Individual differences in initial sensitivity to ethanol are strongly related to the heritable risk of alcoholism in humans. To elucidate key molecular networks that modulate ethanol sensitivity we performed a systems genetics analysis of ethanol-responsive gene expression in brain regions of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, a highly diverse family of isogenic mouse strains before and after treatment with ethanol. Acute ethanol altered the expression of ~2,750 genes in one or more regions and 400 transcripts were jointly modulated in all three. Ethanol-responsive gene networks were extracted with a powerful graph theoretical method that efficiently summarized ethanol's effects. These networks correlated with acute behavioral responses to ethanol and other drugs of abuse. As predicted, networks were heavily populated by genes controlling synaptic transmission and neuroplasticity. Several of the most densely interconnected network hubs, including Kcnma1 and Gsk3-beta, are known to influence behavioral or physiological responses to ethanol, validating our overall approach. Other major hub genes like Grm3 and Nrg3 represent novel targets of ethanol effects. Networks were under strong genetic control by variants that we mapped to a small number of chromosomal loci. Using a novel combination of genetic, bioinformatic and network-based approaches, we identified high priority cis-regulatory candidate genes, including Scn1b, Gria1, Sncb and Nell2. The ethanol-responsive gene networks identified here represent a previously uncharacterized intermediate phenotype between DNA variation and ethanol sensitivity in mice. Networks involved in synaptic transmission were strongly regulated by ethanol and could contribute to behavioral plasticity seen with chronic ethanol. Our novel finding that hub genes and a small number of loci exert major influence over the ethanol response of gene networks could have important implications for future studies regarding the mechanisms and treatment of alcohol use disorders.

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.

Background: The current clinical practice of factor VIII (FVIII) prophylaxis revolves around converting patients with severe hemophilia A, hereafter simply referred to as hemophilia, phenotype (defined as plasma factor VIII coagulant activity [FVIII:C] <1 IU dL-1) to moderate hemophilia phenotype (defined as plasma FVIII:C from 1–5 IU dL-1). However, a wide inter-individual variation in bleeding tendency is observed despite changes in plasma FVIII:C (pharmacokinetic [PK] changes). Therefore, monitoring FVIII prophylaxis by global hemostasis biomarkers (pharmacodynamic [PD] response) can potentially be beneficial. Objective: To conduct appropriate PK/PD modeling using plasma FVIII:C and global hemostasis (platelet function and blood viscoelastic) biomarkers in severe hemophilia. Methods: Nine non-bleeding severe hemophiliacs (plasma FVIII:C <1 IU dL-1) with variant bleeding tendency (5 frequent bleeders and 4 infrequent bleeders) were infused with a recombinant factor FVIII (rFVIII) prophylactic dose (mean = 32.1 international units per kilogram [IU kg-1]). Blood was collected at baseline and 0.5-, 1-, 2-, 4-, 8-, 12-, 24- and 48-hours (h) post-dose for plasma FVIII:C, platelet function (platelet contractile force [PCF], clot elastic modulus [CEM] and force onset time [FOT]) and blood viscoelastic (reaction-time [R], kinetics-time [K] and maximum amplitude [MA]) biomarkers and activated partial thromboplastin time (aPTT). Non-compartmental analysis (NCA) was performed using standard methods. Compartmental analysis (CA) and PK/PD modeling were performed by non-linear regression. Correlation and analysis of variance (ANOVA) were used to explore the role of clinically relevant modifiers of bleeding tendency, as appropriate. ANOVA was used to assess inter-group differences in pertinent PK and PD parameters. P value <0.05 significance level was pre-specified for all statistical tests. Results: Mean (±SD) volume of distribution at steady state (Vss), total clearance (CLtot) and terminal half-life (t1/2) from NCA were 40.5 (±11.2) milliliter per kilogram (mL kg-1), 2.9 (±1.2) milliliter per hour per kilogram (mL h-1 kg-1) and 11.6 (±6.2) h, respectively. Mean (±SD) Vss and CLtot from the one-compartment body model (CA) were 39.6 (±8.9) mL kg-1 and 3.1 (±1.3) mL h-1 kg-1, respectively. The mean (±SD) baseline effect (E0) and slope from the PK/PD linear modeling were: for aPTT, 48.9 (±4.4) seconds (sec) and -0.025 (±0.009) second deciliter per international unit (sec dL IU-1), respectively; for PCF, 0.3 (±0.3) kilodynes (kdynes) and 0.008 (±0.004) kilodynes deciliter per international unit (kdynes dL IU-1), respectively; and for CEM, 0.0 (±0.0) kilodynes per square centimeter (kdynes cm-2) and 0.032 (±0.016) kilodynes deciliter per international unit per square centimeter (kdynes dL IU-1 cm-2), respectively. The mean (±SD) maximum effect (Emax) and half the maximum effective concentration (EC50) from the PK/PD sigmoidal Emax model were: for FOT, 70.1 (±16.9) % reduction and 87.8 (±31.4) IU dL-1 for FOT, respectively; for R, 74.9 (±26.0) % reduction and 68.5 (±28.4) IU dL-1, respectively; and for K, 73.2 (±36.4) % reduction and 67.2 (±29.0) IU dL-1, respectively. MA was not PK/PD modeled due to its low sensitivity. Conclusions: Plasma FVIII:C remained ≥1 IU dL-1 over the prophylactic interval. FOT and R were the most sensitive biomarkers at lower plasma FVIII:C. PCF and CEM were more sensitive than K and aPTT at lower plasma FVIII:C. MA was the least sensitive biomarker. Correlation and inter-group differences did not reach statistical significance (small sample size). These results may be used to assess risk of bleeding and dose-optimize FVIII prophylaxis in severe hemophilia.

Fullerenes are carbon nanospheres that can be solublized by the addition of polar chemical groups to the carbon cage, forming fullerene derivatives. One specifically derivatized fullerene compound, termed C70-Tetragylocolate (C70-TGA), has been shown to stabilize mast cell responses in vitro thus we hypothesized it may have an effect on mast cell-driven diseases such as asthma and systemic anaphylaxis. To observe the effects of C70-TGA on systemic anaphylaxis, mice were subjected to a model of passive systemic anaphylaxis. In this model, mice were injected with DNP-specific IgE 16 hours prior to challenge, then treated with C70-TGA. Immediately prior to DNP challenge, mice were subjected to a second injection of C70-TGA. Following DNP challenge, body temperature was recorded and blood was collected for quantitation of histamine levels. Treatment with C70-TGA significantly reduced body temperature drop associated with systemic anaphylaxis and serum histamine levels. To observe the effects of C70-TGA on chronic features of asthma in vivo, we utilized a heavily MC influenced model of asthma pathogenesis. Mice were sensitized by intraperitoneal (i.p.) injection of ovalbumin (OVA) in saline, challenged intranasally (i.n.) with OVA, and one of two treatment strategies was pursued. In one, C70-TGA was given i.n. throughout disease development. In the other, C70-TGA was given following an initial set of challenges to allow disease to develop prior to treatment; mice were then re-challenged with OVA to assess the effect on established disease. We found that C70-TGA treatment significantly reduced airway inflammation and eosinophilia and dramatically reduced bronchoconstriction in either model. Cytokines IL-4 and IL-5 and serum IgE levels are significantly reduced in C70-TGA treated animals. Interestingly, we also saw an increase in the anti-inflammatory eicosanoid 11, 12-epoxyeicosatreinoic acid (11,12-EET) in the BAL fluid, suggesting the involvement of this mediator in C70-TGA inhibition. Further experiments utilizing an inhibitor of 11,12-EET formation (6-(2-Propargyloxyphenyl)hexanoic acid) and a structural analog of 14,15-EET (14,15-EE-5(Z)-E) in vivo indicate that these mediators are closely associated with C70-TGA mediated inhibition as their inhibition reverses the anti-inflammatory effects of C70-TGA. Importantly, mice did not exhibit any acute toxicity following C70-TGA treatment and liver and kidney function were normal. Collectively, these results show that the fullerene C70 derivative C70-TGA is capable of dampening severe allergic responses including systemic anaphylaxis, airway inflammation, and bronchoconstriction. The mechanism of inhibition is through the upregulation of the anti-inflammatory EETs, which may dampen mast cell degranulation in vivo, thus contributing to the inhibitory effect of C70-TGA on allergic disease.

The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft into the presynaptic terminus and plays a critical role in maintaining the normal function of dopaminergic neurons. DAT is the major target of widely abused psychostimulant drugs, including cocaine and amphetamine. DAT also figures into disease states, and it is a target for therapeutic drugs. It is known that cathinone and methcathinone, β-keto analogs of amphetamine and methamphetamine, respectively, produce pharmacological actions similar to amphetamine. Cathinone and methcathinone analogs are recently gaining in popularity on the clandestine market (e.g. ‘bath salts’). Cathinone and methcathinone analogs as well as their amphetamine and methamphetamine counterparts were synthesized and examined at the hDAT expressed in Xenopus oocytes. One of the two major constituents of ‘bath salts’ (i.e., mephedrone) produced an electrophysiological signature similar to the dopamine releasing agent S(+)-amphetamine while the other major constituent (i.e., MDPV) produced an electrophysiological signature similar to the dopamine re-uptake inhibitor cocaine.

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.

There continues to be a need for more effective analgesics. The α2-adrenoceptor (AR) agonist clonidine is an analgesic whose use is severely limited by undesirable side effects. meta-Chlorophenylguanidine (MD-354), an agent developed in our laboratory, selectively potentiates the antinociceptive effects of clonidine in a biphasic manner. Mechanistic studies suggest that both 5-HT3 receptor and α2-AR mechanisms are involved. To further evaluate mechanisms underlying the analgesia-potentiating effect of clonidine by MD-354, pharmacological studies using more established 5-HT3 receptor agonists: meta-chlorophenylbiguanide (mCPBG) and centrally-acting SR57227A, and non-selective α2-adrenoceptor ligand TDIQ, administered alone and in combination with clonidine, were conducted in mouse antinociceptive assays. None of the examined analogs produced an antinociceptive effect when administered alone. Nevertheless, mCPBG potentiated the antinociceptive actions of clonidine in a monophasic manner and the effect was antagonized by the 5-HT3 receptor antagonist tropisetron but not by tropisetron methiodide, suggesting that potentiation is, at least in part, due to a central 5-HT3 receptor mechanism. SR57227A did not alter the antinociceptive actions of clonidine. TDIQ was found to potentiate the analgesic actions of clonidine in a synergistic manner (as determined by an isobolographic analysis) and the effect was blocked by α2-AR antagonists (BRL-44408, imiloxan, ARC-239; α2A-, α2B-, and α2C-AR antagonists, respectively). This supports the hypothesis that MD-354 could be potentiating the analgesic actions of clonidine via an α2-AR agonist mechanism. In order to explore the role of the ring nitrogen atoms and the chloro substituent of conformationally-constrained rotamers of MD-354, analogs of 2-amino-7-chloro-3,4-dihydroquinazoline, with a varying number of nitrogen atoms in the ring were synthesized. Preliminary binding affinity results indicated that the ring nitrogen atoms are essential for 5-HT3 receptor binding. In attempt to explain the varied binding and functional activity of MD-354 at α2-ARs, 3D homology models of α2A-, α2B- and α2C-AR were generated and docking studies of the low-energy rotamers of MD-354 were conducted. The present studies support a role for the involvement of 5-HT3 receptors and α2-ARs in antinociception. Analgesic adjuvants with a dual mechanism of action such as MD-354 might represent a promising avenue to pain treatment.

For many years it has been known that the injection of antigen bound to an antibody leads to more than a 1000-fold increase in antigen specific antibody response. This observation holds true for IgE, which is dependent upon CD23 expression, as this enhancement is not present in mice deficient in CD23. It also has been shown that when mice are injected with IgE-antigen complexes also display an increase in antigen specific T cell proliferation. While there are published studies that demonstrate a role for B cell derived exosomes in the activation and proliferation of T cells, none have focused upon the potential role of CD23 as a molecular basis for this phenomenon, at least in the context of allergy and asthma. This thesis provides direct evidence that B cell-derived exosomes possess co-stimulatory molecules, including CD80 and CD86, which act in concert with CD23 to induce T cell proliferation, at least in vitro. This is due to, or enhanced by, the exosomal transfer of the antigen or peptide to T cells. Importantly, the antigen transfer is dependent upon the availability of IgE and the expression of CD23.

The objective of this study was to evaluate parents’ preferences of the appearance and attire of orthodontists. Six subjects were asked to pose for photographs wearing various combinations of attire (casual, scrubs, white coat, formal), hairstyle and nametag. Survey participants were presented with choice sets and asked to select the most and least preferred provider photographs. A total of 77 parents of orthodontic patients participated in the computer-based survey. The results indicated that there were significant differences due to provider sex (P = 0.0013), provider age (P < .0001), dress (P < .0001), nametag (P = 0.0065) and hair (P < .0001). The most preferred providers were the younger female and the older male. Formal attire or scrubs was the most preferred style of dress. There was also a preference for the use of a nametag and for the provider to have his/her hair in a controlled style.

As esthetic improvement continues to be a primary goal of orthodontic patients and practitioners, it is important to understand the soft tissue movements that occur during the transition from rest to smile. Advances in technology allow capture of 3-dimesional photographs. The purpose of this study was to quantify the soft tissue changes that occur upon smiling and to compare changes between males and females using 3-dimensional photography. Fifty-four participants had resting and smiling photographs taken with the 3dMDface camera system. The two images were registered on stable facial surfaces, landmarks were placed, and measurements were recorded. Three-dimensional changes occurred in the following regions of the face in both males and females: eyes, ears, nose and lips. Intercommissure width of the lips was shown to increase more in females than males, but males showed increased mobility in landmarks at the base of the nose.