Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 190-204). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Background and objectives: Growing bodies of evidence demonstrate that adipocyte fatty acid binding protein (A-FABP) mediates the pathogenesis of atherosclerosis through its direct impacts on macrophages. Loss-of-function study was conducted by utilizing peritoneal macrophages derived from A-FABP knockout (KO) mice, to investigate the role of A-FABP in autophagy and macrophage foam cell formation. Key findings: 1. No morphological changes between the peritoneal macrophages derived from A-FABP knockout (KO) or their wild-type (WT) littermates. 2. Foam cell formation was successfully induced by the treatment of acetylated low-density lipoproteins (LDL) in peritoneal macrophages derived from A-FABP WT and KO mice. 3. LDL treatment induces autophagy in peritoneal macrophages from both A-FABP WT and KO mice. 4. The extent of LDL-induced autophagy is reduced in peritoneal macrophages of WT mice and is accompanied by increased lipid droplet accumulation when compared with A-FABP KO mice. Conclusions: A-FABP is a suppressor of autophagy and contributes to the attenuation of cholesterol efflux, subsequently resulting in enhancement of lipid droplets accumulation in peritoneal macrophages. A-FABP mediates the formation of macrophage foam cell via the suppression of autophagy. The results suggest that A-FABP is a potential therapeutic target to suspend the progression of atherosclerosis and remit the atherosclerotic lesion. / published_or_final_version / Medicine / Master / Master of Medical Sciences

The mouse node is the structural equivalent of Spemann's Organizer. The transcription factor Foxa2 (HNF3beta) is required for node and notochord specification and its enhancer has been characterized to a 500bp element that drives the expression of a reporter gene to the node and notochord in transgenic embryos. / The aim of the study was to identify sequence elements, within this enhancer, required to drive node/notochord specific-expression. Since, in the Xenopus organizer, Siamois activates organizer-specific gene expression, it was tested whether this factor could activate expression from the Foxa2 node-specific enhancer. Using deletion analysis, the response element was mapped to a 33bp region and it was shown that this element was both necessary and sufficient for reporter gene activation by X-Siamois . Furthermore, it was shown that X-Siamois can bind this DNA element and two sequence motifs required for binding and transactivation by X-Siamois were identified. Preliminary results suggest that the 33bp element, within the 500bp enhancer, is required for the maintenance of expression in the notochord of transgenic mice.

No description available.

In this study the mechanisms of Ca²⁺-induced-Ca²⁺-release, effects of membrane depolarizations and the actions of pharmacological intracellular Ca²⁺-modulators were examined in cultured rat dorsal root ganglion (DRG) neurones. The whole cell configuration of the patch clamp technique was used to record action potentials, action potential after-potentials and voltage-activated calcium currents, (I_Ca), calcium-activated chloride currents, (I_CI(Ca)), and non-selective cation currents, (I_CAN), under current and voltage clamp recording conditions, respectively. A sub population of DRG neurones expressed action potential after-depolarizations and I_CI(Ca)tail currents which were due to activation of Ca²⁺-activated Cl^- channels as a result of Ca²⁺ entry. I_CANwas dominantly activated due to Ca²⁺ release from intracellular stores evoked by pharmacological Ca²⁺-releasing agents such as caffeine, ryanodine and dihydrosphingosine. Calcium-activated conductances were identified by estimating reversal potentials of the activated currents, using selective pharmacological blockers and extracellular ionic replacement studies. Calcium-dependence of activated currents was also examined by using high concentration of intracellular Ca²⁺ buffer, EGTA, to prevent elevation of intracellular Ca²⁺-levels and by rapidly buffering raised intracellular Ca²⁺ using intracellular 'caged Ca²⁺ chelator', diazo-2. The involvement of intracellular Ca²⁺- stores was examined by performing experiments in Ca²⁺-free extracellular recording medium and pharmacologically inhibiting release of Ca²⁺ from intracellular stores, using dantrolene. Ryanodine had complex actions on DRG neurones, which reflected its ability to mobilize Ca²⁺, deplete Ca²⁺ stores, and inhibit Ca²⁺ release channels. Ryanodine inhibited action potential after-depolarizations and I_CI(Ca)tail currents by interacting with intracellular stores and preventing amplification of Ca²⁺ signalling by CICR. It was found that CICR observed under physiological conditions in rat DRG neurones involves intracellular Ca²⁺stores which were sensitive to ryanodine. In addition to ryanodine sensitivity these intracellular Ca²⁺ stores could be mobilized by caffeine and dihydrosphingosine.

This thesis gives insight into the way that transcription of the insulin gene is regulated by nutrients. This is achieved primarily by characterising a MAP kinase pathway which links glucose metabolism to the activation of a beta cell transcription factor IUF1. An understanding of the precise mechanisms by which nutrients control beta cell function may be invaluable for the development of artificial cell lines that can be used for gene replacement therapy. A study of the E2 element of the rat II promoter illustrated that at least three factors bound to the region. These were identified as IUF1 (complex D5), USF (complex D4) and an uncharacterised factor D3. IUF1 is a beta cell specific transcription factor that has been implicated previously in glucose responsive insulin gene transcription. IUF1 binds to the insulin promoter in response to high levels of extracellular glucose. USF has been shown to be involved in the carbohydrate responsive transcription of various hepatic genes. The recently characterised stress activated (Reactivating Kinase) MAP kinase pathway was clearly shown to be involved in mediating the link between glucose metabolism within the beta cell and the binding activity of IUF1. Phosphorylation of the factor serves to induce an alteration in protein structure, which converts the factor to an active form that shows a high affinity for its DNA binding site, thus activating transcription. The RK pathway may prove to be a crucial link between nutrient metabolism and the activity of other physiological processes.

The triglyceride-free fatty acid (TG-FFA) cycle was studied in white adipose tissue. The major aims of the study were 1) to see if the rate of TG-FFA cycling (i.e. FFA reesterification) and the sensitivity properties (see Newsholme and Crabtree, 1976, Biochem. Soc. Symp. 41, 61-109) were affected by various treatments, and 2) to measure the rate of cycling in vivo and assess its contribution to the metabolic rate of an animal. There are two ways of estimating the rate of TG-FFA cycling; the first is based on the release of glycerol and FFA from the tissue, and the second on the synthesis of the glycerol and FFA moieties of triglyceride. Experimental agreement between the two methods is very good. It is shown that the rate of TG glycerol synthesis can be estimated by measuring the incorporation of tritium from tritiated water into the TG-glycerol moiety; this method is used to study the TG-FFA cycle in vivo. Experimental results indicated that the rate of TG-FFA cycling in white adipose tissue in vitro and in vivo is affected by various short- and long-term treatments. However, the reesterification of FFA in adipose tissue can only account for perhaps ~1% of the basal metabolic rate of a mouse, and perhaps 4% of the increase in osygen consumption observed in fenoterol-treated mice. The equations of Newsholme and Crabtree (1976) describing the sensitivity properties of substrate cycles are extended and used to show that the TG-FFA cycle increases the sensitivity of control of FFA release from adipose tissue. The degree of sensitivity attainable is variable depending on the treatment used. The use of tritiated water for estimating TG-FFA cycling is tentatively extended to brown adipose tissue. It is suggested that the rate of cycling could be used as an indicator of sympathetic activity in brown and white adipose tissue.

This thesis is concerned with structural aspects of the recognition and effector functions of antibody molecules. The recognition process is investigated in the dinitrophenyl (DNP) binding mouse IgA produced by the myeloma MOPC 315. The studies on effector functions utilize a DNP binding mouse hybridoma IgG2a to examine the role of N-glycosylation in IgG. The combining site of protein 315. The involvement of tyrosyl residues in the combining site of protein 315 was examined by preparing specifically nitrated NO₂-Tyr-33_H and NO₂-Tyr-34_L derivatives of the Fv fragment of this protein. The ionizations of tnese derivatives were studied in the presence and absence of various DNP-ligands. Perturbations to the nitrotyrosine ionizations were found to be caused by the side chains of certain of these ligands, allowing an indication of the distance of these tyrosines from the bound hapten. On examination of the compatibility of these data with the model of the combining site of protein 315 proposed by Dower <en>et al. (1977) (Biochem. J. 165, 207-225) it was found that while the location of Tyr-33_H is consistent with this model, the position of Tyr-34_L is not. A remodelled combining site using the modified ring-current treatment of Perkins and Dwek (1980) (Biochemistry 19, 245-258) is presented. This allows a better rationalization of the nitration data and of previous experimental observations on protein 315. The role of the conserved C 2 domain oligosaccharide of IgG. This was examined by a functional comparison of native IgG with an aglycosylated IgG preparation. Aglycosylation was acheived by cell culture of the hybridoma cells in the presence of the glycosylation inhibitor tunicamycin. The conditions for preparation and purification of this aglycosyl IgG are described. Aglycosylated IgG is found to be correctly assembled as an H₂L₂ unit. It retains the antigen binding and Staphylococcal protein A binding abilities of the native glycosylated molecule. Using an assay system designed specifically to overcome certain problems in comparing Clq binding to different preparations of IgG it was found that the aglycosylated preparation showed only slightly reduced affinity for Clq. In addition the aglycosylated IgG is able to activate bound Cl. The above results are consistent with the structure of the Fc region being only minimally altered in the absence of oligosaccharide. The structural integrity of the aglycosylated molecule may be compromised however, as its ability to bind to monocyte Fc receptor is significantly reduced. In addition the aglycosylated molecule becomes much more susceptible to proteolytic digestion. A computational model-building analysis of the quaternary structure of Fc allows an explanation of at least some of the effects of aglycosylation in terms of reduced conformational stability of the C_H2 domains.