screen for active factorial effects from such experiments that utilizes the potential outcomes framework and is based on sequential posterior predictive model checks. One advantage of the proposed method lies in its ability to broaden the standard definition of active effects and to link their definition to the population of interest. Another important aspect of this approach is its conceptual connection to Fisherian randomization tests. As in the literature in design of experiments, the unreplicated case receives special attention and extensive simulation studies demonstrate the superiority of the proposed Bayesian approach over existing methods. The unreplicated case is also thoroughly explored. Extensions to three level and fractional factorial designs are discussed and illustrated using a classical seat belt example for the former and part of a stem-cell research collaborative project for the latter. / Statistics

The posterior parietal cortex (PPC) has been reliably implicated in visuospatial attention, such that the dorsal regions (dPPC) are associated with voluntary ‘top-down’ attention, whereas the ventral regions (vPPC) are associated with automatic ‘bottom-up’ attentional processes. The Attention-to-Memory model (AtoM: Ciaramelli, Grady, & Moscovitch, 2008) has suggested that it also plays a similar role in memory retrieval, suggesting that the PPC mediates a domain-general attentional process. Furthermore, domain-generality of attentional processes may account for differences in perception and memory function accompanying old age. This study examined domain-generality by determining the shared variance in performance of tasks thought to recruit top-down and bottom-up attentional processes mediated across both domains. Results clearly suggested generality across domains in top-down processing; and in bottom-up processing, depending on its operationalization. Ageing was characterized by an absence of shared variance across domains and slower reaction times during bottom-up attentional reorienting only in perception.

The posterior parietal cortex (PPC) has been reliably implicated in visuospatial attention, such that the dorsal regions (dPPC) are associated with voluntary ‘top-down’ attention, whereas the ventral regions (vPPC) are associated with automatic ‘bottom-up’ attentional processes. The Attention-to-Memory model (AtoM: Ciaramelli, Grady, & Moscovitch, 2008) has suggested that it also plays a similar role in memory retrieval, suggesting that the PPC mediates a domain-general attentional process. Furthermore, domain-generality of attentional processes may account for differences in perception and memory function accompanying old age. This study examined domain-generality by determining the shared variance in performance of tasks thought to recruit top-down and bottom-up attentional processes mediated across both domains. Results clearly suggested generality across domains in top-down processing; and in bottom-up processing, depending on its operationalization. Ageing was characterized by an absence of shared variance across domains and slower reaction times during bottom-up attentional reorienting only in perception.

<p>It has been suggested that primate brains are inherently biased towards gathering and processing the social information present in the world. In fact, the neural network that mediates our engagement with the external world - the default mode network (DMN) ¬- is strongly convergent with the neural circuitry for social cognition. The posterior cingulate (PCC) is believed to be a key node in both the DMN and in social cognition. Human and non-human primate studies have demonstrated a role for the PCC in outcome monitoring: it tracks rewards, subjective values of choices, task engagement and global choice strategies. It is also implicated in social cognition. Human studies show that PCC activity varies with the recall of autobiographical memories and exposure to social stimuli. While several electrophysiological studies explicate the response of PCC neurons to non-social outcome monitoring and valuation, there is a lack of similar studies for social valuation. This thesis is concerned with characterizing the neuronal responses in the PCC to social stimuli and determining whether social valuation occurs in the PCC in a manner similar to that previously described for non-social outcomes. I recorded the single unit activity of neurons in the PCC of rhesus macaques while they performed behavioral tasks that required attending to the faces of high-status or low-status individuals. Monkeys valued the faces of high-status individuals more than low-status individuals, though they were equally likely to identity and overtly attend to faces of both social classes. This differential valuation of face stimuli was represented in the firing activity of PCC neurons, with higher neuronal activity seen in response to subordinate faces as compared to dominant ones. Cells in the PCC did not track the individual identity of the presented faces. Furthermore, neuronal activity in the PCC predominantly tracked social value, and not non-social reward delivery as previously reported. Neuronal activity also predicted task engagement, with higher firing rates being predictive of a decrease in task engagement. To summarize, the PCC is biased towards social information processing, and neuronal activity in the PCC tracks social category information and the level of task engagement.</p> / Dissertation

Techn. University, Diss., 2007--Darmstadt.

Thesis (Ph. D.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains ix, 140 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 123-137).

Thesis (Ph.D.) - University of Queensland, 2003. / Includes bibliography.