Pain is the primary reason why patients seek medical care and there is a great unmet need for the development of pain relieving medications. The treatments that are currently available either have limited efficacy or are accompanied by a multitude of unwanted side effects. However, discovering novel therapeutics for the treatment of pain has been challenging. Part of the reason for this may be that that the ways in which pain is assessed in the preclinical setting are different from the way that it is evaluated clinically in human trials. The most common method for evaluating pain in preclinical models is to measure responses to evoked stimuli. However, a change in the threshold of response to evoked pain likely does not measure whether the unpleasant component of pain has actually been reduced. The most clinically relevant question for pain is whether the treatment actually makes the patients "feel better". Here, we demonstrate that the aversiveness of pain can be captured using motivated behavior to seek pain relief. We used conditioned place preference (CPP) to establish that animals with ongoing pain will seek a context that has been paired with effective pain relief, likely as a result of negative reinforcement. These studies allowed for mechanistic investigation. Our results show that: 1) effective pain relief can be achieved by either blocking noxious peripheral input or by directly attenuating pain related unpleasantness in the brain, and 2) pain relief is rewarding and activates the reward circuitry. These studies provide a basis for development of a future platform for drug discovery for pain.

Cocaine dependence is a major health concern worldwide, but despite this high rate of abuse there are currently no approved therapies for cocaine dependence. Replacement pharmacotherapies are one possible approach for treating cocaine dependence, and identification of such therapeutics for cocaine abuse is the long-term goal of this research. Cocaine binds to, and inhibits uptake at the dopamine (DAT), serotonergic (SERT) and noradrenaline (NET) uptake transporters, but studies have shown that cocaine produces its strong behavioural and positive reinforcing effects through inhibition of the DAT. To this end a great number of diverse, non-selective DAT-inhibiting compounds have been investigated as potential cocaine replacement therapies. It was the initial objective of this research to determine whether the behavioral profile of a novel, selective DAT inhibitor, D-84, fit with that thought for an ideal cocaine replacement therapy. Results indicated that D-84 stimulated locomotor activity, incompletely generalized to the cocaine cue in discrimination tests, attenuated cocaine-self-administration and was self-administered. These observations provide a profile consistent, although perhaps not ideal, with one possible treatment strategy for cocaine dependence. Although it is well established that cocaine predominantly produces its abuse-related effects through inhibition of the DAT, recent evidence suggests that inhibition at the SERT may have modulating effects on the pharmacology of cocaine-like compounds. The second part of this dissertation investigated what effects that increasing SERT inhibition had on the cocaine-like behavioural effects of DAT inhibitors, as a method of determining the fruitfulness of incorporating this feature into future drug candidates to improve them. RTI-55 (DAT Ki 2.7 nM SERT Ki 3 nM) and GBR-12909 (DAT Ki 4.3 nM SERT Ki 73 nM) were selected based on their high and intermediate SERT inhibitory effects, respectively. They were compared in behavioural studies with D-84, which is considered to be a selective DAT inhibitor. The results indicated that although increasing SERT inhibition attenuated locomotor activity effects, it had less effect on cocaine-like discriminative stimulus and reinforcing effects, at least with the doses tested

Several classes of antimetabolites have been developed for the treatment of cancer, including numerous inhibitors of nucleotide biosynthesis. N-(phosphonacetyl)-L-aspartate (PALA) and hydroxyurea (HU) are two antimetabolites that inhibit nucleotide biosynthesis; PALA inhibits de novo pyrimidine synthesis and HU inhibits the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. Due to the similar mechanisms, it was thought that cancer cells would respond similarly to HU and PALA treatment. However, studies in this dissertation revealed strikingly different responses to either HU or PALA treatment in HCT116 cells. A cytoprotective S-phase arrest was activated upon HU treatment while PALA treatment failed to activate the S-phase checkpoint, resulting in p53-dependent apoptosis. The checkpoint effector kinase, Chk1, was not significantly phosphorylated during PALA treatment due to a failure to recruit ATR, the upstream kinase, to chromatin sites. The post-translational modifications of p53, phosphorylation of serines 46 and 392, suggested that PALA treatment promotes the accumulation of a transcriptionally active p53 while HU does not. ChIP analysis showed that p53 bound to pro-apoptotic promoters, therefore activating p53-dependent apoptosis during PALA treatment. To gain more insight into these differential cellular responses, we developed a tandem-affinity purification (TAP) tagged p53 cell line in which a TAP tag was inserted into the C-terminus of the endogenous p53 genetic locus through homologous recombination. This technology allows purification of p53 with its protein binding partners at endogenous expression levels. The tagged p53 accumulated and bound to promoters in response to DNA damage similar to the untagged p53, suggesting that the TAP tag did not interfere with the normal cellular functions of p53. Using mass spectrometry, we can identify the different p53 protein binding partners in response to PALA or HU treatment. We can also determine the variable pattern of post-translational modifications on different drug-stabilized p53 and determine which modifications are responsible for promoting apoptosis versus cytoprotective arrest. We can then exploit the identified proteins and post-translational modifications in the development of new chemotherapeutic agents.

CP47,497 and other synthetic cannabinoid compounds were incipiently synthesized as research tools to investigate the mechanisms by which marijuana affects the brain and to aid in the development of therapeutic agents. Recently, these cannabinoid compounds have resurfaced in the designer drug market, marketed as “herbal incense products” (HIPs). Their popular use has resulted in an alarming rate of reported adverse effects and toxicities. Current legislation classified CP47,497 and several other synthetic cannabinoids compounds as Schedule I agents, but abuse of these compounds persists with serious consequences to public health and safety. In vivo studies examining the behavioral consequences of abused synthetic cannabinoids are limited. As a result, the goals of this research were to elucidate the acute and chronic pharmacological effects of CP47,497 and to develop a bioanalytical method for CP47,497 drug detection in mice. Cannabimimetic effects were evaluated in well-established in vivo models, the tetrad paradigm and drug discrimination assay. The tetrad test is comprised of four outcome measures sensitive to the primary psychoactive cannabinoid present in marijuana, delta-9-tetrahydrocannabinol (THC): catalepsy (bar test), antinociception (tail withdrawal latency), hypothermia, and decreases in spontaneous locomotor activity. While many pharmacological agents can produce one or a subset of these tetrad effects, drugs that activate CB1 receptors produce characteristic effects in all four parameters. An HPLC/MS/MS method was developed and confirmed the presence of CP47,497 in brain. We investigated whether CB1 receptors mediate the pharmacological effects of CP47,497. Cumulative dose-response experiments determined CP47,497 is more potent than THC in vivo in using multiple behavioral assays. Complementary pharmacological (CB1 receptor antagonist, rimonabant) and genetic (CB1 (-/-) mice) approaches were used to investigate whether CB1 receptors mediate the effects of CP47,497. Rimonabant (3 mg/kg or 10 mg/kg, depending on independent measure) blocked all cannabinoid-like pharmacological effects of CP47,497. Supporting these findings, CB1(-/-) mice were resistant to cannabimimetic effects of CP47,497. CP47,497 fully substituted for THC in the drug discrimination assay, with a potency of more than 5 times that of THC. Collectively, these results indicate that CP47,497 is markedly more potent (i.e. 5-8 fold) than THC, and its repeated administration produces tolerance to the cataleptic, antinociceptive, hypothermic and hypolocomotor effects in mice, with significant presentation of somatic withdrawal signs (paw flutter and head shakes) upon drug cessation. These findings are consistent with the high incidence of adverse events in humans abusing synthetic cannabinoids.

Methamphetamine is a psychomotor stimulant that produces hyperactivity and euphoria and can lead to drug-seeking and abuse. An estimate from 2005 put the cost of methamphetamine abuse to society at an estimated 23.4 billion dollars. One of methamphetamine's effects is activation of glial cells and associated neuroinflammatory activity in the periphery and CNS. Glia are responsible for maintaining calcium homeostasis, neuroplasticity, immune activity, and cell signaling. Activation of glia and neuroinflammation are becoming recognized as links in drug abuse-related behavior. The goal of the present work was to assess the ability of ibudilast, AV1013, and minocycline, three glial cell modulating compounds, to attenuate responding in rodent procedures that model different aspects of methamphetamine abuse-related behavior. First, Ibudilast (1.8, 7.5, 13 mg/kg) and AV1013 (10, 30, 56 mg/kg) were examined for their effects on methamphetamine-induced (3 mg/kg) locomotor activity and sensitization in mice, the latter thought to involve neurocircuitry common with drug relapse. Ibudilast and AV1013 dose-dependently attenuated methamphetamine-induced locomotor activity and its sensitization. Second, ibudilast (1, 7.5, 10 mg/kg), AV1013 (1, 10, 30 mg/kg), and minocycline (10, 30, 60 mg/kg) were examined for their effects on methamphetamine self-administration (0.001, 0.03, and 0.1 mg/kg/inf) in rats. All three compounds significantly reduced methamphetamine (0.03 mg/kg/inf) self-administration. Results suggested baseline self-administration rate as a possible determinant of these effects; however, follow-up tests with ibudilast while controlling for baseline response rate dismissed this possibility. Additional follow-up tests identified whether the attenuation of operant response rates was specific to methamphetamine-maintained behavior. Using a behavioral economic approach, all three test compounds were determined to also affect non-nutritive banana pellet-maintained responding when the baseline strengths maintained by methamphetamine and banana pellet delivery were matched. Finally, ibudilast was evaluated for its effects on methamphetamine discrimination in rats, a procedure thought to model clinical subjective effects. Ibudilast (1, 7.5, 10 mg/kg) did not significantly modify methamphetamine’s discriminative stimulus effects when trained at either 1 or 0.56 mg/kg. These results support the hypothesis that attenuation of glial cell activity and neuroinflammation may be linked to some abuse-related behaviors of methamphetamine, reinforcing their examination as novel targets for treating methamphetamine abuse.

In general, ionizing radiation promotes cytoprotective autophagy in a majority of breast tumor cells. Previous studies from our laboratory indicated that radiation (5x2 Gy) induces cytoprotective autophagy in MCF-7 cells. In the current work, inhibition of autophagy by silencing of Beclin-1 in MCF-7 cells resulted in an increase in sensitivity to radiation based both on cell number and clonogenic survival; however, there was no increase in apoptosis and the basis for this sensitization is currently under investigation. Unexpectedly, enhancement of autophagy by silencing of Bcl-2 also led to an increase in sensitivity to radiation, possibly through the conversion of cytoprotective to cytostatic autophagy. In contrast to the MCF-7 cells, radiation (5x2 Gy) induces non-protective autophagy in Hs578t cells. Interference with autophagy through silencing of Beclin-1 or induction of Bcl-2 did not alter radiation sensitivity in the Hs578t cells. Since the induction of cytoprotective autophagy can represent an impediment to radiation therapy, it is important to understand the types of autophagy that occur in response to radiation in specific cellular settings and whether interference with autophagy can increase sensitivity to different forms of cancer treatment.

These studies were completed to examine how status epilpeticus seizure characteristics are modulated during post-juvenile brain development. This may determine if postnatal age in rats is a better identifier of stages of post-juvenile brain development. The first study fully detailed the acute discrete seizure phase of the rat pilocarpine-SE model. Results for this study showed that Racine behavioral severity score, spike frequency, and seizure severity during the acute discrete seizure phase change in relation to post-juvenile brain developmental stages. The second study fully detailed early and late patterns of status epilepticus. Results for this study displayed modulation of time in pattern, spike frequency, and relative delta power for seizure pattern during post-juvenile ages. The third study displayed modulation of diazepam efficacy during post-juvenile ages. The data suggest characteristics in the acute discrete seizure pliase, chronic SE phase, and therapeutic window of SE change in relation to age during post-juvenile brain development. This establishes that age is a better estimator of developmental stage than animal bodyweight.

DNA double-strand breaks (DSB) with chemically modified end-termini are the most significant lesions resulting from radio/chemotherapeutic intervention of cancer and non homologous end-joining (NHEJ) factor Artemis nuclease has been implicated in the repair of such breaks. To examine whether the resolution of terminally blocked DNA DSBs is the biologically relevant function of Artemis, Artemis deficient fibroblasts were stably complemented with wild type or an endonuclease deficient D165N mutant Artemis. Physiological levels of wild type (WT) Artemis completely restored DSB repair proficiency and resistance to γ-radiation, bleomycin, and neocarzinostatin. Cells expressing the D165N mutants remained as chemo/radiosensitive and as repair deficient as parental cells, with persistent γ H2AX and 53BP1 foci that increased in size 6-18 hour post irradiation. These persistent foci co-localized with DNA double strand break repair factor Mre11 and also with promyelocytic leukemia protein (PML). Further, in vitro studies have revealed that DNA-PK dependent Artemis endonucleolytic activity may play a role in the repair of commonly found oxidative base damage; 8-oxoguanine (8-oxoG), a hallmark of complex DSBs. However, majority of DNA DSBs are repaired in an Artemis independent manner, and recently discovered, DNA end-specific nuclease, Metnase is a candidate enzyme for repair of such breaks. To study the role of Metnase in resolution of 3ʹ-blocked termini, several substrates mimicking such breaks were constructed. A 3ʹ-phosphoglycolate moiety on longer overhangs (4 and 6 bases) altered specificity and stimulated Metnase-mediated cleavage of the terminal 3 nucleotides. However, an 8-oxoG residue at the single-strand/double-strand border did not affect specificity or extent of cleavage. Metnase preferentially cleaved ssDNA-overhang of a partially duplex substrate, and the cleavage increased with increase in length of 3ʹ-overhangs. A D483A mutation in Metnase completely abrogated Metnase cleavage activity towards DNA ends. These results suggest that Metnase may resolve oxidatively damaged DNA ends to facilitate repair while Artemis is required for the resolution of more complex DNA DSBs that persist for longer times and are not amenable to repair by other NHEJ factors.

A targeted approach to the development of antifolate therapies has been sought for many years. Central to the success of such development is an understanding of the molecular mechanisms dictating the sensitivity of cells to antifolates and the fundamental differences of these processes between normal and neoplastic phenotypes. This dissertation addressed transcriptional mechanisms and cell-signaling events responsible for the efficacy of antifolate therapies. Transcriptional processes and cell signaling pathways are often aberrant in neoplastic tissues, providing a potential point of distinction between a normal and neoplastic cellular state. Folylpolyglutamate synthetase (FPGS) catalyzes the formation of poly-γ-glutamate derivatives of folates and antifolates, which permits intracellular retention and accumulation of these compounds. The mouse fpgs gene uses two distant promoters to produce functionally distinct isozymes in a tissue-specific pattern. We questioned how the two promoters were differentially controlled. An analysis of DNA methylation and histone post-translational modifications across the length of the mouse fpgs gene showed that epigenetic mechanisms contributed to the tissue-specific control of the upstream (P1), but not the downstream (P2) fpgs promoter. RNAPII complexes and general transcription factors were present over P1 only when P1 was transcribed, but these components were present over P2 in most tissues, and promoter-proximal pausing was evident in brain. Clear promoter occlusion was found over P2 in liver. These studies concluded that tissue-specific coordination of dual promoters required multiple interacting controls. The mammalian target of rapamycin (mTOR) controls protein translation initiation, and is central to a cell-signaling pathway rich in tumor suppressor and oncogenic proteins. mTOR dysregulation is a common feature of several human cancers and inhibition of this protein has been sought as an ideal cancer drug target. We have determined that antifolates inhibiting the two folate-dependent steps of purine synthesis (GART or AICART) activate AMP-dependent protein kinase (AMPK) and inhibit mTOR. The mechanism of AMPK stimulation appears to be mediated by either nucleotide depletion (GART inhibitors), or ZMP accumulation (AICART inhibitors). These studies discovered a new mechanism for antifolates that surprisingly defines them as molecularly targeted therapeutics.

Status epilepticus (SE) is a major clinical emergency resulting in continuous seizure activity that can cause brain injury and many molecular and pathophysiologic changes leading to neuronal plasticity. The neuronal plasticity following SE-induced brain injury can initiate epileptogenesis and lead to the ultimate expression of acquired epilepsy (AE), characterized clinically by spontaneous, recurrent seizures. Epileptogenesis is the process wherein healthy brain tissue is transformed into hyperexcitable neuronal networks that produce AE. Understanding these alterations induced by brain injury is an important clinical challenge and can lend insight into possible new therapeutic targets to halt the development of AE. Currently there are no means to prevent epileptogenesis following brain injury; thus, the elucidation of mechanisms of epileptogenesis will be useful in preventing the long-term clinical sequela. It has been demonstrated in vivo that calcium (Ca2+) dynamics are severely altered during SE and that elevations in intracellular Ca2+ ([Ca2+]i) in hippocampal neurons are maintained well past the duration of the injury itself (Ca2+ plateau). Here we report that similar changes in [Ca2+]i are observed in the hippocampal neuronal culture model of SE-induced AE. As an important second messenger, the maintenance of a Ca2+ plateau following injury can lead to several changes in gene expression, neurotransmitter release, and overall, neuronal plasticity. Thus, changes in post-SE [Ca2+]i and Ca2+ homeostasis may be important in understanding epileptogenesis and eventually preventing the progression to chronic epilepsy. This dissertation examines the development and maintenance of the Ca2+ plateau after SE and demonstrates the novel finding that pharmacological modulation of [Ca2+]i following SE may inhibit epileptogenesis in vitro.