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.

Axonal pathology is a major contributor to impaired motor, sensory and cognitive dysfunction associated with multiple sclerosis particularly with the progressive forms of the disease. However, the early pathologic events responsible for axonal deterioration remain unclear. It is well recognized that maintaining proper axonal function is intimately related to proper establishment and maintenance of axonal domains such as the node of Ranvier and the axon initial segment (AIS). Numerous laboratories, including ours, have investigated the mechanisms that regulate node of Ranvier formation and maintenance. These studies have shown that node of Ranvier formation and maintenance require myelin contact. Interestingly, many of the same proteins that cluster at the node of Ranvier also cluster in the AIS; however, the mechanisms responsible for AIS clustering appear to be unique to the AIS as myelin contact is not required and the mechanisms appear to be intrinsic to the neuron. Determining how the AIS is developmentally generated is vital to a complete understanding of the AIS function. However, more in line with understanding the pathobiology of MS, our laboratory is interested in identifying the mechanisms responsible for the maintenance and restoration of AIS integrity and function. To achieve this goal, we have exploited the cuprizone toxicity model. This model results in a consistent course of demyelination followed by remyelination of layer V of the cerebral cortex. Using a combination of immunocytochemistry and confocal microscopy, we have analyzed AIS integrity as evidenced by the clustering of ankyrinG, a prominent initial segment protein. Our findings indicate that the number of AIS is not decreased following myelin loss. In addition, AIS length and surface area are not changed following demyelination. These findings are important as they suggest that myelin is not required for the maintenance of initial segment organization.

Objective: The aim of this study was to (1) assess the orthodontic patient and practitioner use and preferences of social media, and (2) to investigate the potential benefit of social media in marketing and communication strategies in orthodontic practices. Material and Methods: A survey was distributed to all participants, which included orthodontists from the American Association of Orthodontists (AAO) and patients/parents from the VCU Orthodontic Clinic and Private Practices throughout the United States. The participants were asked to answer questions related to their use of social media as well as their perceptions of usage of social media in the orthodontic practice. Results: 76% of orthodontists, 71% of the VCU Practice participants and 89% of the Private Practice participants used social media, with the highest preference for Facebook among all of the participants. Orthodontist’s posed information more often in the morning and afternoon (40% and 56%, respectively) and patients used social media mainly in the evening (76%). The most commonly used marketing strategies in the orthodontic practice were social media and a practice website (76% and 59%, respectively). Social media and practice websites were positively related to new patient starts (P=0.0376, P=0.0035, respectively). Newspapers were negatively related to new patient starts (P=0.0003). Conclusions: Social media use was more common in females and younger adults and facebook was the most commonly used social media site among all of the participants. Orthodontists posted information on social media websites mainly in the morning and afternoon, while patients spend more time on social media sites in the evening. Newspaper advertisements were negatively related to new patient starts. Facebook and Twitter were positively related to new patient starts when used as a marketing and communication tool in the orthodontic practice.

The Reporting of Supplement Use by Dental Patients on Their Medical History Questionnaire Objectives: The goals of the study were three fold. Estimate the prevalence of supplement use by dental patients. Determine if the design of a medical history form influences the prevalence of supplement use reported. Determine whether or not patients are aware of supplement side effects and interactions with medications. Methods: Patients were randomly allocated to either a standard medical history form or the same form with additional questions about supplement use. After completing the initial forms, a survey containing questions about supplements was filled out by both groups. For investigating differences between groups, logistic regression and analysis of variance were used depending on the type of outcome variable. Results: Two hundred and nine patients participated in the study. The mean number of supplements reported by patients was influenced by the type of health history questionnaire given to the patient. Specifically asking about supplements versus not asking at all resulted in nearly double the number of supplements reported by the patient (mean of 1.53 when asked, 0.76 when not asked, p< 0.0001). Patient age and income were related to number of supplements used per patient. The two oldest age categories (50–65 and >65) reported a mean number of supplements used of 2.82 and 2.72, respectively versus the youngest age group (<30 years old) which reported a mean of 1.05 (p<0.05). The highest income level (>$75,000 per year) reported the lowest number of supplements per patient of 0.56 versus the other income levels (p<0.02), which reported mean supplemental use ranging from 2.28 to 2.71. Additionally, the majority of the subjects (69 %) were not aware of the side effects and interactions of supplements with medications. Conclusion: Patients tend not to report supplement use on the medical history questionnaire unless they are directly asked and the majority of patients are not aware of interactions with medications. Patient income and age have an effect on the frequency of supplement use.

The current understanding of the distribution and frequency of periapical pathoses include biopsies submitted by all specialists and general dentists. As a result, they do not accurately reflect the distribution seen by endodontists. This retrospective chart review aims to determine the prevalence of periapical pathoses and associated demographics from biopsies submitted by endodontists over 30 years. All biopsy reports submitted to the Virginia Commonwealth University Oral Pathology Diagnostic Service from January 1, 1983 to December 31, 2012 were reviewed. Only reports submitted by verified endodontists and those with a periapical location were included. The following data was recorded from each report: submission date, referring endodontist, sex, age, race, biopsy location, tooth number, and histologic diagnosis. Results were calculated using chi-square and logistic regression analysis (significance p<0.05). Meeting the inclusion criteria were 9,777 biopsy reports for an overall distribution of 24.11% radicular cysts, 73.54% periapical granulomas, 1.66% scars, and 0.70% other pathoses. Findings include a significant association between sex, location, and diagnosis. An association with race, age, or location (left/right) was not seen. Significantly more radicular cysts were seen in males and in the anterior maxilla. Conversely, significantly more periapical granulomas were seen in females and in the posterior quadrants. Significantly more other diagnoses were found in the anterior mandible and more scars in the anterior maxilla. Overall, approximately ¾ of biopsies submitted for evaluation by endodontists are diagnosed as periapical granulomas and ¼ as radicular cysts. Other pathoses and scars make up less than 3% of diagnoses. Funding was provided through the AAE Resident Research Grant.

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.

Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.

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.

Vital pulp therapy is the group of procedures indicated to maintain pulp vitality allowing for continued root development and apical closure in the presence of pulp exposure due to caries or trauma. Complete maturation results in stronger tooth structure that is more able to withstand occlusal forces. Historically, calcium hydroxide (Ca(OH)2) was the material of choice for a vital pulpotomy. Recently mineral trioxide aggregate (MTA) has been used as a pulp sealing material because of its biocompatibility and its ability to induce hard tissue barrier formation. The purpose of this retrospective study was to evaluate the clinical and radiographic outcome of vital pulp therapy procedures performed with MTA. All patients receiving MTA pulpotomies and MTA pulp caps at Virginia Commonwealth University School of Dentistry Graduate Endodontic Practice between November 30, 2009 and August 15, 2013 were recalled and evaluated for presence or absence of clinical symptoms, pulp vitality, radiographic evaluation of continued root development, dentin bridge formation or pulp canal obliteration. Results were analyzed descriptively. At time of treatment caries was found to be the most common etiologic factor. Fifty-seven percent of cases were symptomatic at time of treatment (43% asymptomatic). Forty-seven percent of the teeth presented with immature apices (53% with radiographically closed apices). Overall recall rate was 50%. At recall all teeth were clinically asymptomatic. Forty seven percent of teeth tested normally to cold (33% were non-responsive to cold) at recall. Pulp canal obliteration was seen in 6 cases, 4 molars and 2 anterior teeth. In conclusion, MTA pulpotomy and MTA direct pulp caps are a predictable treatment modality for young vital permanent teeth affected by caries or trauma.