Thesis (Ph. D.)--Bob Jones University, 1974. / Typescript. Bibliography: leaves 282-307.

2,3,7,8-Tetrachlorodibenzo-para-dioxin (TCDD) is found as a contaminant of certain herbicides. Vietnam veterans and others exposed to this dioxin have suffered a wide range of serious health problems, many of which appear to be associated with the toxic effects of dioxins demonstrated by experiments in animal systems. The mechanism by which 2,3,7,8-TCDD exerts its toxic effects is most likely based on microsomal enzyme induction, yet the exact workings of the mechanism remain to be discovered. This paper is the result of an extensive survey of the recent scientific literature describing research on the biological effects of dioxins, specifically, 2,3,7,8-TCDD and its possible effects on human health.

BCL6, a protooncogene, is most commonly involved in diffuse large cell lymphomas (DLCL) which is the most common form of non-Hodgkin lymphoma. BCL6 recruits a number of transcriptional corepressors including BCL6 corepressor (BCoR) to function as a transcriptional repressor. BCoR mutation causes two X-linked allelic disorders termed as Oculofaciocardiodental (OFCD) and Lenz microphthalmia syndrome, which are characterized by ocular, dental, cardiac and skeletal anomalies. The involvement of BCL6 in the development of the eye has not been reported so far. Here, we report that BCL6 is essential for eye development in Xenopus embryo prior to the neurula stage. We found BCL6 function is required for eye development prior to stage 11 and loss-of-function studies of BCL6 showed a reduction in expression of Rx, Pax6 and Otx2, which play vital roles at the early stage of eye development, in the anterior neural plate. These findings suggest that BCL6 regulates the expression of major eye field transcriptional factors (EFTFs) and controls eye development during the early stages of Xenopus development. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the requirements for the degree of Master of Science. / Fall Semester, 2010. / October 18, 2010. / Xenopus, Eye, BCL6 / Includes bibliographical references. / Yoichi Kato, Professor Directing Thesis; Choogon Lee, Committee Member; Branko Stefanovic, Committee Member; Jamila I Horabin, Committee Member; Wu-Min Deng, Committee Member.

Many animals, including humans, choose their source of nutrition based on the nutritive value and the flavor (i.e, odor, taste and texture) of available foods. Sweet taste is one of the more potent orosensory stimuli that contributes to food choice. Laboratory animals develop preferences for neutral or aversive tastes and flavors by associating them with the taste of sugars and non-caloric sweeteners. Learning how these preferences develop would aid in understanding how and why specific foods are selected over others. Given the wide availability and variety of sweetened foods and beverages in modern society, the formation and persistence of learned food preferences by consumers may contribute to health issues such as diabetes and obesity. Conditioned flavor-taste preference (CFTP) learning is a form of associative learning in which a rat comes to prefer a neutral flavor paired with a preferred taste. Experimentally, one flavor (the conditioned stimulus or CS+; e.g., cherry or grape Kool-Aid) is paired with the sweet and highly preferred taste of fructose (F; the unconditioned stimulus or US) while a second flavor (the CS-) is paired with the less preferred taste of saccharin (S) on 1-bottle conditioning days (CS+/F or CS-/S). The acquisition of the learned preference is then assessed with a 2-bottle preference test in which both flavors mixed with saccharin (CS+/S and CS-/S) are presented simultaneously. While CFTP learning is well known, it has not been well characterized. The olfactory and gustatory associative brain regions necessary for CFTP learning are unknown. Dopamine receptors have been implicated, but otherwise it is not known which neurotransmitters or receptors mediate CFTP. In order to identify the associative neural substrates that are involved in CFTP learning, three approaches were taken; behavioral, pharmacological and molecular assays. To precisely characterize the behavioral acquisition and expression of a CFTP, lickometers were used to determine the pattern of drinking in rats. During 1 or 2-bottle preference tests, total intake, bout size, bout number, lick rate and first minute licks were analyzed. The pattern of drinking was examined under 3 conditions: 1. During expression of unconditioned preferences for 8% fructose over 0.2% saccharin. The unconditioned preference for fructose over saccharin was slow to develop, but was seen in significantly greater total intake, bout size, and first minute licks for fructose by the fourth preference test. 2. During the pairing of Kool-Aid flavors with either 8% fructose or 0.2% saccharin. CS-/S total intake and bout size was significantly greater than CS+/F during conditioning, but a preference for the CS+ flavor was seen in the third and fourth 2-bottle preference test days with significantly greater total intake and bout size of CS+/S vs. CS-/S. 3. During long-term presentations of Kool-Aid mixed with two different concentrations of saccharin (0.2% vs 0.05%) as the unconditioned stimuli. Total intake, bout size and bout number were significantly greater for the flavor mixed with the high concentration of saccharin over the low concentration of saccharin. during conditioning. During 2-bottle preference tests when both flavors were mixed with the low concentration of saccharin, total intake and bout size were significantly greater for the CS+. The increases in lick rate and bout size observed in all 3 experiments suggest that fructose is more palatable than saccharin, and a high concentration of saccharin is more palatable than a low concentration. A change in relative palatability of the Kool-Aid flavors is conditioned by association with the high palatability tastes; greater intake of the conditioned flavor is mediated by increased bout size. These results suggest that flavor preference learning interacts with both orosensory processes and satiety processes (i.e. prolonged bout size) to elevate intake of the preferred flavor. The N-methyl-D-aspartate (NMDA) glutamate receptor (NR) is a candidate mediator in olfactory and taste learning (Barkai and Saar, 2001; Jimenez and Tapia, 2004). To determine if NR is involved in CFTP, systemic MK-801, a non-competitive NR antagonist, was administered prior to conditioning and prior to expression. To determine the glycinergic contribution to NR activation in CFTP, systemic D-cycloserine, an agonist at the NR glycine-binding site, was administered prior to conditioning and reversal learning. While vehicle-treated rats acquired a preference for CS+/S over CS-/S, CFTP learning was completely blocked in MK-801-treated rats. The effect of MK-801 was specific to CFTP acquisition, because follow-up experiments demonstrated that MK-801 did not induce a conditioned taste aversion, cause state-dependent learning, or affect CFTP expression. In a second approach, rats were injected with DCS (15 mg/kg) 60 min prior to daily conditioning. In contrast to MK-801, administration of DCS prior to conditioning enhanced CFTP learning (but not reversal conditioning). These results demonstrate that NR neurotransmission is critical for CFTP learning. Furthermore, enhancement of CFTP learning by DCS suggests that endogenous levels of glycine or D-serine may be a limiting factor in CFTP learning. To determine the activation of neural populations during associative CFTP learning, c-Fos immunohistochemistry was used to illuminate the differential patterns of cellular activation. To eliminate the potential confounds of food restriction, restricted drinking sessions and potential postingestive effects that may effect c-Fos activation, rats were conditioned using a highly preferred concentration (0.2% saccharin) and a lesser preferred concentration of saccharin (0.05% saccharin) as the unconditioned stimuli. In order to standardize exposures, stimuli were applied by intraoral infusion. C-Fos immunolabeling was visualized within the brain after an intraoral infusion of either CS+ or CS- flavors (e.g. grape and cherry Kool-Aid) in combination with greater or lesser preferred taste US (e.g. 0.2% or 0.05% saccharin). Neuronal activation was assessed in forebrain sites (e.g. gustatory cortex, amygdala, and lateral hypothalamus). There was no difference in intraoral intake between all experimental groups in both conditioned and unconditioned rats, and extensive c-Fos activation was evoked in the olfactory, gustatory and learning relays of all experimental groups. Analysis of the differential patterns of c-Fos immunolabeling among unconditioned rats revealed a significant increase in c-Fos immunolabeling in the basolateral nuclei of the amygdala after intraoral infusions of dH2O compared to unconditioned rats after intraoral infusions of CS+/0.2% saccharin or CS-/ 0.055 saccharin. Therefore, the basolateral amygdala may be involved in the unconditioned response to sweetened flavors, or in the association of flavor with sweet tastes. Among conditioned rats, there was a trend towards greater in c-Fos immunolabeling in the lateral habenula of rats after intraoral infusions of CS+ vs the CS- or saccharin alone. Therefore, the lateral habenula, which is part of the accumbens-ventral tegmental area reward pathway, may be involved in the discrimination of learned preferences. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2007. / August 24, 2007. / Olfactory Conditioning, Associative Learning, Serine, Fructose, Glycine, NMDA, Ingestive Behavior, C-Fos Immunolabeling, Saccharin / Includes bibliographical references. / Thomas A. Houpt, Professor Directing Dissertation; Barbara G. Licht, Outside Committee Member; Michael Meredith, Committee Member; Zuoxin Wang, Committee Member; James M. Fadool, Committee Member; Scott J. Steppan, Committee Member.

Hepatitis C virus (HCV) infection leads to acute and chronic liver disease for thousands of people every year. With the absence of an effective treatment method, new classes of anti-HCV therapies are needed. Cyclosporine (CsA) and its derivatives potently suppress HCV replication. Recently, CsA resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA resistant HCV replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of a siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection. In addition, we selected mutant viruses that were able to infect CyPA-knock down cells. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding by nuclear magnetic resonance. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype. Four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials. This approach could be applied broadly to other cofactors in other viruses as large numbers of cellular cofactors important for viral infection have been discovered since the advent of genome-wide small interfering RNA screening. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / June 25, 2010. / Hepatitis C viurs, Cyclophilin, Cyclosporin A, NS5A, NS5B / Includes bibliographical references. / Hengli Tang, Professor Directing Dissertation; Branko Stefanovic, University Representative; Hank W. Bass, Committee Member; Kenneth H. Roux, Committee Member; Fanxiu Zhu, Committee Member.

Genetic variation is a dominant feature in the study of evolution. The presence of variation is embedded at every level, from a single individual all the way to different species. Understanding the mechanisms that allow variation to be introduced and maintained is crucial to our understanding of nature. If empirical data and observations provide the substrate needed for such understanding, the role of theory is instead to train our intuition and help experimenters in formulating testable hypotheses. Here I use mathematical modeling as a tool to investigate the effects of genetic variation at different levels. I begin by looking at variation expressed at the level of an individual. Enzyme isoforms are a pervasive presence in many species. They can be produced by different genes, different alleles or by alternative splicing of a single gene. I examine the potential benefit provided by the presence of enzyme isoforms in the Drosophila circadian rhythm. I show that controlling isoform proportion can be a powerful mechanism to reduce the effects of variations in the values of system parameters, thereby increasing system robustness. My second research project looked at genetic variation at the population level. Gamete recognition proteins are central to reproduction. They determine gametic compatibility both within and between species. In marine broadcast spawners an astonishing number of variants have been found for these proteins. The mechanism responsible for this variation is thought to be sexual conflict. This conflict arises because each sex has different and opposing interests at fertilization. I investigate how sexual conflict and spawning behavior can drive the evolution of recognition protein polymorphism and promote gametic disequilibrium by assortative mating. I conclude by looking at variation at the species level. Marine broadcast spawners need to synchronize the release of their gametes to increase the chance of successful fertilization. Highly synchronized gamete release can result in polyspermy. Polyspermy is the fusion of multiple sperm with a single egg at fertilization, making an unviable zygote. A possible strategy to avoid polyspermy is to shift spawning time. I show how a population where spawning time is the phenotype under selection can split in two reproductively isolated populations in sympatry. / A Dissertation Submitted to the Department of Biological Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2008. / May 12, 2008. / Genetic Variation, Speciation, Enzyme Isforms, Broadcast Spawners, Mathematical Models / Includes bibliographical references. / Thomas F. Hanasen, Professor Co-Directing Dissertation; David Houle, Professor Co-Directing Dissertation; Richard Bertram, Outside Committee Member; Peter Beerli, Committee Member; Don R. Levitan, Committee Member.

Under normal physiological conditions, trypsin remains inactive as trypsinogen inside the pancreas. Upon entering the small intestine, trypsinogen is converted to active trypsin. Acute pancreatitis is caused by premature activation of trypsinogen and the digestion of the pancreas. Up to now the exact initiating mechanism of this premature activation is still not clear. In these experiments, pH fluctuations, Ca2+ concentration changes and trypsin activity inside pancreatic zymogen granules were monitored. The effects of possible pharmacological inhibitors were also assessed. The results show that a sustained increase of Ca2+ in the cytosol can trigger K+ influx into zymogen granules (ZGs) via a Ca2+-activated K+ channel (ASKCa). This influx of K+ then mobilizes bound Ca2+ by Ca2+/K+ ion-exchange to increase free Ca2+ concentration in the ZGs and also mobilizes bound H+ by H+/K+ ion-exchange to decrease the pH in the ZGs. Both the increase of free Ca2+ concentration and the decrease of pH in the ZGs will facilitate trypsinogen autoactivation and stabilize active trypsin. Moreover these investigations show that the ASKCa in the membrane of ZGs may be a small conductance Ca2+-activated K+ channel (SKCa channel), because it can be activated by 300 nM [Ca2+] and inactivated by apamin (100 nM) and TEA (20 mM). / A Thesis Submitted to the Department of Biological Science in Partial Fulfillment of the Requirements for the Degree of Master of Science. / Summer Semester, 2004. / April 21, 2004. / Acute Pancreatitis, Matrix, Ion Exchange, Zymogen Granule / Includes bibliographical references. / Wei-Chun Chin, Professor Co-Directing Thesis; George Bates, Professor Co-Directing Thesis; Thomas Keller, Committee Member; Laura Keller, Committee Member.

The voltage-gated potassium channel, Kv1.3, carries 60-80% outward current in the olfactory bulb (OB) primary output neuron, the mitral cell. Gene-targeted deletion of the Kv1.3, produces lean mice that have supernumerary and heterogeneous glomeruli and an increased ability to detect and discriminate odors. These skinny and "super-smeller" phenotypes exhibited by Kv1.3-null mice, suggest a role for Kv1.3 in both olfactory and metabolic sensitivity. To understand how Kv1.3 gene-targeted deletion affect mitral cell electrical sensitivity, the current evoked properties of mitral cells from Kv1.3-null and wildtype (WT) mice were evaluated by current-clamp slice electrophysiology. Gene-targeted deletion of Kv1.3 resulted in an increased action potential firing frequency, modified action potential kinetics, and a more depolarized resting membrane potential. These results suggest that decreased expression or conductance of Kv1.3 will increase the sensitivity of mitral cells to stimulation. There are several natural modulators of Kv1.3 activity including brain derived neurotrophic factor (BDNF), insulin, adenosine triphosphate (ATP) and reactive oxygen species (ROS) that are also important metabolism related factors. ATP and ROS are products of glucose metabolism. Heterologously expressed Kv1.3 channels were found to be sensitive to D- but not L-glucose via voltage-clamp electrophyology. Mitral cells have previously been shown to be sensitive to BDNF and insulin via modulation of Kv1.3 activity, glucose sensitivity has not been examined. To understand how changing glucose concentrations affect mitral cell electrical sensitivity, the current evoked properties of mitral cells from Kv1.3-null and wildtype (WT) mice were evaluated in the presence of 0, 5, 10 and 20 mM D-glucose by current-clamp slice electrophysiology. Like the hypothalamus, the OB contained two populations of glucose-sensitive mitral cells; glucose excited and glucose inhibited. Mitral cells from Kv1.3-null mice were insensitive to changing glucose concentrations. To explore the correlation between Kv1.3, metabolism, and olfaction, 11 week old mice were maintained on a moderately high-fat diet (MHF, 32% fat) for 26 weeks. Following treatment, the systems physiological parameters of body weight, respiration, locomotion, and ingestive behaviors were quantified in a custom-designed, computer-interfaced, metabolic chamber. Diet-induced obese (DIO) mice exhibited a 47% increase in body weight where as Kv1.3-null mice were resistant to DIO as a result of a significant increase in basal metabolic rate linked to the MHF challenge. Bilateral olfactory bulbectomy (OBX) in a Kv1.3-null background yielded mice that were no longer resistant to DIO resulting in weight gain. OBX actually prevented the MHF-induced increase in basal metabolic rate in these Kv1.3-null mice. Selective, olfactory bulb specific blockade of Kv1.3 resulted in a selective upregulation of basal or light phase metabolic rate in MHF and, to a lesser extent, control fat-diet (CF) treated WT mice. These results suggest that natural changes in the sensitivity of the OB by modulation of Kv1.3 via energetically important molecules such as insulin and glucose, could contribute to the body's natural metabolic response to fat intake. Kv1.3 is also involved in weight and metabolic modulation in a genetic model of obesity. Gene-targeted deletion of Kv1.3 in a mouse model of genetic obesity, the melanocorton receptor 4-null (MC4R-null) mouse, reduced body weight by decreasing fat deposition and subsequent fasting leptin levels, without changing overall growth, fasting blood glucose, or serum insulin. Light-phase mass-specific metabolic rate and locomotor activity were not affected by genetic deletion of Kv1.3 in MC4R-null mice but dark-phase locomotor activity and mass-specific metabolism were significantly increased resulting in increased total energy expenditure. These results suggest that not only is the olfactory system modulated by energy availability, the olfactory system can also modulate energy balance. One way for the system to accomplish this is to modulate the sensitivity of the olfactory system output, the mitral cells, via Kv1.3 regulation. / A Dissertation Submitted to the Department of Biological Science, Program in Neuroscience in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Fall Semester, 2010. / August 16, 2010. / Glucose Sensitivity, Metabolism, Potassium Channel, Kv1.3, MC4R, Olfaction, Obesity / Includes bibliographical references. / Debra Ann Fadool, Professor Directing Dissertation; Michael Roper, University Representative; Michael Meredith, Committee Member; J. Michael Overton, Committee Member; Paul Q. Trombley, Committee Member; Laura Keller, Committee Member.

The fields of community ecology and evolution are theoretically tightly linked, but in general, community ecologists discount evolution in describing the dynamics of present-day community patterns. Yet, evolution in response to strong selection pressure might affect species interactions and alter ecological patterns on a relatively short time scale. Conversely, evolutionary studies are typically limited to examining the evolution of traits in response to, at most, one other species. In more complex communities, higher-order indirect effects emerge that might have significant effects on how species evolve. Evolution in a community context provides more insight into how species evole in natural communities. This dissertation focuses on the evolution of a ciliated protozoan, Colpoda sp. that lives within the water-filled leaves of the purple pitcher plant (Sarracenia purpurea). The evolution of several traits was measured in response to the direct effects of predators (specialist mosquito larvae that live in the same inquilines community), competitors (another co-occuring ciliated protozoan) and indirect effects that emerge when both predators and competitors are present. Two traits (cell size and population growth rate) evolved in response to predation, resulting in predator avoidance and tolerance, respectively. The evolution of these traits resulted in a significant decrease of the ecological effect of predators in only 12 days (approximately 40 prey generations), but less than one predator generation. These same two traits, as well as cyst production, evolved in response to competition. However, evolution in response to the indirect effects that occurred when both predators and competitors were present was stronger and in the opposite direction of direct effects. The result was little net evolution in response to the sum of direct and indirect effects. The importance of these indirect effects suggests that evolution in a community context may proceed much differently from that predicted by the simple additive effects of multiple species. To fully understand how species evolve in natural communities, complex interactions between multiple species must be fully understood. Similarly, to explain ecological patterns, it is imperative to account for the constant evolution of species in response to the suite of other species in their environment. / A Dissertation Submitted to the Department of Biological Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Summer Semester, 2010. / April 28, 2010. / Effect Size, Tolerance, Resistance, Prey, Colpidium, Mosquito Larvae, Rapid Evolution, Selection Colpoda, Competition, Predation, Indirect Interactions, 18S rRNA, Character Displacement, Convergence, Divergence / Includes bibliographical references. / Thomas E. Miller, Professor Co-Directing Dissertation; Don R. Levitan, Professor Co-Directing Dissertation; J. Anthony Stallins, University Representative; David Houle, Committee Member; Jeanette L. Wulff, Committee Member.

Recent years have seen a dramatic worldwide increase in allergies and asthma that can now be described as reaching epidemic proportions. Food allergies affect approximately 2% of the adult population and up to 6% of the pediatric population. Tree nut allergies, in particular, affect about 0.5% of the US population. Five commonly consumed tree nuts in the US are almonds, walnuts, cashews, pistachios, and pecans, all of which are allergenic to a subset of the consuming population. Among the proteins thus far associated with tree nut allergies include the seed storage proteins belonging to the 2S albumin and 11S globulin gene families. Complementary DNA expression libraries were created from English walnut and cashew nut embryos. To identify the 2S albumin and 11S globulin genes in both nuts, the libraries were used either directly as targets for degenerative primers in PCR 'fishing' experiments or transferred to nitrocellulose membranes and screened with nut allergenic patient sera. Once the genes were identified and amplified, they were modified with restriction enzymes, ligated into an expression vector, and expressed as fusion proteins. These purified fusion proteins were used to screen for walnut and cashew nut-allergic patient IgE-reactivity in direct immunoblots and to identify the native counterparts of these proteins in crude nut extracts via inhibition immunoblots. Synthetic overlapping peptide libraries were created using the SPOTs technology and the linear IgE-binding epitopes of each allergen subsequently determined by screening with pooled patient sera. For the walnut 2S albumin and cashew 11S globulin the most reactive epitopes were examined further and amino acids critical or influential for IgE binding identified. Some reactive peptides appeared to be conserved in sequence position and composition among the 2S albumins and 11S globulins of the walnut, cashew, and other previously characterized plant allergens when analyzed in comparative epitope maps. Structural motifs that might explain the conservation of these antibody-binding regions among the proteins were identified through homology modeling experiments. Largely unexplored in terms of allergen epitope characterization are those of a conformational nature, comprised of amino acids distant in the proteins primary structure but adjacent once the protein folds. A chimeric molecule-based strategy for identifying the existence and proposed location of a conformational IgE-binding epitope was successfully carried out for the cashew 11S globulin. / A Dissertation Submitted to the Department of Biological Science in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Spring Semester, 2006. / January 27, 2006. / Allergy, Tree Nuts, Cashew, Walnut / Includes bibliographical references. / Kenneth H. Roux, Professor Directing Dissertation; Shridhar K. Sathe, Outside Committee Member; Peter G. Fajer, Committee Member; Thomas C. S. Keller, III, Committee Member; Kenneth A. Taylor, Committee Member.