2005 Projects
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Item Open Access The effect of over-expression of ζ-crystallin on glutaminase mRNA stability(Colorado State University. Libraries, 2005) Propst, Keri J., author; Taylor, Lynn, author; Lee, Yeon, author; Curthoys, Norman P., authorDuring metabolic acidosis, increased renal catabolism of glutaminegenerates ammonium and bicarbonate ions to partially restore normal acid-basebalance. The remaining carbons derived from glutamine are then used to synthesizeglucose. This adaptive response is sustained in part by a pH-responsive increase inglutaminase (GA) that results from selective stabilization of the GA mRNA.Previous studies have shown that the 3’-UTR of the GA mRNA contains a pHresponseelement that consists of a direct repeat of an eight-base AU sequence andthat this element binds ζ-crystallin with high affinity and specificity. Increasedbinding of this protein during metabolic acidosis may initiate the pH-responsivestabilization of the GA mRNA. A tetracycline-responsive expression system (tet-off) was developed to test the effect of over-expression of ζ-crystallin on the expression and the stability of the GA mRNA. Two constructs, pcDNA 3.1-βG-GA–Hygro and pTRE2-ζ-crystallin, were created. The pcDNA 3.1/Hygro vector is designed for high-level,constitutive expression in mammalian cell lines and contains the selectable marker,hygromycin. A chimeric βG-GA cDNA segment that encodes β-globin and the 3’-UTR of the GA mRNA was inserted into the pcDNA 3.1/Hygro vector. Theconstruct, pTRE2-ζ-crystallin contains the tet-responsive element (TRE) that drivesthe expression of ζ-crystallin. The two plasmids were co-transfected into 8C cellsthat express high levels of the tTA protein that binds to and activates transcriptionfrom the TRE only in the absence of doxycycline (Dox). Clonal cell lines wereselected with hygromycin. These cells were grown in the presence and absence ofDox and screened with ζ-crystallin specific antibodies to identify clonal lines thatexhibit a large induction of ζ-crystallin when grown in the absence of Dox. RNAisolated from the selected line was quantified using Real-Time RT-PCR. Theresulting data demonstrate that over-expression of ζ-crystallin does not increase GAmRNA levels.Item Open Access Effect of dominant negative MKK expression on pH-induced increases in PEPCK mRNA levels(Colorado State University. Libraries, 2005) O'Hayre, Morgan, author; Taylor, Lynn, author; Curthoys, Norman P., authorAcid-base homeostasis is essential for survival. When metabolic acidosis is induced by factors such as prolonged starvation, severe shock, high protein diet, or uncontrolled type I diabetes, the kidneys act to compensate for the decreasing pH. Renal catabolism of glutamine, which is sustained through increased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glutaminase (GA), is activated during metabolic acidosis. Mitogen activated protein kinase kinases three and six (MKK3 and MKK6) are thought to play roles in the signal transduction pathway that lead to enhanced PEPCK and glutaminase activity. To examine the potential roles of MKK3 and MKK6, LLC-PK1-FBPase+ cells were stably transfected with dominant negative (dn) forms of either or both kinases. Expression of the transgenes was controlled by a Tetracycline-responsive promoter element (TRE). Doxycycline (dox) is used to inhibit transcription by preventing the tTA transcription factor from binding to the TRE. The absence of dox then enables transcription and turns on expression of the mutated kinase. Western blots were performed on extracts of clonal cell lines to determine the levels of the MKK isoforms as well as the levels of p38 and phosphorylated p38 in LLC-PK1-FBPase+ cells grown in both the absence and presence of dox. Northern blots were also performed to determine the effect of dnMKK expression on levels of PEPCK mRNA. Expression of both dominant negative kinases, but not the expression of either dnMKK3 or dnMKK6 alone, blocked the acid-induced increases in the levels of PEPCK mRNA and the Anisomycin stimulated increases in levels of phosphorylated p38.Item Open Access Evaluation of Aedes aegypti densonucleosis (AeDNV) infection in adult mosquito mortality(Colorado State University. Libraries, 2005) Plake, Emily M., author; Doehling, Malena, author; Bray, Stacey, author; Carlson, Jon, author; Suchman, Erica, authorAedes aegypti densonucleosis virus (AeDNV) has long been considered a likely agent in biologic control against arthropod-borne diseases. Extensive studies have been done to evaluate infection and pathogeneses in Aedes aegypti larvae and pupae, but only primary studies have been preformed in adults. The primary studies on adult mortality produced a two part mortality curve that showed high levels of early mortality, low levels of intermediate mortality and high levels of late mortality. It was hypothesized that the high levels of early mortality were due to high levels of virus infection, whereas the high levels late mortality were due to old age. In this study we analyzed the mosquitoes that died early and late in the mortality assay with quantitative PCR to determine virus titers in individual mosquitoes. We found that there is no significant difference between titer level of mosquitoes that died in the early part of the curve (Day 17-29), which had an average of 1.264 x 1011 genome equivalents per ml and those that died in the late part of the curve (Day 45-54), which had an average of 2.255 x 1011 genome equivalents per ml.Item Open Access Effects of body condition scores and fat depots on gene expression of fatty acid synthase in horses(Colorado State University. Libraries, 2005) Doran, Lynn A., author; Yemm, R., author; Dickinson, C. E., author; Hossner, Kim L., authorThe current study was designed to examine the expression of fatty acid synthase (FAS) genes in horse adipose tissue. Subcutaneous and visceral fat samples were collected from Quarter horses with obese or normal body condition scores and four fat samples were taken from clinical admission horses that were diagnosed with Cushing's disease at the C.S.U. Veterinary Teaching Hospital. Total RNA was isolated from the fat samples and analyzed by ribonuclease protection assay. A nonisotopic ribonucleic probe for fatty acid synthase (FAS) mRNA was constructed from sheep RNA and was utilized for analysis. The results from the ribonuclease protection assays on the four horses with both visceral and subcutaneous fat samples were analyzed to determine the effects of body condition scores and fat depots on the level of adipose tissue FAS mRNA expression. No difference in FAS gene expression was seen in horses with normal (BCS=3) or obese (BCS=5) body condition scores. The expression of FAS in visceral versus subcutaneous fat depots was numerically but not statistically significant. The data suggest that an ovine probe for FAS mRNA can be effectively used in horses. Although no differences were observed between fat depots, body condition score or Cushing's horses, use of larger sample numbers may provide significant results.Item Open Access Ocular dominance and subjective color perception: a study using the Fechner-Benham Visual Illusion(Colorado State University. Libraries, 2005) Kellogg, Jessica M., author; O'Shea, Geoffrey, authorPast research has demonstrated differences in visual processing between the dominant, or preferred eye, and the non-dominant, or less preferred eye. Researchers have accounted for these differences according to either physiological mechanisms (Porac & Coren, 1976) or behavioral preferences (Mapp, Ono, & Barbeito, 2003). The behavioral preferences account can be eliminated by examining how the eyes process visual illusions which are not expected to be influenced by prior experiences. One particular illusion, the Fechner-Benham Color Illusion, involves disks featuring black and white designs which, when rotated, produce the illusion of various subjective colors within the disks (Rosenblum, Anderson, & Purple, 1981). It was hypothesized that when viewing the Fechner-Benham Illusion with the nondominant eye, participants would exhibit longer latencies for the onset of color andwould report fewer colors compared to viewing with the dominant eye and with binocular vision. The underlying physiological mechanisms that are often regarded in subjective color perception are either described high in the visual information hierarchy, in a neurophysiological site, thus eliminating the retinal level (Rhollec & Vi�not, 1999;Robinson, 1896). Or, antagonistically, they are described as taking place within theretina in terms of an uneven distribution of photoreceptor cells (Jarvis, 1977).Item Open Access The relationship between nicotine and neurophysiology in schizophrenia(Colorado State University. Libraries, 2005) Nolan, Megan, author; Nicksic, C., author; Tassi, M., author; Davalos, Deana, authorAbnormalities of the nicotinic cholinergic system in the brain have been noted in a number of clinical disorders. The higher than average rate of smoking in clinical populations has been theorized as being related to abnormalities in this system. In schizophrenia, the rate of smoking surpasses that of other clinical populations (approximately 80-90% compared to 45-70%). The high rate of smoking, evidence of genetic linkage of schizophrenia to specific nicotinic receptors, and evidence for positive neuropsychological effects of nicotine, all suggest that nicotinic cholinergic mechanisms may play a pathophysiological role in schizophrenia. To assess whether nicotine could normalize early neurophysiological processing in schizophrenia, we studied a measure that has repeatedly been shown to be impaired in this population. The mismatch negativity (MMN) paradigm is an electrophysiological index that has gained interest in recent years as an endophenotype of schizophrenia. MMN measures "preattentive" physiological processes and is elicited by an infrequent change in a repetitive sound. The utility of MMN to assess change in response to pharmacological challenge has been identified by other researchers. However, MMN deficits do not appear to improve with the use of either conventional or atypical medications. Improvements associated with nicotine would suggest a novel change in physiological processing that is unique to nicotinic agonists. To assess the effects of nicotine challenge on MMN amplitude and latency, controls and individuals diagnosed with schizophrenia were administered nicotine gum versus placebo gum during two visits. Subjects underwent a baseline recording on each of the two visits and an additional recording following administration of either nicotine or placebo. Participants were played a series of tones (standard ISI between tones was 500 ms, deviant ISI of 250 ms occurred on average every 20th interval). The average amplitude of MMN waveform elicited by the deviant interval was significantly larger following nicotine administration compared to placebo condition in both the controls and the schizophrenia patients (p<.02). In addition, a significantly greater improvement was noted in the schizophrenia group compared to the controls (p<.05). Finally, symptom checklists suggest that nicotine may alter certain mood states. These results are consistent with the idea that pharmacological agents targeting nicotinic receptors may provide unique physiological benefits that are not addressed by current medications.Item Open Access Potential role of cholesterol in the migration of neurons containing gonadotropin-releasing hormone(Colorado State University. Libraries, 2005) Wadas, Brandon, author; Edelmann, M., author; Spivak, I., author; Feinstein, E., author; Schwarting, G., author; Intlekofer, K., author; Tobet, Stuard, authorSignaling by Sonic Hedgehog (Shh) is instrumental in the development of midline facial and forebrain structures. Signaling by Shh can be dependent upon conjugation with cholesterol. Structural abnormalities related to cholesterol depletion may be a result of a failure of Shh signaling. Disorders resulting in cholesterol depletion are often characterized in part by developmental malformations, including holoprosencephaly. Neurons that synthesize gonadotropin releasing hormone (GnRH; controls the reproductive axis) originate in the nasal compartment and migrate into the brain along a route that may depend upon proper Shh signaling. The current study was conducted to assess whether cholesterol-depleted enzyme Dhcr24-/- mice would affect the unique migration of GnRH neurons as they migrate to the brain.Item Open Access Social distance and diversity(Colorado State University. Libraries, 2005) Barton, Moriah, author; Metcalfe, Michelle, author; Downey, Eleanor Pepi, authorThe authors created a survey using the Bogardus Social Distance scale to answer the following: What are the tolerance levels of college students from a predominately white society towards people of different ethnicities, races, religions, and sexual orientation? and What factors can influence the tolerance levels?Item Open Access Characterizing a mosquito gut bacterial community(Colorado State University. Libraries, 2005) Kemp, Janet A., author; Lang, Gail M., author; DuTeau, Nancy M., authorMosquitoes are common vectors of devastating diseases that infect both humans and animals. Methods of control, such as insecticides and vaccines, are losing their effectiveness. Alternative strategies need to be considered. Paratransgenesis is one method of blocking transmission of disease from mosquito to host. Paratransgenesis is the genetic engineering of symbiotic or commensal bacteria to produce products that block pathogen transmission. This research project looked at characterizing the bacterial community of adult female Aedes aegypti to find candidates for paratransgenesis. We identified 72 diverse culturable bacterial isolates present in the midgut and on the exoskeleton of this mosquito. We examined the difference between the midgut bacterial community of blood-fed and sugar-fed female adults. We also found that approximately 10% of the total bacterial species found in the midgut are culturable, which is consistent with studies from other natural bacteria consortia.Item Open Access ERP study of metaphor processing(Colorado State University. Libraries, 2005) Gibson, Linzi M., author; Schmidt, Gwen L., authorWhat is the role of the right hemisphere in processing metaphors? One theory suggests that metaphor processing in the RH is part of amore general function of the RH, coarse semantic coding (Beeman,1998). The LH mediates semantic relationships between words havinghigh semantic feature overlap, whereas the RH activates a broadersemantic network of more distantly related words.Item Open Access Characterization of the bacterial midgut community of wild-caught Aedes aegypti(Colorado State University. Libraries, 2005) Shimizu, Julia, author; DuTeau, Nancy M., authorIt is estimated that 10 to the 14th power microbes make the human gut home, outnumbering the total number of cells in the body. The importance of microbes in the digestive system of mammals is fairly well known. However the microbial flora inhabiting the gut of insects, particularly mosquitoes, is less well studied. Understanding the microbial flora of the mosquito may provide valuable information for understanding why some mosquitoes are more effective vectors for human disease than others. Genetic alteration of these gut microbes may also lead to novel methods for controlling mosquito populations or blocking disease transmission. In this study, the microbial gut community of field-caught larval Aedes aegypti (the major vector of yellow fever and dengue) was analyzed. PCR products of bacterial 16S ribosomal RNA genes, amplified from larval DNA extracts, were cloned. 68 clones were then digested with Msp1. 57% of the clones had unique fragment patterns, while the other 43% shared one of 11 patterns, suggesting that the mosquito gut contains a diverse consortium of bacteria.Item Open Access Influence of early exercise on bone mineral density in equine distal third metacarpal condyles(Colorado State University. Libraries, 2005) Zimmerman, Chelsea A., author; Duesterdieck, K., author; Kawcak, Chris E., authorAlthough musculoskeletal injuries have a large impact on the horse industries, little is known as to the effects of early exercise. Prior research suggests that repeated loading and exercise leads to an increase in higher density, low quality bone that is slow to repair. However, adapting the musculoskeletal system to high intensity exercise early may lead to less injuries in the equine metacarpophalangeal joint (MCP), a common site of fracture in Thoroughbred (TB) racehorses. The objective of this study was to determine bone density patterns in the equine MCP joints of young TBs to evaluate the effects of early exercise.Item Open Access Activation of muscle spindle afferents increases force fluctuations in the knee extensor muscles(Colorado State University. Libraries, 2005) Coellen, Eric T, author; Cuaresma, Lindsay S., author; Intlekofer, Karlie A., author; Tracy, Brian L., authorThe purpose of this project was to examine the effect of acute tendon vibration on fluctuations in force during contractions of the knee extensor muscles in young healthy subjects.