Astrocytes are one type of glia in the central nervous system, and they provide metabolic and structural support to neurons. Following injury astrocytes undergo an injury stress response known as reactive gliosis. Gliosis is characterized by both morphological and functional changes including increased cell migration. Astrocyte migration is seen in multiple disease states including glaucoma, age-related macular degeneration and glial scar formation. Migration relies upon multiple signaling pathways, many of which are activated by increases in calcium. One such source of calcium influx is the transient receptor potential vanilloid 1 (TRPV1) channel, which has a high calcium conductance and is expressed by astrocytes. Using a wound healing model, I found that antagonism of TRPV1 reduced both astrocyte migration and calcium influx following injury. Increases in calcium can drive cytoskeletal remodeling to facilitate migration, and I found that TRPV1 antagonism reduced alpha-tubulin intensity and induced retraction of both actin and alpha-tubulin in astrocytes following injury. My results suggest that in astrocytes TRPV1 is activated by injury, and that this activation contributes to injury-induced migration through an influx of calcium and subsequent cytoskeletal remodeling. By better understanding the events that underlie astrocyte migration, we can target astrocyte reactivity and migration to promote neuronal survival.

Synaptic transmission is characterized by exocytotic events which mediate the release of chemical transmitters to facilitate neuronal communication. Inhibitory presynaptic GPCRs act as feedback regulators limiting transmitter release from presynaptic terminals via the actions of their Gβγ subunits. Although Gβγ subunits have been shown to regulate exocytosis through direct interaction with the exocytotic machinery, relatively little is known about which G protein heterotrimers exist in vivo, the specificity of this interaction or its physiological consequences. The hypothesis proposed in the present study is that endogenous Gβγ subunits exhibit specificity when interacting with SNARE proteins to modulate synaptic transmission. To address this, efforts were made to examine the expression of different G protein isoforms throughout the CNS, study the functional specificity of α2A adrenergic receptor mediated Gβγ/SNARE interactions, and develop compounds which would allow its modulation. Targeted proteomics studies demonstrated a wide distribution of most G protein isoforms across brain regions and at synaptic terminals with distinct localization patterns observed for different Gβ and Gγ subunits. Investigation into the functional selectivity of SNARE modulation revealed that α2A adrenergic receptors exhibit specificity when interacting with Gβγ subunits as only a subset of Gβ and Gγ isoforms were coimmunoprecipitated with the receptor following stimulation. Further, lead compounds were developed which had an effect on the Gβγ/SNARE interaction, suggesting it may be possible to target this association directly through the use of selective protein-protein modulators. Taken together, the data presented in this study contribute to a better understanding of G protein signaling within the CNS as well as the role of specificity in α2A adrenergic receptor modulation of SNARE function.

PHARMACOLOGY Hypo-osmotic Effects in Cardiovascular Controls: Hepatic and Renal Mechanisms Underlying the Osmopressor Response. Patients with baroreflex impairment experience a robust increase in blood pressure (BP) after 16 oz. of water consumption. This increase in BP was also significant in healthy elderly subjects though with smaller magnitude but absent in healthy young adults. Using the sino-aortic mouse model, the pressor response to water was replicated to a similar fashion as seen in humans. This response to water was named the Osmopressor Response (OPR) because osmolality played a significant role in the initiation and maintenance of BP elevation. One of the molecular mediators of this response was the transient receptor vanilloid potential (TRPV4). These channels were highly expressed in the nervous system, the gut, the liver and the kidneys. Hypotonicity was sensed in the liver to trigger the OPR, and the presence of TRPV4 in this region was required for the response to occur. One of the gastrointestinal nerves that innervates the liver is the splanchnic nerve that feeds into the celiac ganglion. Mice that underwent celiac ganglionectomy was still able to trigger the OPR, however, the duration of BP elevation was significantly less than in sham mice, indicating the important role of the celiac ganglia in the maintenance of the response. The renal nerves also play a crucial role in the OPR because mice that had bilateral renal nerve denervation had significantly diminished OPR compared to WT mice. Although the OPR was initially only observed in baroreflex-impaired patients and elderly, water ingestion and its effect on BP might be substantial. Water ingestion beyond thirst has been used therapeutically in conditions such as: the orthostatic hypotension of autonomic neuropathy, the orthostatic hypotension in multiple system atrophy (MSA); and possibly in postprandial angina pectoris. The American Red Cross now recommends drinking 16 oz. of water before blood donation to prevent syncope. Approved___________________________Date___________ David Robertson, MD.

The goal of this project is to better understand the relationship between cardiac sodium channel dysfunction and cardiomyopathy. Mutations in the gene SCN5A, encoding the cardiac sodium channel, typically cause ventricular arrhythmia or conduction slowing. Recently, SCN5A mutations have been associated with heart failure combined with variable atrial and ventricular arrhythmia. Here we present the clinical, genetic and functional features of an amiodarone-responsive multifocal ventricular ectopy-related cardiomyopathy associated with a novel mutation in a NaV1.5 voltage sensor domain. A novel, de novo SCN5A mutation (NaV1.5-R225P) was identified in a boy with prenatal arrhythmia and impaired cardiac contractility followed by postnatal multifocal ventricular ectopy suppressible by amiodarone. We investigated the functional consequences of NaV1.5-R225P and noted that mutant channels exhibited significant abnormalities in both activation and inactivation leading to large, hyperpolarized window- and ramp-currents that predict aberrant sodium influx at potentials near the cardiomyocyte resting membrane potential. Mutant channels also exhibited significantly increased persistent (late) sodium current. This profile of channel dysfunction shares features with other SCN5A voltage sensor mutations associated with cardiomyopathy and overlapped that of congenital long-QT syndrome. Amiodarone stabilized fast inactivation, suppressed persistent sodium current and enhanced frequency-dependent rundown of channel availability. Comparisons with other cardiomyopathy-associated NaV1.5 voltage sensor mutations revealed a pattern of abnormal voltage dependence of activation that manifested in large, hyperpolarized ramp currents near resting membrane potentials as a shared molecular mechanism of the syndrome. Because the sodium gradient is critical to many processes in the myocyte, we endeavored to determine if expression of R814W or R222Q could affect calcium regulation within cardiomyocytes expressing the mutant channels. To test this, we produced lentiviral vectors capable of transducing isolated rabbit ventricular myocytes. Subsequent experiments examining calcium levels in myocytes transduced with exogenous NaV1.5 or mutant channels revealed little to no difference among all the parameters tested. Therefore, we conclude, that in these experiments there is no alteration of calcium handling resulting from overexpression of the mutations R222Q or R814W compared to WT channels.

Inward rectifier potassium (Kir) channels are a family of two transmembrane-spanning potassium selective ion channels. Kir channels are found in all kingdoms of life where they play essential roles in regulating numerous physiological functions. Mutations affecting Kir channels result in various diseases ranging from cardiac, neurological, renal and metabolic defects. Therefore, Kir channels represent important therapeutic targets. However, the Kir channel small-molecule pharmacology remains limited, which has impeded progress toward understanding their integral physiology and druggability. Since the emergence of insecticide-resistance in mosquito populations against current vector control agents, such as pyrethroids, efforts to decrease the transmission of vector-borne diseases are becoming less efficacious. As a result, we are facing a critical need for novel control agents. In mosquitoes, Kir channel functions remain largely unknown and their insecticidal potential unexplored. In an effort to expand the small-molecule pharmacology of Kir channels, we employed a broad array of experimental techniques, including molecular biology, fluorescence-based high-throughput screening, conventional and automated patch clamp electrophysiology, in conjunction with medicinal chemistry and mosquito bioassays to discover, optimize and characterize new modulators of Kir channel functions for the development of new therapeutics and insecticides.

The dietary polyphenol curcumin shows great promise as an anti-cancer and anti-inflammatory agent. While many cellular targets of curcumin have been identified, the chemical mechanisms whereby these targets are affected remain unclear. Orally administered curcumin undergoes metabolic conjugation with glucuronic acid and reduction of the double bonds along the heptadieneone chain. We have recently described oxidative transformation of curcumin in vitro leading to a dioxygenated bicyclopentadione derivative as the final product. We proposed that the oxidative metabolites of curcumin are direct mediators of its biological effects. I have synthesized [14C2]curcumin and used it to detect and isolate ten novel oxidative transformation products, including four reaction intermediates. The products were identified by UV spectroscopy, mass spectrometry, and a combination of 1D and 2D NMR methods. Mechanistic studies on the incorporation of 2H and 18O into the oxidative metabolites during reactions with 2H2O, H218O, and 18O2, enabled us to determine the mechanism of oxidative transformation of curcumin. I further showed that the phenolic glucuronide of curcumin, a major in vivo metabolite, undergoes enzymatic oxidative transformation in vitro to generate the bicyclopentadione-glucuronide, suggesting the same reaction mechanism as curcumin. Our studies for the first time determined a complete profile of the products of curcumin oxidative transformation and detailed their mechanisms of formation. The isolation and identification of the oxidative products will allow for future studies in assessing their involvement in the biological activities of curcumin. Implicating the oxidative products as bioactive agents provides a novel paradigm for understanding how curcumin exerts its pleiotropic therapeutic effects. Furthermore, oxidative metabolites of curcumin could represent new lead compounds with improved efficacy, and the possibility to overcome the pharmacokinetic limitations of dietary curcumin.

Mutations in SCN5A encoding the cardiac voltage-gated sodium channel (NaV1.5) can result in severe life-threatening cardiac arrhythmias such as long QT syndrome (LQTS). However, the molecular basis for arrhythmia susceptibility in early developmental stages remains unclear. Our lab has shown prominent expression of a fetal-expressed splice variant of the cardiac sodium channel (fetal NaV1.5) in human fetal and infant hearts. We hypothesized that mutations in SCN5A expressed in the fetal NaV1.5 may result in more severe functional effects vs expression in adult NaV1.5. Electrophysiological studies were conducted on heterologously expressed WT or mutant adult or fetal NaV1.5. We compared the functional effects of SCN5A mutations associated with early-onset LQTS with that of a mutation associated with typical onset LQTS (delKPQ) expressed in the context of fetal NaV1.5. We have shown that early onset LQT3 mutations exhibit equal or more severe functional consequences such as persistent sodium current (INa) in the fetal NaV1.5 vs expression in the adult NaV1.5. Typical onset mutation, delKPQ, demonstrated an attenuated gain of function in fetal NaV1.5. In addition to these findings, we elucidated the molecular mechanism of calmodulin (CaM) mutations associated with neonatal LQTS on NaV1.5. We hypothesized that mutant CaM would evoke an increased persistent INa associated with LQTS. Electrophysiological studies were conducted on WT adult or fetal NaV1.5 co-expressed with WT or mutant calmodulin. We observed that CaM-D130G co-expressed with fetal NaV1.5 exhibits a significantly greater persistent INa vs co-expression with WT CaM. This increase in persistent INa was not observed for CaM-D130G co-expressed with the canonical NaV1.5 and required the presence of high calcium. We also show that CaM mutations caused slowing of calcium-dependent inactivation of L-type calcium channels. We conclude that developmentally regulated alternative splicing of SCN5A contributes to the genetic risk for prenatal life-threatening cardiac arrhythmias. We also conclude that the voltage-gated cardiac sodium channel is not consistently involved in the molecular mechanism of LQTS associated with mutations in calmodulin.

PHARMACOLOGY Nanonetworks as Innovative Platforms for Therapeutic Solubilization and Delivery David Michael Stevens Dissertation under the direction of Professor Eva M. Harth Solubility remains the biggest obstacle in the development of new therapeutics and is the primary cause for clinical failure of promising drugs. The high lipophilicity of many chemotherapeutics and peptides imposes a major challenge for systemic administration and drug efficacy. Recent interest of pharmaceutical companies to apply nanoformulations stems from the interest to improve solubility, specificity, and efficacy for current, off-patent, and shelved drugs rather than creating new therapies. Numerous approaches have been investigated including poly(lactic-co-glycolic acid) (PLGA) formulations, lipid-based micelles, and pegylation of proteins, but these efforts often fall short of expectations due to rapid drug release, the use of non-degradable materials, and accumulation and toxicity in the liver. To overcome these obstacles, practical approaches have been developed for the formation of biodegradable nanoparticles and hydrogels via crosslinking reactions. Polyester nanoparticles, or nanosponges, are degradable, biocompatible networks synthesized using developed intermolecular crosslinking chemistries and are capable of encapsulating the therapeutic while enhancing the drugs solubility in aqueous solution, and the crosslinking density of the nanosponge can be adjusted to allow customized drug release rates. The availability of functionalities such as allyl and amine groups on the surface of the particles allows for targeting ligand attachment for targeted drug delivery applications. Having the abilities of organ-specific delivery and adjustable drug release rates allows the tailoring of this drug delivery platform to meet the specific needs of various applications. The same concept of using crosslinking chemistries to form nanosponges can be used to form hydrogel materials under concentrated conditions, and these biodegradable hydrogels are capable of tunable swelling, drug encapsulation, and adjustable drug release rates. Methods to synthesize predictable and defined polymer precursors have been developed which allows for complete customization of the resulting nanosponges and hydrogels that can be used for various applications including cancer, diabetes, and bone healing. Approved _________________________________________________ Date_________ Eva M. Harth, Ph.D.

<p>Inositol phosphates (IPs) are versatile metabolites that play important roles in multiple cellular processes. They have been considered signaling messengers that relay extracellular signals via a wave of their production and allosteric regulation of downstream targets. In addition to this classical role, recent studies have revealed that certain IPs can also function as protein structural cofactors. However, except for the two plant hormone receptors TIR1 and COI1, these IP binding proteins have neither sequences nor functions in common. Therefore, to test whether other cellular proteins are also subjected to this type of regulation and whether an IP binding motif exists, more proteins that bind IPs in a similar manner need to be identified. Via a proteome-wide biochemical screen, two yeast proteins were found to contain IP₆ as an integral component. One is the N-terminal acetyltransferase A complex (NatA), and the other one is Tif31 (or Clu1). IP₆ binding was also observed in NatC, another N-terminal acetyltransferase. The bioinformatics analysis and mutagenesis study showed that tandem tetratricopeptide repeats (TPRs), the only common structural element of NatA and Tif31, were responsible for coordinating IP₆. This mechanism of IP<sub>6<sub> binding is conserved in the fly homologs of these proteins. </p><p>NatA is one of the enzymes that acetylate the &alpha;-amino groups at protein N-termini. This widespread protein modification affects a wide range of cellular processes. IP₆ was shown to be essential for yeast NatA <italic>in vitro</italic> thermostability and for some but not all functions of the protein in cells grown under temperature stress. Other multiple phosphate-containing molecules including IP₅ species and the bacterial alarmone ppGpp were found to bind NatA and partially compensate for the lack of IP₆. IP₆ also binds the human NatA homolog. This binding is crucial for hNatA complex formation, <italic>in vitro</italic> and <italic>in vivo</italic> activities, and ability to rescue NatA-deficient phenotypes when it is expressed in yeast. Therefore, IP₆ acts as a molecular glue that brings hNatA (and hNatE) subunits together. The other protein found in our screen, Tif31, is important for normal mitochondrial morphology and distribution. In cells that cannot produce IP₄, IP₅ and IP₆, Tif31 levels were significantly decreased and these cells exhibited severe mitochondrial aggregation. Tif31 mutants that cannot bind IP₆ showed a reduction in cellular levels, a shift to high molecular weight complexes or aggregates, and inability to rescue <italic>tif31</italic>&delta; mitochondrial phenotype. This study established the vital role of IP₆ and IP₅ in maintaining Tif31 stability and Tif31-mediated regulation of mitochondrial distribution.</p><p>Collectively, this dissertation discovered two proteins that use IP₆ as a structural cofactor. For the first time, a conserved IP₆ binding motif has been shown to be present in certain TPR-containing proteins. Via tight binding to these proteins, IP₆ stabilizes their structures or subunit interaction. This research provides mechanistic evidence for the interplay between IP biology and N-terminal acetylation as well as between IP biology and mitochondrial morphology.</p> / Dissertation

It is now well established that the functional importance of the coronary circulation is to provide an adequate amount of nutrition (mainly oxygen) to meet the needs of the heart. The efficiency of the coronary circulation depends therefore upon two main factors: (1) the rate of coronary blood supply, i.e. the rate at which nutrition is supplied to the heart, and (2) the nutritional demands of the heart, i.e. the need of the heart for nutrition in order to meet its work requirements.