"Oxidative stress is a type of cellular stress that can damage and kill cells. While it is naturally occurring, many non-natural substances found in our environment can also induce the formation of reactive oxygen species (ROS), which then cause oxidative stress within the cell. Oxidative stress has been shown to be involved in the death of neurons in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The primary causes for these diseases are still unknown; however, we do know oxidative stress plays a primary role in their development. In conditions where oxidative stress is present, adenosine receptor expression has been upregulated and has played a cytoprotective role, but the specific mechanism of action is unknown. In this thesis, oxidative stress was studied in a model eukaryote, Saccharomyces cerevisiae, and the effects of the expression of the human A1 and A2A receptors upon stress response was examined. Oxidative stress was induced by the addition of hydrogen peroxide at concentrations of .5 mM, 1 mM, and 2 mM. The growth of cells expressing either A1-GFP R or A2A-GFP R at the varying hydrogen peroxide concentrations were compared to the parental cells. Confocal microscopy was performed to determine the receptor expression levels, and to confirm the expression of the receptors via their GFP tag. Immunoblots were also performed to assess the receptor expression level at the differing hydrogen peroxide concentrations. A ROS assay was also performed to show the presence of ROS and oxidative stress in the cells. No significant increase in receptor level expression or localization for either A1 R or A2A R at the varying hydrogen peroxide concentrations was found. The data did show trends indicating that A2A receptors may help process the oxidative stress better than A1 receptors and that A2A receptor containing cells had a shorter doubling time. However, more research on this subject should be performed in the future. However, the concentration of hydrogen peroxide should be greatly increased for further experiments in S. cerevisiae in order to better differentiate the trends observed." / 1 / Bryan Goldman

archives@tulane.edu / G protein-coupled receptors (GPCRs) are the largest family of proteins in humans and are expressed widely throughout the body. GPCRs consist of seven-transmembrane helices that bind extracellular ligands to initiate intracellular downstream signaling via interaction with G proteins, and function in many short and long-term responses in the body, including taste, immune function, and sugar sensing. Extracellular binding and the coupled downstream signaling pathway means that GPCRs are ideal drug targets for many diseases, making them of great interest to the pharmaceutical industry. Some GPCRs have been crystallized in an effort to better elucidate the structure-function relationship to aid in the design of novel therapeutics. The adenosine A2A receptor (A2AR) is a GPCR that has been crystallized bound to agonist, antagonist, and G protein. Although these crystal structures are informative in regards to A2AR structure when associated with binding partners, all current crystal structures truncate nearly 100 amino acids of the C-terminus. As a crystallization strategy, this truncation makes sense considering the C-terminus is long and unstructured. However, truncating roughly 25% of the protein, as well as making other point mutations calls into question the authenticity of the crystal structures in reflecting functional receptor and thus their potential value for therapeutic design. Beyond structural studies, biophysical characterization of drug binding to receptors in vitro to predict efficacy in vivo has shifted away from measures of affinity and selectivity and towards determination of kinetic rates. Kinetic rate constants in combination with affinity and drug residence time are thought to be better predictors of drug behavior in vivo. For these reasons, this thesis focuses on experiments to characterize A2AR kinetic rate constants. Previously, our lab showed that truncating the A2AR C-terminus reduced downstream cAMP signaling in mammalian cells, although where the effect on the signaling pathway occurred was not determined. Here, we report that truncation of the C-terminus ablates receptor association to Gαs, the first step in signaling. In this work, A2AR ligand binding kinetics, stability, and association to Gαs are characterized to better delineate the importance of interactions between receptor and stimuli in a way that is impactful to drug design. / 1 / Kirsten Swonger Koretz

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that leads to destruction of articular cartilage and subchondral bone at the synovial joints. Clinically, RA is characterized by swelling, tenderness and destruction of synovial joints, which results in severe disability and premature mortality. In the RA disease state, inflammation in the synovial compartment is regulated by a complex cytokine and chemokine network, including tumor necrosis factor α (TNFα), which has been clinically demonstrated a key mediator of RA pathogenesis. TNFα can be found in elevated levels in the synovial fluid and serum of RA patients and the role of the cytokine in both the inflammation and bone destruction of RA suggests it is important in the understanding of disease progression as well as the development of therapeutic targets. Many of the biological processes that mediate RA, including bone turnover and cartilage resorption, involve signaling pathways that are mediated by adenosine and its receptors. The A2B adenosine receptor (A2BAR) is highly expressed in the synoviocytes of RA patients and the receptor has a similar expression profile in humans and mice. The goal of this thesis was to use a mouse model of RA to understand how the A2B adenosine receptor modulates TNFα and other destructive enzymes that contribute to the progression of the disease. A collagen antibody-induced arthritis (CAIA) mouse model was used to determine the effect of A2BAR ablation on systemic and joint-specific TNFα expression. Comparable arthritic conditions were observed in CAIA mice of both A2BAR knockout (KO) and wild-type (WT) genotypes and the absence of the A2BAR gene did not result in any observable differences in the gross arthritic state created in each genotype. Immunohistochemistry analysis of TNFα expression in mouse paws revealed that paw joints from CAIA A2BAR KO mice exhibited more robust TNFα staining compared to CAIA WT specimens of the same treatment duration. ELISA analysis of the serum showed that only CAIA A2BAR KO mice had greater serum production of TNFα at day 10 after induction of arthritis. TNFα and matrix metalloproteinase-9 mRNA expression were also elevated in KO CAIA knee joints in comparison to WT CAIA knee joints; however, WT CAIA mice were found to have higher levels of aggrecanase mRNA compared to KO mice. These results suggest that while the loss of A2BAR activity leads to a hyper-inflammatory state, the A2B adenosine receptor alone is not responsible for the progressive inflammation of the synovial joints associated with rheumatoid arthritis.

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 29-32.

Obesity is a significant health care problem, affecting more than one third of the United States population and is an important risk factor for Type 2 Diabetes Mellitus (T2D). Adipose tissue expansion results in the recruitment and accumulation of macrophages, which secrete proinflammatory cytokines that impair insulin signaling. Adenosine regulates inflammation by signaling through G-protein coupled receptors (GPCRs), such as the A2b adenosine receptor (A2bAR). Recently a role for adenosine receptors has been described in the differentiation of osteoblasts and adipocytes. This thesis tests the hypothesis that the A2bAR regulates adipose tissue dynamics at the level of preadipocyte differentiation and macrophage inflammation. This thesis showed that activation of the A2bAR inhibited preadipocyte differentiation. A2bAR-induced adipocyte inhibition was dependent on the expression of Krüppel-like factor 4 (KLF4), which is important for stem cell maintenance and renewal. A2bAR knockdown enhanced adipogenesis in vitro and A2bAR knockout (KO) mice had more adipocytes as compared to wild type (WT) mice, suggesting enhanced adipogenesis in the absence of the A2bAR. The translational potential of this work is strengthened by the previous finding of elevated A2bAR expression in adipose tissue of obese individuals as well as our new finding of a close correlation between the expression of A2bAR and KLF4 in adipose tissue of obese individuals. A2bAR KO mice have impaired insulin resistance, in part due to reduced levels of insulin receptor substrate-2 (IRS-2). Proinflammatory cytokines have been shown to reduce IRS-2 levels. Given the role of the A2bAR in regulating inflammation, the contribution of A2bAR signaling in macrophages to insulin resistance was elucidated. Transgenic mice that express A2bAR only in macrophages were generated. Intriguingly, restoration of A2bAR signaling in macrophages ameliorated insulin resistance, glucose tolerance, and fat and liver tissue insulin signaling. As expected, tissue and plasma proinflammatory cytokine levels were reduced to that of WT mice. This suggested that the protective effect of A2bAR signaling on insulin resistance was due in large part to A2bAR control of macrophage cytokine expression. This thesis highlights the importance of A2bAR signaling in adipogenesis and in regulating inflammation in the setting of obesity and T2D.

L'adénosine est un nucléoside ubiquitaire issu de la déphosphorylation de l'ATP qui est libéré par les cellules endothéliales et les myocytes lors de l'hypoxie, de l'ischémie ou du stress oxydatif. Elle exerce un contrôle puissant sur les systèmes nerveux, immunitaire et cardiovasculaire par l'intermédiaire de quatre récepteurs membranaires : A1R, A2AR, A2BR et A3R. La compréhension de l'implication du système adénosinergique dans le système cardiovasculaire implique la possibilité technique d'un dosage de l'adénosine endogène et la quantification de l'expression de ses récepteurs. L'adénosine ayant globalement une action hypotensive (via les récepteurs A2AR) et chronotrope négative (via les récepteurs A1R), nous nous sommes intéressés à son implication chez les patients présentant des syncopes neurocardiogéniques, la bradycardie et l'hypotension étant 2 signes cardinaux dans ce syndrome. Les manifestations cliniques de cette affection peuvent être reproduites par le test d'inclinaison (HUT) et/ou le test à l'ATP. Dans un premier temps nous avons réalisé des dosages d'adénosine plasmatiques chez ces patients au moment d'un test d'inclinaison. Les concentrations en adénosine étaient élevées chez les patients présentant un test positif. Par la suite, nous avons comparé les concentrations en adénosine plasmatiques et l'expression des récepteurs A2A en fonction du résultat du test d'inclinaison et du test à l'ATP. / Adenosine is a ubiquitous nucleoside that comes from the dephosphorylation of ATP and which is released during hypoxia or oxidative stress, by endothelial cells and myocytes. Adenosine interacts on its cell surface receptors, namely A1R, A2AR, A2BR and A3R, to exert physiological effects on target tissues. Our knowledge about the adenosinergic system was improved because of our ability to measure adenosine plasma levels and to quantify its receptors expression. Because adenosine, via A1 or A2A receptor activation leads to bradycardia and hypotension, we first tried to understand the implication of the adenosinergic system in patients with neurocardiogenic syncope (NMS for neutrally mediated syncope). Indeed, this syndrome is characterized by relative or absolute bradycardia associated with a drop in blood pressure and a loss of consciousness. The symptomatology can be reproduced by the tilt test (HUT) or by the intravenous administration of ATP (ATP test). First, we measured adenosine plasma levels in patients with NMS just before and during HUT. We found that adenosine plasma levels were higher in patients with a positive HUT. Then, we compared adenosine plasma levels and the expression of A2A receptors in patients with NMS depending on the result of HUT and ATP-test. We found that elevated adenosine plasma levels and A2A receptors overexpression were associated with positive HUT. On the opposite, low adenosine plasma levels and normal expression of A2A receptor were associated with positive ATP test.

Adenosine and adenosine analogues are recognised as cardioprotective agents due to the responses that they induce through the activation of myocardial adenosine receptors. Antioxidants such as nitroxide radicals have also been found to possess cardioprotective properties in biological systems, namely through their ability to scavenge the oxygen-based free radicals that are potentially damaging to tissues and cells. It was envisaged that the linking of an antioxidant moiety to adenosine would produce an adenosine analogue that activates adenosine receptors and also scavenges oxygen-derived free radicals in the body. Consequently, one aim of this project was to synthesise a series of adenosine analogues that possessed a nitroxide or a phenolic antioxidant at the N6 position of the adenosine skeleton. Allosteric ligands have several advantages over orthosteric ligands as potential therapeutic agents, and research into the allosteric enhancement of adenosine receptors is a burgeoning field. It was envisaged that the linking of an antioxidant moiety to an allosteric enhancer would produce a compound that enhances the response of endogenous activation of adenosine receptors and also scavenges oxygen-based free radicals in the body. Consequently, a second aim of this project was to synthesise a series of allosteric enhancers of the A1 adenosine receptor that possessed antioxidant capability endowed by a nitroxide or a phenolic antioxidant functionality. This project has resulted in the synthesis and characterisation of 19 novel N6 substituted adenosine analogues, and additionally 12 novel derivatised thiophenes. Each of the target compounds was tested for its ability to bind to each of the adenosine receptor subtypes and some analogues were found to be potent and selective adenosine receptor agonists.

In the present study, we investigated the mechanism of adenosine-induced apoptosis in Drosophila imaginal disc cell line using the overexpression and silencing of several candidate genes. Our results indicate that the cell death is associated with the activity of c-Jun N-terminal kinase (JNK).

Adenosine is a ubiquitous metabolite with multiple physiological functions in organisms. In this thesis, I studied the effect of extracellular adenosine on Huntington´s disease (HD) model Drosophila melanogaster. I show that extracellular Adenosine signaling mitigates HD pathology by observing three main types of symptoms of the disease in Drosophila. The results suggest that the mechanism involves Drosophila melanogaster adenosine receptor signaling.