Status epilepticus (SE) is a type of neurological injury characterized by continuous seizure activity and can lead to molecular and pathophysiological alterations leading to plasticity changes. SE can lead to the development of AE by the process of epileptogenesis, which is a phenomenon that describes the transformation of normal brain tissue into a hyperexcitable neuronal population. It has been demonstrated both in vivo and in vitro that calcium (Ca2+) dynamics are severely altered during and after SE, and these changes play a major role in the progression of epileptogenesis. It has also been reported that preventing the rise in intracellular Ca2+ ([Ca2+]i) immediately following injury (the Ca2+ plateau) prevents the plasticity changes and ultimate development of epilepsy. Currently, there are no treatments available that can be administered following an injury to prevent the development of AE. Therefore it is clinically important to develop a therapy that can be administered after an injury to block epileptogenesis. Hypothermia is a potential therapeutic intervention. Hypothermia is used clinically to provide neuroprotection following various neurological insults such as stroke and traumatic brain injury (TBI). However, no studies have been performed to evaluate the therapeutic potential of hypothermia following SE. Hypothermia provides protection via multiple mechanisms, one of which includes modulating excitotoxic neurotransmission. It is believed to reduce Ca2+ influx by reducing NMDA receptor activation. It is unclear how hypothermia affects Ca2+ through other modes of entry. This dissertation evaluates the effects of hypothermia on the Ca2+ plateau and demonstrates the novel finding that hypothermia induced post-SE blocks the development of the Ca2+ plateau and reduces the development of AE.

Nanotechnology has recently emerged as a strong contributor toward research efforts to develop targeted systems of drug delivery in cancer therapy. Our work investigates the therapeutic potential of Targeted Charge-Reversal Nanoparticles (TCRNs), a novel nanoparticle with in vitro evidence of nuclear drug delivery. Using M12 prostate cancer cells, MDA-MB-231 breast cancer cells, and modified derivatives of these cell lines, we investigated the ability of Folic Acid-tagged TCRNs to deliver Nile Red and Dimethyl Indole Redfluorescent (DiR) fluorescent dyes to the nucleus of cells using confocal microscopy and in vivo biphontonic imaging using Xenogen® Technology. Confocal imaging with the SCP28 derivative of MDA-MB-231 cells shows nuclear association of the TCRNs over time, although specific nuclear deposition was unclear. Biophotonic imaging with M12 and SCP28 xenograft tumors in athymic nude mice shows retention of TCRNs in animals out to 7 days with minimal localization of TCRNs to tumor tissues. Our findings suggest that further characterization and manipulation of both the cells and the nanoparticle is necessary in order to make definitive claims regarding the TCRN’s ability to deliver fluorescent dyes, and eventually therapeutic compounds, to the nucleus of cells.

The ATP sensitive potassium channel (KATP) in mouse colonic smooth muscle cell is a complex containing a pore forming subunit (Kir6.1) and a sulfonyl urea receptor subunit (SUR2B). These channels are responsible for maintaining the cellular excitability of the smooth muscle cell which in turn regulates the motility patterns in the colon. We used whole-cell voltage-clamp techniques to study the alterations in these channels in smooth muscle cells in experimental model of colitis (colonic inflammation). Colitis was induced in BALB/C mice following an intracolonic administration of trinitrobenzene sulfonic acid (TNBS). KATP currents were measured at Vh -60 mV in high K+ external solution. The dose-response to levcromakalim (LEVC), a KATP channel opener, was significantly shifted to the left in the inflamed smooth muscle cells. Both the affinity and maximal currents induced by LEV were enhanced in inflammation. The EC50 in control was 6259 nM (n=10) and 422 nM (n=8) in inflamed colon while the maximal currents were 9.9 ± 0.71 pA/pF (60 μM) in control and 39.7 ± 8.8 pA/pF (3 μM) following inflammation. Similar to LEVC, KATP currents activated by sodium hydrogen sulfide (NaHS) (10-1000 μM) were significantly greater in inflamed compared to controls. In control cells, pretreatment with 100 µM NaHS shifted the EC50 for LEV-induced currents from 2838 nM (n=6) to 154 nM (n=8). These data suggest that NaHS can act as an allosteric modulator for LEV-induced KATP currents. Decreased colonic motility may result from enhanced KATP activation by increased release of H2S in colitis.

The α7 nicotinic acetylcholine receptor (nAChR) subtype is abundantly expressed in the central nervous system (CNS) and in the periphery. Positive allosteric modulators (PAMs) of the α7 increase the response to an agonist and are divided into two types depending on whether they also decrease desensitization of the receptor (type II) or not (type I). Therefore, this study aims to investigate whether the enhancement of endogenous α7 nAChR function will result in a beneficial effect in nociceptive, inflammatory and chronic neuropathic pain models. While NS1738 and PNU-120596 were not active to reduce acute thermal pain, measured by hot-plate and tail-flick tests, only PNU-120596 dose-dependently attenuated paw-licking behavior in the formalin test. Our results with selective (MEK) inhibitor U0126 argues for an important role of extracellular signal-regulated kinase (ERK1/2) pathways activation in PNU-120596’s antinociceptive effects in formalin-induced pain. The α7 antagonist MLA, via intrathecal and intraplantar administration, reversed PNU-120596’s effects, confirming PNU-120596’s action through central and peripheral α7 nAChRs. Tolerance to PNU-120596 was not developed after chronic treatment of the drug. Furthermore, mixtures of PNU-120596 and choline, an endogenous α7 nAChR agonist, synergistically reduced formalin-induced pain, while interactions of non-antinociceptive doses of PNU-120596 and PHA-543613, a selective α7 nAChR agonist, or nicotine resulted in antinociception. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, -hypomotility and –antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via α7 nAChRs. In the carrageenan inflammatory test both NS1738 and PNU-120596 significantly reduced thermal hyperalgesia, while only PNU-120596 significantly reduced edema. Importantly, PNU-120596 reversed established thermal hyperalgesia and edema induced by carrageenan. In the chronic neuropathic pain (CCI) model, PNU-120596 had long-lasting (up to 6 hrs), dose-dependent anti-hyperalgesic and anti-allodynic effects after a single injection, while NS1738 was inactive. Subcutaneous and intrathecal administration of MLA reversed PNU-120596’s effects, suggesting the involvement of α7 nAChRs. Finally, PNU-120596 enhanced an ineffective dose of selective agonist PHA-543613 to produce anti-allodynic effects in the CCI model. Our results show a fundamental in vivo difference between type I and II α7 nAChR PAMs, and demonstrate type II’s potential for the treatment of chronic inflammatory pain.

Substantial evidence in the literature shows that tobacco smoking has complex and divergent effects on inflammatory bowel diseases (IBD). It ameliorates ulcerative colitis (UC); whereas it aggravates the risk of Crohn’s disease (CD) and affects the disease course and severity. Studies have shown that nicotine has a positive influence on symptoms of UC. Also, it is demonstrated that nicotinic acetylcholine receptor, especially α7 subunit plays an essential component in the vagus nerve-based cholinergic anti-inflammatory effects. In the present study, we explored the effect of nicotine and α7 nicotinic agonists treatment in the DSS colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. Methods: Different groups of C57BL6 mice, as well as α7, α5, and β2 nicotinic receptor knock out mice, and their littermates wild-type nicotinic receptor male adult mice were given DSS solution freely in the drinking water for 7 consecutive days after which tap water was given on the 8th day. We measured a Disease Activity Index (DAI) that includes body weight loss, blood presence in stools, stool consistency, local rectal irritation and length of the colon. The mice were then sacrificed on day 8 to allow examination of the entire colon. Disease severity and colon tissue histology and inflammatory markers including colonic myeloperoxidase (MPO) and colonic tumor necrosis factor-α (TNF-α) were evaluated. Levels of MPO and TNF-α were determined by enzyme-linked immunosorbent assay analysis of the homogenized colon samples. The effect of oral, subcutaneous, mini pump nicotine, and oral cotinine treatments were examined on experimental colitis induced by 2.5% DSS in mice. In addition, we measured the plasma levels of the nicotine and cotinine in our treatment protocols. Results: The DSS 2.5% model of colitis is easily induced in mice. Administration of low doses of oral nicotine (12.5 and 25 μg/ml), but not high doses in DSS-treated mice displayed a significant decrease in disease activity index value, total histological damage scores, as well as colonic level of TNF-α compared to the control group. However, the anti-inflammatory effect of nicotine was not seen with chronic s.c., mini pump nicotine or oral cotinine administration. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Moreover, neither nicotine nor cotinine reversed colon length shortening in DSS-treated mice, except with the 0.5 mg/kg s.c. dose of nicotine. There was no change in MPO activity among the groups treated with oral or s.c. nicotine. Cotinine oral administration on its own failed to show a significant effect in the DSS model of colitis. α7 KO mice displayed a significantly increased in DAI value starting from day 4 till day 8, histological damage scores and TNF-α levels of were increased significantly compared to their littermate WT mice. Moreover, pretreatments with PHA-543613 (8 mg/kg), a selective α7 agonist, and choline chloride (40 ug/ml), an α7 nAChR natural agonist, significantly reduced clinical parameters in DSS-treated mice; however, they slightly inhibited the increase in the colonic TNF-α levels compare with vehicle DSS-treated mice. Moreover, PNU-120596 (3 mg/kg), a positive allosteric modulator for α7 nAChRs, significantly reduced DAI value and total histological damage score in DSS-treated mice. Conclusion: Results obtained from this study highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model. Also, these data suggest that α7 nAChR has a protective role in colitis with narrower therapeutic index. Data obtained from this study further understanding of the effect of nicotine in UC and may contribute in the development of new pharmaceutical designs for targeting nAChRs for the treatment of ulcerative colitis.

Breast cancer is the most commonly observed cancer type in women and is the second leading cause of cancer death in women. Radiation can be used to debulk tumors prior to surgery as well as to treat patients after surgery and/or chemotherapy. Previous studies from our laboratory have shown that the anti –malarial drug chloroquine sensitizes breast cancer cell lines to radiation by suppression of autophagy which is a conservative catabolic process that can be cytoprotective. The scientific literature has demonstrated that many tumor cell systems undergo cytoprotective autophagy and that pharmacological or genetic inhibition of autophagy leads to other modes of cell death such as apoptosis. Acridine orange staining was used for determination of acidic vacuole formation, an indication of autophagy and DAPI/TUNEL staining was used to identify apoptotic cells. Our studies in Hs578t breast tumor cells show the lack of sensitization by chloroquine upon autophagy inhibition with minimal apoptosis when cells are treated with 5 × 2Gy radiation. The extent of apoptosis was not increased upon autophagy inhibition by Chloroquine as determined by DAPI/TUNEL assays and quantified by Flow Cytometry using AnnexinV/PI. The potential role of senescence in the effects of radiation in the Hs578t cells was determined with the use of β-Galactosidase dye staining for senescence. It appears from these studies that autophagy need not to be cytoprotective in all breast cancer cell lines. Additional studies are in progress to effort to identify the factors that might distinguish between cytoprotective and non-cytoprotective autophagy.

Alcohol use and abuse has many harmful effects, especially to children exposed prenatally, including fetal alcohol spectrum disorders (FASDs). The disabilities due to fetal alcohol exposure continue throughout life and cause major financial burdens to society. The molecular mechanisms underlying FASDs are not well understood. We have taken a genetic approach to characterize ethanol’s effect on changing a discrete cell fate decision during embryogenesis in the nematode, Caenorhabditis elegans (C. elegans). Our preliminary data suggest that ethanol can affect the development of AWC neurons, a pair of olfactory neurons in C. elegans. We suggest that lipids can protect AWC neurons from ethanol’s effects. Importantly, we show that altering the metabolism of triacylglycerols (TAGs) can rescue this cell fate change in behavioral assays. By identifying molecular causes of fetal alcohol damage in humans we hope to be able to develop a greater understanding of how to prevent these detrimental effects.

DNA DSBs are most toxic to cells because they can lead to genomic rearrangements and even cell death. Most DSBs induced by ionizing radiation or radiomimetic drugs such as calicheamicin and bleomycin, bear 3′-phosphate or 3′- PG moieties that must be removed to allow subsequent gap filling and ligation. DSBs can be repaired by two main pathways: the homologous recombination (HR) pathway and the non-homologous end-joining (NHEJ) pathway, NHEJ is the primary repair pathway in mammalian cells. While HR repairs single strand breaks (SSBs) or DSBs accurately by using an undamaged copy of the sequence mostly at late S phase and G2 phase, the NHEJ pathway repairs DSBs without the requirement for sequence homology in a processing that may be error-free or error- prone and is most active at G1 phase. TDP1 is a DNA repair enzyme in both pathways, It associates with DNA SSB repair proteins XRCC1 and DNA ligase III and plays a role in processing of topoisomerase I- mediated SSBs. Our early results suggested that TDP1 also can remove protruding 3’- PG and other 3’ blocks from DSBs ends in vitro. A homozygous H493R mutation in the active site of TDP1 causes spinocerebellar ataxia with axonal neuropathy (SCAN1), a rare autosomal recessive genetic disease with neurological symptoms including peripheral neuropathy. DNA damage and misrepair can be determined by measuring the incidence of chromosomal aberrations such as rings, breaks, dicentrics, acentric fragments, and translocations in metaphase cells, and micronuclei in interphase cells. To assess the possible role of TDP1 in DSB repair in intact cells, the radiosensitivity of SCAN1 cells was determined by using a dose-fractionation method of irradiation. The data indicated that, when exposed to fractionated radiation doses, the SCAN1 cells were more sensitive than normal cells. Moreover, following treatment of cells with calicheamicin, SCAN1 cells showed a significantly higher incidence of dicentric chromosomes, acentric fragments, and micronuclei compared to normal cells, indicating that calicheamicin-induced DSBs were repaired less accurately and less efficiently, or more slowly in SCAN1 cells than in normal cells. All these results are consistent with a role for TDP1 in repair of 3’-PG DSBs in vivo. Oxidative stress is thought to induce replicative senescence and DNA damage in mouse embryo fibroblasts (MEFs). To determine the possible roles of oxidative stress on Tdp1-deficient MEFs, Tdp1-knockout MEFs and normal MEFs were cultured in 20% oxygen (atmospheric) and 3% (physiological) oxygen. The data from growth assays indicated that normal MEFs showed replicative senescence in 20% oxygen but not in 3% oxygen. Tdp1-knockout MEFs showed very poor growth compared to Tdp1 normal MEFs in both oxygen conditions, clearly suggesting an influence of repair of Tdp1 on oxidative stress induced DNA-DSBs in MEFs. Taken together, our results indicated that TDP1 is capable of removing protruding 3’-PG from DSB ends in intact cells. Moreover, DSBs induced by oxidative stress were repaired more slowly or inefficiently in MEFs when Tdp1 is absent, resulting in cell cycle arrest and poor cell growth.

It has been reported that an individual’s initial level of response to a drug might be predictive of his or her future risk of becoming dependent, thus basal gene expression profiles underlying those drug responses may be informative for both predicting addiction susceptibility and determining targets for intervention. This dissertation research aims to elucidate genetic risk factors underlying acute alcohol and nicotine dependence phenotypes using mouse genetic models of addiction. Phenotyping, brain region-specific mRNA expression profiling, and genetic mapping of a recombinant inbred panel of over 25 mouse strains were performed in order to identify quantitative trait loci (QTL) harboring candidate genes that may modulate these phenotypes. Previous BXD (B6 x D2) behavioral studies performed in our laboratory identified an ethanol-induced anxiolysis-like QTL (Etanq1) in the light dark box (LDB). We hypothesized that genetic variation within Nin (a gene within the Etanq1 support interval involved in microtubule-anchoring) may modulate anxiolytic-like responses to acute ethanol in the LDB as well as other preclinical models of anxiety, the elevated plus maze (EPM), and marble burying (MB) task. Molecular studies have allowed us to confirm cis regulation of Nin transcript levels in the NAc. To elucidate potential mechanisms mediating Etanq1, the pharmacological tools, diazepam and HZ166 (a benzodiazepine derivative) were utilized to interrogate whether GABAA receptor activation modulates ethanol’s anxiety-like behaviors in the LDB. We show that the LDB phenotype, percent time spent (PTS) in the light following a brief restraint stress, is not being modulated through direct activation of GABAA α2/α3 receptor subunits. To genetically dissect Etanq1 as well as parse the ethanol anxiolytic-like phenotype, we have assayed 8 inbred strains, selected based on genotypes at Nin, in various preclinical models of anxiety. Principal components analysis of these behavioral data suggests that the gene(s) modulating the ethanol anxiolytic-like component in the LDB do not overlap with similar phenotypes in the elevated plus maze (EPM), nor the MB phenotype. Furthermore, site-specific delivery of an sh-Nin lentivirus into the NAc of D2 mice revealed that Nin may modulate one LDB endophenotype, latency to enter the light side of the LDB, which loaded as a part of the “anxiolysis” principal component. These data strongly imply that basal neuronal Nin expression in the NAc is important for acute ethanol anxiolytic-like behavior, perhaps through a novel mechanism involving synaptic remodeling. In separate behavioral QTL mapping studies, we hypothesized that genetic variation regulating expression of Chrna7 modulates the reward-like phenotype, conditioned place preference (CPP), for nicotine. We provide evidence for genetic regulation of Chrna7 across the BXD panel of mice and through pharmacological and genetic behavioral studies, confirm Chrna7 as a quantitative trait gene modulating CPP for nicotine in mice. Microarrays, followed by network analyses, allowed us to identify a genetically co-regulated network within the nucleus accumbens (NAc), differentially expressed in mice null for Chrna7, which was similarly correlated in the BXD panel of mice. Our network and molecular analyses suggest a putative role for Chrna7 in regulating insulin signaling in the NAc, which together, may contribute to the enhanced sensitivity to nicotine observed in strains of mice that lack or have low mRNA levels of Chrna7 in the NAc. Overall, this research has elucidated and confirmed new genetic risk factors underlying alcohol and nicotine dependence phenotypes and has enabled a better understanding of the neurogenomic bases of alcohol and nicotine addiction. Future studies that further investigate the signaling pathways and/or gene interactions involving Nin and Chrna7 may lead the field to new candidates for pharmacotherapies that may be tailored for use in individuals with susceptible genotypes. Supported by NIAAA grants P20AA017828 and R01AA020634 to MFM, NIDA T32DA007027 to WLD, and NIDA R01DA032246 to MFM and MID.

The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis.