Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

Ischemic stroke is characterized by a disruption of blood supply to a part of the brain tissue, which leads to a focal ischemic infarct. The expression and activity of MMP-9 is increased in ischemic stroke and is considered to be one of the main factors responsible for damages to the cerebral vasculature, resulting in compromised blood-brain barrier (BBB) integrity. However, the regulatory mechanisms of MMP-9 expression and activity are not well established in ischemic stroke. Since hypoxia/ischemia and reperfusion generates reactive oxygen species (ROS), I hypothesize that ROS is one of factors involved in up-regulation of MMP-9 expression in brain cells and ROS-mediated effect may occur via MAPK signaling pathway. My study has provided the evidence that ROS is responsible for an increase in MMP-9 expression in astrocytes mediated via MAPK-AP1 signaling pathway. Preliminary studies with an in vitro model of the BBB suggest that inhibition of MMP-9 is a critical component of reducing ROS-induced BBB permeability.

Our laboratory has previously demonstrated that experimental peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan in Sprague Dawley rats, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). Increased P-gp functional expression was associated with a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. The present study examined whether the PIP-induced increase in P-gp functional expression was due to changes in intracellular trafficking (i.e., localization of P-gp), mediated by changes in the association of P-gp and caveolin-1, a key trafficking protein. These studies also determined if the drug diclofenac, a non-steroidal antiinflammatory (NSAID) that is commonly administered in conjunction with opioids during peripheral inflammatory pain (PIP), altered or modulated P-gp functional expression providing evidence of a drug-drug interaction. Confocal microscopy and subcellular fractionation revealed that under conditions of PIP, the disassembly of high-molecular weight P-gp-containing structures result in an increase in P-gp ATPase activity and changes in the localization of P-gp. Western blot analysis demonstrated further an increase in P-gp expression in rat brain microvessels following PIP induction and also after diclofenac treatment alone in the absence of PIP. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased radiolabeled- morphine uptake into the brain. This concurrent administration of NSAIDs and opioids in the presence of a pathophysiological stressor (i.e., pain/inflammation) may result in clinically significant drug-drug interactions that may impair the desired pharmacologic response and analgesic effects of opioids. Such interactions can lead to significant modifications to pain management in clinical settings. Therefore: The central hypothesis of this work is that the pathophysiological stressor peripheral inflammatory pain (PIP) and the pharmacological agent diclofenac modulate P-glycoprotein functional expression at the BBB. This hypothesis may be broken down further into two parts: 1) PIP induced changes in P-gp functional expression are mediated via changes in Pgp intracellular trafficking. 2) The non-steroidal anti-inflammatory drug Diclofenac, a drug commonly used to treat pain, modulates P-gp functional expression at the BBB thus decreasing morphine uptake into the CNS.

No description available.

Glycosylation may be a general strategy for the transport of biologically active neuro(glyco)peptides into the brain. With that in mind, a series of modified DAMGO analogues were synthesized and subjected to conformational analysis, and in vitro and in vivo studies related to opioid analgesia. Those studies will help to determine the balance of carbohydrate and peptide, to reach maximum BBB transport; in other words this is a study to test the biousian hypothesis. 1) The μ-agonist DAMGO was altered by incorporating moieties of increasing water solubility into the C-terminus, including carboxamide and simple glycosides. The hydrophilic C-terminal moieties were varied from glycinol in DAMGO to L-serine amide (LYM100), L-serine amide β-D-xyloside (LYM50), L-serine amide β-Dglucoside (LYM110), L-serine amide β-lactoside (LYM147). Two phosphopeptides LYM1311 and LYM1312 were synthesized with the phosphate group attached to Lserine amide at the C-terminus. Conformational analysis experiments included: 1HNMR, diffusion, variable temperature experiments to find the temperature coefficient, circular dichroism, 2DNMR noesy and tocsy, and molecular modeling. The peptides associate with SDS micelles with a strong electrostatic component. The SDS micelles stabilized the β-turn that is nascent in water. CSI (chemical shift indexes), temperature coefficients and circular dichroism do not give much insight into the structural conformation. 2D NMR analysis followed by molecular modeling confirmed a β-turn preferred conformation. No specific type of β-turn could be assigned to the DAMGO analogs. 2) Antinociceptive mouse tail-flick studies were performed, and opioid binding was determined. Analgesic potency (i.v.) increased, passing through a maximum (A₅₀ ≈ 0.2 μmol/Kg) for LYM100 & LYM50 as membrane affinity vs. water solubility became optimal, and then dropped off (A₅₀ ≈ 1.0 μmol/Kg) for LYM110 & LYM147 as water-solubility dominated the molecular behavior. Correlation of i.v. A₅₀ values with estimated hydrodynamic values (glucose units) for the glycoside moieties, or the hydrophilic/hydrophobic Connolly surface areas (A₅₀ vs e^(-Awater/Alipid)), provided U-shaped or V-shaped curves, as predicted by the “biousian hypothesis.” The μ-selective opioid agonism was maintained upon modifications at the C-terminus. The optimal “degree of glycosylation” that achieves the maximum degree of transport for the DAMGO peptide message seems to be between the peptide with the carboxamide C-terminal group and the xyloside.

<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>

<p><font size="3"> <p>The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.<font size="3">The aim of this study was to investigate </font><i><font size="3" face="Times New Roman,Times New Roman"><font size="3" face="Times New Roman,Times New Roman">in vitro </font></font><font size="3">effects of pure and street MA &quot / tik&quot / on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. <font size="3">Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo&reg / luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P&lt / 0.05 was denoted as significant. <font size="3">Results of this study showed that:&nbsp / <font size="3">1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P&gt / 0.05). <font size="3">2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P&le / 0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P &le / .0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P&le / 0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P&le / 0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. <font size="3">In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA. </font></font> <p>&nbsp / </p> </font>&nbsp / </font></font></font> <p>&nbsp / </p> </i></p> </font></p> <p>&nbsp / </p>