Doctor of Philosophy

dissertationNewts have an amazing ability to regenerate lost structures and injured tissues. After a complete transection injury, which paralyzes the lower half of the animal, the newt spinal cord regenerates in as little as 4 weeks and re-establishes controlled movement. We have developed new methods of visualizing the cellular and molecular events of spinal cord regeneration and have used these to define six stages of axon regeneration. We also find that axon regeneration appears to be enabled, in part, because the lesion environment is permissive. The extracellular matrix is made up of canonically permissive and inhibitory proteins, but it remains loose and is not dense like it is in mammalian lesions. Meningeal cells and glia are most closely associated with regenerating axons and, instead of forming barriers to axon regeneration as they do in mammals, they appear to assist the regenerative process. Amputated limbs are regenerated in about 7 - 10 weeks. After amputation, mature cells de-differentiate and form a proliferating pool of progenitor cells under the amputation plane called the regeneration blastema. The blastema then organizes itself into a new limb. Little is known about what molecular factors drive blastema formation and growth. We have identified a novel newt chemokine, NvCXCL, that is highly upregulated during limb regeneration and may play a role in this process. NvCXCL is expressed in de-differentiating tissues and the blastema, stimulates cells of mouse fibroblastic and myoblastic cell lines to proliferate, and mildly induces the mouse fibroblasts to migrate, although not in a directed fashion. This work establishes a detailed baseline of the events occurring during spinal cord regeneration and identifies a novel chemokine that may be involved in the formation and growth of the limb blastema. Further insights into the mechanisms driving limb and spinal cord regeneration will be greatly aided by the development of more powerful genetic tools. If the dream of regenerative medicine is to become a reality, it is imperative that we learn all we can about how nature has already derived a solution for regenerating injured structures and organs in this exceptional animal

Precise tracking of the Magellan and Pioneer Venusorbiters by same-beam interferometry. Part 1: Dataaccuracy analysis

Simultaneous tracking of two spacecraft in orbit about a distant planet by two widely separated Earth-based radio antennas provides more-accurate positioning information than can be obtained by tracking each spacecraft separately. A demonstration of this tracking technique, referred to as same-beam interferometry (SBI), is currently being done using the Magellan and Pioneer 12 orbiters at Venus. Signals from both spacecraft fall within the same beamwidth of the Deep Space Station antennas. The plane-of-sky position difference between spacecraft is precisely determined by doubly differenced phase measurements. This radio metric measurement naturally complements line-of-sight Doppler. Data was first collected from Magellan and Pioneer 12 on August 11-12, 1990, shortly after Magellan was inserted into Venus orbit. Data were subsequently acquired in February and April 1991, providing a total of 34 hours of same-beam radio metric observables. Same-beam radio metric residuals have been analyzed and compared with model measurement error predictions. The predicted error is dominated by solar plasma fluctuations. The rms of the residuals is less than predicted by about 25 percent for 5-min averages. The shape of the spectrum computed from residuals is consistent with that derived from a model of solar plasma fluctuations. This data type can greatly aid navigation of a second spacecraft when the first is well-known in its orbit

What Everything Has to Do With Everything: Professional Development Convergence

With so many changes taking place in education and so little professional development time available, it is important to think creatively about ways to bring your staff together to learn about technology integration, media literacy, and 21st century skills. To do this, we must think about how these skills converge with other initiatives in our districts and take advantage of what little time we have to keep our staff moving forward. Find out how Wayzata uses this philosophy to maximize our professional development time. See how we are using online opportunities to bring our staff together outside of the standard work day

Zika Virus-induced Acute Myelitis and Motor Deficits in Adult Interferon αβ/γ Receptor Knockout Mice

Zika virus (ZIKV) has received widespread attention because of its effect on the developing fetus. It is becoming apparent, however, that severe neurological sequelae, such as Guillian-Barrë syndrome (GBS), myelitis, encephalitis, and seizures can occur after infection of adults. This study demonstrates that a contemporary strain of ZIKV can widely infect astrocytes and neurons in the brain and spinal cord of adult, interferon α/β receptor knockout mice (AG129 strain) and cause progressive hindlimb paralysis, as well as severe seizure-like activity during the acute phase of disease. The severity of hindlimb motor deficits correlated with increased numbers of ZIKV-infected lumbosacral spinal motor neurons and decreased numbers of spinal motor neurons. Electrophysiological compound muscle action potential (CMAP) amplitudes in response to stimulation of the lumbosacral spinal cord were reduced when obvious motor deficits were present. ZIKV immunoreactivity was high, intense, and obvious in tissue sections of the brain and spinal cord. Infection in the brain and spinal cord was also associated with astrogliosis as well as T cell and neutrophil infiltration. CMAP and histological analysis indicated that peripheral nerve and muscle functions were intact. Consequently, motor deficits in these circumstances appear to be primarily due to myelitis and possibly encephalitis as opposed to a peripheral neuropathy or a GBS-like syndrome. Thus, acute ZIKV infection of adult AG129 mice may be a useful model for ZIKV-induced myelitis, encephalitis, and seizure activity

Effect of a Type II Collagen Fragment on the Expression of Genes of the Extracellular Matrix in Cells of the Intervertebral Disc

Knowledge of factors regulating the turnover, repair, and degeneration of the intervertebral disc (IVD) is lacking. Although type II collagen (CII) fragments accumulate in the degenerative IVD, little is known of how they affect the degenerative process. A better understanding of the cellular interactions with fragments of matrix molecules are a key factor in promoting therapies for degenerative disc diseases. In the present study, we have investigated the effect of the CII (245-270) peptide on the expression of matrix molecules, proteinases, and interleukin genes in cells of the IVD. Cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) of adult bovine tails were cultured up to 8 days in the absence (control) or presence of the CII (245-270) peptide. RT-PCR was used to analyze the expression of the different genes. Exposure of these cells to the CII (245-270) peptide led to a transient up-regulation of the aggrecan gene in AF cells while this up-regulation was maintained for a longer time in NP cells. The fragment also enhanced a transient up-regulation of the type II collagen gene in AF cells but had no effect in NP cells. The peptide enhanced transiently the expression of matrix metalloproteinase (MMP)-1 and cathepsin K genes in both AF and NP cells whereas it increased MMP-13 expression only in NP cells. The peptide up-regulated tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and TIMP-3 gene expression on day 1 in AF cells but had very little effect on their expression in NP cells. Finally, the CII (245-270) peptide had no effect on IL-6 expression while IL-1α was not expressed in these cells. In conclusion, our results showed that the CII (245-270) peptide differentially alter the expression of genes in bovine AF and NP cells and suggest that degradation products of collagen may be involved in the regulation of IVD homeostasis

Prostaglandin PGE2 at very low concentrations suppresses collagen cleavage in cultured human osteoarthritic articular cartilage: this involves a decrease in expression of proinflammatory genes, collagenases and COL10A1, a gene linked to chondrocyte hypertrophy

Suppression of type II collagen (COL2A1) cleavage by transforming growth factor (TGF)-β2 in cultured human osteoarthritic cartilage has been shown to be associated with decreased expression of collagenases, cytokines, genes associated with chondrocyte hypertrophy, and upregulation of prostaglandin (PG)E2 production. This results in a normalization of chondrocyte phenotypic expression. Here we tested the hypothesis that PGE2 is associated with the suppressive effects of TGF-β2 in osteoarthritic (OA) cartilage and is itself capable of downregulating collagen cleavage and hypertrophy in human OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with a wide range of concentrations of exogenous PGE2 (1 pg/ml to 10 ng/ml). COL2A1 cleavage was measured by ELISA. Proteoglycan content was determined by a colorimetric assay. Gene expression studies were performed with real-time PCR. In explants from patients with OA, collagenase-mediated COL2A1 cleavage was frequently downregulated at 10 pg/ml (in the range 1 pg/ml to 10 ng/ml) by PGE2 as well as by 5 ng/ml TGF-β2. In control OA cultures (no additions) there was an inverse relationship between PGE2 concentration (range 0 to 70 pg/ml) and collagen cleavage. None of these concentrations of added PGE2 inhibited the degradation of proteoglycan (aggrecan). Real-time PCR analysis of articular cartilage from five patients with OA revealed that PGE2 at 10 pg/ml suppressed the expression of matrix metalloproteinase (MMP)-13 and to a smaller extent MMP-1, as well as the proinflammatory cytokines IL-1β and TNF-α and type X collagen (COL10A1), the last of these being a marker of chondrocyte hypertrophy. These studies show that PGE2 at concentrations much lower than those generated in inflammation is often chondroprotective in that it is frequently capable of selectively suppressing the excessive collagenase-mediated COL2A1 cleavage found in OA cartilage. The results also show that chondrocyte hypertrophy in OA articular cartilage is functionally linked to this increased cleavage and is often suppressed by these low concentrations of added PGE2. Together these initial observations reveal the importance of very low concentrations of PGE2 in maintaining a more normal chondrocyte phenotype

Zika Virus Infection Causes Temporary Paralysis in Adult Mice With Motor Neuron Synaptic Retraction and Evidence for Proximal Peripheral Neuropathy

Clinical evidence is mounting that Zika virus can contribute to Guillain-Barré syndrome which causes temporary paralysis, yet the mechanism is unknown. We investigated the mechanism of temporary acute flaccid paralysis caused by Zika virus infection in aged interferon αβ-receptor knockout mice used for their susceptibility to infection. Twenty-five to thirty-five percent of mice infected subcutaneously with Zika virus developed motor deficits including acute flaccid paralysis that peaked 8-10 days after viral challenge. These mice recovered within a week. Despite Zika virus infection in the spinal cord, motor neurons were not destroyed. We examined ultrastructures of motor neurons and synapses by transmission electron microscopy. The percent coverage of motor neurons by boutons was reduced by 20%; more specifically, flattened-vesicle boutons were reduced by 46%, and were normalized in recovering mice. Using electromyographic procedures employed in people to help diagnose Guillain-Barré syndrome, we determined that nerve conduction velocities between the sciatic notch and the gastrocnemius muscle were unchanged in paralyzed mice. However, F-wave latencies were increased in paralyzed mice, which suggests that neuropathy may exist between the sciatic notch to the nerve rootlets. Reversible synaptic retraction may be a previously unrecognized cofactor along with peripheral neuropathy for the development of Guillain-Barré syndrome during Zika virus outbreaks

Consequences of \u3ci\u3ein utero\u3c/i\u3e exposure to Zika virus in offspring of AG129 mice

Zika virus (ZIKV) can cause various diseases in offspring after congenital infection. The purpose of this study was to identify disease phenotypes in pups exposed to ZIKV in utero. Female interferon-α/β, -γ receptor knockout mice (AG129) were infected intraperitoneally with ZIKV 7.5 days’ post coitus (dpc). Viral RNA, antigen and infectious virus were detected in some, but not all, maternal and fetal tissues at various times during gestation. Fetuses of infected dams had significant intrauterine growth restriction (IUGR), which was more pronounced as females neared parturition. Pups born to infected dams were significantly smaller and had significantly shortened skull lengths, as determined by measurement with a caliper and by micro-CT analysis, as compared with age-matched controls. Growth rates of exposed pups after birth, however, was similar to sham-exposed offspring. Viral RNA was detected in pups of infected dams after birth. A lower survival rate was observed in neonates exposed to ZIKV in utero. A mortality rate of over 50%, attributed to consequences of ZIKV infection, occurred after birth in pups born to infected dams. A transient hearing loss was observed in some animals exposed to virus in utero. No motor deficits or cognitive deficits were detected using running wheel or viral paresis scoring assays. Abnormalities in offspring included smaller size, shorter skull length and increased neonatal mortality, while the only functional deficit we could detect was a low incidence of transient hearing loss

Significance of Elevated Blood Metal Ion Levels in Patients with Metal-on-Metal Prostheses: An Evaluation of Oxidative Stress Markers

It is widely known that cobalt and chromium ions can enhance the production of reactive oxygen species, known to be damaging to cells by disturbing their redox status and then generating oxidative stress. The aim of the present study was to determine if increased metal ion levels induce a state of oxidative stress in patients with metal-on-metal (MM) hip arthroplasty. Results indicated that there was no significant difference in the concentration of oxidative stress markers (total antioxidants, peroxides, and nitrated proteins) in the patients with MM bearings compared to patients without prostheses. The activity antioxidant enzymes was stable (catalase and glutathione peroxidase) or slightly decreased (superoxide dismutase and heme oxygenase-1) over time. This work is the first to determine the biological effects of metal ions released from MM hip implants with regards to mid-term systemic oxidative stress and showed that the increased levels of Co and Cr ions are not associated with significant oxidative stress damage in the plasma of patients with these implants