Breast cancer is the second leading cause of cancer death in women according to the American Cancer Society. The standard treatment regimen for breast cancer involves ionizing radiation combined with surgery and chemotherapy. Ionizing radiation induces a complex signaling response in cells resulting in either growth arrest, senescence or cell death, and the cell killing after exposure to radiation results largely from DNA double-strand breaks (DSBs). There are two main mechanisms in mammalian cells responsible for repairing the DSBs; the primary mechanism is non-homologous end joining (NHEJ) and the secondary mechanism is homologous recombination (HRR). Previous studies showed that breast tumor cells depend mainly on NHEJ for repairing induced DNA damage. XRCC4 and XLF are two essential proteins in the NHEJ process. The interaction between XRCC4 and XLF (also called Cernunnos) is responsible for stimulating ligase IV for rejoining DNA ends. A single mutation on the XLF-binding interface of XRCC4 at M61, F106, M59 or D58 has been shown to disrupt its interaction with XLF and thus inhibiting NHEJ. Therefore, it is proposed that small natural cyclic peptides that bind to the XLF interface of XRCC4 near M61 and F106 can be identified through an mRNA display in vitro selection, and these peptides will inhibit NHEJ and thereby radiosensitize breast tumor cells. We have synthesized five DNA libraries that produced mRNA-peptide fusions containing a trillion unique peptide sequences that will be used for the selection of peptide inhibitors of the XRCC4/XLF interaction, and we have verified their randomness. Tagged wild-type and mutant versions of the head domain of XRCC4 protein, containing the XLF binding site, were successfully purified, and the wild-type version was applied to initial stages of selection of inhibitory peptides by mRNA display. The percentage of the mRNA-peptide fusions that bound to the XRCC4157 after the first round was 2.1%. The recovery after the second and third rounds was 1.14% and 2%, respectively. Results obtained thus far, although preliminary, suggest that the mRNA display method can be successfully applied to the XLF/XRCC4 interaction.

CB1 cannabinoid receptors are G-protein-coupled receptors that mediate the central nervous system (CNS) effects of marijuana and endocannabinoids. Recently, cannabinoid receptor interacting protein 1a (CRIP1a) was discovered as a novel protein that binds to the CB1 receptor C-terminus and inhibits CB1 receptor activity without affecting CB1 expression. This thesis investigated the functions of CRIP1a by characterizing the first CRIP1a knockout (KO) mouse line. The absence of CRIP1a was confirmed in KO mice using quantitative PCR and immunoblotting. I hypothesized that CRIP1a KO mice would exhibit enhanced CB1 receptor-mediated G-protein activity in the CNS, as well as cannabimemetic phenotypes and enhanced sensitivity to cannabinoid agonists in vivo. Results showed increased CB1 agonist-stimulated G-protein activity in the amygdala of CRIP1a KO relative to WT mice, but not in cerebellum, hippocampus or spinal cord. CB1 receptor levels did not differ between genotypes in in any region examined. Interestingly, CRIP1a KO mice exhibited an anxiolytic-like phenotype and decreased nociceptive sensitivity in vivo, but did not differ from WT mice in tests of motor activity or coordination. Surprisingly, sensitivity to agonist-induced antinociception, hypothermia, catalepsy or motor incoordination did not differ between genotypes. Our findings suggest that CRIP1a could play a selective role in modulation of anxiety by endocannabinoids, and this action could be mediated through the amygdala. Thus, CRIP1a might serve as a future pharmacological target for studying and treating anxiety disorders.

The misuse of prescription opiates is on the rise with combination therapies (e.g. acetaminophen or NSAIDs) resulting in severe liver and kidney damage. In recent years, cannabinoid receptors have been identified as potential modulators of pain and rewarding behaviors associated with cocaine, nicotine and ethanol in preclinical models. Furthermore, activation of cannabinoid 2 (CB2) receptors on immune cells through the inhibition of monoacylglycerol lipase (MAGL), results in increased 2-arachidonylglycerol (2AG) production and analgesia in animal models. Yet, few studies have identified whether mu opioid and CB2 receptor agonists act synergistically to inhibit chronic pain while reducing unwanted side effects including reward liability, or if it could ameliorate the excruciating pain that is poorly managed with by opiates in bone cancer patients. We determined if analgesic synergy exists between the mu-opioid agonist morphine and the selective CB2 agonist, JWH015, or the inhibitor of MAGL, MJN110, in rodent models of acute and chronic inflammatory, post-operative, neuropathic, and cancer-induced bone pain (CIBP) using isobolographic analysis. We also investigated if the MOR-CB2 agonist combination decreased morphine-induced conditioned place preference (CPP) and slowing of gastrointestinal transit. Next, we examined whether JWH015 or the inhibition of MAGL decreased the release of pro-inflammatory mediators by activating nuclear factor kappa enhancer of activated B cells (NFkB), as dysregulation of NFkB is observed in various cancers. Here we show that MOR + CB2 agonism results in a significant synergistic inhibition of preclinical pain while significantly reducing opioid-induced unwanted side effects. The opioid sparing effect of CB2 receptor agonism strongly supports the advancement of a MOR-CB2 agonist combinatorial pain therapy for clinical trials.

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a major dose-limiting side effect of several anticancer drugs. The prevalence of CIPN ranges from one-third to two-thirds of cancer patients. CIPN can persist for months to years after completion of chemotherapy. Despite the efficacious use of paclitaxel in the treatment of tumors, it can induce many sensory symptoms, such as paresthesia, numbness, tingling and burning pain, and mechanical and cold allodynia, which typically are present in the hands and feet. Similar to other types of chronic pain, paclitaxel-induced CIPN is comorbid with depression and anxiety in cancer survivors, and paclitaxel induces changes in affect-like behavior in cancer-free animal models, suggesting that paclitaxel can cause long-lasting changes in mood, reducing the quality of life. While adjuvant therapies, such as duloxetine, tricyclic antidepressants, and gabapentin are prescribed to treat CIPN symptoms, none of these compounds can consistently reverse or prevent the development of CIPN. With no FDA-approved medication to treat CIPN, the purpose of the dissertation was to: i) characterize and develop a mouse model of paclitaxel-induced CIPN, ii) identify putative targets for CIPN treatment, and iii) test novel compounds for their ability to prevent and reverse CIPN in C57BL/6J mice. In the first Aim, we demonstrate that paclitaxel induces time- and dose-dependent hypersensitivity (mechanical and cold), which is potentiated by combination therapy with the chemotherapeutic carboplatin. In addition, paclitaxel-treated mice show changes in affect-like behaviors (anxiety-like, depression-like). In the second Aim, we used the prototypic nicotinic receptor (nAChR) agonist nicotine to reverse or prevent paclitaxel-induced mechanical hypersensitivity and degeneration of Intra-Epidermal Nerve Fibers (IENFs). Further, we discovered that nicotine’s antinociceptive effects in this mouse model of CIPN are mediated by the nicotinic receptor subtype α7. The third Aim used genetic and pharmacological approaches to dissect the role of α7 on the development and maintenance of paclitaxel-induced CIPN. Null mutant α7 mice (KO) hastens the onset, increases the magnitude, and delays the recovery of paclitaxel-induced mechanical hypersensitivity, as compared to littermate wildtype controls, whereas the selective α7 silent agonist R-47 to reverses and prevents paclitaxel-induced CIPN in C57BL/6J mice. We also examined the impact of R-47 on the paclitaxel-induced reduction of intraepidermal nerve fiber (IENF), as well as microglial morphology in the dorsal horn of the spinal cord. The data show that R-47 prevents paclitaxel-induced changes in microglial morphology and mechanical hypersensitivity behavior, without producing tolerance upon repeated administration. Finally, R-47 induces preference using the conditioned place test in paclitaxel-treated mice but vehicle-treated animals, suggesting that R-47 is a viable candidate for ongoing, spontaneous pain, with limited risk of abuse potential. Overall, these results support that the α7 nAChR subtype is an important target for the treatment and prevention of CIPN.

Pain is the most common and debilitating medical problem for which patients seek medical care. Opioids remain the gold standard in the treatment of pain but are limited by poor side effect profiles such as emesis, constipation, dependence/addiction and respiratory depression. Despite a myriad of analgesic compounds on the market, tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof, nearly two thirds of the chronic pain patients report inadequate pain relief; therefore, a new approach in the development of pain management is necessary. In recent years, the N-Type voltage gated calcium channel (CaV2.2) has become an attractive target in the treatment of chronic pain. Ziconotide, a selective CaV2.2 blocker, has been FDA approved in the United States for the treatment of severe chronic pain that is refractory to other treatments, but due to its profound side effect profile (nausea/vomiting, somnolence, vertigo, muscle spasms, myalgia, insomnia, anxiety, tremor, memory impairment and induced psychiatric disorders), the use of Ziconotide is severely limited. Mapping of the CaV2.2 interactome led to the identification of novel regulatory proteins, including collapsin response mediator protein 2 (CRMP2). Initially identified as an intracellular protein in the specification of axon/dendrite fate and axonal outgrowth, it is now known that this protein can regulate the activity of CaV2.2 and hence may be a critical regulatory node in pain modulation. Here, I describe a novel peptide aptamer derived from CRMP2, designated CaV2.2 binding domain 3 (CBD3), which when fused with the HIV transactivator of transcription protein (TAT), created tat-CBD3, which was able to significantly reverse thermal and mechanical hypersensitivity induced by the surgical incision on the plantar surface of the left hind paw in rats, a pre-clinical model of post-operative pain. Additionally, tat-CBD3 significantly attenuated thermal hypersensitivity induced via intraplantar injection of carrageenan, a model of acute inflammatory pain. Furthermore, the administration of tat-CBD3 did not produce any rewarding behaviors as measured by the conditioned placed preference (CPP) paradigm, nor did the administration of tat-CBD3 produced any motor coordination deficits measured using the rotarod performance test. Moreover, the addition of a 14-carbon myristate (myr) group to the parent peptide, myr-tat-CBD3, had increased efficacy in the attenuation of paw incision and carrageenan induced thermal/mechanical hypersensitivities when compared to the parent peptide (tat-CBD3). These types of novel compounds that lack unwanted side effects and addiction propensities are urgently needed to relieve individuals suffering from chronic pain.

Objectives: When used as a cardioplegic solution, Custodiol® HTK solution is typically administered in a single-dose, allowing the operation to be performed continuously. This is an advantage over alternative cardioplegic solutions that may have to be re-administered every 20-30 minutes. Although Custodiol is widely used as a cardioplegic solution in Europe, its use for myocardial protection remains an off-label indication in the United States. Thus, the aim of this study is to compare the efficacy of Custodiol to standard 4:1 blood cardioplegia in adult cardiac cases. METHODS: This study was a single-center retrospective review of prospectively collected data. Adult cardiac cases performed between November 2011 and August 2013 using Custodiol® were compared to cases using standard Plegisol® 4:1 blood cardioplegia. Twenty-six primary intra-operative and post-operative endpoints were compared including 30-day mortality, 30-day hospital readmission, prolonged mechanical ventilation time, and renal failure. RESULTS: Of the 229 cases identified, 63 cases used Custodiol and 166 used 4:1 blood cardioplegia. Demographics were similar in both groups with a mean patient age of 65.27±15.07 years for Custodiol and 66.72±12.85 years for 4:1 blood cardioplegia. The average cardiopulmonary bypass time for Custodiol and 4:1 blood cardioplegia was 124.76±61.45 and 137.93±54.05 minutes respectively. The Custodiol group had a greater incidence of prolonged ventilation (>24 hours), 20.6% versus 15.1% respectively, and this approached statistical significance with a p value of 0.052. Intra-operative blood usage was significantly higher in the Custodiol group compared to the blood cardioplegia group, with 44.4% of patients receiving fresh frozen plasma during the operation compared to only 25.3% in the blood cardioplegia group (p=0.005). The results revealed no statistically significant difference in 30-day mortality, 30-day hospital readmission, renal failure, and stroke. CONCLUSION: Despite the distinct advantage of long-term ischemic tolerance, Custodiol use was associated with an increased requirement for fresh frozen plasma during the perioperative period when compared to blood cardioplegia.

Migraine headache is one of the most common neurological disorders. While the mechanisms contributing to migraine pathophysiology have yet to be fully elucidated, the disproportionate number of post-pubescent, pre-menopausal women affected suggests a central role for female hormones, such as estrogen. The mechanism(s), however, by which estrogen contributes to migraine have yet to be deciphered. Cortical spreading depression (CSD) is associated with "Classic Migraine", now referred to as migraine with aura. Here we use a well-established animal model for migraine with aura to test the putative role of estrogen in the development of CSDs in awake and freely moving female rats. Beta estradiol administration in ovariectomized female rats resulted in a significant increase in CSD episodes over a 12-hour recording period. Additionally, beta estradiol administration in these rats promoted migraine-associated behavior, significantly reducing exploratory behavior (i.e., number of vertical rearing episodes) when compared to vehicle-treated controls. Critically, the increase in CSD episodes was completely abolished with pre-administration of ICI 182,780 a pure alpha and beta estrogen receptor antagonist. ICI 182,780administration also blocked beta estradiol-induced migraine-associated behaviors, restoring vertical rearing episodes to baseline levels. These data illustrate that an increase in estrogen levels in an animal that no longer produces estrogen (postmenopausal characteristic) can promote the development of CSDs. These data suggest that an estrogen receptor-mediated mechanism may drive episodes of migraine with aura and highlight the need for further investigation into estrogen's role in migraine.

A role for gene variants in regulating the pharmacokinetics of systemically available toxicants has not yet been established. A panel of 18 genetically-diverse inbred mouse strains was used to determine the range of total exposure kinetic parameters in blood and bone marrow following a single oral administration of benzene (100 μg/kg) to male and female mice. Large ranges in several pharmacokinetic parameters were found when data from blood and bone marrow were analyzed. AUC and CL_F pharmacokinetic parameters in blood and bone marrow pharmacokinetics were strikingly different as were these parameters in males and females. Final clearance (CL_F) was found to be the most statistically robust pharmacokinetic parameter as it accounted for exposure of the matrix (AUC) and normalized for dose variations among the strains. The CL_F values in blood and bone marrow used for haplotype association mapping showed 331 and 164 quantitative trait loci with statistical significance, respectively (male mice; -logP>4). Two loci were found to be shared between males and females QTL bone marrow data sets and one common locus was found for male blood and bone marrow data. No overlap was found among blood QTL in males and females (or between blood and bone marrow data from females). Protein and mRNA expression data for the primary benzene-metabolizing enzymes CYP2E1 and UGT1A6 showed very little strain-dependent variation. Strain dependent differences in mRNA levels of NQO1 and MPO were small but statistically significant, as were those for GAPDH and β2-microglobulin. These data demonstrated that polymorphisms with the greatest contribution toward overall variations in systemic exposures occurred in genes encoding for non-metabolic proteins. While exposure does not equate to toxicity, identification of the genes regulating distribution and clearance may be useful for investigating host susceptibility to toxic effects following benzene exposure. This research was supported in part by the NIEHS NTP Grant N01ES45529, NIEHS Toxicology and Toxicogenomics Training Grant (5T32ES007091-29), NIEHS/NTP Division of Intramural Research, and Southwest Environmental Science Center Grant P3ES06694.

The studies in this dissertation research were conducted to investigate the possible mode of action by which a brominated flame retardant, 2, 2-Bis (bromomethyl)-1, 3-propanediol (BMP) causes genotoxicity. Binding of BMP to DNA and BMP induced DNA strand breaks were investigated in SV-40 immortalized human uroepithelial cells (UROtsa) as an in vitro model for the bladder (a tissue that developed cancer after two year exposure to BMP in rodents). Results showed binding of [¹⁴C]-BMP equivalents to DNA increased with increased exposure time and concentration of [¹⁴C]-BMP. Comet analysis indicated BMP significantly increased the extent of DNA strand breaks at 1 and 3 h of incubation. However, strand breaks were repaired by 6 h of incubation. The DNA damaging effects of BMP at 1 h was concentration dependent. Compared with the parent compound, BMP-glucuronide (the predominant metabolite of BMP) bound less to DNA and produced less DNA strand breaks in UROtsa cells. Evidences that the BMP induced strand breaks were the result of an oxidative stress include: a concentration and time dependent increase in ROS generation; increased expression of Nrf2 and HSP70; complete attenuation of BMP induced DNA strand breaks by the antioxidant, NAC; and the presence of the oxidized base 8-OHguanine. UROtsa cells appear to be target cells for BMP because, as compared to rat hepatocytes (non-target cells), these cells lack the ability to detoxify BMP via glucuronidation and also because they are deficient in glutathione, a major intracellular antioxidant molecule. Both of these genotoxic events, DNA binding and oxidative DNA damage may, in part, contribute to BMP carcinogenicity observed in rodents. The relevance of current results to humans is remained to be established.

As a major public health problem affecting more that 76.5 million Americans, chronic pain is one main reason why people seek medical attention. It is a pathological nervous system disorder that persists for months or years. Sensitization of nociceptive neurons in the dorsal horn of the spinal cord is crucial in the development of allodynia and hyperalgesia. The work presented in this thesis will focus on spinal protein kinase M zeta (PKMζ)-mediated plasticity and GABAergic disinhibition as spinal amplification mechanisms that orchestrate persistent changes in the dorsal horn of the spinal cord. As a result of central sensitization following peripheral nerve injruy, GABAergic disinhibition occurs due to an alteration in Cl- homeostasis via reduced KCC2 expression and function. Intrathecal administration of acetazolamide (ACT), a carbonic anhydrase inhibitor, attenuated neuropathic allodynia and spinal co-adminitation of ACT and midazolam (MZL), an allosteric modulator of the benzodiazepine class of GABAA receptors, synergistically inhibited neuropathic allodynia. Further studies concerning the impact of altered Cl-homeostasis via reduced KCC2-mediated Cl-extrusion capacity on the analgesic efficacy and potency of GABAA receptor agonist and allosteric modulators revealed that there is a differential regulation of the agonists and allosteric modulators at the GABAA receptor complex when Cl-homeostasis is altered. Another spinal amplification mechanism leading to central sensitization is PKMζ-mediated spinal LTP. In model of persistent nociceptive sensitization, allodynia induced by IL-6 injection or plantar incision was abolished by both the inhibition of protein translation machinery and PKMζ inhibitor, ZIP. However, only PKMζ inhibition prevented the enhanced pain hypersensitivity precipitated by a subsequent stimulus after the initial hypersensitivity had resolved, asserting that spinal PKMζ underlies the maintenance mechanisms of persistent nociceptive sensitization. Also, these results confirmed that the initiation mechanisms of persistent sensitization parallel LTP initiation mechanisms and the maintenance mechanisms of persistent sensitization parallel LTP maintenance mechanisms. Taken together, these results indicate that these amplification mechanisms drive a chronic persistent state in these models such that inhibition of these spinal amplication mechanisms will serve as an effective approach in the quenching chronic pain hypersensitivity in chronic pain models.