Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "May 1999." Typescript. Vita. Includes bibliographical references (leaves 108-124). Also available on the Internet.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Human A2A adenosine receptors. Includes bibliographical references (115-135). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2005" Includes bibliographical references.

Human embryonic stem cells (hESCs) have been a useful tool in the study of the embryo development and could be used by drug developing companies to create disease models and assist in the production of new medicines. One of the models that has been studied before, is the development of the pancreas. Scientists have obtained mixed results so far in the generation of functional pancreatic  cells from hESCs. We studied the differentiation potential of hESCs. As purinergic signalling is involved in may physiological processes, including cell proliferation and differentiation, a study of purinergic signalling in hESCs would help us deeper understand the hESC physiology. In order to study the purinergic profile of hESCs we established a culture system that allowed the transfer and attachment of pluripotent hESC colonies on glass coverslips. We then studied the functional purinergic profile of hESCs and found that they do not express functional P2X1 receptors, but they do express functional P2Y6 receptors, which might be implicated in the hESC differentiation. In parallel to these studies, we developed a reporter gene lentivirus, where the mouse Pdx-1 promoter area controlled the expression of a reporter fluorochrome, eGFP. We managed to generate a functional lentivirus, however, further analysis is needed in order to be able to use it in developmental studies. Finally, we tested the hypothesis that glucose affects the differentiation of hESCs towards pancreatic endoderm. Our preliminary results suggested that glucose does affect the differentiation potential of hESCs.

Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

Morphine is one of the most widely used drugs for the treatment of pain but its clinical efficacy is limited by adverse effects including persistent constipation and colonic inflammation. Morphine-induced colonic inflammation is facilitated by microbial dysbiosis and bacterial translocation. In this study, we demonstrate that secondary inflammation and persistent constipation are modulated by enteric glia. In chronic morphine treated mice (75 mg morphine pellet/5 days), ATP-induced currents were significantly enhanced in enteric glia isolated from the mouse colon myenteric plexus. Chronic morphine resulted in significant disruption of the colonic epithelium and increased Il-1β in the myenteric plexus. The increase in ATP-induced currents, IL-1β expression and ATP release were also observed in isolated glia treated with lipopolysaccharide (LPS) consistent with bacterial translocation as a potential mediator of chronic morphine-induced inflammation. These effects of LPS were reversed by carbenoxolone, a connexin43 hemichannel blocker. In-vivo treatment with carbenoxolone (25 mg/kg) prevented 1) ATP-induced currents in enteric glia, 2)the decrease in neuronal density, and 3) colonic inflammation in chronic morphine treated mice. Inhibition of connexin43 in enteric glia also reversed morphine mediated decrease in gastrointestinal transit. These findings indicate that bacterial translocation-induced enteric glial activation and inflammation is a significant modulator of morphine-related constipation.

by Vincent, Wai-ip Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 128-134). / Abstract also in Chinese. / Chapter Chapter I. --- Literature Review / Chapter I.1. --- "Structure, functions and general physiology of equine sweat gland" / Chapter I.1.1. --- Ultrastructure of equine sweat gland --- p.1 / Chapter I.1.2. --- Functions and physiology of equine sweat gland --- p.3 / Chapter I.1.3. --- Experimental studies on equine sweat gland by functional approaches --- p.4 / Chapter I.1.4. --- Hormonal and neuronal regulation of sweat secretion in equidaes --- p.5 / Chapter I.1.5. --- Possible role(s) of extracellular ATP in equine sweat gland epithelia --- p.6 / Chapter I.1.6. --- Measurement of electrogenic anion secretion by short-circuit current (Isc) technique --- p.8 / Chapter I.2. --- Classification of purinergic receptors and its existence in biological systems / Chapter I.2.1. --- Functional classification of purinergic receptors --- p.13 / Chapter I.2.2. --- Basic structure of G-protein coupled P2Y receptors --- p.17 / Chapter I.2.3. --- Physiological function and significance of purinergic receptors --- p.20 / Chapter I.3. --- Objectives of study --- p.22 / Chapter Chapter II. --- Methods and Materials / Chapter II.l. --- Culture technique of the equine epithelial cells --- p.23 / Chapter II.2. --- Conventional short-circuit current (Isc) measurement technique / Chapter II.2.1. --- Introduction --- p.25 / Chapter II.2.2. --- Preparation of permeable supports and electrodes --- p.25 / Chapter II.2.3. --- Experimental set up and measurement of Isc --- p.28 / Chapter II.2.4. --- Measurement of Isc during experiment --- p.30 / Chapter II.3. --- Measurement of intracellular free calcium ( [Ca2+ ]ii) by microspectrofluorimetry / Chapter II.3.1. --- Preparation of cells --- p.31 / Chapter II.3.2. --- The set up and procedures for experiment --- p.31 / Chapter II.4. --- Simultaneous measurement of changes in [Ca2+ ]i and Isc / Chapter II.4.1. --- Experimental set up and manipulation --- p.35 / Chapter II.4.2. --- Other preparations before experiment --- p.37 / Chapter II.5. --- Material and solutions used for experiment / Chapter II.5.1. --- Culture media and enzyme --- p.40 / Chapter II.5.2. --- Chemicals and Drugs --- p.40 / Chapter II.5.3. --- Preparation of solution for experiments --- p.42 / Chapter II.6. --- Statistical analysis --- p.44 / Chapter Chapter III. --- Results / Chapter III.l. --- Effects of nucleotides on transepithelial ion transport / Chapter III.1.1. --- Basic electrophysiological properties of cultured equine sweat gland epithelia --- p.45 / Chapter III. 1.2. --- Short-circuit current (Isc) induced by nucleotides --- p.45 / Chapter III. 1.3. --- Identification of ion species responsible for the change in Isc --- p.50 / Chapter III. 1.4. --- Effects of chloride channels blockers on the UTP-induced Isc --- p.51 / Chapter III.2. --- Signal transduction mechanisms of P2Y-nucleotide receptors / Chapter III.2.1. --- The involvement of Gi-proteins --- p.56 / Chapter III.2.2. --- Effect of BAPTA on the increases in Isc induced by nucleotides --- p.58 / Chapter III.2.3. --- Study of P2Y-receptor mediated increase in [Ca2+]i --- p.62 / Chapter III.3. --- Characterization of the P2Y subtype(s) by cross desensitization experiments / Chapter III.3.1. --- Autologous desensitization experiments --- p.70 / Chapter III.3.2. --- Classical cross desensitization experiments --- p.70 / Chapter III.3.3. --- Characterization of the ATP-insensitive P2Y-receptor --- p.80 / Chapter III.3.4. --- Interaction between ATP and bradykinin --- p.87 / Chapter III.4. --- Simultaneous measurement of [Ca2+]i and Issc / Chapter III.4.1. --- Effect ofUDP and ADP --- p.89 / Chapter III.4.2. --- Correlation of Isc and [Ca2+]i --- p.92 / Chapter III.4.3. --- Cross desensitization experiments --- p.97 / Chapter III.5. --- Evidence of a [Ca2+]i-independent Isc-component induced by nucleotides / Chapter III.5.1. --- The time course of the ΔRf and ΔISC --- p.102 / Chapter III.5.2. --- Effect of ionomycin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.3. --- Effect of thapsigargin on the ΔISC and ΔRf induced by nucleotides --- p.110 / Chapter III.5.4. --- Effect of thapsigargin in nominal Ca2+-free solution --- p.115 / Chapter Chapter IV. --- Discussion / Chapter IV. 1. --- Role of extracellular nucleotides in epithelial tissues --- p.119 / Chapter IV.2. --- Characterization of an ATP-insensitive P2Y-nucleotide receptor --- p.120 / Chapter IV.3. --- Expression of the novel ATP-insensitive receptor on a functionally polarized epithelia --- p.122 / Chapter IV.4. --- Involvement of a [Ca2+]i -independent Isc induced by nucleotides --- p.124 / Chapter Chapter V. --- References --- p.128

INTRODUCTION: The cornea forms the anterior-most barrier of the eye, consisting of a non-keratinized pseudostratified squamous epithelium, a collagen-based stroma, and an endothelium. It is completely avascular, yet the most densely innervated structure in the human body. The sensory nerves project from the ophthalmic branch of the trigeminal cranial nerve into the limbal/stromal interface. From there, the nerves branch and ascend into Bowman’s membrane, a basal lamina delineating the epithelium from the stroma, and project into the epithelium as free nerve endings. Injury to the corneal epithelium can potentially lead to impaired vision if the wound healing process is not properly initiated. Immediately after injury, nucleotides such as ATP are released and bind to purinergic receptors known to be located in epithelial cell membranes, thereby initiating epithelial cell migration to close the wound. Malfunctions in the interactions between the corneal nerves and their epithelial counterparts during the wound healing process are thought to contribute to the attenuated wound healing characteristic of diabetes. However, the precise nature of these interactions, how they facilitate wound healing, and how they are impaired in diabetes, is not well understood. OBJECTIVES: Previously, our lab has shown that a member of purinergic family receptors (P2X7) is localized in the basal epithelial cells and becomes relocated to the leading edge of the wound after injury. When the relocation is inhibited, migration is attenuated. Additionally, it is known that diabetic mouse models display slower wound healing rates. The present study has three aims: (1) to replicate the characteristic sub-basal whorl organization of the corneal nerves in organ-cultured corneas; (2) to elucidate the connections between patterns of corneal innervation and purinergic receptor expression; and (3) to understand how these patterns interact to facilitate normal wound healing and how these interactions are disrupted in a diabetic model. METHODS: Our approach was to use immunohistochemistry of dissected mouse and to visualize the tissue using confocal microscopy. Sensory innervation profiles from diet induced obesity (DIO) mouse corneas and their wildtype C57Bl6 counterparts were compared in unwounded and wounded tissue. To image the nerves a methanol fixation protocol was optimized to examine the sub-basal plexus and the apical nerves. Corneas were dissected, stained with beta III-tubulin, which identifies nerves, and with an antibody to the P2X7 purinergic receptor, which is expressed in the epithelium and nerves. Trephine induced epithelial abrasion injuries were made on separate DIO and control models to compare re-epithelialization and re-innervation between the diseased and healthy states. Corneas were imaged using a Zeiss LSM 700 laser scanning confocal microscope and optical images were taken through the cornea over a distance averaging 115 microns. Corneas were imaged using a macro tiling plugin, stitching 3x3 optical z-stacks into composite images. The 3x3 tiles were created to image the central whorl, as well as the peripheral nerve fibers. Co-localization of P2X7 and betaIII tubulin were determined by thresholding using ImageJ/FIJI software. RESULTS: The elegant organization of the centralized sub-basal whorl of the control mouse was disrupted in the DIO mouse cornea, appearing fragmented and incomplete. Analysis of 7.5 and 15 wk corneas showed the whorl to be present at 7.5 wks. Average apical nerve fiber projection length was decreased in DIO cornea. Yet, analyses at each epithelial layer demonstrated overall increased apical nerve density in the DIO corneas as compared to control while sub-basal nerve density decreased dramatically. Stromal nerves remained equivalent. P2X7 did co-localize to the large stromal nerve fibers but it was difficult to show the localization along the sub-basal nerve plexus. However in cross-section images, P2X7 displayed an intracellular polarity, and was present along the apical surface of the columnar basal epithelial cells lining the basement membrane. This localization may suggest the presence of P2X7 expressing sensory nerves, which may be ideally poised for communication with the basal cells after injury. CONCLUSIONS: These data support the hypothesis that there is indeed a difference between diabetic and control corneal innervation. While wound healing differences due to the interaction between sensory nerves and the localization of P2X7 in epithelium at the leading edge remain to be fully elucidated, the novel finding of P2X7 expression in corneal nerves confirms a potential role of purinergic receptor and nerve coordination in conducting the wound healing response.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 108-125). Also available on the Internet.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.