The field of research into endothelial progenitor cells is complex and difficult to interpret. There are a number different investigators working on specific subpopulations that are isolated in unique ways using unique or semi-unique cell surface antigens. Each population is, additionally, assayed in a specialized way corresponding to a certain tissue or disease process. All of these variables make it difficult to determine what cell populations might be important in a particular tissue or disease state, and greatly complicate comparisons between studies and cell populations. These studies sought to develop methods for: (1) analyzing definitive EPC populations and (2) comparing them to one another in the context of ocular neovascularization. The first aim of this work was to develop quantum dot-coded EPC subpopulations and to assess their recruitment to neovascular tufts. This was done using quantum dot nanocrystals conjugated to acetylated low density lipoproteins. Labeled endothelial progenitor cells were then tracked for incorporation into a model of laser-induced choroidal neovascularization. The second aim was to develop high throughput, in vitro methods to analyze the angiogenic capacity of EPCs using quantum dot coded subpopulations. This analytical tool aids in separating the individual aspects of EPC functions: (1) homing to angiogenic sites, (2) incorporating into and forming capillary tubes, and (3) proliferating into neovascular lesions. A unique system of using a parallel plate flow chamber was developed to assess EPC homing capabilities. Using this system, the EPC subpopulation CD133+/CD34+ was shown to be highly capable in terms of all three EPC functions. The third aim was to determine the role of endoglin in oxygen-induced retinopathy (OIR) using the methods developed to analyze the angiogenic capacity of EPCs. Endoglin was studied in the context of retinal neovascularization, and determined to have a pro-angiogenic role, largely through its control of cell proliferation in EPCs. Using an antibody to block endoglin in an OIR model resulted in the dose dependent inhibition of neovascular area, which inhibited additively with VEGF-directed treatments.

Human subjects with impaired baroreflex function cannot buffer rises or falls in blood pressure (BP), thus allowing BP effects of endogenous or environmental stimuli that previously escaped detection to emerge dramatically. Studies in these patients led us to discover that water ingestion induces a robust increase in BP and vascular resistance. This project explores the mechanism of waters cardiovascular effects using a mouse model of baroreflex impairment. We show that the pharynx, esophagus, and stomach are not critical sites for waters pressor action, and that plasma volume expansion does not contribute significantly to the rise in BP observed after water ingestion. We also show that the increase in BP after water ingestion is mediated through sympathetic nervous system activation, and appears to be independent of the effects of renin and angiontensin. Genetic knockout mouse models were used to investigate the potential role of several candidate molecular mediators. The osmosensitive transient receptor potential vanilloid 4 channel (TRPV4) was found to be an important component of the response. Although portal osmolality decreased after water ingestion in both wild-type and Trpv4-/- mice, only the wild-type animals showed a pressor response. The same volume of physiological saline failed to elicit an increase in BP, suggesting osmolality as the stimulus. The osmopressor response to water, and TRPV4 thus appear to be new factors now implicated in the physiology of BP regulation.

Mercury accumulates in fish-eating populations. Glial cells have diverse functions including providing nutrition[1], maintaining CNS homeostasis, removing pathogens, inducing neuronal differentiation and mediating CNS immune responsiveness. Dysfunction of glial cells is contributed to MeHg-induced brain damage. We hypothesized that 1) the cellular responses to MeHg are cell-type specific; 2) differences exist in the uptake of MeHg between astrocytes and microglia, leading to differential temporal cellular responses. I tested the unique response of primary glial cells to MeHg exposure at physiologically relevant concentration. The results are presented in this dissertation. Firstly, I established the methodology to separate a large amount of rat primary microglial cells with high purity (>95%) from mixed glial cell culture. Secondly, I investigated the toxic effects of MeHg on primary glial cells as well as their cellular response to acute MeHg exposure at environmentally relevant concentrations (0.1µM~ 5µM). The results showed MeHg treatment caused a concentration- and time- dependent microglial cell death, intracellular ROS generation and GSH depletion. I analyzed the dynamic changes of nuclear factor erythroid- derived 2- like 2 (NFE2L2) in both cytosolic fraction and nuclear fraction. My results suggested that the increase in Nrf2 protein level and the subsequent nuclear translocation are regulated by ROS in both glial cell types. However, Nrf2 changes in astrocytes occurred on a protracted time scale. Thirdly, the effects of Nrf2 on its downstream gene expression and cell viability post MeHg exposure were further studied, using short hairpin RNA (shRNA) approach. Finally the responses of rat primary microglial cells post MeHg treatment were compared with those of rat primary astrocytes. In conclusion, our study has demonstrated that microglial cells are more sensitive than astrocytes to MeHg. Their regulation kinetics differ, therefore allowing astrocytes and microglial cells to play different roles in mediating MeHg toxicity.

Coronary artery disease accounts for 54% of all cardiovascular disease in the United States. To provide novel therapeutic targets for the prevention and treatment of coronary artery disease, my dissertation project focused on understanding the molecular mechanisms regulating coronary vessel formation during embryonic development. In particular my project focused on understanding the role of the Type III TGF-beta Receptor in regulating the behavior of epicardial cells, the precursors of coronary vessels. It was previously established that the loss of TGFâR3 in mice leads to embryonic lethality due to failed coronary vessel formation but the cellular and molecular mechanism had not been established. My project focused on identifying this mechanisms through in vitro and in vivo analysis, and uncovered that the loss of this receptor deregulates processes such as proliferation, apoptosis and invasion, which are essential to the formation of coronary vessels. We conclude from my thesis that mice lacking this receptor die in utero due to the limited number of cells available to participate in the process of coronary formation.

Proteostasis is maintained by a complex network of genes and processes which includes core synthesis and degradation machineries as well as chemical and protein chaperones. Much of what is known about the function and organization of the proteostasis network stems from analyzing how cells respond to genetic or environmental perturbation of proteomic integrity. Recent evidence points to a critical role for the proteostasis network in survival of hypertonic environments, but the proteotoxic effects of hypertonic stress remain largely undescribed. Employing the many experimental advantages of the nematode C. elegans, we provide the first detailed description of the nature and extent of protein damage caused by hypertonic stress. Misfolding and aggregation of diverse reporters and endogenous proteins are rapid and widespread in vivo. Additionally, we demonstrate that acclimation of C. elegans to a mild hypertonic environment activates unknown proteostasis activities capable of preventing aggregation during extreme hypertonic stress. To define novel aspects of the hypertonic stress response and extend our understanding of cellular proteostasis strategies, we employ genetic and pharmacological approaches in determining the mechanism by which hypertonic acclimation enhances proteostasis. We hypothesize that chemical chaperones, protein chaperones, proteolysis machineries, and/or protein synthesis must be involved. Surprisingly, hypertonicity- or mutation-induced accumulation of glycerol, an organic osmolyte widely believed to act as a chemical chaperone in vivo, does not directly ameliorate protein damage during stress or aging. Protein chaperone expression is not transcriptionally upregulated. Further, hypertonic stress actually reduces protein degradation, an effect not reversed by acclimation. We demonstrate for the first time that suppression of protein translation during an environmental stress directly enhances proteostasis by preventing aggregation of extant proteins. Combined with recent observations that inhibition of translation extends lifespan and occurs naturally in response to other proteotoxic stressors, this finding suggests that translational reprogramming represents a conserved mechanism by which cells reduce the population of nascent, damage-prone proteins to enhance the availability and effectiveness of pre-existing chaperones.

Mutations in inhibitory GABAA receptor γ2 subunit gene, GABRG2, have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS). This dissertation elucidated the function of three epilepsy associated GABRG2 mutations, IVS6+2T->G, Q40X, and S443delC, which are found in both GABRG2 gene coding and non-coding regions. They impairing GABAA receptor biogenesis by multiple mechanisms including reducing subunit mRNA stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. Aminoglycosides treatment could treat seizures induced by GABRG2(Q40X) mutation, but the treatment for epilepsies induced by other GABRG2 mutation is under investigation.

Arrestins bind G protein-coupled receptors and more than 100 non-receptor partners, regulating various signaling pathways and cellular functions. The interactions of many proteins (e.g., Src, JNK3, ERK½, Mdm2, etc.) with receptor-bound arrestin localize these molecules to receptor-rich membranes. Our recent finding that arrestins bind microtubules and recruit signaling proteins to the cytoskeleton prompted us to investigate whether arrestins affect cell motility and morphology. Here we describe a novel function of arrestins, their direct effect on focal adhesion dynamics. We demonstrate excessive spreading of cells lacking both non-visual arrestins, which is substrate-independent, evident on both fibronectin and poly-D-lysine. Reduced activity of small GTPases RhoA and Rac1 in arrestin-deficient cells is only partially responsible for the cell spreading phenotype. Increased adhesion, reflected by elevated activity of focal adhesion proteins paxillin and focal adhesion kinase, underlies the exaggerated spreading of arrestin-null cells and their reduced motility. The absence of arrestins greatly increases the size and lifespan of focal adhesions, indicating that arrestins are necessary for rapid focal adhesion turnover. Focal adhesions in arrestin-deficient cells are insensitive to microtubules, suggesting that arrestins likely mediate the induction of focal adhesion disassembly upon microtubule regrowth. Overexpression of WT arrestins and their receptor binding-deficient mutants in arrestin-null cells rescues the phenotype, demonstrating that regulation of focal adhesion dynamics by arrestins is receptor-independent. This is the first demonstration that arrestins play a direct role in focal adhesion dynamics.

THE ROLE OF CA2+/CALMODULIN-DEPENDENT KINASE II IN NORMAL AND ABNORMAL EARLY POSTNATAL DEVELOPMENT RICHARD GUSTIN Dissertation under the direction of Professor Roger J. Colbran Ca2+/calmodulin-dependent kinase II (CaMKII) is known to play important roles in mechanisms underlying molecular memory and has been shown to be misregulated in adult rodent models of altered learning and memory. However, the regulation and role of CaMKII during development in vivo is poorly understood. In this dissertation I have taken advantage of three rodent models of genetic (Angelman Syndrome and CaMKIIα-Thr286Ala knock-in) and environmental (Early-Life Stress) perturbation to assess the role of CaMKII during development. I was able to show that maternal imprinting of UBE3A in a mouse model of Angelman Syndrome (AS) was not brain region specific, but extended throughout the brain as well as in peripheral tissues, presumably contributing to the broad neurological and peripheral phenotypes of the AS mice and human AS patents. Studies with this AS mouse model led me to hypothesize that the previously reported CaMKII misregulation was potentially due to deficient maternal care provided by AS dams. This led to the adoption of an early-life stress (ES) paradigm in mice, where I was able to establish that CaMKII was misregulated in the ES mice. The ES animals also showed synaptic transmission deficits lasting into adulthood that may account for some of the previously reported behavioral deficits in this model. Phosphorylation of CaMKIIα at Thr286 renders the kinase autonomously active. To specifically test the role of CaMKIIα-Thr286 phosphorylation during development I used a transgenic mouse model, CaMKIIα-Thr286Ala knock-in mouse, where I was able to identify an early-life novel object recognition deficit associated with CaMKII misregulation and altered localization. Overall, I determined that genetic and environmental perturbations that alter CaMKIIα regulation during development also alter synaptic transmission and learning and memory long-term. Furthermore, I have shown that altering the ratio of specific CaMKII isoforms that make up the holoenzyme is able to cause differential association with protein complexes that can have significant effects on learning and memory.

Studies described in this thesis identify a novel mechanism for α4s regulation of PP2Ac stability. α4 binds the PP2A catalytic subunit (PP2Ac) and the microtubule-associated E3 ubiquitin ligase MID1, and through unknown mechanisms can both reduce and enhance PP2Ac stability. We show MID1-dependent monoubiquitination of α4 triggers calpain-mediated cleavage at the F255-G256 bond in α4 releasing α4 from MID1. The cleavage of α4 switches its activity from protective to destructive towards PP2A, and results in the increase in phosphorylation of the microtubule-associated protein (MAP) tau. This regulatory mechanism appears important in MAP-dependent pathologies as levels of cleaved α4 are decreased in Opitz Syndrome (OS) and increased in Alzheimers disease (AD), disorders characterized by MAP hypophosphorylation and hyperphosphorylation, respectively. These findings indicate that regulated inter-domain cleavage controls the dual functions of α4, and dysregulation of α4 cleavage may contribute to OS and AD.

Prostaglandin E2 (PGE2) is a lipid autacoid, which is an oxidative metabolite of arachidonic acid synthesized by cyclooxygenase and prostaglandin E synthases. PGE2 is proposed to mediate in part the pathophysiology of hypertensive and diabetic kidney diseases. To probe the structure and function of a cell surface receptor for PGE2, single amino acid substitutions of the mouse EP3 receptor were generated, each having a single cysteine-to-alanine missense mutation. These studies demonstrated a critical disulfide bond between the first and second extracellular loops of mouse EP3 at position C107 and C184. Receptors substituted at either of these cysteines had attenuated expression and were functionally inactive. To study EP1 and EP3 receptors in mouse models of chronic kidney disease, selective antagonists of EP1 and/or EP3 were synthesized and characterized. A 4-chlorophenylsulfonamide was a functional antagonist of EP1 and EP3, lacked the off-target activity of the lead antagonist, and was efficiently metabolized in vitro to a small subset of metabolites. Subcutaneous injection of this compound prolonged plasma exposure of this antagonist sufficiently to maintain receptor coverage with once daily injection. Subcutaneous injection of this antagonist significantly blunted the vasopressor response of EP1 and EP3 agonists in mice. Thus, this represents a novel, selective EP1 and EP3 antagonist that is bioavailable in mice and suitable for use in studies of chronic kidney disease.