http://hdl.handle.net/1974/788 2013-05-26T02:41:37Z http://hdl.handle.net/1974/7942 Title: OPIOIDS AND GLIA: INVESTIGATING THE MECHANISMS THROUGH WHICH ULTRA-LOW DOSE OPIOID ANTAGONISTS MODULATE OPIOID TOLERANCE AND HYPERALGESIA. Authors: Mattioli, THERESA ALEXANDRA Abstract: Ultra-low doses (ULD) of the opioid receptor antagonists, naloxone and naltrexone, augment the analgesic actions of morphine, block the induction of tolerance, and reverse established tolerance by an unknown mechanism. Preclinical studies demonstrate that chronic morphine administration induces spinal gliosis and that inhibition of gliosis prevents the development of analgesic tolerance to opioids. Thus, this thesis investigated the inhibition of spinal gliosis as a mechanism by which ULD antagonists attenuate analgesic tolerance and opioid-induced hyperalgesia. Immune cell activation is implicated in the etiology of morphine tolerance and intrathecal catheterization, a technique commonly used to study the spinal effects of drugs, causes profound gliosis. Thus, the first study investigated the effects of catheter-induced gliosis on acute and chronic morphine analgesic tolerance. Catheterization-induced gliosis did not alter antinociceptive responses to acute intrathecal morphine; however, tolerance to chronic morphine was exacerbated in catheterized rats compared to sham and surgery-naïve controls. The potentiation of analgesic tolerance to chronic morphine by spinal gliosis provided evidence that glia modulate opioid analgesia; therefore, inhibition of opioid-induced activation of glia was explored as a potential mechanism by which ULD antagonists prevent tolerance. The second series of experiments reported morphine-induced activation of spinal microglia and astrocytes was blocked by co-administering ULD naltrexone with morphine. These findings prompted us to elucidate the specific molecular target through which ULD antagonists attenuate opioid analgesia. Activation of glial Toll-like receptor 4 (TLR4) induces gliosis and may contribute to analgesic tolerance and/or morphine-induced hyperalgesia (MIH). Antagonism of TLR4 by the opioid receptor-inactive (+) stereoisomer of naloxone was identified as a potential mechanism by which ULD antagonists modulate opioid analgesia. Tolerance and MIH developed in mice expressing non-functional TLR4 and in wildtype controls. Analgesic tolerance was stereoselectively blocked by ULD (-)naloxone, whereas MIH was blocked by both naloxone enantiomers. Collectively, these studies demonstrate analgesic tolerance and MIH occur through distinct mechanisms. ULD naloxone attenuates analgesic tolerance likely via an opioid receptor-mediated mechanism that is TLR4-independent. ULD antagonists do not attenuate tolerance via inhibition of spinal gliosis as hypothesized. In contrast, ULD antagonists prevent MIH by inhibiting opioid-induced gliosis in an opioid receptor- and TLR4-independent manner. Immune cell activation is implicated in the etiology of morphine tolerance and intrathecal catheterization, a technique commonly used to study the spinal effects of drugs, causes profound gliosis. Thus, the first study investigated the effects of catheter-induced gliosis on acute and chronic morphine analgesic tolerance. Catheterization-induced gliosis did not alter antinociceptive responses to acute intrathecal morphine; however, tolerance to chronic morphine was exacerbated in catheterized rats compared to sham and surgery-naïve controls. The potentiation of analgesic tolerance to chronic morphine by spinal gliosis provided evidence that glia modulate opioid analgesia; therefore, inhibition of opioid-induced activation of glia was explored as a potential mechanism by which ULD antagonists prevent tolerance. The second series of experiments reported morphine-induced activation of spinal microglia and astrocytes was blocked by co-administering ULD naltrexone with morphine. These findings prompted us to elucidate the specific molecular target through which ULD antagonists attenuate opioid analgesia. Activation of glial Toll-like receptor 4 (TLR4) induces gliosis and may contribute to analgesic tolerance and/or morphine-induced hyperalgesia (MIH). Antagonism of TLR4 by the opioid receptor-inactive (+) stereoisomer of naloxone was identified as a potential mechanism by which ULD antagonists modulate opioid analgesia. Tolerance and MIH developed in mice expressing non-functional TLR4 and in wildtype controls. Analgesic tolerance was stereoselectively blocked by ULD (-)naloxone, whereas MIH was blocked by both naloxone enantiomers. Collectively, these studies demonstrate analgesic tolerance and MIH occur through distinct mechanisms. ULD naloxone attenuates analgesic tolerance likely via an opioid receptor-mediated mechanism that is TLR4-independent. ULD antagonists do not attenuate tolerance via inhibition of spinal gliosis as hypothesized. In contrast, ULD antagonists prevent MIH by inhibiting opioid-induced gliosis in an opioid receptor- and TLR4-independent manner. Description: Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2013-04-25 15:06:50.731 2013-04-25T04:00:00Z http://hdl.handle.net/1974/7722 Title: THE ADIPOCYTE AND ENDOTHELIAL CELL-SPECIFIC ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA IN BREAST TUMOURIGENESIS Authors: Reid, ALEXIS Abstract: Peroxisome proliferator-activated receptor (PPAR)γ plays a role in tumorigenesis. Previous studies with PPARγ(+/-) mice suggest PPARγ normally suppresses dimethylbenz[a]anthracene (DMBA)-induced breast, and other, tumor progression. Since many cell types associated with the mammary gland express PPARγ, each with unique signaling pathways, the present study aimed to define which tissues are required for PPARγ-dependent anti-tumor effects. Conditional adipocyte and endothelial cell-specific PPARγ knockout mice (PPARγ-A KO and PPARγ-E KO respectively) were used to evaluate whether PPARγ signaling normally acts to prevent DMBA-mediated breast tumour progression in a stromal cell-specific manner. Twelve week old PPARγ KO mice and their congenic wildtype (WT) controls were randomly assigned to one of two treatment groups. All mice were treated by gavage once/week for 6 weeks with 1 mg DMBA and maintained on a normal chow diet. At week 7, mice in each group were divided into those continuing normal chow, and those receiving a PPARγ ligand (ROSI, 4 mg/kg/day) supplemented diet for the duration of the 25 week study, and monitored weekly. Tumour and tissue samples were collected at necropsy, and portions of each were fixed and frozen for future analysis. In both PPARγ-A KOs and PPARγ-E KOs versus PPARγ-WT mice, malignant mammary tumor incidence was significantly higher and mammary tumor latency was decreased. DMBA+ROSI treatment reduced average mammary tumor volumes by 50%. Gene expression analyses of mammary glands by qRT-PCR and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared to PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. In the PPARγ-E KO mice, both treatment groups saw a significant increase in thymic tumour incidence, a finding not established before with the study of other stromal cell knockout mice. These studies provide the first direct in vivo evidence that PPARγ signalling in stromal adipocytes and endothelial cells attenuates DMBA-mediated breast tumourigenesis. This study supports a protective effect of activating PPAR gamma as a novel chemopreventive therapy for breast cancer. Description: Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-24 11:28:17.668 2013-01-04T05:00:00Z http://hdl.handle.net/1974/7673 Title: The Effect of Prenatal Ethanol Exposure on DNA Methylation and TGF-β1, SHH and Wnt3a Transcription Regulating Factors Within the Developing Hippocampus of the Guinea Pig Authors: SONDY, YVONNE Abstract: One of the most frequently reported deficits seen in individuals with Fetal Alcohol Spectrum Disorder (FASD) is impairments in learning and memory, which is likely attributed to the teratogenic effects of ethanol on the developing hippocampus. TGF-β (transforming growth factor-β), hedgehog and Wnt signaling pathways have been identified as high probability candidate pathways associated with brain deficits seen in FASD. Increasing evidence indicates that ethanol may induce changes in DNA methylation that could alter transcription regulating factors within signaling pathways critical in brain development. The purpose of this study was to test the hypotheses that prenatal ethanol exposure during i) the first trimester-equivalent period, or ii) throughout the entire gestational period induces changes in DNA methylation and alters the transcription/translation of TGF-β1, SHH (sonic hedgehog) and Wnt3a within the developing hippocampus. Pregnant Dunkin-Hartley-strain guinea pigs were assigned to one of three groups: ethanol (4 g/kg maternal body weight), isocaloric-sucrose/pair-feeding, or no treatment. Embryonic telencephalon tissue (which gives rise to the hippocampus) and fetal hippocampus were collected at gestational day (GD) 23 or GD 65, respectively. GD 23 ethanol-exposed and nutritional control embryos exhibited decreased crown-rump and head lengths. GD 65 ethanol-exposed fetuses exhibited decreased body and brain weights compared with the control groups. Ethanol exposure during the first trimester-equivalent period, but not during the entire gestational period, resulted in an increase in global DNA methylation. First trimester-equivalent ethanol exposure did not alter TGF-β1, SHH and Wnt3a gene expression within the GD 23 telencephalon. However, ethanol exposure throughout the entire pregnancy led to an increase in the expression of all three genes within the GD 65 hippocampus. No change in TGF-β1 protein was seen in the hippocampus of ethanol-treated fetuses. Post-translationally modified (ptm) SHH, but not unmodified SHH protein, was decreased in the hippocampus of ethanol-exposed fetuses. A decrease in unmodified, but not ptm Wnt3a protein, was observed in both ethanol-exposed and nutritional control hippocampus. These results suggest that prenatal ethanol exposure may affect hippocampal development through alterations in i) DNA methylation as shown at early gestation and ii) the expression of transcription regulating factors, especially SHH, as shown at term. Description: Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-03 12:36:33.035 2012-12-03T05:00:00Z http://hdl.handle.net/1974/7597 Title: CHARACTERIZING VALPROIC ACID-INDUCED DNA DOUBLE STRAND BREAK REPAIR Authors: Cutler, Geoffrey Lloyd Abstract: The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications. Description: Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-10-15 11:06:30.613 2012-10-15T04:00:00Z