http://digital.library.wisc.edu/1793/7951 http://minds.wisconsin.edu:80/retrieve/7803 http://digital.library.wisc.edu/1793/7951 http://digital.library.wisc.edu/1793/37689 TRPV1 mediates matrix metalloproteinase expression in spinal cord injury-induced chronic neuropathic pain Tilghman, Jessica Neuropathic pain (NP) is common following spinal cord injury (SCI). Evidence exists for the involvement of many processes in NP development following SCI (Cain et al, 2007). Our past studies identified increased expression of nociceptive genes, including vanilloid receptor-1 (TRPV-1) following SCI in rats. Recent studies have described the matrix metalloproteases (MMPs) role in many disease processes involving the central nervous system. There is evidence that MMPs may be involved ini the pathway initiated by TRPV-1 activation. Our current study investigates the role of TRPV-1 receptor activity in the regulation of MMP-2 and following SCI. We hypothesize that 1) SCI will cause TRPV-1 upregulation and a corresponding increase in gene expression of MMP-2. The increased MMP-2 levels promote the manifestation of NP and 2) inhibition of MMP-2 by expression will attenuate NP onset. The results from our studies have potential use in the development of novel treatments for post SCI NP. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. Wed, 29 Oct 2008 22:58:59 GMT http://digital.library.wisc.edu/1793/37516 Neuronal lysosome permeabilization dynamics in ischemia He, Shuhan The mechanism in which lysosomes are damaged by free radicals during neuronal ischemia is examined in rat hippocampus. There are two hypotheses. The free radicals can degrade the lysosome membrane through liquefaction; all of the intralysosomal proteins would be able to penetrate the membrane, albeit at slower rates for larger proteins. The second mechanism is that pores are created; the proteins within the lysosome that are smaller than the pores can escape while those larger than the pores would not due to the robust nature of the undamaged membrane. Acid Phosphatase (67kda) and Beta-Galactosidase (540 kDa) has been identified as rat intralysosomal neuronal proteins that can be detected as proxies for leakage. Our initial results indicate that the damage undergoes the pore mechanism of damage based on preferential acid phosphatase leakage. 16 p. Wed, 29 Oct 2008 22:58:59 GMT http://digital.library.wisc.edu/1793/37514 LEF/TCF family transcription factors are important in the upregulation and restricted expression of zic genes in the dorsal neural tube Webert, Kyle C. A central question in biology is how different cell-types arise from an initially one-celled embryo to form an organism. Many cellular differences arise through differential gene expression in space and time via the action of transcription factors (TFs). Zic genes are TFs that contribute to the patterning of the developing vertebrate nervous system, but little is known about how their transcription is regulated. Consensus scoring across several vertebrate species identified possible loci for TF binding sites within a previously identified stretch of zic regulatory DNA, cis-regulatory module D5. The three most promising sites were mutated and injected in constructs upstream of eGFP reporters into zebrafish embryos at the one cell stage. Expression was observed in F1 transgenics by in vivo fluorescence at approximately 24 hrs. The mutation of the LEF TF binding site suggests that Zic expression is upregulated and restricted to the dorsal neural tube by action of LEF TFs. 14 p. Wed, 29 Oct 2008 22:58:59 GMT http://digital.library.wisc.edu/1793/37512 The Role of PGC-1alpha in Adipocyte Differentiation and Fat Mobilization Braun, Kristina H. Calorie Restriction (CR) without malnutrition extends mean and maximal lifespan in multiple species, although the mechanism is unknown. CR has a dramatic effect on adipose tissue morphology, gene expression and energy metabolism. The concept of adipose tissue as an endocrine organ has come into focus recently, raising the possibility that the whole-body beneficial effects of CR are initiated in adipose tissue. Transcription co-activator PGC-1alpha is a key regulator of energy metabolism. CR influences components of the PGC-1alpha pathway, resulting in PGC-1alpha activation in adipose tissue. We set out to determine whether PGC-1alpha plays a role in the regulation of adipocyte function. Here we show the effect of manipulation of PGC-1alpha activity on two parameters of adipocyte function; differentiation and fat mobilization. First, we show that pharmacological inhibition of PGC-1? pathway accelerates differentiation, and activation of PGC-1alpha pathway impairs differentiation. Second, pharmacological activation of PGC-1alpha in mature adipocytes increases fat mobilization. Third, genetically increased levels of PGC-1alpha also inhibit differentiation and fat storage. This study demonstrates that PGC-1alpha plays a role in the processes of differentiation and fat mobilization in cultured adipocytes. These data are consistent with involvement of PGC-1alpha in the mechanism of adipose remodeling induced by CR. 9 p. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37510 The role of offspring in mitigating extreme stress in lactating female mice Homer, Jennifer Mothers of many species find great comfort in their offspring, which in turn has been found to alleviate mild stress in the mother. In the face of a life-threatening event, however, do offspring still act as effective stress reducers? Using mice as test subjects, I investigated how variable exposure time to pups affects a mother's anxiety levels after being subjected to a major stressor. It was found that exposure to pups alleviates fear and anxiety in lactating females after an extreme stressor. Additionally, the mothers who recovered without their pups in the original home cage, with only the smell of their pups, experienced the least amount of stress reduction and the highest anxiety levels compared to the other treatment groups. We believe this absence of pups in the home cage created a "double stressor", which added to the stress created by the major stressor, ultimately leading to a greater increase in anxiety. 18 p. Wed, 29 Oct 2008 22:58:59 GMT http://digital.library.wisc.edu/1793/37496 The Biochemical Characterization of a Translational Repressor Luo, Xinwei Sarah Iron regulatory proteins (IRP) are crucial post-transcriptional regulators of iron metabolism. They influence the synthesis of proteins involved in iron uptake, storage and use by binding to their mRNAs. IRP1 stands out particularly with its ability to incorporate a [4Fe-4S] cluster to double function as the enzyme, cytosolic aconitase (c-acon). Since RNA binding and c-acon activity are mutually exclusive, the incorporation of this Fe-S cluster serves as a regulatory switch between the two forms that is dependent on iron availability in cells. This project aims to characterize the biochemical properties of IRP1 in order to understand the finely tuned translational regulation it performs. Polysome profiles were used to isolate different forms of IRP1 based on their presence in actively translating or repressed pools of mRNA. Functional assays like aconitase activity assays and RNA-binding assays were used for biochemical characterization of these forms. This study establishes baseline activities for these assays and also demonstrates that IRP1 differentially regulates the mRNAs of ferritin, the iron storage protein, and ferroportin, which exports iron from the cell. My research will allow further understanding of IRP1 as an agent of regulatory control. 23 p. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37482 Inhibition of the Escherichia coli replication cycle by a mutant RecA protein Li, Hao Abstract: A conserved [K/R]x[K/R] motif is found at the subunit-subunit interface in bacterial RecA protein filaments. In the Escherichia coli RecA protein, this motif is made up of residues K248 and K250. Both of these residues have a role in ATP hydrolysis (in trans across the interface) and in the transmission of conformational information across the interface. A K250R mutation creates a RecA protein that promotes both ATP hydrolysis and DNA-strand exchange at a six-fold lower rate than the wild type protein. E. coli strains overexpressing this mutant RecA protein also grow much slower than wild type strains. Suppressor mutations appear quickly, and most suppressor mutations inactivate RecA (leading to high levels of UV sensitivity). One suppressor mutation, A11V, did not inactivate the RecA protein, but instead produced a RecA variant that allowed normal rates of growth under normal growth conditions, while partially restoring UV resistance. We found that this suppressor mutation has no effect on the rate of ATP hydrolysis of the mutant RecA K250R protein, but has slightly better strand exchange activity than RecA K250R. We also found that RecA K250R/A11V has a higher rate of disassembly than RecA K250R, showing that this suppression mutation allowed the double mutant RecA nucleoprotein filament to be more dynamic. Our hypothesis is that the [KR]x[KR] motif, especially RecA residue K250 is directly involved in RecA functions. Through mutational studies, we can get a better understanding of the role of this residue in RecA-catalyzed reactions. 23 p. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/35955 Matching between dietary preferences and digestive capacity in passerine birds Kohl, Kevin It has been argued that evolutionary shifts in diet preferences are paired with changes in digestive physiology, including digestive enzyme activities. For example, a previous study in birds (Martinez del Rio 1990) found interspecific variation in enzyme activities that matched species? feeding guilds (frugivore, omnivore, nectivore, etc.). To enhance the findings of this study, we measured the activities of pancreatic and intestinal enzymes in six avian species where diet patterns have been extensively studied. The adaptive modulation hypothesis states that enzyme activities should match the relative levels of substrate present in an animal?s diet so as not to waste biosynthetic energy or cell membrane space with unneeded enzymes. There were no differences in aminopeptidase-N, trypsin, or chymotrypsin activities between species with different percent protein in their diets. However, consistent with the adaptive modulation hypothesis, we found that species with a higher percent starch in their diets had higher maltase, sucrose, and amylase activities. However, future studies may need to be conducted to differentiate long-term, evolutionary matching from rapid modulation of enzyme levels. 28 p. Wed, 29 Oct 2008 22:58:59 GMT http://digital.library.wisc.edu/1793/23667 Delta-opioid receptor (DOR) activation prolongs respiratory motor output during oxygen-glucose deprivation (OGD) in neonatal rat spinal cord in vitro Freiberg, Sara M. Severe reduction of blood flow to the brain results in oxygen-glucose deprivation (OGD) and neuronal damage. DOR activation is neuroprotective during OGD in the cerebellum and cortex, but it is not known whether DOR activation protects spinal motor circuits that contribute to breathing. To address this question, a split-bath in vitro neonatal rat brainstem/spinal cord preparation was used to record spontaneous respiratory motor output from cervical (C4-C5) and thoracic (T4-T7) ventral spinal roots. A plastic barrier at spinal segment C1 allowed oxygenated solution to continuously bathe the brainstem while the solution on the spinal cord was switched from oxygenated to OGD (0 mM glucose, bubbled with 95% nitrogen/5% carbon dioxide) with or without DADLE (1.0 uM, DOR agonist). After OGD solution was applied to the spinal cord (n=7), cervical and thoracic motor output was abolished at 25.8 ± 2.0 and 26.4 ± 1.6 min, respectively. However, when DADLE was applied 10 min prior to and during spinal OGD solution application (n=8), cervical and thoracic motor output was abolished at 41.5 ± 4.9 min (p = 0.014) and 57.0 ± 4.2 min (p = <0.001), respectively. These data suggest that spinal DOR activation protects both cervical and thoracic respiratory motor output during OGD. 2 p. (abstract and cover sheet only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author’s wishes Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/23547 Gaze-evoked blinks in rhesus monkeys Hintz, Adam S. Gaze-evoked blinks, blinks that occur during rapid movements of the head and eyes, are believed to be the result of a shared mechanism that is responsible for a blink occurring with a saccade. In humans, it has been found that blinks are more likely to occur with larger saccades. Saccades accompanied with a blink are also more likely when attentional demands are low. Saccades associated with a reflex blink elicited by an air puff in monkeys have been shown to be slower than saccades without a blink. The purpose of the present study was to see if monkeys tested on visually-guided and memory-guided saccade tasks would show the same behavior in gaze-evoked blinks as humans. In this study, similar findings were found of gaze-evoked blinks in monkeys as were found in humans. Gaze-evoked blinks also slowed saccades as was found in reflex blinks elicited by a puff of air. 13 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8152 Determining the impacts of residential lakeshore development and various physical factors on largemouth bass (Micropterus salmoides) growth rates Guarascio, Matthew J. Development of residential lakeshore properties can have several effects on aquatic ecosystems including the thinning of littoral coarse woody habitat. Previous studies have shown that reduction of littoral coarse woody habitat negatively affects largemouth bass (Micropterus salmoides) growth rates. In order to validate these results and attempt to find the mechanism through which bass growth rates are reduced, we surveyed 16 lakes in Vilas, County Wisconsin on a gradient of conductivity and development. Regressions of size-specific growth rates with lake area (p < .01) and maximum depth (p < .05) were negatively correlated and significant. A negative correlation was observed between mean largemouth bass (Micropterus salmoides) growth rate and development, between lake categories of low development low conductivity and high development high conductivity, although this trend was not statistically significant. Our research confirms that extensive residential lakeshore development may reduce the growth rates of bass as demonstrated by Schindler (2000). 21 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8150 Studies on the heat stability of egg yolk antibody (lgY) Bobeck, Elizabeth A. Nutritionally and economically-important heat-labile proteins, including enzymes, hormones, and antibodies (Ab), lose a substantial amount of activity following industrial processing. Protection of these heat-sensitive bioactive molecules is needed in order to realize expanded markets for these biologics. Using a model of heat-labile proteins, Ab to phospholipase A2, and a sensitive detection system for Ab binding (ELISA), a pilot steam chamber was designed and constructed to develop methods of encapsulating proteins. After modification of the pilot chamber, it was shown that water plays a key role in Ab destruction. Samples (Ab, trehalose-encapsulated Ab, and industry standard) dried with drierite before steam treatment retained 100% activity after 60 seconds in 92-93C in a sealed 15ml centrifuge tube, while samples not dried but sealed prior to steam treatment lost activity (Ab retained 72.24% activity, trehalose-encapsulated Ab retained 74.03% activity, and industry standard retained 42.26% activity). A hydrophobic protein matrix (HPM) was developed. 41.73% binding activity remained in 1% Ab in pasta matrix, 0.94% remained in Ab in egg matrix, and 4.5% remained in industry standard after 60s in 92-93C in unsealed containers. This RPM may protect against steam-induced losses by protecting Ab from water. 59 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8144 Comparison of non-heme liver iron and iron metabolism protein levels in superoxide dismutase 1 knock-out mice versus superoxide dismutase 1 wild-type mice Bittner, Tara Iron is an important micronutrient that is necessary for multiple cellular functions. However, iron levels must be tightly regulated in order to prevent iron-deficiency and iron-toxicity. There are many proteins involved in iron metabolism. This study focuses on iron-regulatory proteins 1 and 2 (IRP1/2), ferritin, and transferrin receptor protein (TfR). IRPs are key iron sensors that bind to iron response elements (IREs) located on mRNA when the IRP Fe-S clusters are removed, regulating translation or stability of mRNA. Ferritin is the storage form of iron. Ferritin levels must increase in iron-sufficient or overloaded conditions to store the excess iron in a safe form. TfR takes up iron and moves it into a free iron pool for utilization by the body. TfR levels must increase in iron-deficient conditions to mobilize iron to necessary tissues. When IRP binds to a ferritin IRE, ferritin mRNA translation is inhibited causing ferritin protein levels to decrease. However, when IRP binds to TfR IRE, TfR mRNA is stabilized causing TfR protein levels to increase. Reactive oxygen species (ROS) such as the superoxide anion have been known to destabilize the Fe-S cluster in IRP, possibly leading to IRP degradation in high concentrations of ROS. Superoxide dismutase 1 (SOD1) reacts with the superoxide anion to yield safer complexes (hydrogen peroxide and water). I hypothesized that SOD1 knock-out (KO) mice would have increased non-heme liver iron and ferritin levels as well as decreased TfR and IRP1/2 levels due to the increased levels of superoxide anions removing the Fe-S clusters, decreasing IRP/IRE TfR and ferritin mRNA binding. My data supported my hypothesis. I used a non-heme liver assay, hematocrit measurements, and western blots to measure the levels of non-heme liver iron and protein levels in KO and WT male mice (C57BU61 background) at ages 8 weeks, 2.5-3 months, 4 months, 6 months, and 12 months. 23 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/8130 E. coli contaminated drinking water in rural Uganda: Using results to make an impact Klink, Jenna In January 2006, I worked with Community Based Integrated Nutrition (COBIN) to perform water quality testing and design & distribute health questionnaires to 40 households in the Lyantonde and Kiruhura districts of rural Uganda. Two types of water microbiology tests were used to test families' drinking water for E.coli contamination: the Colilert MPN Test made by IDEXX Laboratories (presence/absence test) and the E.coli Count Petrifilm made by the 3M Company (quantification of E.coli). 46 total water samples were taken directly from homes. Over 90% of unboiled water samples were contaminated with E.coli. All samples from rainwater collection tanks were negative for E.coli. There were boiled samples that were positive for E.coli, most likely because after boiling the water, it was poured back into the same container that the water was collected with. This showed the need for multiple interventions. In January 2007, additional water testing was performed (samples taken straight from the source), and we provided clean clay pots with covers for water storage to village health workers at workshops titled "Home-based water purification trainings." 12 p. Sun, 29 Oct 2006 22:58:59 GMT http://digital.library.wisc.edu/1793/7983 The effects of 1,25 D3 on the mouse osteopontin promoter Strasburg, Annalyse Vitamin D (1,25 D3) is an important transcriptional regulator of many genes including our gene of interest, osteopontin. Osteopontin is a known regulator of bone calcification and mineralization and is involved in signaling in the immune response. We looked at osteopontin expression in mouse MC3T3-El preosteoblast cells in response to 1,25 D3. We also tested how pathways known to crosstalk with 1,25 D3 modify mOPN expression. These include MNAR, Wnt3a conditioned media, PTH, and dexamethasone. Additionally, we checked how ZK159222, an antagonist to VDR with partial agonist activity, effects mOPN expression. The osteopontin promoter was cloned into a POL3 reporter vector and transfected into MC3T3-El cells. A luciferase assay normalized by a tl-gal assay was used to measure gene expression. 1,25 D3 induces activity of the mOPN promoter. ZK159222, MNAR, Wnt3a conditioned media, PTH, and dexamethasone were not found to effect mOPN promoter activity in the presence of 1,25 D3. 15 p. Sun, 29 Oct 2006 22:58:59 GMT