http://digital.library.wisc.edu/1793/6662 Senior Theses http://minds.wisconsin.edu:80/retrieve/5875 http://digital.library.wisc.edu/1793/6662 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/1793/37686 Identification of QTLs for voluntary running activity in mice for the CBA/J and DBA/2J mouse strains Trammell, Sam Our goal is to understand the genetic factors related to voluntary running activity. Voluntary running activity in humans and mice is known to be a heritable behavioral trait. The genetic factors influencing voluntary running activity may be associated with human diseases, such as obesity. Therefore, identifying these factors may not only further our understanding of the behavior at the molecular level but also better our understanding of related disorders. We have found that CBA/J and DBA/2J inbred mouse strains have different voluntary running activities. We used quantitative trait locus (QTL) analyses to locate the genetic loci responsible for this difference. As a quantitative trait, we measured voluntary running activities in the parental mouse lines, their F1, and F2 progeny. We also identified genetic markers distinguishing CBA/J and DBA/2J alleles. We genotyped 176 F2 mice. We performed QTL analyses to identify loci associated with the phenotype. We identified significant QTLs on chromosomes 5 and 6 (LOD scores=3.697 and 5.047). We identified interactions between loci on chromosomes 3 and 6 (LOD score-6.737) and between loci on chromosomes 5 and 6 (LOD scores=8.063). In this study, we identified multiple loci influencing voluntary running activity in mice, providing a guideline for future identification of responsible genes. 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/1793/37683 The role of dynamin and Rho1 during cytokinesis in C. elegans Amodeo, Amanda Cytokinesis is the final phase of cell division in which a single cell is physically separated into two daughter cells. This is accomplished by constricting the acto-myosin contractile ring and inserting new membrane along the furrow. The large GTPase dynamin is essential for cytokinesis. Cynamin is required for endocytosis and also regulates the dynamics of the actin cytoskeleton. We hypothesize that dynamin may regulate the acto-myosin contractile ring. In order to test this hypothesis, we use in vivo microscopy to shed light on the role of DYN-1 in regulating actin dynamics by using GFP::RHO-1 as a marker. These experiments yield insight into the establishment and regulation of the contractile ring during cytokinesis. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37680 Large scale production of stable hemolymph juvenile hormone binding protein Arifin, Trina To better understand the pathways of the insect juvenile hormone (JH) action, milligram amounts of pure hemolymph juvenile hormone binding protein (hJHBP) are required. Hence the goal of this study is to scale up the system obtaining for purified and stable hJHBP. A drosophila S2 cell line was previously established in the laboratory and stably transfected with a plasmid containing the coding region of hJHBP. The construct included a metallothionein promoter which induces the recombinant JHBP (rJHBP). Purification of the protein produced involved column chromatography using a HisBind resin (Novagen). The production, purification and cleavage of rJHBP had to be significantly modified from previous results to obtain a functional Manduca sexta hJHBP. 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/1793/37677 Probing the chemistry in the active site of P4H Pua, Khian Hong Prolyl 4-hydroxylase (P4H), a mononuclear, non-heme FeII, 2-oxoglutarate and O2 dependent enzyme, plays an integral role in the biosynthesis of collagen. It catalyzes the post-translational hydroxylation of proline residues that leads to the stabilization of the collagen triple helix. Mononuclear, non-heme FeII, 2-oxoglutarate and O2 dependent enzymes are known to catalyze a wide variety of reactions including oxidative aromatic ring cleavage, cis-dihydroxylation of double bonds, and the hydroxylation of aliphatic C-H bonds. Non-heme FeII enzymes which carry out oxidative halogenations have recently been discovered. Halogenase SyrB2 and hydroxylase P4H are related enzymes, both 2-oxoglutarage and oxygen dependent. It is thought that both the halogenations and hydroxylation reactions proceed via a similar mechanism, each performing an extremely difficult reaction, abstracting a hydrogen atom from an aliphatic carbon, to allow the formation of a halogenated or hydroxylated product. Here we report that replacing the aspartate residue in the active site of P4H with alanine, so that the active site now mimics that of the halogenase SyrB2 was not successful in converting P4H to do halogenations chemistry. In addition, switching the relative positions of residues involve in the active site in wild-type P4H results in the loss of hydroxylase activity and no gain in halogenase activity when critical residues in P4H wer substituted to mimic that of a halogenase. 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/1793/37674 In vitro screenings predict polymer ability to facilitate RNAi through its use as a delivery vehicle of siRNA Scarpace, Katie Gene therapy techniques have emerged in recent decades as a method of combating genetic disorders. One prospective branch of gene therapy is using RNA Interference (RNAi) to regulate gene expression. RNAi employs small interfering RNA (siRNA) to knockdown gene expression posttranscriptionally. The challenge for scientists is figuring out how to deliver the siRNA to target cells. Many groups are currently researching siRNA delivery techniques. It is hypothesized that in vitro screening of delivery techniques by measuring gene expression knockdown and viability will predict delivery success in vivo. For my senior thesis I will be studying a delivery mechanism of foreign siRNA and using in vitro screening techniques to analyze siRNA delivery by measuring gene knockdown and technique toxicity. It can be concluded that in vitro screening can predict in vivo delivery success. 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/1793/37671 Cleavage and Polyadenylation Specific Factor 6 (CPSF6): A possible protein mediator for CARM1 and pre-MRNA splicing Ang, SiangYun Coactivated associated methyltransferase 1 (CARM1), an enzyme that regulates cellular processes via arginine methylation, has been suggested to play a role in RNA processing and alternative splicing. The focus of this project is to identify if cleavage and polyadenylation specific factor 6 (CPSF6), a protein essential for pre-mRNA3'-end processing, is a substrate of CARM1. We hypothesize that CPSF6 may bridge CARM1 to mRNA splicing machinery and CPSF6 itself is modified. The results suggest that CPSF6 is an in vitro substrate of CARM1 and the regions on CPSF6 methylated by CARM1 were narrowed down. Further research involves examining the in vivo interaction of CARM1 and CPSF6 and further mapping of CARM1 methylation sites on CPSF6. We will also determine whether CPSF6 is important in CARM1-mediated splicing process and whether CPSF6 methylation by CARM1 regulates dynamic association of CPSF6 with the spliceosome. Since currently CARM1 regulation of alternative splicing is not well defined, this project will provide insight into this mechanism 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37668 Inflammatory response and extracellular matrix turnover following pulsed dyed laser treatment of vocal fold and vocal fold fibroblasts Ya, Lin The 585-nm pulsed dye laser (PDL) is commonly utilized for the treatment of vascular and epithelial vocal fold lesions; however there are anecdotal reports that it may improve scar outcomes, perhaps by altering extracellular matrix (ECM) turnover; in certain patients. The purpose of this study was to evaluate the effects of PDL treatment on a selected group of inflammatory and procollagen/collagenase genes in normal rat vocal folds and vocal fold fibroblast cells. Vocal folds and fibroblasts were treated with 5-50J/cm2 PDL fluence and harvested at several time points. The mRNA expression profiles of TGF-Beta1, COX-2, IL-1Beta, IL-6, MMP-13, Procollagen I and Procollagen III were examined using real time RT-PCR. Our results indicated changes in inflammatory response alongside alterations in procollagen/collagenase expression, suggesting that PDL treatment does alter gene expression of fibroblasts within the focal fold ECM. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37665 Pre-steady state product formation rates for mutant and wild type toluene 4-monooxygenases Hauser, Andrew David This study measures the pre-steady state product formation rate for toluene 4-monooxygenase (T4MO). T4MO catalyzes the NADH and O2 dependent hydroxylation of toluene to form p-cresol. By using alternative substrates with altered benzene ring substitution patterns, the hypothesis that the chemical mechanism of hydroxylation by T4MO is an electrophilic aromatic substitution will be tested. T4moH has been expressed and purified with active site mutants to investigate the role of specific amino acid side chains during catalysis. Rapid chemical-mix quench was used to obtain the product which was analyzed with gas chromatography. The percent turnover for nitrobenzene and chlorobenzene was well below 100% in both the wild-type (wt) and T201A mutant. The percent turnover of both toluene and methoxybenzene in the wt and T201A mutant was almost 100%. It was observed that each alcohol group was added to the para position of each substrate and that the rate of product formation was higher in mutant than the wild-type. 1 p. (abstract only). YOU CANNOT OPEN THE COMPLETE PAPER. It is not available to the public, in accordance with the author's wishes. Mon, 29 Oct 2007 22:58:59 GMT http://digital.library.wisc.edu/1793/37518 Validation of the Combs-Goeser in vitro procedure to test if it improves repeatability and precision of neutral detergent fiber (NDF) digestibility measurements to allow improved near-infrared reflectance spectroscopy (NIRS) calibration Goldschmidt, Stephanie Abstract: More precise and accurate estimates of NDF digestibility (NDFD) are needed to better predict forage energy utilization by dairy cattle. Currently, NDFD measurement accuracy and repeatability are weak due to two facets, lab-to-lab variability and run-to-run variability, making it a poor tool for feed formulation. The objective of this research was to validate the Combs-Goeser in vitro fiber digestion procedure that was developed and the concurrent Near-Infrared Reflectance Spectroscopy (NIRS) calibration that is based upon the wet data from this procedure. I hypothesize that the new in vitro method will significantly reduce variation in measurement of NDFD, and this will allow improved NIRS calibration. This new procedure and concurrent NIRS calibration was validated against the in vitro procedures and calibrated NIRS procedures currently used by major commercial forage testing laboratories. A more precise and repeatable NDFD procedure would have great impact on the dairy industry in Wisconsin and the Midwest by allowing nutritionists and producers to more accurately predict forage digestion and ultimately milk production from forage crops. 25 p. 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/1793/37506 The Effect of Education Programs on the Knowledge and Attitudes about Snakes in San Isidro de Upala, Costa Rica Gramza, Ashley I interviewed 30 people in San Isidro de Upala, Costa Rica, to reveal their knowledge and attitudes about snakes. I found that many people hated and feared snakes because they assumed incorrectly that many or all snakes in the area were venomous. I then administered an education program designed to improve people's knowledge and attitudes about snakes. The program included information on the biology, identification, and ecological importance of snakes. I also explained how to safely respond to snake encounters. Before-and-after comparisons of responses to questionnaires measuring knowledge and attitudes showed that education programs made people more knowledgeable about snakes. Increased knowledge has been linked to positive attitudes. If people have positive attitudes towards snakes, they will be less likely to kill them; therefore helping to preserve the biodiversity of Costa Rica. 28 p. Mon, 29 Oct 2007 22:58:59 GMT