Browsing by Subject "Biology"
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Item A Novel Approach to Inquiry-Based Learning Models in the Sciences: Utilization of Case Presentations and Patient Encounter Workshops in High School Life Science Classrooms(2004-07-01) Whitaker, Lekeisha R.; Rustin E. Reeves; Victoria Rudick; Rouel RoqueA Novel Approach to Inquiry-Based Learning Models in the Sciences: Utilization of Case Presentations and Patient Encounter Workshops in High School Life Science Classrooms. Lekeisha R. Whitaker. Abstract. A novel approach to inquiry-based learning models is needed in a curriculum that reflects changing demographics, societal demands, and diverse cultural background. The proposed module which is designed to teach the urinary system to Fort Worth Independent District high school biology students as outlined in the Texas Essential Knowledge and Skills (TEKS) guide, represents application of this novel tool in the classroom. It is designed to be implemented in inquiry-based learning classrooms that function to encourage the natural process of inquiry throughout grade levels. The proposed module takes advantage of media resources, introductory case reports, and patient encounter workshops that outline diet, disease, and health disparities as a way to capture student interest in content relevant material, engage students in the daily lessons, and invoke long-term retention of basic life science concepts. Through future research and testing of module design and effectiveness in the classroom, the proposed approach to inquiry-based learning may serve to optimize student understanding of fundamental science concepts and diet and disease as it relates to normal body function, with implications that may affect change in lifestyle. Furthermore, the proposed learning module, if effective, may also serve as a template to be used to teach other life science subjects.Item Carotid Baroreflex of Leg Vasculature(2004-07-01) Keller, David Melvin; Peter A. Raven; H. Fred Downey; Patricia A. GwirtzKeller, David Melvin, Carotid Baroreflex Control of Leg Vasculature. Doctor of Philosophy (Biomedical Science), July 2004; 110 pp; 5 tables; 10 figures; bibliography. The carotid baroreflex (CBR) exerts control of arterial blood pressure primarily as a result of changes in total vascular conductance. In humans, understanding CBR control of the vasculature supplying a given vascular bed, such as the leg, remains unclear. Furthermore, it appears that metabolic attenuation of sympathetic vasoconstriction may modulate the CBR of the vasculature supplying contracting skeletal muscle during exercise. However, the balance between baroreflex-mediated vasoconstriction and the mechanisms responsible for the metabolic attenuation has not been fully elucidated. Therefore, the purpose of the investigations within this dissertation was to: i) explain CBR control of leg vascular conductance (LVC) and the relationship between changes in LVC and muscle sympathetic nerve activity at rest and during one-legged knee extension exercise, ii) examine the CBR control of the vasculature supplying an exercising leg and a non-exercising leg during exercise, and iii) demonstrate the role of the ATP-sensitive potassium channel in contributing to the metabolic attenuation of CBR-mediated vasoconstriction in the vasculature supplying contracting skeletal muscle. In the first investigation, we demonstrated: i) the stimulus response relationships for CBR control of LVC and MSNA at rest and during two intensities of one-legged knee extension exercise; ii) that CBR control of LVC was preserved during exercise; iii) that the attenuation of CBR-mediated vasoconstriction was no different between 7W and 25W exercise in the vasculature supplying an exercising leg; and iv) that the contribution of changes in LVC to CBR changes in mean arterial pressure was no different from rest to exercise in both the exercising leg and the non-exercising leg. In the second investigation, we examined the role of the ATP-sensitive potassium channel in modulating sympathetically-mediated vasoconstriction at rest and during exercise in the vasculature supplying an exercising leg and a non-exercising leg. The attenuated vasoconstrictor response to the carotid baroreceptor stimulated hypotension observed in the vasculature supplying an exercising leg was partially restored two to four hours after the oral ingestion of glyburide (5mg). This finding indicates that ATP-sensitive potassium channel activation plays a primary role in the effects of functional sympatholysis during leg exercise in humans. We further demonstrated that CBR control of MAP was not altered by oral glyburide administration in healthy subjects.Item Cellular and Molecular Mechanisms that Distinguish the Effects of Progestorone and Medroxyprogesterone Acetate on Neuroprotection(2006-07-28) Kaur, Paramjit; Goldfarb, Ronald; Singh, Meharvan; Agarwal, NeerajKaur, Paramjit. Cellular and Molecular Mechanisms That Distinguish the Effects of Progesterone and Medroxyprogesterone Acetate on Neuroprotection., Doctor of Philosophy, (Pharmacology and Neuroscience), July, 2006, 203 pp., 5 illustrations, 20 figures and bibliography. Women have a higher prevalence for Alzheimer’s disease (AD) than men, suggesting that the precipitous decline in gonadal hormone levels following the menopause may contribute to the risk of developing AD. However, principal results from the Women’s Health Initiative concluded that women taking conjugated equine estrogens combined with medroxyprogesterone acetate (MPA, tradename: Prempro) incurred more harmful than beneficial outcomes versus the placebo group (Rossouw et al., 2002). This dissertation was aimed at determining if the discrepancy between basic science reports and these clinical studies could have been due to the synthetic progestin, MPA. I hypothesized that P4 and MPA differed in their ability to protect against the excitotoxic/oxidative insult, glutamate. Further, I proposed that this difference in neuroprotective potential would be reflected in the difference in the ability of these hormones to elicit key effectors of two neuroprotection-associated signaling pathways, the ERK/MAPK and P13-Kinase pathways. Finally, studies were initiated to evaluate the potential importance of BDNF (brain-derived neurotrophic factor) in mediating the protective effects of P4. I used organotypic explants of the cerebral cortex, and found that both P4 and MPA elicit the phosphorylation of ERK and Akt, two signaling pathways implicated in neuroprotection, with maximal phosphorylation occurring at a concentration of 100 nM. Interestingly, P4 protected against glutamate- induced toxicity however, while an equimolar concentration of MPA (100nM) did not. Further, P4 resulted in an increase in BDNF, while MPA did not. Our data bring into question the relevance of using MPA as a component of hormone therapies in postmenopausal women, and instead, argue that the relevant progestin for use in treating brain-related disorders is progesterone. Collectively, the data presented here suggest that P4 is protective via multiple, and potentially related mechanism, and importantly, its neurobiology is different from the clinically used progestin, MPA.Item Effects of Testosterone on Obesity-Related Cardiac Hypertrophy and Fibrosis(2009-08-01) Wilson, Ana Kaye; Joan F. Carroll; James L. Caffrey; Robert T. MalletWilson, Ana Kaye. Effects of testosterone on obesity-related cardiac hypertrophy and fibrosis. Master of Science (Integrative Physiology), August 2009, 71 pp, 3 tables, 6 figures. Both testosterone and obesity are known to increase renin-angiotensin system activity, leading to cardiovascular dysfunction. This study determined the interactive effects of obesity and testosterone on left ventricular hypertrophy and cardiac fibrotic factors. Male New Zealand White rabbits were fed a lean or 10% added fat diet. After 12 weeks, fat-fed rabbits exhibited increased left ventricular weight (6.05±0.16 vs. 4.75±0.10 g, respectively, p≤0.05) and cardiomyocyte cross-sectional area compared to lean rabbits (372.3±19.0 vs. 305.0±13.4μm2, respectively; p≤0.01). These effects were attenuated by both castration and treatment with the angiotensin type 1 receptor blocker, losartan. Obese rabbits did not exhibit increased myocardial collagen as expected. However, castration and losartan treatment increased matrix metalloproteinase-2 (MMP-2) activity in obese rabbits. Despite the effects of castration hypertrophy and MMP-2 activity, castration did not attenuate plasma renin activity of aldosterone. These data suggest that testosterone contributes to obesity-related left ventricular hypertrophy and decreases collagen degradation, independent of renin activity.Item Evaluation and Validation of Tecan Genios Microplate Reader for Quantification and Normalization of Family Reference DNA Samples(2007-08-01) Fuqua, Lauren; John Planz; Arthur Eisenberg; Joseph WarrenIn 2001, the Texas State legislation established the Texas Missing Persons DNA Database (TMPDD) at the University of North Texas System Center for Human Identification Laboratory. Texas was the first state to participate in the missing persons section of the federal (FBI) database titles Combined DNA Index System or CODIS. Two indices of CODIS include the Unidentified Human Remains index and the Relatives of Missing Person index. Medical specimens, such as bone marrow or blood, or personal items used only by the missing person, such as a toothbrush or hairbrush, are ideal for identifying human remains through comparison of DNA profiles; although, DNA samples can be taken from family members to help locate missing persons or identify remains. DNA profiles from family reference samples, such as blood or buccal swabs from a close relative, are analyzed and uploaded into CODIS to allow federal, state, and local crime laboratories to exchange and compare profiles to missing persons electronically. At the University of North Texas Health Science Center, family reference samples, missing person reference samples, and unidentified human remains are analyzed to obtain DNA profiles for comparison. This research project involves a method that is proposed to improve the efficiency of DNA analysis for family reference samples. At the UNT System Center for Human Identification laboratory, the family reference samples are extracted in batches of 86 using the Tecan Freedom EVO® 100 extraction robot with the DNA IQ™ extraction kit from Promega Corporation. The DNA IQ™ extraction process is used in conjunction with the EVO® 100 robot in order to obtain a consistent amount of total extracted DNA; although, substantial variation has been detected in the output DNA quantity delivered. A considerable percentage (~20%) of samples exceed the optimal input template DNA amount required for successful amplification using the Applied Biosystems AmpFʅSTR® kits. A method of normalizing these samples was needed to bring the standard input DNA range within the optimal analytical range of the Applied Biosystems 3130 Genetic Analyzers and GeneMapper™ ID software. The ultimate objective of this internship practicum was to improve the efficiency of DNA analysis for family reference samples by using the Tecan GENios microplate reader in conjunction with an OliGreen® assay to estimate DNA quantity with the aim of using the quantification values to normalize family reference samples into an ideal input range for genetic analysis.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Function of Differentially Expressed Intracellular Calcium Channels in Retinal Neurons(2008-05-01) Nixon, Everett Sheldon; Peter Koulen; Raghu Krishnamoorthy; Rong MaNixon, Everett, Function of differentially expressed intracellular calcium channels in retinal neurons. Doctor of Philosophy (Pharmacology and Neuroscience), May, 2008, pp154, 17 illustrations. The retina, a specialized part of the central nervous system (CNS) is the innermost layer of the eye responsible for capturing light and converting the light response into a signal that can be transmitted through the optic nerve and onto the brain for interpretation. The ability of the retina to perceive light is dependent on its sensory neurons and the neural circuitry present that initiate the primary stage of processing the image being visualized, which then transmits an electrical signal down the optic nerve to the brain for processing and ultimately visual perception. In the vertical pathway of the visual process that involves the photoreceptor cells, bipolar cells and the ganglion cells, glutamate is the main excitatory neurotransmitter. Communication between these cells is dependent upon the release of glutamate into the synaptic region within both the outer plexiform layer and inner plexiform layer, a process that is Ca2+ regulated. In neurons, Ca2+ regulates a plethora of processes such as gene expression, cell death, synaptic plasticity and neurotransmitter release since it serves as a critical intracellular messenger. In view of the involvement of Ca2+ in a variety of physiological processes, it is essential for the intracellular Ca2+ concentration to be tightly regulated within neuronal cell. Regulation of Ca2+ signaling within retinal neurons can occur via inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) and ryanodine receptors (RyRs). These receptors are involved in the release of Ca2+ from the intracellular stores such as the endoplasmic reticulum (ER) into the cytosol. IP3Rs and RyRs contribute substantially to cytosolic free Ca2+ concentration transients and thereby play an important role in neuronal function. The purpose of the study was to determine the role of mGluRs, IP3Rs and RyRs in increasing intracellular Ca2+ levels in retinal neurons as related to signaling and neurotransmitter release. The present study provides experimental evidence for the following mechanisms: -Activation of mGluR8 in photoreceptor cells reduced cytosolic Ca2+ concentration by inhibition of the voltage gated Ca2+ channels on the plasma membrane. –The distribution of IP3R and RyR isoforms was associated with cytosolic Ca2+ transients and the IP3R induced transients occurs by activation of group I mGluRs. –In rod bipolar cells, the main increase in cytosolic Ca2+ concentrations during depolarization is due to Ca2+ release from internal stores via activation of RyR. The results of the present study contribute to the understanding of intracellular Ca2+ signaling in retinal neurons and Ca2+ signaling mechanisms. This is of relevance for identifying mechanisms controlling neurotransmitter release and possible pharmacological targets in neurodegenerative retinal diseases characterized by Ca2+ dyshomeostasis.Item Heredity In Relation to Evolution and Animal Breeding(D. Appleton and Company, 1911-01-01) Castle, WilliamItem Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork(2000-12-01) Shade, Debra L.; Pang, Iok-Hou; Yorio, Thomas; Dillon, GlennShade, Debra L., Interleukin-1Alpha-Mediated Signaling Mechanisms in the Human Trabecular Meshwork. Doctor of Philosophy (Biomedical Sciences/Pharmacology), December, 2000, 140 pp., 13 tables, 30 figures, references, 156 titles. This research provides important insights into the means by which interleukin-1alpha (IL-1α) regulates TM cell functions and enhances aqueous outflow, thus lowering IOP. The studies reported herein represent the first known characterization of the central role of the AP-1 transcription factor pathway in IL-1α-mediated production of proMMP-3 by TM cells, as well as the first known evidence that IL-1α can also enhance TM phagocytosis. Using these results as a stepping stone, this research has furthermore led to the identification of “AP-1 activators” as a novel compound class which may be useful in the treatment of glaucoma; it also points to the potential for compounds which regulate MEK, p38, and PKCμ activity as additional means of treatment. Based on these results, it is postulated that such compounds would be expected to lower IOP via upregulations of MMP production, followed by ECM degradation, and potentially, enhanced clearance of degraded ECM via phagocytosis.Item Local Enkephalins Modulate Vagal Control of Heart Rate(2001-05-01) Jackson, Keith E.; James L. Caffrey; H. Fred Downey; Michael W. MartinJackson, Keith E., Local Enkephalins Modulate Vagal Control of Heart Rate. Doctor of Philosophy (Biomedical Sciences), May 2001; 112pp; 7 tables; 22 figures; bibliography, 99 titles. Endogenous opioids, such as enkephalins, were first investigated for their ability to modulate pain. A body of evidence now supports opioid actions in many facets of regulation, including the cardiovascular system. Our laboratory is particularly interested in the ability of opioids to modulate autonomic function. Specifically, the role of the endogenous encephalin, methionine-enkephalin-arginine-phenylalanine (MEAP) was investigated to determine its ability to modulate parasympathetic function in the canine. To investigate MEAP’s response in the sinoatrial (SA) node a novel application of microdialysis was employed, whereby microdialysis was employed, whereby microdialysis probes were fabricated as described by Dr. David Van Wylen (38), and implanted in the SA node. After implantation of the probe, there was a significant attenuation of vagal function during the nodal application of MEAP. Specifically, vagally mediated bradcardia was reduced as compared to control, during the nodal application of MEAP. This inhibition of the vagus by MEAP was blocked by naltrindole, a selective delta antagonist. These data suggested that the vagolytic effects of MEAP were elicited via a delta opioid receptor. To test the hypothesis that MEAP’s effects were elicited through a delta opioid receptor mechanism, selective agonists and antagonists for the opioid receptors were utilized. An attenuation of vagal bradycardia was only observed during the infusion of a very selective delta opioid receptor agonist, deltorphin. A mu and kappa agonist showed no significant differences from control. Deltorphin was observed to elicit vagolytic effects in a similar concentration range as MEAP. However, deltorphin was more efficacious that MEAP. There was a significant attenuation of the deltorphin and MEAP’s vagolytic effects, during the co-infusion of the selective delta antagonist, naltrindole. The mu and kappa antagonists were both ineffective. These data further demonstrate that the observed vagolytic effect is linked to a delta opioid receptor. Endogenous MEAP. A series of experiments were undertaken to determine if endogenous MEAP could be demonstrated in the SA node and is so, was it similarly vagolytic. A preconditioning-like protocol was performed to produce intermittent local nodal ischemia to increase the local concentration of endogenous MEAP. The resulting MEAP was measured and was observed to be elevated during the periods of local nodal ischemia and return to control during reperfusion. Contrary to expectations an augmentation of vagal function was observed, during vagal stimulation. The augmented vagal bradycardia was only observed during ischemia, when MEAP was elevated and returned to control during each subsequent reperfusion. Therefore, there was a correlation between elevated MEAP concentrations and augmented vagal bradycardia. The delta antagonist, naltrindole, prevented the augmented vagal response, during nodal ischemia Glibenclamide, a selective KATP channel blocker, partially reversed the augmented vagal response. These data confirm that delta opiate receptors are involved in the augmented vagal bradycardia and that the mechanism may involve the activation of a KATP channel.Item Mechanisms of Right Ventricular Oxygen Supply/Demand Balance in the Concious Dog(2000-06-01) Hart, Bradley; H. Fred Downey; Patricia A. Gwirtz; James L. CaffreyHart, Bradley Joe. Mechanisms of Right Ventricular Oxygen Supply/Demand Balance in the Conscious Dog Doctor of Philosophy (Biomedical Sciences), August,2000, 119 pp, 4 tables, 13 figures, references, 79 titles. No data exist in the literature describing the myocardial oxygen supply/demand relationship of the right ventricle in a conscious, anaesthetized animal. A novel technique developed in our laboratory enables us to collect right ventricular (RV) venous blood samples from conscious dogs to determine RV myocardial oxygen consumption (MVO2). RV oxygen supply/demand balance was examined in conscious dogs, chronically instrumented to measure right coronary blood flow (RCBF), segmental shortening (%SS) and RV pressure (RVP) during increases and decreases in RV myocardial oxygen demand. Right ventricular MVO2 and O2 extraction (O2E2) were determined; RCBF, RVP, dP/dt, and %SS were recorded concomitantly. Acute increases in RV MVO2 were accomplished by atrial pacing (200 beats/min), increasing RV afterload by 65%, infusion of isoproterenol (0.1 μg/kg/min, i.v.), and by conducting a submaximal exercise routine (70-75% of maximum VO2). An acute decrease in RV MVO2 was created by propranolol administration (1 mg bolus, i.c.). During acute increases in RV MVO2, the extraction reserve is utilized primarily; flow is not affected in the absence of direct vasodilatory effects of the intervention. A decrease in RV oxygen demand is associated with a further increase in the RV extraction reserve. Since RV O2E increases linearly with increases in RV MVO2, these data show that changes in RV venous O2 tension can occur with little or no change in RCBF. LC resistance is very sensitive to alterations in LC venous pO2; therefore, there appear to be significant differences between the left and right ventricles concerning the matching of oxygen supply with myocardial oxygen demand.Item Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed(2000-08-01) Pearlman, Eric Brian; Barbara Barron; Patricia A. Gwirtz; Michael L. SmithPearlman, Eric B., Met-Enkephalin-Arg-Phe (MERF) and Metabolism of MERF Across the Canine Heart Vascular Bed. Master of Science (Biomedical Science), August, 2000, 37 pp., 3 tables, 11 figures, references, 20 titles. Methionine enkephalin arginine phenylalanine (MERF) has been shown to be co-stored with catecholamines in vesicles. The catecholamines appear to decrease the degradation rate of 3H-MERF in vitro. The aim of this study is to investigate the spillover and metabolism of MERF across the canine heart vascular bed. I hypothesize that 3H-MERF is either degraded in the plasma or taken up and degraded by the heart. I further hypothesize that the exogenous catecholamine, isoproterenol, inhibits or reduces the rate of MERF degradation. Mongrel dogs were anesthetized and instrumented to record cardiovascular parameters, infuse 3H-MERF, and obtain blood samples across the heart. Blood samples were taken before and after stopping 3H-MERF infusion to evaluate kinetics, show steady state, and test the effect of treatments. Steady state concentration of 3H-MERF was observed after 30 min of infusion. Chromatography separated intact from degraded 3H-MERF. Three experimental groups were used: control, propranolol plus isoproterenol, and propranolol only. Blockade of β-receptors was necessary to prevent changes in coronary blood flow. Propranolol bolus (0.2 mg/kg) was administered IV at 50 min. 3 μg/min isoproterenol or 0.5 ml/min normal saline was infused starting at 70 min until the end of sample collection. The 3H-MERF venous-arterial (V-A) difference prior to treatment was negative, indicating degradation in the plasma or uptake and degradation by the heart. The 75 min V-A difference was used to calculate the effect of the infusions on the degradation or uptake of the 3H-MERF; this value was unchanged by any treatment. Spillover of 3H-MERF was significantly lower in the propranolol + isoproterenol dogs (p [less than] 0.05) compared to propranolol only treatment at 75 min. Heart rate was significantly lower for the propranolol only group compared to control. Blood pressure and change in coronary flow were unchanged. In conclusion, isoproterenol does not affect the metabolism of 3H-MERF across the canine heart vascular bed. Propranolol, however, does increase the intact 3H-MERF in the plasma, but additional β adrenergic blockade agents need to be investigated to determine the mechanism by which this takes place.Item Molecular Cloning, Expression, and Regulation of the Na+/Myo-Inosiotl Cotransporter Gene(1996-08-01) Zhou, Cheng; Chaitin, Michael; Easom, Richard; Garner, MargaretZhou, Cheng, Molecular Cloning, Expression, and Regulation of the NA+/Myo-Inositol Cotransporter Gene. Doctor of Philosophy (Biomedical Sciences), August 1996. Mammalian cells respond to osmotic stress by accumulation of high concentrations of intracellular osmolytes. Osmotic-induced accumulation of the osmolyte, myo-inositol (MI), is achieved through activation of the NA+/MI cotransporter. Hypertonic stress results in elevated NA+/MI cotransporter mRNA abundance and transcription rate, and increased transporter activity. The goals of this dissertation are to establish the osmoregulation of the NA+/MI cotransporter gene expression in lens cells, and to investigate the transcriptional regulation of the NA+/MI cotransporter gene. Expression of the Na+/MI cotransporter in cultured bovine lens epithelial cells (BLECs) was demonstrated by RT-PCR amplification and Northern blot analysis. Hypertonic stress resulted in induction of the NA+/MI contransporter mRNA abundance in cultured BLECs. The induction patterns of the NA+/MI cotransporter and aldose reductase mRNA abundance by hypertonic stress indicated that osmoregulation of MI and sorbitol accumulations were regulated in concert. Accumulation of MI is an early-onset protective system, which is suppressed by the elevated sorbitol, the late-onset protective system. 5’-RACE analysis indicated that multiple transcription start sites were utilized in controlling of the expression of the NA+/MI cotransporter. Osmotic stress resulted in preferential utilization of a hypertonic promoter a. The bovine NA+/MI cotransporter gene was cloned and analyzed. The regulation of the Na+/MI cotransporter expression was investigated by transient transfection assays using promoter-luciferase constructs. Although multiple promoters were functional in cultured BLECs, only the hypertonic promoter a was osmotically responsive. Characterization of this osmotic-responsive element(s) between -536 to -300 bp upstream of the hypertonic transcription start site a. The studies presented in this dissertation refined the osmoregulation of the Na+/MI cotransporter gene expression. Hypertonicity induces MI accumulation by activation of an osmotic-responsive promoter. The consequences of the activation of this promoter lead to more cotransporter mRNA, more cotransporter protein, and higher transporter activity, resulting in accumulation of a higher concentration of intracellular Mi.Item Molecular Regulation of Interferon Gamma in 2B4-Activated Natural Killer Cells: Functional Role in Tumor Rejection(2001-11-01) Johnson, Lori Ann; Mathew, Porunellor A.; Goldfarb, Ronald H.; Dimitrijevich, S. DanNatural killer cells are a third population of lymphocytes, distinct from T and B cells. NK cells are non-MHC-restricted cytotoxic effector cells which are effective against intracellular pathogens, virally-infected cells and tumor cells. 2B4 is a natural killer cell receptor originally identified in the mouse as a surface molecule involved in non-MHC-restricted killing and enhancement of IFN-γ secretion. The human and rat homologues of 2B4 have recently been cloned in our laboratory. Interferon gamma (IFN-γ) is a cytokine with potent anti-viral and anti-proliferative effects. In addition, this cytokine acts as a global immune regulator by regulating gene expression and serving to attract other immune cells. In this work, we establish the function of human 2B4 in a NK cell line, YT. We have shown that human 2B4 activation induces cytolytic function and enhances IFN-γ release in YT cells. Additionally we show that 2B4’s regulation of IFN-γ occurs at the transcriptional level, both through mRNA stability and increased promoter activity. We also demonstrate that several regions in the IFN-γ promoter respond to 2B4 activation and IFN-γ both separately and together in the rejection of metastatictumor cells in C57B7/6 mice. Our results confirm that both 2B4 and IFN-γ are critical in the rejection of metastatic tumor cells. Through the use of activating monoclonal antibodies, our studies indicate that 2B4’s anti-tumor activity is through IFN-γ as well as through cytolytic function of NK cells.Item N-Acylethanolamine Signaling in Neurons(2008-12-01) Duncan, Raymond Scott; Koulen, Peter; Simpkins, James; Forster, MichaelDuncan, Raymond S., N-acylethanolamine signaling in neurons. Doctor of Philosophy (Biomedical Sciences), December 2008, 356 pp., 1 table, 70 illustrations, bibliography, 576 titles. Neurodegenerative diseases including Alzheimer’s disease are and will continue to be significant health problems as the aging population increases. The maintenance of neuronal calcium homeostasis has been a focus in degenerative disease research for many years. Within the last several years, lipids that activate cannabinoid receptors, and thus called cannabinoids, have gained recognition as neuroprotectants in models of neurodegenerative diseases. A subset of these cannabinoids, the N-acylethanolamines (NAEs), includes the well characterized neuroprotective lipid, arachidonylethanolamine. Other NAEs, such as palmitoylethanolamine (PEA), are more abundant in neurons and do not activate cannabinoid receptors, suggesting other targets for these lipids exist. Since non-cannabinoid NAEs rapidly accumulate after neuronal injury, it is likely they play a role in cellular responses to injury. Interestingly, some NAEs can alter intracellular Ca2+ signaling, but the underlying mechanism of action remains unclear. I hypothesized that the non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by reducing intracellular calcium release. I determined that HT22 cells and cultured mouse cortical neurons express proteins involved in NAE signaling, thus warranting the use of pharmacological inhibitors of these proteins in subsequent neuroprotection studies. Using HT22 cells, I determined that PEA exhibitis antiproliferative effects and neuroprotects against oxidative stress. In addition, I determined that PEA facilitates the nuclear translocation of putative protective proteins that can be regulated by Ca2+ through a mechanism not involving cannabinoid receptor activation. These findings led me to hypothesize that PEA alters release of Ca2+ from intracellular stores. To test this hypothesis, I determined that our cell models express inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) both of which are intracellular Ca2+ channels elevated in response to oxidative stress. I determined that treatment of HT22 cells with PEA reduced intracellular Ca2+ release elicited by chemical depolarization with KCI. My results suggest that non-cannabinoid NAEs, such as PEA, protect the hippocampal cell line, HT22, from oxidative stress in part by activating putative neuroprotective signaling proteins and by reducing intracellular calcium release.Item Neural Control of the Carotid Baroreflex During Exercise(2000-05-01) Gallagher, Kevin Matthew; Peter B. Raven; Stephen R. Grant; H. Fred DowneyGallagher, Kevin Matthew, Neural Control of the Carotid Baroreflex During Exercise. Doctor of Osteopathic Medicine/Doctor of Philosophy (Biomedical Sciences), May 2000; 151 pages; 13 tables; 19 figures; bibliography; 161 titles. Carotid baroreflex (CBR) function is reset upward and rightward to the prevailing blood pressure during dynamic and static exercise. Feedforward central neural inputs (central command) and negative feedback from skeletal muscle (exercise pressor reflex) both contribute to the resetting. The purpose of this investigation was to identify the individual roles of central command and the exercise pressor reflex in the resetting of the CBR during dynamic and static exercise. First, it was necessary to determine which receptor group that comprises the exercise pressor reflex, chemically-sensitive (chemoreceptors) or mechanically-sensitive (mechanoreceptors) receptors, was primarily involved in the regulation of the cardiovascular system. We observed the cardiovascular responses during exercise to individual action of the chemoreceptors and the mechanoreceptors. We demonstrated an increased mean arterial pressure (MAP) response to mechanoreceptor activation that was not identified during chemoreceptor stimulation. This finding suggested that the mechanoreflex was the primary exercise pressor mediated of arterial blood pressure during exercise. To identify the role of central command on CBR resetting, a second investigation increased central command by partial neuromuscular blockade during dynamic and static exercise. Resetting of CBR control of heart rate (carotid-cardiac; CSP-HR) and MAP (carodtid-vasomotor; CSP-MAP) during control exercise was further reset upward and rightward by increased central command without alterations in sensitivity. In conclusion, central command, a feedforward mechanism, was actively involved in the resetting of the CBR during exercise. To investigate the role of the exercise pressor reflex on CBR function, a third investigation activated by the exercise pressor reflex with the application of medical anti-shock trousers (MAS) during dynamic and static exercise. From control exercise, carotid-vasomotor function was further reset upward and rightward by the application of MAS trousers while CSP-HR function was only reset rightward. Sensitivity of the CSP-MAP and CSP-HR function curves were unaltered. The negative feedback mechanism of exercise pressor reflex, primarily mediated by mechanoreceptors, appeared to act as a modulator of CBR resetting during exercise.Item Opioid and Nitric Oxide Interaction in the Control of Heart Rate(2002-12-01) Farias III, Martin; James Caffrey; Fred H. Downey; Patricia GwirtzFarias III, Martin, Opioid and Nitric Oxide Interaction in the Control of Heart Rate. Doctor of Philosophy (Biomedical Sciences), December 2002, 130 pp, 2 tables, 30 figures. Understanding of the role endogenous opioids play as modulators of parasympathetic function has increased. The endogenous opioid, methionine-enkephalin arginine phenylalanine (MEAP) attenuates vagal control of heart rate when delivered by microdialysis directly in the canine sinoatrial node. This effect was mimicked by the δ-2 agonist, deltorphin-II indicating involvement by a δ-opioid receptor. The nodal delivery of the δ-antagonist naltrindole abolished the effect of deltorphin-II, further supporting the delta character of the receptor. Although the findings suggested that the opioid receptor mediating vagolysis was delta in character, the exact subtype of δ-receptor remained in question. Selective agonist and antagonists for δ-1 and δ-2 opioid receptors were employed to determine which subtype of δ-receptor mediated MEAP vagolysis. In these experiments, vagolysis produced by the nodal delivery of MEAP was unaltered by the highly selective δ-1 antagonist BNTX but abolished by the δ-2 antagonist, naltriben. Nodal delivery of deltorphin-II attenuated vagal bradycardia similar to MEAP while δ-1 agonists, DPDPE and TAN-67 failed to interrupt vagal bradycardia. TAN-67 actually improved vagal transmission and this effect was reversed by BNTX. These data indicate that δ-2 opioid receptors in the sinoatrial node and vagolytic and support the presence of vagotonic δ-1-opioid receptors in the same location. Nitric Oxide/Opioid Interaction. The hypothesis that intranodal nitric oxide synthase (NOS) modulates vagal transmission and that MEAP attenuates vagal bradycardia via the interruption of the NOS-cGMP pathway was tested. The general (L-NAME) and neuronal (7-nitroindazole) NOS inhibitors each attenuated vagal bradycardia and both effects were reversed by adding excess of the NOS substrate, L-arginine. These findings suggested that nNOS was a necessary component of vagal bradycardia in the canine sinoatrial node. Various probes of the NOS-cGMP pathway (L-arginine, SNAP, cGMP, and IBMX) were employed to determine if MEAP interrupted this pathway to produce vagolysis. The delivery of MEAP into the sinoatrial node for sixty minutes exerted a consistent vagolytic effect during vagal simulations. When MEAP was combined with a NOS pathway components, the vagolytic effect was reversed after 15-45 minutes of treatment. These findings suggested that MEAP exerted its effect by interacting with the NOW-cGMP system. The site of convergence maybe cAMP since the phosphodiesterase inhibitor, IBMX (by allowing the accumulation of cAMP) reversed the vagolytic effect of MEAP. To rule out a postjunctional effect, MEAP and the NOS inhibitors were combined with the direct acting muscarinic agonist, methacholine. The bradycardia produced by methacholine was unaltered by MEAP or nNOS inhibitors. This suggested that the effect of NOS inhibitors and MEAP were prejunctional. In summary, the cumulative findings suggest that MEAP, by activating δ-2-opioid receptors, attenuated vagal bradycardia prejunctionally, through modulating the cAMP component of the NOS-cGMP pathway in the canine sinoatrial node.Item Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing with the Potential for Automation(2002-05-01) Marshall, Pamela L.; Eisenberg, Arthur; Martin, Michael W.; Wordinger, Robert J.Marshall, Pamela. Optimization of Spermatozoa Capture During the Differential Extraction Process for STR Typing With the Potential for Automation. Master of Science (Forensic Genetics). May, 2002. In 1998, within the United States, it is estimated that a rape occurred every 2.3 minutes. In 1995, according to the Bureau of Justice Statistics, an estimated 350,000 rapes or sexual assaults (R/SA0 were experienced by persons age 12 or older. Of the estimated 100,000 R/SA reported, there were only approximately, 25,000 cases analyzed by crime labs nationwide. The majority of crime laboratories throughout the U.S., especially those in major metropolitan cities, have a significant backlog of unresolved R/SA cases. With the implementation of the Convicted Offender Database (CODIS), it is essential that all R/SA cases by analyzed, especially those lacking a known suspect. The comparison of the short tandem repeat (STR) profiles derived from sperm DNA recovered from evidentiary material with CODIS samples would provide the police with critical investigative leads resulting in the identification of the assailant. The goal of this research is to develop a cellular sorting method for the isolation of sperm cells from sexual assault samples which will: 1) take advantage of differentiating features (extracellular antigenic sites) for complete separation of cell types, 2) provide a more efficient means of sperm recovery, increasing DNA yield from the male fraction, and 3) ensure the DNA isolation process is compatible with the amplification of the CODIS core STR loci. Overall, the proposed technique will increase the probability of success in the analysis of sexual assault case samples. (NIJGrant #: 2000-IJ-CX-K009).Item Peripheral and Central Muscarinic Cholinergic Receptors in Arterial Blood Pressure Regulation(1999-12-01) Wray, David Walter; Shi, Xiangrong; Gwirtz, Patricia A.; Raven, Peter B.Wray, David Walter, Peripheral and Central Muscarinic Cholinergic Receptors in Arterial Blood Pressure Regulation. Master of Science (Biomedical Sciences), December, 1999, 70 pp., 7 tables, 8 illustrations, references, 83 titles. This study was designed to test the hypothesis that an age-related vagal dysfunction compromises arterial blood pressure (ABP) regulation. Changes in heart rate (HR) and ABP during lower body negative pressure (LBNP) were compared between ten elderly (≥60 yrs) and ten young (≤30 yrs) adults. A separate, young group (n=10) was also assessed following muscarinic cholinergic (MC) blockade with atropine (central and peripheral receptor blockade) or glycopyrrolate (peripheral receptor blockade) to simulate vagal dysfunction. During the onset of LBNP -40 too, orthostatic hypotension (OH) was observed in both the older subjects and the post-blockade younger subjects, with a diminished HR response. Furthermore, the reflex response to hypertensive stimuli was augmented in the post-blockade younger subjects, also associated with a diminution in HR response. We concluded that age-related or pharmacologically stimulated vagal dysfunction compromises ABP regulation during hypotensive and hypertensive stimuli, and that the difference between atropine and glycopyrrolate was insignificant.Item Predictors of Complicated Staphylococcus Aureus Bacteremia: A Retrospective Validation Study(2008-04-01) Krishnamurthy, Pramod; Fischbach, Lori; Cardarelli, Roberto; Coggin, Claudia S.Krishnamurthy, P., Predictors of Complicated Staphylococcus aureus Bacteremia (SAB): A Retrospective Validation Study. Master of Public Health (Epidemiology), April 2008, 57 pp, 9 tables, 1 illustration, bibliography, 39 titles. SAB often has a complicated clinical course and it is important to identify those at risk for complications to guide management. We conducted a validation study of a clinical prediction tool that uses a scoring system to predict the likelihood of developing complicated SAB. Chapter I is a review of background literature and rationale for our study. Chapter II has sections describing the study design, methods, eligibility criteria, statistical analysis and a summary of the results. We observed significantly higher complications among patients with SAB in our validation study. The prediction tool is not a valid predictor of complicated SAB and we recommend better prediction models to accurately predict complications of SAB.