The developmental psychopharmacology of cannabinoids is poorly understood and little is known about the developmental consequences of repeated exposure to cannabinoid antagonists. In these experiments, male Long-Evans rats were treated with SR141716A, a cannabinoid antagonist, throughout adolescence and allowed unrestricted access to food. Control groups were treated with vehicle during the same developmental period and allowed either unrestricted access to food or were pair-fed with a member of the SR-treated group. Motivation to work for food was measured in progressive-ratio sessions at varying levels of food deprivation. For rats that consumed fewer calories throughout adolescence, whether because of pharmacological intervention or food-restriction, motivation was not significantly related to the level of food deprivation. Additionally, the SR-treated group ate more of a novel, palatable food than the vehicle-treated group. Finally, the SR-treated group was generally more motivated to work for food than the pair-fed group, irrespective of the level of deprivation.

Many links exist between cocaine abuse and stress. The literature and laboratory studies in rats suggest that this could be because stress increases the reinforcing efficacy of cocaine. Using male Long-Evans rats, experiments in this thesis tested effects of footshock on the reinforcing efficacy of cocaine using a progressive ratio schedule of reinforcement. They also examined effects of footshock on the reinforcing efficacy of a half-maximal dose of cocaine. Finally, they tested the effects of footshock on cocaine self-administration in rats initially resistant to acquisition of cocaine self-administration. Footshock did not increase reinforcing efficacy of cocaine on a PR schedule of reinforcement, nor did it enhance sensitivity to a half-maximal dose of cocaine. Footshock did, however, cause acquisition of cocaine self-administration in acquisition-resistant rats. Therefore, while footshock stress may be capable of sensitizing acquisition-resistant rats to the reinforcing efficacy of cocaine, it does not appear that it significantly increases the reinforcing efficacy of cocaine in rats with a history of cocaine self-administration.

A senescence-like growth arrest succeeded by rapid recovery of proliferative capacity is observed in MCF-7 breast tumor cells exposed to fractionated radiation (5 x 2Gy) alone. Exposure to the vitamin D3 analog EB 1089 (100nM) prior to irradiation converts the initial growth arrest response to cell death in part through the inhibition of radiation-induced senescence and promotion of both apoptotic and autophagic cell death. More importantly, EB 1089 was shown to profoundly reduce the rate of recovery following fractionated irradiation. The effect of EB 1089 on the temporal response to radiation is also observed in MCF-7 cells expressing caspase 3, but not in cells where p53 function is abrogated. EB 1089 does not increase radiation-induced DNA damage or inhibit DNA repair, as measured by both the alkaline unwinding assay and 53BPl fociformation. EB 1089 inhibits radiation-induced down-regulation of myc; however, doxycyclin-induction of myc does not mimic the radiosensitizing effects of EB 1089. EB1089 increases radiation-induced reactive oxygen species (ROS) generation; however, both free radical scavengers, N-acetyl cysteine (NAC)and reduced glutathione (GSH), fail to attenuate the radio-sensitizing effects of EB 1089. These studies rule out increased radiation-induced DNA damage, inhibition of DNA repair, inhibition of myc suppression,or increased ROS generation as the mechanism(s) responsible for the radiosensitizingeffects of EB 1089. However, EB 1089 does cause an increase in radiation-induced ceramide generation in the cell, while the ceramide synthase inhibitor, fumonisin B1, inhibits apoptosis and increases cell viability following treatment with EB 1089 plus radiation. Signaling pathways that promote ceramide generation and autophagic cell death may provide insights as to the mechanisms underlying the interaction(s) between EB 1089 or vitamin D3 and radiation in breast tumor cells.

The majority of research on the endogenous opioid system has focused on various pain assays and the efficacy of different opioid agonists. However, minimal attention has been focused on the effects of gonadal hormones and their impact on the opioid peptide system. The present study was designed to determine the effects of modulation of gonadal hormones on the opioid receptor protein levels in Complete Freund's Adjuvant (CFA)-treated arthritic and non-arthritic male and female Lewis rats. Midbrain and spinal cord tissues were collected for comparison of the μ, δ, and κ receptor protein levels in arthritic and non-arthritic animals. Male gonadectomy did not dramatically impact opioid receptor protein levels, whereas female gonadectomy resulted in significant increases in opioid receptor protein levels. Furthermore, it was determined that MOR protein levels were greatest in male rats, while KOR protein levels were greatest in female rats regardless of arthritis condition or gonadal hormones.

Alcohol abuse is a problem in our society. There are few treatments available, in part due to the unclear molecular mechanisms of ethanol’s effects. Human studies indicate that there is a genetic component influencing disease susceptibility, and that an individual’s initial response to alcohol can predict their development of addiction. We have taken a forward genetics approach to study one component of initial response, acute functional tolerance (AFT), in Caenorhabditis elegans. We identified bet11, a mutation that causes animals to be defective in the development of AFT. Genetic analysis suggested that the gene that bet11 disrupts participates in a synthetic genetic interaction with an unlinked natural allelic variant in another gene that alters ethanol response. We also examined the role of lipid membrane composition in the response to ethanol. Identification of modulators that are responsible for alcohol-induced responses will provide a greater understanding of the mechanisms that cause alcohol-related diseases.

Monoamine releasers constitute a class of compounds that promote release of dopamine, serotonin, and/or norepinephrine. These compounds have a range of different uses in the medical setting, including treatment of attention deficit hyperactivity disorder, narcolepsy, and obesity. A major limitation of many of these compounds (i.e. amphetamine, methamphetamine, phenmetrazine) is their propensity for abuse; however, not all monoamine releasers are abused (i.e. fenfluramine). The goal of this dissertation was to examine pharmacological determinants of abuse-related effects produced by monoamine releasers in two preclinical assays in rats: intracranial self-stimulation (ICSS) and drug discrimination. First, this work confirmed and expanded upon previous findings that selectivity for promoting release of dopamine versus serotonin is one determinant of abuse-related effects produced by monoamine releasers. This was accomplished by determining the behavioral effects of 11 different compounds that ranged in their selectivity for dopamine versus serotonin, and a correlation was found between the ability of a compound to facilitate ICSS and the selectivity of that compound for releasing dopamine versus serotonin. These data were then submitted to a rate-dependency analysis. Here, we found that all compounds produced rate-dependent effects, but that the profile of these effects varied with a compound’s selectivity for dopamine versus serotonin. Next, the mechanism by which serotonin exerts it response rate-decreasing effects was investigated - specifically, the hypothesis that the 5HT2C receptor mediates serotonin’s abuse-limiting effects of monoamine releasers was tested. The data collected suggest that the 5HT2C receptor contributes to, but is not exclusively responsible for, the abuse-limiting effects produced by serotonin release. Finally, selectivity for norepinephrine versus dopamine was examined as a potential determinant of monoamine releaser abuse liability; results from these studies suggest that release of both dopamine and norepinephrine are required for expression of abuse-related effects in assays of ICSS and drug discrimination. These data provide a systematic examination of the determinants of the abuse-related effects produced by monoamine releasers and may contribute to development of medications with reduced abuse potentials.

The idea that humans and laboratory animals can become physically dependent on marijuana or its primary psychoactive constituent, delta-9-tetrahydrocannabinol (THC), is gaining acceptance. However, there are no currently approved pharmacotherapies to treat cannabinoid withdrawal. The objective of this thesis was to evaluate whether elevating endogenous anandamide levels using mice lacking fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide metabolism, would ameliorate THC dependence. Mice were treated subchronically with a low or high THC dosing regimen and challenged with the CB1 receptor antagonist, rimonabant, to precipitate withdrawal. Following subchronic THC treatment, rimonabant precipitated a significant increase in paw flutters that was dependent on THC dose. However, FAAH-/- mice displayed a similar magnitude of withdrawal responses as wild type control mice, regardless of subchronic dosing regimen. Finally, rimonabant was equipotent in precipitating withdrawal responses in both genotypes. Collectively, these results demonstrate that FAAH-/- and +/+ mice show identical THC dependence, thus arguing against the notion that elevating anandamide levels through FAAH suppression will reduce cannabinoid withdrawal.

Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3'-PG (PO4CH2COOˉ) or 3'-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3'-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3'-PGs, in either the presence or absence of magnesium, to give a 3'-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3'-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3'- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl.

Folic acid is an essential vitamin utilized in the one-carbon metabolism pathway for the synthesis of purine and thymidine nucleotides, which are necessary for cell growth and proliferation. As a result, the enzymes that participate in the metabolism of folic acid have been good targets for cancer chemotherapy. Folylpoly-γ-glutamate synthetase (FPGS) is an enzyme in the folate metabolism pathway that catalyzes the addition of glutamic acid to the naturally occurring folates, thereby allowing the retention of folate cofactors in cells. Similarly, in the case of cancer chemotherapy, antifolates, such as Lometrexol and Tomudex are retained in cells through the activity of FPGS. Consequently, any single nucleotide polymorphisms (SNPs) that exist in the fpgs gene may decrease or increase the cytotoxicity of antifolates and, ultimately, the clinical response rate to antifolate therapy. The goal of this project is to define the position and frequency of single nucleotide polymorphisms (SNPs) in the mRNA made from the fpgs gene from peripheral blood of one hundred normal individuals. Six Polymerase Chain Reaction (PCR) primers were designed to amplify the gene as three overlapping pieces and four primers were designed for sequencing of the three PCR products. In this study, we found polymorphic sites at nucleotides 64, 123, 253, 423, 1334 and 1781. The majority of the samples (49/88) expressed rnRNA with point mutations on at least one allele at base 64, while 8 samples had a SNP at base 123. At nucleotide 123, 6 samples expressed the heterozygote GIA genotype, and one sample expressed the homozygote A/A allele at this site. At nucleotide 423, two samples expressed a G allele and also the common C allele. While the SNPs at nucleotide 64, 123, and 423 caused a silent or conservative mutation in the gene, in sample 82, a mutation C253T produced an amino acid change from an arginine to tryptophan, which may cause a functional change in the fpgs protein, due to the significant change in size and charge of the wild type amino acid. Similarly, sample 26 expessed a homozygote T/T allele at nucleotide 1334 instead of the common C/C allele expressed in the remaining samples. This point mutation caused a valine to alanine amino acid change. We also detected a SNP that is expressed after the stop codon in sample 40.

Approximately 200 million men and 100 million women smoke worldwide. In the United States, an estimated 25.9 million men (23.9 percent) and 20.7 million women (18.1 percent) are smokers. The commencement of smoking at a young age is thought to increase addiction liability, decrease the probability of successful cessation, and correlate with a higher number of cigarettes smoked per day. Studies from the World Health Organization indicate that between 80,000 and 100,000 children start smoking every day worldwide. These statistics suggest that adolescence is a critical phase for developing nicotine dependence. The work in this dissertation contributes to the further understanding of this unique developmental period. Our research shows that various aspects of nicotine dependence are both age- and sex-dependent. We observed age- and sex-related differences in both nicotine reward and withdrawal models that imply a heightened vulnerability for adolescents. In addition, we have investigated possible behavioral and molecular mechanisms which may underlie the elevated vulnerability to dependence. The data illustrate that while behavioral mechanisms only play a minor role in the differences seen in reward and withdrawal, molecular mechanisms appear to have a greater contribution. Specifically, increased nicotinic receptor function is likely to be a substantial contributor to age-related disparities. In addition, nicotine is one of the first and most commonly abused drugs in adolescence and is known to be a strong predictor of subsequent alcohol and other drug abuse. Our research investigated the effects of adolescent nicotine exposure on both nicotine and cocaine dependence in adulthood. We found that exposure to nicotine during the early phase of adolescence affects both nicotine reward and withdrawal in adulthood. Moreover, this exposure also bears impact on other drugs of abuse such as cocaine. In summary, our data suggest that early adolescence is the most critical period for becoming dependent to nicotine and that early experimentation with nicotine may lead to enhanced vulnerability to dependence on more illicit drugs of abuse. It is imperative that we understand why adolescents have a heightened susceptibility to nicotine dependence so that better smoking cessation therapies and prevention messages can be developed for this age group.