<p>Regulation of the flux-generating enzyme complex pyruvate dehydrogenase (PDHc) was examined in the context of its physiological function in human skeletal muscle. In the first two studies, the role of PDHc in intramuscular fuel selection and the mechanisms regulating PDHc transformation from its inactive form (PDHb) to its active form (PDHa) and PDHa activity were examined. In a third study, the role of PDHc in muscle lactate production during exercise and recovery was assessed and the factors controlling transformation to PDHa were also examined.</p> <p>In the first study, 5 subjects were examined during rest and cycling exercise at 75% of VO₂max after 3 days of consuming a low-carbohydrate (LCD) or high-carbohydrate (RCD) diet In the second study, 8 subjects were examined at rest and during cycling exercise at 40% and 80% VO₂max while they were infused with sodium acetate (ACE) or sodium bicarbonate (BIC). At rest, consumption of a LCD and ACE infusion increased the intramuscular acetylCoA-to-CoASH ratio and citrate, as a result of increased oxidation of available fat fuels and acetate, respectively. Elevation of the acetylCoA-to-CoASH ratio and citrate inhibited PDHa and phosphofructokinase activity, respectively. Consequently, the rates of pyruvate oxidation by PDHa and pyruvate production by glycolysis were reduced preventing the oxidation of intramuscular glucose. The resting results of these two studies were consistent with the operation of a reciprocal cycle of glucose and fat oxidation for intramuscular energy production. The events leading to glucose restriction were initiated by changes in acetylCoA accumulation which caused an increase in the acetylCoA-to-CoASH ratio and citrate concentration which induced the transformation of PDHa to PDHb and inhibition of glycolytic flux, respectively.</p> <p>In contrast, during exercise at 40%, 75% and 80% VO₂max, transformation from PDHb to PDHa was determined by Ca²⁺ and was not restricted in any of the conditions except the LCD condition. Lower PDHa activity and incomplete transformation to PDHa occurred in this condition, placing a restriction on intramuscular glucose utilization. However, the acetylCoA-to-CoASH ratio actually decreased in this condition, suggesting that the lower rate of transformation was independent of alterations in the acetylCoA-to-CoASH ratio. In contrast, when transformation to PDHa was not limited in any other condition there was an increase in the acetylCoA-to-CoASH ratio which should have limited transformation. Thus, during exercise, the restriction on muscle glucose oxidation occurred only after a LCD indicating that this reciprocal cycle of fuel selection and utilization was also operating during exercise and that PDHc transformation was an integral part of its operation. However, during exercise, the regulatory factors controlling PDHa differed from those that determined the activity of this enzyme at rest. Lower PDHa during exercise in the LCD condition was consistent with an increase in the PDH-kinase/PDH-phosphatase activity ratio, in the 3 day period before exercise, increasing the occupancy of monophosphate esters on both the transformation site and the inhibitory sites of the Elα components. Incomplete removal of these additional phosphate esters by PDH-phosphatase during exercise may have then resulted in lower PDHa and glucose conservation later on during exercise.</p> <p>In a third study, the role of PDHa in muscle lactate production during exercise and recovery was examined and the factors controlling PDHc transformation were determined in 7 subjects. During repeated 30 second bouts of maximal isokinetic cycling exercise, complete transformation to PDHa occurred concurrently with muscle lactate accumulation and increased mitochondrial oxidation, indicating that lactate production was not dependent on the development of tissue hypoxia. Instead, during exercise muscle lactate production resulted from a rate of glycolytic pyruvate production that was greater than PDHa activity. Conversely, during recovery, net lactate oxidation occurred as the lactate dehydrogenase equilibrium shifted more toward pyruvate production and PDHa remained partially active due to attenuation of the acetylCoA-to-CoASH ratio, reductions in the ratios of NADH-to-NAD and ATP-to-ADP and elevated concentrations of hydrogen ion and pyruvate.</p> <p>The present studies extend the analysis of PDHc regulation from in vitro and in situ studies to human skeletal muscle in vivo. The findings of the present studies suggest that the during muscle contraction the most important factor regulating PDHc transformation in human muscle is probably Ca⁺, while the other regulatory factors perform secondary roles. In contrast, during rest and recovery from maximal exercise, PDHc transformation to PDHa and PDHa activity are determined by the intramuscular ratios of acetyICoA-to-CoASH, NADH-to-NAD, ATP-to-ADP and the concentrations of hydrogen ion and pyruvate.</p> / Doctor of Philosophy (PhD)

One of the most fundamental aspects of extracorporeal membrane oxygenation (ECMO) is maintaining proper anticoagulation management in order to prevent hemorrhagic or thrombotic events. Anticoagulation on ECMO is most commonly achieved with the use of unfractionated heparin to maintain a minimum anticoagulation level as monitored by activated clotting time (ACT). Heparin's main effect is exerted by binding to and potentiating antithrombin III. Many factors may contribute to a sub-therapeutic ATIII level that may decrease the effectiveness of heparin. A retrospective record review was performed on all adult ECMO patients at the University of Arizona Medical Center between 2008 and 2011, in order to determine optimal ATIII levels for maintaining proper anticoagulation. In addition, we investigated correlations between ATIII levels and hemorrhagic and/or thrombotic events. Variables measured include, ACTs, heparin dose, ATIII dose, ATIII levels, blood product use, and adverse events. Thirty-five patients received ATIII over the course of the ECMO run. Six patients did not receive ATIII and they were found to have used significantly more blood products than those who did receive ATIII. Also, heparin dose dropped significantly 24h after the first dose of ATIII. There is a significant positive correlation between the amount of ATIII given per day and the amount of packed red blood cells transfused per day. The results suggest an ideal therapeutic range of ATIII dosing, where lack of or too much ATIII administration can lead to excessive bleeding.

Breast cancer, which in advanced stages often leads to bone metastasis, is the most frequent malignant tumor and the second deadliest form of cancer among women in the U.S. Skeletal metastasis is associated with imbalanced bone remodeling and eventual bone fracture that contributes to incapacitating pain and loss of mobility. Bone cancer pain remains a significant health problem due to the limited repertoire of analgesics available to treat this pain without negatively influencing the quality of life and "bone health" of the patient. Bone cancer results in a marked influx of pro- and anti- inflammatory hematological cells into the medullary cavity resulting in activation of nociceptors that express cytokine and chemokine receptors. Thus, blockade of these factors may result in a significant attenuation in bone cancer pain. The sustained release of cytokines by both primary tumor cells and invading leukocytes into the tumor microenvironment shapes the immune response to tumor invasion and ultimately mediates the shift in immune balance to the predominantly immunosuppressive state seen with late stage disease. Activation of cannabinoid receptor 2 (CB2), found on immune cells but not neuronal cells, has been shown to inhibit the release of cytokines from leukocytes; this inhibition plays an important role in CB2 agonist's ability to inhibit pain without producing the CNS side effects commonly associated with CB1. Cannabinoids have also been demonstrated in a number of cancer models to modulate the tumor microenvironment via effects specific to the tumor cells as well as regulation of invading leukocytes. Here, we show that the CB2 specific agonist JWH-015 mediates inflammatory factors in vitro and in vivo in the femoral intramedullary cavity in a murine model of bone cancer while simultaneously attenuating breast cancer induced bone pain and promoting overall health of the bone microenvironment. Further, we demonstrate JWH-015's ability to positively modify the systemic balance of regulatory to effector lymphocytes as well as modulate the suppressive function of regulatory T lymphocytes. We also show that JWH-015 attenuates breast cancer cell proliferation in vitro in a concentration dependent manner. Finally, utilizing a murine in vivo bioluminescence model, we demonstrate that JWH-015 treatment not only attenuates primary tumor growth, but also rate of metastasis. Taken together, these data establish CB2 as an innovative therapeutic target across multiple stages of breast cancer.

ANTINOCICEPTIVE EFFECTS OF MONOAMINE REUPTAKE INHIBITORS IN ASSAYS OF PAIN-STIMULATED AND PAIN-DEPRESSED BEHAVIOR By Marisa B. Rosenberg A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Advisor: Sidney Stevens Negus, Ph.D. Professor, Department of Pharmacology/Toxicology Noxious stimuli can produce pain-related stimulation of some behaviors (e.g. withdrawal responses) and depression of other behaviors (e.g. feeding, locomotion, responding maintained by many types of positive reinforcement). Monoamine reuptake inhibitors are used clinically to treat depression and to manage some types of pain. This study examined the antinociceptive properties of a variety of monoamine reuptake inhibitors selective for SERT, NET and DAT in complementary assays of acute pain-stimulated and pain-depressed behaviors. Intraperitoneal injection of dilute lactic acid (1.8% in a volume of 1ml/kg) was used as a noxious stimulus to stimulate a stretching response and to depress intracranial self-stimulation (ICSS) of the median forebrain bundle. All eight monoamine reuptake inhibitors produced an antinociception-like blockade of acid-stimulated stretching, but only compounds with prominent DA reuptake inhibition (SDRIs RTI-113 and bupropion and the TRI RTI-112) were able to block acid-depressed ICSS, although these effects were produced only at doses that also produced an abuse-related facilitation of control ICSS. Selective or mixed-action inhibitors of 5-HT and NE failed to block acid-induced depression of ICSS. In a separate group of rats, citalopram was also tested using a repeated dosing regimen (10 mg/kg x 3 doses) shown previously to produce antidepressant effects in a forced-swim test in rats. As with acute administration, repeated citalopram decreased acid-stimulated stretching but failed to block acid-induced depression of ICSS. Taken together, these results suggest that SSRIs, SNRIs and S+NRIs produce relatively non-selective depression of all behavior rather than a selective blockade of sensory sensitivity to noxious stimuli. Conversely, dopamine reuptake may be able to block sensory detection of noxious stimuli. Additionally, these results suggest that assays of pain-depressed behavior can provide new insights on analgesia-related effects of monoamine reuptake inhibitors.

Abstract The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells. The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. However, our studies indicated that stilbene 5c works in a p53-independent manner. Exposure of P53-null HCT116 cells to stilbene resulted in a similar sensitivity as in p53-wild type HCT116 cells. We found that autophagic vacuoles were formed in response to stilbene 5c in p53-null HCT116 cells as well. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death.

The high co-morbidity between alcohol (ethanol) and nicotine abuse suggests that nicotinic acetylcholine receptors (nAChRs), which are thought to underlie nicotine dependence, may also be involved in alcohol dependence. A genomic region that encodes the Alpha5* nAChR subtype has recently been shown to be associated with alcohol dependence phenotypes in humans. Therefore, the aim of this study was to determine the role of Alpha5* nAChRs in ethanol-responsive behaviors upon acute administration in mice as well as in their drinking behavior. We conducted tests in mice lacking the Alpha5 coding gene (Chrna5) in ethanol-induced hypothermia, hypnosis, anxiolysis, and conditioned place preference. We also assessed drinking behavior in these mice using models of voluntary ethanol consumption, two-bottle choice preference and intermittent access, as well as acute binge drinking behavior in the Drinking-in-the-Dark paradigm. Our results showed that deletion of the Alpha5 gene enhanced acute behaviors, including ethanol-induced hypothermia, hypnosis recovery time, and the anxiolytic-like response in mice. We also found that Alpha5 gene deletion resulted in decreased ethanol CPP, but had no effect on ethanol consumption in either model of drinking behavior tested under normal conditions. However, we discovered that under conditions of stress from multiple daily injections of saline or nicotine, Drinking-in-the-Dark intake was reduced in Alpha5 null mutant mice. We also examined the role of Beta2* nAChRs due to the tendency of the Beta2 subunit to be co-expressed with this subtype, which also plays an important role in nicotine dependence. Our results showed that pharmacological and genetic manipulation of Beta2* nAChRs modulated some acute alcohol-responsive behaviors, namely, hypnosis, recovery-time and the anxiolytic-like response produced by ethanol, but did not modulate ethanol drinking behavior in mice. These studies provide evidence that Alpha5* subtypes and Beta2* subtypes, which play a critical role in nicotine dependence, also play a role in acute ethanol-responsive behaviors in vivo, thus supporting studies in humans that nicotine and alcohol dependence share common genetic components.

Hyperhomocysteinemia (hHcys), an important pathogenic factor contributing to the progression of end-stage renal disease (ESRD), has been shown to activate NOD-like receptor protein 3 (NLRP3) inflammasomes and cause podocyte dysfunction and glomerular sclerosis. hHcys induces aggregation of the three inflammasome components – NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 – and its activation is indicated by increased caspase-1 activity and secretion of interleukin-1β (IL-1β). The aims of the present study sought to elucidate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated redox signaling in hHcys-induced NLRP3 inflammasome activation, to dissect the contribution of common endogenous reactive oxygen species (ROS) including superoxide (O2•−), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radical (•OH), and to explore the molecular mechanisms by which the NLRP3 inflammasome senses changes in oxidative stress through thioredoxin-interacting protein (TXNIP). Specific inhibition of the gp91phox subunit of NADPH oxidase markedly reduced Hcys-induced caspase-1 activity and IL-1β production in cultured podocytes. Concurrently, gp91phox−/− or administration of a gp91ds-tat peptide also exhibited diminished glomerular inflammasome formation and activation in mice fed a folate-free (FF) diet to induce hyperhomocysteinemia and displayed glomerular protection as shown by prevention of hHcys-induced proteinuria, albuminuria and glomerular sclerosis. Interestingly, dismutation of O2•− by 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl and administration of H2O2 decomposer catalase either in cultured podocytes or hyperhomocysteinemic mice inhibited hHcys-induced NLRP3 inflammasome aggregation and activation. Hyperhomocysteinemic mice also demonstrated a significant increase in glomerular TXNIP binding to NLRP3, confirmed by confocal microscopy, size-exclusion chromatography, and co-immunoprecipitation studies. Blockade of TXNIP by genetic interference or by the calcium channel blocker verapamil prevented this hHcys-induced TXNIP-NLRP3 binding, NLRP3 inflammasome formation and activation, as well as protected hyperhomocysteinemic mice from glomerular dysfunction and damaged morphology. In conclusion, hHcys-induced NADPH oxidase activation is importantly involved in the switching on of NLRP3 inflammasomes in podocytes, where NADPH oxidase-derived O2•− and H2O2 primarily contribute to NLRP3 inflammasome activation. TXNIP binding to NLRP3 is a key signaling mechanism allowing NLRP3 inflammasome to sense these changes in oxidative stress. These findings greatly enhance our understanding of the early pathogenesis of hHcys-induced glomerular sclerosis, which may identify new therapeutic targets for prevention or treatment of ESRD.

A DIRECT AND INDIRECT MECHANISM FOR CCR5 IN OPIOID AND HIV-1 MEDIATED NEURODEGENERATION By Elizabeth M. Podhaizer, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2014 Major Director: Kurt F. Hauser, Ph.D., Professor of Pharmacology & Toxicology Human immunodeficiency virus (HIV)-1 infection currently affects over 34 million people worldwide, and despite the use of cART, the prevalence of HIV-1 associated neurocognitive impairments (HAND) has not declined. Additionally, other co-morbid factors such as the abuse of injection drugs (i.e. heroin, morphine) increase both the frequency and the speed by which patients progress to AIDS. To begin to understand the mechanisms, we chose to examine a pathway, through CCR5, which may act as a convergence point for opioids and HIV-1 proteins. C-C chemokine receptor 5 (CCR5) is an immune receptor involved in physiological processes in the brain in addition to mediating neuroinflammatory signaling events, and it is a co-receptor for HIV-1. CCR5 interacts directly with gp120 to facilitate HIV-1 infection, and may interact indirectly with HIV-1 Tat through convergent signaling mechanisms. Additionally, CCR5 is modified by opioid responses, and so may be central to opioid-HIV-1 interactions that are seen in our model. We hypothesized that CCR5 would mediate the opioid-HIV-1 interaction. We examined both HIV-1 gp120 and HIV-1 Tat, both for interactions with opioids and modification by the CCR5 antagonist, maraviroc. HIV-1 gp120ADA was neurotoxic on its own, but showed no interactions with morphine. However, further probing revealed that morphine can in fact modify the neurotoxic effects of gp120, but that the response is dependent on gp120 strain. We did, however, find that morphine did enhance the neurotoxicity of Tat, which we’ve shown previously, as well as that inhibition of CCR5 can prevent this interactive effect. Additionally, use of CCR5 knockout glia or neurons modified the response and suggests that neurons and glia play different roles in the integration of opioid and HIV-1 signals. Sublethal effects of morphine and Tat were also dampened by maraviroc pretreatment or use of knockout cells, as was the secretion of chemokine ligands. Manipulation of CCR5 showed utility in preventing neurodegenerative effects both to HIV-1 proteins alone as well as to the interactive opioid-HIV-1 signaling responses and suggests that maraviroc, a cART therapeutic used to prevent viral entry, may also aid in reducing the chronic inflammatory state of the CNS that leads to the persistent neurocognitive complications.

Cancer is the second most common cause of death in the US. The most frequently observed cancer type in women is breast cancer. A special type of breast cancer is triple negative (TNBC) cancer that is characterized by lacking three receptors: estrogen, progesterone and human epithelial growth factor (HER 2). The HS578t breast cell line is a model of TNBC that also has a mutation of the p53 protein. Ionizing radiation is used widely in the clinic to debulk tumors before surgery as well as post-surgery to eliminate residual tumor cells outside the surgical field. Previous studies from our laboratory showed that inhibition of autophagy does sensitize p53 wild type MCF-7 and ZR-75 breast tumor cells to radiation. However, this is not necessarily the response in all breast cell lines. The Hs578t cells did not appear to be sensitized to radiation after inhibition of autophagy using chloroquine as a pharmacological inhibitor. The present study was designed to build upon these previous findings and further confirm that the Hs578t breast cell line could not be sensitized to radiation through autophagy inhibition. Time course studies showed a reduction of viable cell number upon irradiation of Hs578t breast tumor cells and that both autophagy and senescence were induced. Acridine orange staining was used to examine the acidic vacuole formation while β-galactosidase staining indicated the promotion of senescence. Flow cytometry was used to quantify both autophagy and senescence. Inhibition of autophagy using pharmacological inhibitors such as ammonium chloride, or genetic silencing of autophagy by beclin1, which is a protein initiator of autophagy, did not sensitize Hs578t breast tumor cells to irradiation. It shows from these studies that autophagy is not necessarily cytoprotective in all breast cancer cell lines, which should be considered in current clinical trials designed to sensitize tumor cells to chemotherapy and radiation through inhibition of autophagy.

Microtubule poisons have proven to be effective in the treatment of a variety of malignancies. Although taxol-based derivatives promote microtubule stabilization, there is continuing interest in compounds that, like colchicines, act as microtubule destabilizing agents. Previous work from this laboratory showed that the novel microtubule poison, JG- 03-14, was active against breast tumor cells, promoting autophagic cell death. In the current work, we studied the influence of JG-03-14 on p53 wild type HCT116 colon carcinoma cells. A crystal violet sensitivity assay indicated that JG-03-14 induced growth inhibition, with 75% suppression of growth evident at a concentration of 500 nM. Time course studies of drug effects on cell viability indicated that JG-03-14 also produced cell killing. FACS analysis demonstrated that the HCT-116 cells arrested in the G2/M stage; furthermore, there was evidence of a hyperdiploid population that would be consistent with failure of the cells to divide despite completion of DNA synthesis. Finally, there was evidence of a small sub G0/G1cell population, indicating that the cells were not dying primarily by apoptosis, and suggesting that JG-03-14 induces an alternative mode of cell death. In contrast to cell shrinkage and nuclear fragmentation that was evident after treatment with taxol ( a positive control for apoptosis), DAPI staining of HCT-116 cells treated with JG-03-14 showed intact and enlarged nuclei, again consistent with the absence of apoptosis. Furthermore, there was no evidence of mitotic catastrophe (micronuclei in binucleated cells). Based on previous studies in MCF-7 and MDA-MB231 cell lines that demonstrated a substantial population of autophagic cells, HCT-116 cells were subjected to staining with acridine orange and monodansylcadaverine after treatment with JG-03-14. While control cells tended to show a single large autophagic vesicle closely associated with the cell nucleus, treatment with JG-03-14 resulted in extensive distribution of small acidic vesicles within the cytoplasm, indicative of autophagy. GFP-LC3 transfected cells incubated with JG-03-14 showed punctuated patterns that were also consistent with the promotion of autophagy. Finally, activation of the DNA damage response pathway was ruled out by the lack of induction of p53 and p21 in cells treated with JG-03-14. In summary, our studies indicate that the JG-03-14 induces both growth arrest and autophagic cell death in HCT116 colon carcinoma cells. The possibility of an alternative mode of cell death induced by JG-03-14 makes it a potentially usefull candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. Our result also suggested that JG-03-14 failed to induce bone marrow toxicity adding to its potential for clinical use.