Department: Pharmacology and Toxicology ![[clear]](close-x.png "Remove this limiter")
35 matches in the database.
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1.
Alghamri, Mahmoud.
Enhanced Angiotensin II-Induced Cardiac and Aortic Remodeling in ACE2 Knockout Mice.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► Activation of the renin angiotensin system (RAS) is a well-known driving force,…
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▼ Activation of the renin angiotensin system (RAS) is a well-known driving force, governing the aggravation and progression of CVD and associated target organ damage. Angiotensin (Ang) converting enzyme 2 (ACE2), a new member within the RAS family, was first cloned from the ventricle of patient with heart failure. ACE2 was shown to be upregulated under pathological conditions. We hypothesize that loss of ACE2 negatively affects cardiac function and exacerbates hypertrophic cardiomyopathy and aortic remodeling in response to Ang II. Eight weeks old ACE2 knock out (KO) and wild type (WT) mice were infused with Ang II s.c. (1000 ng/kg/min for 4 weeks) using osmotic pumps. Blood pressure (radiotelemetry), cardiac function (echocardiography) and cardiac/aortic structural damage (histology) were examined. At baseline before Ang II, ACE2 KO displayed a normal phenotype for cardiac function and blood pressure. After 4 weeks of Ang II infusion, the mean arterial pressure (MAP) was increased (~40% in WT and ~33% in ACE2 KO) with no differences between groups. However, ACE2 KO responded differently to the increased MAP. Cardiac function analysis revealed severe myocardial dysfunction shown by the lowered EF% (31% vs. 13%) as well as FS% (30% vs 6%) in ACE2 KO vs WT, respectively. In addition, the cardiac dysfunction was associated with hypertrophic cardiomyopathy shown by the increased left ventricular wall thickness as well as heart weight/ body weight ratio. Moreover, collagen staining in the myocardium and aorta revealed higher collagen percentage in ACE2 KO which indicates cardiovascular remodeling. Furthermore, results showed enhanced oxidative stress in the myocardium and aorta of Ang II infused ACE2 KO compared to Ang II WT. In conclusion, ACE2 attenuates myocardial maladaptive hypertrophy and cardiovascular remodeling in response to long term Ang II stimulation.
Advisors/Committee Members: Morris, Mariana.
Subjects: Pharmacology; Toxicology
Keywords: pharmacology; toxicology
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2.
Alshahrani, Saeed.
Role of Na+K+2Clâ» Co-transporters in Insulin Secretion.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► The objective of this study is to investigate the role of intracellular…
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▼ The objective of this study is to investigate the role of intracellular chloride concentration ([Clâ»]i) in insulin secretion in vivo and in vitro. The maintenance of high [Clâ»]i in insulin-secreting pancreatic β-cells plays an important role in glucose-induced depolarization and insulin secretion. Insulinotropic glucose concentrations i.e., >6 mM induces electrical activity in β-cells which results in insulin secretion. This electrical activity is mainly due to electrogenic efflux of Clâ» from β-cells through volume-regulated anion channels (VRAC). The solute carrier family 12 group A member 1 (Slc12a1) and 2 (Slc12a2) encode several splice variants of the kidney-specific and the ubiquitous isoforms, respectively, of bumetanide (BTD)-sensitive Na+-dependent K+2Clâ»co-transporters. The Slc12a2 co-transporter, also known as NKCC1, is involved in the maintenance of a high [Clâ»]i in β-cells and its pharmacological inhibition impairs insulin secretion in response to glucose in vivo and in vitro. We have used mice lacking both functional alleles of the Slc12a2 gene (NKCC1 KO) to demonstrate that absence of NKCC1 correlates with better fasting glycaemia and improved glucose tolerance when compared to wild-type mice (NKCC1 WT). These observations correlated with increased insulin secretion in response to glucose in vivo and in vitro in primary culture of islets. Further, morphometric analysis of pancreas obtained from NKCC1 KO mice had increased β-cell mass. Moreover, administration of BTD to NKCC1 KO mice worsened their glucose tolerance. This effect of BTD in vivo correlated with a direct inhibition of insulin secretion by BTD from islets lacking functional Slc12a2 genes, therefore suggesting the presence of a BTD-sensitive mechanism involved in insulin secretion, which is different from Slc12a2. We used reverse transcription coupled to the polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence analysis to determine the molecular expression pattern of Slc12a1 and Slc12a2 genes in β-cells. We demonstrate that these cells express a specific splice variant of NKCC1 and the "kidney-specific" BTD-sensitive Slc12a1 gene. Altogether, our results provide for the first time evidence for a functional role of these co-transporters on insulin secretion. Conclusions: 1) Functional alleles of the Slc12a2 gene are not necessary for insulin secretion 2) Bumetanide impairs insulin secretion and glucose homeostasis in vivo 3) Bumetanide impairs insulin secretion by a direct effect on insulin secreting β-cells 4) Pancreatic β-cells express specific splice variants of Slc12a2 and Slc12a1
Advisors/Committee Members: Di Fulvio, Mauricio.
Subjects: Pharmacology
Keywords: β-cell, Insulin Secretion, NKCC1, NKCC2
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3.
Carlson, Cataleya.
In Vitro Toxicity Assessment of Silver Nanoparticles in Rat Alveolar Macrophages.
Degree: MS, Pharmacology and Toxicology, 2006, Wright State University
► The present study was conducted to assess the toxicity of nanosized silver…
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▼ The present study was conducted to assess the toxicity of nanosized silver particles (Ag-15nm, 30nm and 55nm) in rat alveolar macrophages. For toxicity evaluations, cellular morphology, mitochondrial function (MTT assay), membrane leakage (LDH assay) and reactive oxygen species generation (ROS) were assessed after a 24h under control and exposed conditions. The morphological appearance of control and exposed cells were observed by an inverted, phase contrast microscope and the uptake of nanoparticles was observed using the CytoViva Ultra Resolution Imaging (URI) system. Morphological images captured at 100x magnification demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, and most of the cells were detached from the bottom of the cultured plates. It was also noted that agglomerates of nanoparticles were surrounded by macrophages; while some attached to the cell membrane. Further analysis of CytoViva URI system images exhibit the uptake of agglomerates into macrophages. The results of the biochemical studies revealed that cells exposed to Ag-15nm and 30nm for 24h showed a dose-dependent decrease in mitochondrial function. Calculated EC50 values from MTT data indicate that Ag-15nm and Ag-30nm are more toxic at lower concentrations when compared to Ag-55nm. In correlation with decreased mitochondrial function and subsequent lack of ATP production, cell viability assays also demonstrated a sizeable decrease in the number of viable cells exposed to Ag-15nm and 30nm, including at the lowest dose (5µg/ml). ROS date further supported a size and dose dependent relationship, with a 15.16 ±5.77 fold increase in ROS generation at 50 µg/ml of Ag-15nm. Preliminary apoptosis results reveal a size-dependent increase in caspase 3and7 enzyme activity; an indicator of apoptosis. In conclusion, data reveal that the size of the nanoparticle is a potential contributing factor in exhibiting toxicity, as Ag-15nm is found to be the most toxic when compared to larger nanoparticles (30nm and 55nm). The study also suggests that the mechanism of toxicity is likely due to the generation of reactive oxygen species, inducing oxidative stress and resulting in apoptosis.
Advisors/Committee Members: Hussain, Saber M.
Keywords: in vitro, silver nanoparticles, oxidative stress, macrophages
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4.
Castle, Alicia Brooks.
In-Vitro Biocompatibility of Silver Nanoparticles Anchored on Multi-Walled Carbon Nanotubes.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Silver (Ag) nanoparticles were anchored on the surface of three different multi-walled…
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▼ Silver (Ag) nanoparticles were anchored on the surface of three different multi-walled carbon nanotubes (MWNTs): Ag-MWNTs (pure carbon), Ag-MWNT-COx (carbonyl and carboxyl doped), and Ag-MWNT-CNx (Nitrogen-doped). The chemical reactivity of the nanotubes was MWNTs< COx-MWNTs< CNx-MWNT. Ag-NP anchored on the MWNTs had an average size corresponding to MWNTs 6.64nm +/- 2.25nm; CNx-MWNTs, 13.24nm +/- 3.94nm, and COx-MWNT/Ag 11.75nm +/- 4.65nm. Cellular function and immune response were evaluated to determine biocompatibility of the synthesized nanomaterials (NM) on the human keratinocyte cell line (HaCaT). Cellular assays revealed toxicity after 24h however, full cellular recovery was observed at 48h. These results suggest Ag nanoparticles anchored on MWNTs have the ability to interact with cells without disrupting cellular function.
Advisors/Committee Members: Hussain, Saber.
Subjects: Toxicology
Keywords: silver nanoparticles; Multi-walled carbon nanotubes; functionalization
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5.
Chmura, Douglas F.
Response of Neuroinflammatory and Neurodegenerative Markers Following Sub-lethal Sarin Exposure and Subsequent Treatment via an in-vivo Caspase Inhibitor.
Degree: MS, Pharmacology and Toxicology, 2011, Wright State University
► Organophosphorus nerve agents are amongst the most deadly chemical compounds ever synthesized.…
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▼ Organophosphorus nerve agents are amongst the most deadly chemical compounds ever synthesized. Sarin is an organophosphate (OP) ester that irreversibly forms a phosphoester bond at the active site of acetylcholinesterase and thereby induces a rapid and lethal cholinergic crisis. It remains an active threat to vulnerable civilian populations due to its ease of synthesis and known use by rogue nations and terrorist groups. Death is the most extreme consequence of sarin toxicity. Current treatments fail to provide protection against progressive cognitive impairments years after mild exposure. Q-VD-OPh is an in-vivo caspase inhibitor with potent anti-apoptotic and anti-inflammatory properties. In this pilot study, adult male C57BL/6J mice were subcutaneously injected with 0.5 LD50 sarin followed by an intraperitoneal injection of 20 mg/kg Q-VD-OPh. Mice were sacrificed at a two-day time point followed by MALDI imaging and immunohistochemical analysis of brain sections. Protein mass spectra of tissue sections subjected to organic washes yielded an increase in signal sensitivity compared to untreated sections. A non-significant upward trend in GFAP expression was observed in sarin treated animals in contrast to Q-VD-OPh animals. Statistically significant downward trends in nuclear NF-κB/p50-50 expression were observed in sarin treated animals in contrast to Q-VD-OPh animals. These trends open a window to innovative research paradigms that extend beyond the emphasis of regenerating acetylcholinesterase and managing seizures.
Advisors/Committee Members: Cool, David.
Subjects: Pharmacology; Toxicology
Keywords: pharmacology
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6.
Chodavarapu, Harshita.
Role Of Hyperglycemia And Aldosterone On Renal ACE2 AND Albuminuria In db/db Mice.
Degree: MS, Pharmacology and Toxicology, 2011, Wright State University
► Diabetic nephropathy (DN) is one of the microvascular complications of type II…
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▼ Diabetic nephropathy (DN) is one of the microvascular complications of type II diabetes and a leading cause for the development of end stage renal disease. Renin angiotensin system (RAS) plays a pivotal role in the development and progression of diabetic nephropathy. Hyperglycemia activates RAS and increases tissue and circulating levels of angiotensin II (Ang II) and aldosterone. A new component of RAS, angiotensin converting enzyme 2 (ACE2), has been shown to be renoprotective in early stages of diabetes. The aim of this study is to test the hypothesis that strict glycemic control using rosiglitazone or treatment with mineralocorticoid receptor (MR) antagonist, spironolactone would impart renoprotection in db/db mice via upregulation of renal ACE2. Lean control and db/db mice were fed either rosiglitazone or spironolactone for 10 weeks. Diabetic db/db mice demonstrated hyperglycemia and early onset of microalbuminuria compared to lean control mice. Moreover, elevation in plasma aldosterone levels of db/db mice supports the role of aldosterone in mediating renal injury. Western blot analysis revealed an increase in renal and urinary ACE2 expression of db/db mice. In addition, there was a significant increase in renal and urinary ACE2 activity in db/db mice compared to lean controls. Further, renal mineralocorticoid receptor (MR) and neprilysin (NEP) protein expression increased and decreased respectively in db/db mice compared to controls. Immunohistochemistry demonstrated decreased renal NEP protein expression in db/db mice compared to controls. Periodic acid Schiff stained renal sections of db/db mice showed glomerular basement membrane thickening and mild mesangial expansion than that of lean control mice. Chronic rosiglitazone treatment normalized the blood glucose levels and improved glucose tolerance in db/db mice but did not alter the increased plasma aldosterone levels. In contrast, chronic treatment with spironolactone significantly increased plasma aldosterone levels in lean control and db/db mice without altering blood glucose levels. Urinary albumin excretion rate decreased significantly in rosiglitazone and spironolactone treated db/db mice. However, both rosiglitazone and spironolactone decreased renal ACE2 protein expression and enzyme activity in db/db mice. In addition, urinary ACE2 excretion and enzyme activity were also decreased after treatment with rosiglitazone. Although spironolactone showed no effect on renal MR expression, rosiglitazone significantly decreased the renal MR expression in db/db mice. In addition, rosiglitazone treatment significantly increased renal NEP protein expression in db/db mice. In conclusion, rosiglitazone and spironolactone attenuate albuminuria and impart renal protection independent of ACE2. Upregulation of renal NEP suggests the involvement of alternative Ang (1-7) forming enzyme apart from ACE2 in the renoprotection mediated by rosiglitazone. These findings provide new insights for the possible role of NEP in diabetic renal injury.
Advisors/Committee Members: Elased, Khalid.
Subjects: Pharmacology
Keywords: RAS; Diabetes; Neprilysin; ACE2; Albuminuria
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7.
Chou, Richard M.
Use of Phage Display Libraries to Select For B-cell Receptor-specific Peptides of Chronic Lymphocytic Leukemia Cells.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► Peptides with high affinity to the B-cell receptor (BCR) fused to a…
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▼ Peptides with high affinity to the B-cell receptor (BCR) fused to a toxin could be an effective therapy for Chronic Lymphocytic Leukemia (CLL) patients. We screened captured BCR of a CLL patient with peptides from 7 and 12-mer phage display libraries, using two strategies. Lymphocytes from two patients diagnosed with CLL expressing two different unmutated VH genes (VH 1-3 and VH4-34, respectively) were used. Membrane BCRs were obtained from patient CLL cells by lysis, identified by western blot, semi-quantified and screened with phage libraries. The first strategy involved using patient VH4-34 BCRs which were captured using goat anti-human IgM to deplete bound phages. Unbound-phages were positively screened for those binding to patient VH 1-3 BCRs. Several clones were randomly selected and a sequence consisting of “LLPPAR_” peptide was found in both libraries. A phage clone displaying peptide “LLPPARE” was identified to bind to goat anti-human IgM. By including more goat anti-human IgM negative selections, we identified 3 different phages displaying peptides “GFTFMPA”, “QSRPLLP” and “GLPCCSS”. Clones “GFTFMPA” and “GLPCCSS” showed binding to goat anti-human IgM, while “QSRPLLP” did not. Phage clone “QSRPLLP” showed no binding to human serum IgM but showed binding to both patients' BCRs. “QSRPLLP” peptide binds a common BCR molecule region present in both patients but not present in human serum IgM. This data suggests that it is possible to use a peptide-phage display library to select peptides unique for the BCRs of CLL patients. However, the critical component in making this process patient-specific resides in enhanced discrimination in phage selection.
Advisors/Committee Members: Lopez, Osvaldo.
Subjects: Immunology; Molecular Biology
Keywords: immunology; molecular biology
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8.
Das, Avik.
Ischemic stroke in type II diabetic mice: Deregulation of SDF-1a/CXCR4 axis.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Type 2 diabetes mellitus is a major risk factor for ischemic stroke.…
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▼ Type 2 diabetes mellitus is a major risk factor for ischemic stroke. Also diabetes is associated with poor outcome after stroke. Underlying mechanisms are however not fully understood. Alteration in the expression of the SDF-1a/CXCR4 axis, which is important for ischemic tissue repair, can be a probable cause. In this study, we have determined the expression of SDF-1a/CXCR4 in the brains of type II diabetic mice at basal and in response to ischemic stroke and have investigated a method for overexpression of SDF-1a in the brains of the diabetic mice. Adult male C57BLKS/J mice (db/db) of age 8 weeks were used as the murine model for type II diabetes and their age matched lean littermates served as controls (db/+). Microvascular density was first determined in the cerebral cortex of db/db diabetic mice by immunohistochemical analysis. Focal cerebral ischemia was induced by middle cerebral artery occlusion surgery (MCAO) in type 2 diabetic db/db mice and their controls. 48 hours after surgery, volume of ischemic damage was determined by TTC staining. The expression of SDF-1a and CXCR4 in the ischemic and non ischemic sides of brains of both the groups were determined using western blot and real time RT PCR. The db/db diabetic mice were injected with the vector, adeno associated virus 9 (AAV-SDF-1a) in the brain striatum and the overexpression of SDF-1a was determined by immunohistochemical analysis. Double immunohistochemistry was used to determine the localization of SDF-1a in brain after injection of the vector. The microvascular density in the cerebral cortex was reduced in db/db mice as compared with db/+ mice (p<0.05). Volume of ischemic damage was significantly increased in db/db mice after focal cerebral ischemia (p< 0.01). The levels of SDF-1a expression in both ischemic and non ischemic side of brain were reduced in db/db mice as compared with those in db/+ mice at mRNA (p< 0.01) and protein level (p< 0.01). The amount of CXCR4 expression was significantly reduced only in the ischemic side of the brains of db/db mice at protein level (p=0.001) and at m-RNA level (p=0.001). But in the non ischemic side, the expression of CXCR4 did not show any significant difference between the two groups. Immunohistochemical analysis showed overexpression of SDF-1a in the striatum receiving the microinjection of AAV-SDF-1a and double immunohistochemistry showed SDF-1a to be localized in the glial cells of the cerebral striatum after microinjection. The results indicate that microvascular density is reduced and ischemia induced cerebral damage is enlarged in diabetes which may be linked to the impaired expression of hypoxia regulated SDF-1a/CXCR4 axis after ischemic stroke in diabetes and vector mediated over expression of SDF-1a in the brain can be a novel therapeutic technique for treating ischemic stroke in diabetics.
Advisors/Committee Members: Chen, Yanfang.
Subjects: Biomedical research
Keywords: SDF-1a; CXCR4; EPC; stroke
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9.
Davidson, Molly Elizabeth.
Neurodegeneration and Neuroinflammation in a Mouse Model of Sarin Exposure.
Degree: MS, Pharmacology and Toxicology, 2007, Wright State University
► Sarin is an organophosphorus (OP) ester chemical warfare agent (CWA) that has…
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▼ Sarin is an organophosphorus (OP) ester chemical warfare agent (CWA) that has been used in past terrorist attacks. It remains a threat today because of its ease of manufacture and dispersal. Sarin acts by irreversibly inactivating acetylcholinesterase and interfering with neurotransmission by allowing acetylcholine to build up at neuro-effector junctions, where it continuously elicits a response. Symptoms of sarin toxicity include seizures, hypersecretions, respiratory distress and death in extreme cases. Previous studies on OP poisoning indicate that sarin exposure causes neurodegeneration and neuroinflammation in conjunction with seizure activity. In order to determine the mode of neuronal death and the extent of neuroinflammation induced by sarin exposure, C57Bl/6J mice were first dosed s.c. with 1.5 mg/kg cresylbenzodioxaphosphorin oxide (CBDP) to inhibit the large amount of carboxylesterase found in rodents. One hour later, mice were dosed with 24 ug/kg sarin, a dose which produces approximately 17% lethality. One group of mice was also dosed with 1.7 mg/kg 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, which has been shown by others to decrease different types of neuronal injury. Mice were sacrificed at times ranging from four hours to ten days after sarin administration and left or right brain hemispheres were collected and frozen in isopentane for histological examination. To detect DNA fragmentation, 10um-thick sections were TUNEL-stained using the NeuroTACS II in situ apoptosis detection kit. Activated caspase-3 and interleukin-1beta (IL-1beta) were detected by immunohistochemistry to determine whether apoptotic cell death and neuroinflammation were occurring. Manual cell counts were performed on micrographs of dentate gyrus and CA1 regions of hippocampus, amygdala and piriform cortex in NIH Image. Statistically significant TUNEL labeling was observed at various time points in all brain regions except CA1 hippocampus. Significant increases in caspase-3 staining were observed only in piriform cortex and CA1 hippocampus at 24 hours and four days post-sarin injection, respectively. IL-1beta expression was significantly increased in CA1 hippocampus at four days after sarin injection and in all brain regions except piriform cortex at ten days after sarin exposure. Neither TUNEL nor caspase-3 labeling were significantly decreased with 8-OH-DPAT treatment. The amygdala and dentate gyrus regions showed significant decreases in IL-1beta expression after 8-OH-DPAT treatment. Statistically non-significant upward trends over time in TUNEL labeling and IL-1beta expression were observed. In contrast, caspase-3 expression exhibited a gradual, non-significant decrease at later time points. These trends may be indicative of a decreasing role played by apoptotic cell death and an increasing or persisting role for oncotic cell death and inflammation in sarin neurotoxicity.
Advisors/Committee Members: Cool, David R.
Subjects: Health Sciences, Toxicology
Keywords: sarin, organophosphate, neuroinflammation, neurodegeneration
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10.
Edwards, Jennifer Y.
The Processing of β-Endorphin in Morphine Treated Rats Using SELDI-TOF Mass Spectrometry.
Degree: MS, Pharmacology and Toxicology, 2007, Wright State University
► Endocrine glands secrete peptide hormones that bind to specific receptors, and elicit…
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▼ Endocrine glands secrete peptide hormones that bind to specific receptors, and elicit a response. In the pituitary, prohormone convertases (PC) PC1/3 and PC2 convert inactive prohormones into biologically active peptide hormones. Proopiomelanocortin (POMC) is a precursor molecule that proteolytically cleaves at paired basic residue sites, and produces smaller biologically active peptides, such as adrenocorticotropic hormone (ACTH), α-melanocyte stimulating hormone (α-MSH), and β-endorphin. β-endorphin is an endogenous opioid peptide hormone that plays a vital role in the body’s physiological response to stress, fear, and anxiety. Morphine is an exogenous opioid, used for the treatment of moderate to severe pain and competes with β-endorphins when binding to mu-receptors located on the surface of target cells. Opioids have a high abuse potential leading to the development of tolerance and dependence. The purpose of this research is to analyze the effects of morphine on prohormone processing by examining the β-endorphin peptide hormone spectrum using a Protein Chip Array technology with Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI- TOF-MS). Brain extracts (amygdala, nucleus accumbens, periaqueductal gray, lateral hypothalamus, arcuate nucleus, and paraventricular nucleus) from normal rats (n= 4-6) treated with morphine (75mg/day) or placebo pellet for 24 hours or a seven day treatment were examined in this study. The protein (1ug/uL) was applied to a Weak Cation Exchange (WCX2) ProteinChip, air-dried, and coated with alpha-cyano-4-hydroxy cinnamic acid (CHCA) matrix. SELDI-TOF MS (Ciphergen, LaJolla, CA) was used to analyze the peptide hormone spectrum for changes in peptide expression or processing levels. The results showed that morphine modulates β-endorphin processing at 7 days. Previous studies have shown that PC2 enzyme is primarily responsible for processing β-endorphin1-31 in mice. We conclude that PC2 is down-regulated, which may play a role in regulating the amount of active hormone to prohormone. This process may help the organism maintain homeostasis.
Advisors/Committee Members: Cool, David R.
Keywords: B-endorphin; Morphine; Opioids; Pro-opiomelanocortin (POMC); Prohormone convertase (PC); PC1/3; PC2; SELDI-TOF Mass Spectrometry
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11.
Ellis, David Harold.
2,3,7,8-Tetrachlordibenzo-p-dioxin Mediated Immune Suppression through Interactions at the 3'Immunoglobulin Heavy Chain Regulatory Region Enhancers.
Degree: MS, Pharmacology and Toxicology, 2010, Wright State University
► 2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is a potent toxin which inhibits the antibody response of…
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▼ 2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is a potent toxin which inhibits the antibody response of B cells. The 3'IgHRR which is involved in transcriptional regulation of the heavy-chain polypeptide of antibodies is inhibited by TCDD. The hs1,2 enhancer region, isolated from the 3'IgHRR, is also inhibited while the isolated hs4 is activated by TCDD. This project sought to determine if that dichotomy in effects results from interactions at enhancer-specific binding sites for AhR, thought to mediate transcriptional effects of TCDD, and NFκB, a transcription factor involved in B cell activation. Here, I report a difference in the effect of TCDD on transcriptional activity of the 3'IgHRR and the isolated hs3b/hs4 enhancer pair, in the context of chromatin. I also demonstrate reduced binding of proteins to the hs1,2 and hs4 enhancer sequences containing both AhR and NFκB binding sites compared to single site sequences, suggesting that an interaction between the proteins alters their binding to the enhancers.
Advisors/Committee Members: Sulentic, Courtney.
Subjects: Biology; Immunology; Toxicology
Keywords: 2,3,7,8-Tetrachlordibenzo-p-dioxin; TCDD; IgHRR; NFκB; EMSA; B cell; Immunotoxicology; Immunoglobulin; Immune suppression
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12.
Fernando, Tharu M.
TCDD-induced modulation of the hs1,2 enhancer within the 3’immunoglobulin heavy chain regulatory region.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin that inhibits immunoglobulin (Ig) gene…
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▼ 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental toxin that inhibits immunoglobulin (Ig) gene expression in mice. Transcriptional regulation of the Ig heavy chain (IgH) involves the 3’IgH regulatory region (3’IgH RR). The murine 3’IgH RR consists of four enhancers (hs3A, hs1,2, hs3B, and hs4), which are homologous to the human 3’IgH RR enhancers (hs3, hs1,2, and hs4). In humans, a polymorphism of the hs1,2 enhancer, resulting in a varying number of tandem repeats of a 53 bp sequence, has been correlated with autoimmune diseases like IgA nephropathy and Celiac disease. The repeated sequence contains a kB and DRE binding site. Previous studies have shown that TCDD inhibits the murine 3’IgH RR but activates the hs4 enhancer in a well-characterized mouse B-cell line, CH12.LX. Therefore, the objective of the current study was to determine if TCDD inhibits the murine 3’IgH RR by repressing hs1,2 enhancer activity and if this effect will be mirrored by the human polymorphic hs1,2 enhancer in the CH12.LX model. Using transient luciferase studies and CH12.LX cells that stably express a transgene under the regulation of the hs1,2/hs3A enhancer pair, we have found that indeed the mouse hs1,2 enhancer is inhibited by TCDD in LPS-induced B cells. However the human hs1,2 undergoes a striking activation after TCDD treatment, much like the murine hs4 enhancer. These results suggest a difference in transcriptional regulation between the mouse and human hs1,2 sequence. Mutational analyses determined that DRE, kB, AP-1, and Oct binding motifs found within the human hs1,2 enhancer act in concert to mediate TCDD-induced activation of the human polymorphic hs1,2 enhancer. Since TCDD represents a large class of chemicals found in the environment, diet, and pharmaceuticals, understanding chemical-induced modulation of the 3’IgH RR enhancers may provide a clue to the etiology of certain autoimmune diseases.
Advisors/Committee Members: Sulentic, Courtney.
Subjects: Immunology; Molecular biology; Toxicology
Keywords: 3'IgHRR; polymorphism; Celiac disease; IgA nephropathy; TCDD; aryl hydrocarbon receptor
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13.
Furman, Amanda R.
Evaluation of CM-2,525 as a neuroprotectant against sarin: A comparison with 8-OH-DPAT.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► Exposure to the chemical warfare agent sarin produces long term neurological deficits.…
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▼ Exposure to the chemical warfare agent sarin produces long term neurological deficits. The long term medical consequences could be averted with the development of neuroprotectants to preserve brain function. In our mouse model, the combination of the carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) with the organophosphorus (OP) nerve agent sarin was used to render mice more sensitive to poisoning and reduce the amount of hydrogen fluoride cleaved from sarin during binding to esterases. Since carboxylesterase acts as a scavenger, reducing the levels causes sarin to have a greater inhibition effect on acetylcholinesterase. These smaller doses permitted the use of doses similar to those producing symptoms in humans. Prior work demonstrated that 8-OH-DPAT (DPAT) was neuroprotective when given up to two hours after sarin administration through its secondary pharmacology. The aim of this study was to explore the efficacy of CM-2,525, which acts on part of that secondary pharmacology, as a neuroprotectant and to directly compare its effects with DPAT. Male C57BL/6 mice were administered a toxic challenge of 1.5 mg/kg of CBDP plus the dose of sarin needed to achieve 35% mortality. A dose-response curve for CM-2,535 was determined by administration one minute after the toxic challenge. Male C57BL/6 mice also were administered a toxic challenge followed in one minute by saline or DPAT (1 mg/kg). Functional Observational Battery (FOB) data were collected for each mouse and weight data were collected pre- and post-exposure for 3 days. Treatment with DPAT revealed no benefit on FOB scores and had no effect on weight loss. Low doses of CM-2,525 reduced the FOB scores and the higher doses decreased weight loss. Immunohistological analysis was performed using Glial Fibrillary Associated Protein (GFAP) which increases in sarin treated animals and Neuronal Nuclei (NeuN), a stain for mature neurons that decreases after toxic challenge. Previously we found that treatment with DPAT resulted in a significant decrease in GFAP-labeled cells in the dentate gyrus (DG) and is effective when given two hours after the toxic challenge. In this study it both reduced GFAP and increased NeuN. The higher doses of CM-2,525 significantly decreased GFAP-labeled cells in the amygdala (Amy) and DG regions and increased NeuN-labeled cells in the Amy, piriform cortex, and DG regions. CM-2,525 has efficacy superior to DPAT with effects on weight loss and FOB scores as well as providing neuroprotection and would likely be neuroprotective as long after sarin exposure as DPAT.
Advisors/Committee Members: Lucot, James B.
Subjects: Neurosciences; Pharmacology; Toxicology
Keywords: sarin; neurotoxicity; neuroprotectant; 8-OH-DPAT
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14.
Gut, Chester P. Jr.
Hyperbaric Oxygen in the Prevention of Carbon Monoxide Induced Delayed Neurological Sequelae in Male Sprague Dawley Rats (Rattus norvegicus).
Degree: MS, Pharmacology and Toxicology, 2010, Wright State University
► In Carbon Monoxide (CO) induced Delayed Neurological Sequelae (DNS) clinical signs develop…
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▼ In Carbon Monoxide (CO) induced Delayed Neurological Sequelae (DNS) clinical signs develop 1 to 6 weeks after CO has cleared the body. The aim of this experiment was to develop a model of CO induced DNS which closely mimics “real world” conditions both in exposure and treatment. The model was challenged with hyperbaric or normobaric oxygen, or room air. Basic behaviors were measured by Open Field test on days 1, 7, 14 post exposure and treatment. No significant difference in behavior was observed between exposed and control animals or between treatment groups. Histological analyses showed no DNA or necrotic damage to the basal ganglia, cortex, or hippocampus in CO exposed animals at time of euthanasia regardless of treatment. Data suggest variability in tolerance to CO and development of DNS. Data also indicates that the in vivo COHb half-life is not constant across species.
Advisors/Committee Members: Simman, Richard.
Subjects: Toxicology
Keywords: Carbon Monoxide; Hyperbaric Oxygen; Neurological Sequelae; carboxyhemoglobin half-life; rat
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15.
Harshman, Sean William.
Effects of Chlorpyrifos-oxon on Prohormone Convertase Enzyme Activity.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Organophosphate (OP) compounds have been synthesized for various applications including medicine, pest…
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▼ Organophosphate (OP) compounds have been synthesized for various applications including medicine, pest control and weapons of terror. OPs irreversibly phosphorylate the active site of acetylcholinesterase, the primary target of toxicity, rendering it inactive causing a hypercholinergic state in the post synaptic cleft. However, very little is known about the effects of OPs on secondary targets of toxicity i.e. other serine hydrolases. The prohormone convertase (PC) enzymes, specifically PC1/3 and PC2, are serine hydrolases which are responsible for the processing of pro-opiomelanocortin in the anterior and intermediate lobes of the pituitary. To test the direct interaction between OPs and the PC enzymes, purified PC1/3 and PC2 were treated in vitro with chlorpyrifos-oxon (CHP-O) or vehicle, acetonitrile (ACN), endoproteolyically digested, and analyzed for any evidence of phosphorylation on a MALDI-TOF/TOF mass spectrometer (MS). To further describe the interaction, enzyme assays was performed by pretreating the purified PC1/3 and PC2 with micromolar concentrations of CHP-O over 0.5 h – 4 h. Substrate was added, adrenocorticotropic hormone 1-39 (ACTH 1-39), and analyzed on the MALDI-TOF/TOF MS for processing products and any subsequent reduction in peptide production caused by CHP-O. As a result of these experiments, a statistically significant (p <0.05) reduction in peptide processing was observed for both PC1/3 and PC2 in the presence of CHP-O in vitro. However, evidence of direct interaction of CHP-O on the PC enzymes was inconclusive.
Advisors/Committee Members: Cool, David.
Subjects: Toxicology
Keywords: Prohormone Convertase; PC1/3; PC2; Chlorpyrifos; Chlorpyrifos-oxon; Organophosphate
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16.
Joshi, Amod N.
Analysis of Archived Dried Blood Spots by Mass Spectrometry for Vitamin D and Real-time PCR for its Enzymes and Receptor.
Degree: MS, Pharmacology and Toxicology, 2011, Wright State University
► Vitamin D is an essential micronutrient required for maintenance of the skeletal…
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▼ Vitamin D is an essential micronutrient required for maintenance of the skeletal system and its deficiency leads to diminished availability of calcium for maintaining bone mineralization. Vitamin D deficiency (VDD) during pregnancy and/or in early infancy can lead to rickets and a fragile bone state resulting in fractures with minimal forces. Such multiple unexplained fractures (MUF) present in infants at less than 6 months of age are often misdiagnosed as cases of child abuse. Therefore, it is important to evaluate the status of vitamin D in early infancy. The analysis of dried blood spots (DBS) provides a unique screening method for early and late identification of diseases. The objective of this study was to develop a mass spectrometry (MS) method to analyze vitamin D levels in newborn DBS. We used a methanol-hexane solvent system for the extraction of vitamin D analytes from filter paper spots. The use of methanol-hexane solvent system was evaluated using desipramine HCl as a positive control. We were able to extract desipramine HCl from standard spots spiked with concentrations 8 -5000 ng/ml. We applied this extraction method to control and test DBS with slight modification involving preincubation of DBS in methanol and sodium hydroxide. Our MS results have shown qualitative separation of 25 (OH) D2 (m/z - 413.2) and 25 (OH) D3 (m/z - 401.2) with SNR > 3. The identity of vitamin D analytes was confirmed using MS/MS analysis showing the presence of respective fragment peaks (m/z - 336.2 and 365.2). For quantitative estimation i.e. SNR > 10, we derivatized vitamin D analytes in DBS using 4-Phenyl-1,2,4- triazoline-3,5-dione (PTAD) in a Diels-Alder conjugation reaction. However, both 25 (OH) D2 and 25 (OH) D3 show the formation of identical product ion (m/z- 298.0) making it difficult to quantitate them separately. In addition to MS method development, we also tried to evaluate genetic mutations in vitamin D metabolizing enzymes and vitamin D receptor as a possible cause of VDD in these infants. We extracted genomic DNA and RNA from DBS for mutation scanning using melt curve analysis However, the outcome of such analysis was not successful owing to lack of integrity in genetic material extracted from DBS. In summary we developed a MS method for qualitative detection of vitamin D and also attempted to test genetic mutations as a cause of VDD in infants. Further experiments are necessary to evaluate the use of alternative options of liquid chromatography/gas chromatography (LC/GC) coupled with MS and/or use of atmospheric pressure chemical ionization (APCI) as source of ionization for quantitative assessment of vitamin D.
Advisors/Committee Members: Cool, David.
Subjects: Pharmacology
Keywords: Vitamin D; Mass spectrometry; Dried blood spots; VDR; Mutation
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17.
Joshi, Kaushal V.
Novel Neuroprotectants for Sarin plus CBDP induced convulsions.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Sarin, also known as Sarin (German agent B) is classified as a…
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▼ Sarin, also known as Sarin (German agent B) is classified as a weapon of mass destruction. Sarin (O-isopropyl methyl phosphonofluoridate) is a highly toxic nerve agent originally produced for chemical warfare and has been used in terrorist activities. Sarin is an extremely potent acetylcholinesterase inhibitor with high specificity and affinity for the enzyme. High sarin doses causes death due to anoxia resulting from airway obstruction, weakness of the muscles of respiration, respiratory failure and convulsions. Current treatments are still not effective at protecting against long term effects following exposure. A current approach aims to counteract the increased glutamatergic and cholinergic neurotransmission occurring in sarin neurotoxicity. In vitro and in vivo, serotonin (5-HT) 1A agonist prevented toxicity from glutamate. We determined the neuroprotective capabilities of serotonin (5-HT) 1A agonists as novel pharmacological countermeasures to chemical warfare agents. Rodents have higher amount of carboxylesterase enzyme and requires higher doses of sarin than other species. To address this issue we administered 1.5 mg/kg of CBDP (2-/o-cresyl/-4 H-1: 3: 2-benzodioxa-phosphorin-2-oxide), which specifically blocks carboxyl esterase and makes mouse model comparable to that of human exposure. We determined 1mg/kg dose of serotonin (5-HT) 1A agonists 8-OH-DPAT from dose response curve based on neuroprotection, with toxic challenge of 1.5 mg/kg CBDP and dose of sarin yielding 25-50 % mortality. This mortality rate gave enough number of survivors with seizures and neurodegeneration for reliable baselines. Measurements were mortality, weight loss AChE activity in blood and CNS, functional observational battery (FOB) and histology compared to control and toxic challenge mice. In addition, a time response curve after toxic challenge was determined with 1mg/kg of 8-OH-DPAT at time points of 1, 15, 30, 45, 60 minutes and 2, 4, 6 hours. We observed neuroprotection by 8-OH-DPAT in the dentate gyrus of the hippocampus when administered up to two hours after Sarin. The ability of the combination of serotonin (5-HT) 1A agonist’s dose and time after toxic challenge was tested for its ability to reinstate fear potentiated startle (FPS) response. However this test was invalidated by the response of the control group. DPAT like drugs could be useful in treatment of long term effects produced by sarin induced convulsions.
Advisors/Committee Members: Lucot, James.
Subjects: Behaviorial sciences; Pharmacology; Toxicology
Keywords: sarin; acetylcholineserase; glutamate; CBDP; 8-OH-DPAT; neuroprotection
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18.
Kanakamedala, Keerthy.
ROLE OF ANGIOTENSIN CONVERTING ENZYMES ACE AND ACE2 IN DIABETES INDUCED CARDIOVASCULAR DYSFUNCTION.
Degree: MS, Pharmacology and Toxicology, 2007, Wright State University
► Kanakamedala, Keerthy M.S., Department of Pharmacology and Toxicology, Wright State University,…
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▼ Kanakamedala, Keerthy M.S., Department of Pharmacology and Toxicology, Wright State University, 2007. Role of Angiotensin converting enzymes ACE and ACE2 in diabetes induced cardiovascular dysfunction Cardiovascular disease is a long term complication of diabetes, which remains a leading cause of mortality and morbidity. There is recent evidence for activation of the Renin angiotensin system (RAS) in diabetic animals and humans. Emerging evidence shows that the vasoconstrictor actions of Ang II may be opposed by formation of the vasodilator, Ang (1-7). There is limited data on blood pressure in murine models of type 2 diabetes. The aim is to study the role of angiotensin converting enzymes ACE and ACE2 in diabetes induced cardiovascular dysfunction using type 2 diabetic murine mouse models (db/db mice). Both db/db mice and their controls were implanted with carotid telemetric probes for chronic monitoring of MAP (Mean Arterial pressure), heart rate and activity. At 8-9 weeks age, mice showed hyperinsulinemia, hyperglycemia and increase in body weight compared to their lean controls while MAP was not altered. At an older age (14-15 weeks) there was a significantly increased BP in the db/db mice compared to controls. In young (8 weeks) normotensive mice there was a highly significant increase in plasma ACE activity in db/db mice compared to controls. In contrast there was increased ACE2 activity and decreased ACE activity in kidney in 8 weeks old db/db mice compared to controls. No significant difference between ACE and ACE2 activity was observed in lungs and brain. In addition western blot analysis for ACE/ACE2 protein expression also revealed that there was a significant increase in kidney ACE2 and decrease in kidney ACE protein expression in 8 weeks old db/db mice compared to their lean controls. In addition no difference was observed between lung ACE and ACE2 protein expression. Increased plasma and kidney ACE activity and a decreased kidney ACE2 activity were observed in 24 weeks old db/db mice. In conclusion: 1. Eight weeks’ db/db mice were normotensive despite an increase in plasma ACE activity. 2. There was an age dependent increase in the BP but no change in heart rate (HR) in both young and old mice. 3. ACE2 may play a compensatory mechanism against development of hypertension in db/db mice in the early stage of type2 diabetes. 4. The up regulation of ACE coupled with a down regulation of ACE2 might be the cause for hypertension at a later stage in this murine model of type2 diabetes.
Advisors/Committee Members: Elased, Khalid M.
Keywords: db/db mice, ACE, ACE2, Angiotensin
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19.
Kerns, Scott.
Effects of Direct Mechanical Ventricular Actuation on the Apoptotic Signaling of a Failing Heart.
Degree: MS, Pharmacology and Toxicology, 2010, Wright State University
► Cardiovascular disease accounts for more than 40% of all deaths in the…
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▼ Cardiovascular disease accounts for more than 40% of all deaths in the United States (AHA-2004 report). A non blood contacting ventricular assist device (VAD) can be used to treat heart failure without the complications that arise from blood contacting VADs. This study used cellular markers of heart failure as indicators of heart function in an attempt to assess if direct mechanical ventricular actuation (DMVA) support lessened the impact of heart failure in rabbits. Cell signaling proteins were monitored using enzyme activity measurements and quantitative immunoblotting with antibodies against intrinsic and extrinsic apoptotic pathways during heart failure with and without DMVA support. New Zealand White rabbits were treated as sham or heart failure, with or without DMVA assistance. Animals had heart failure induced by esmolol, with or without DMVA support for 30, 60, or 120 minutes. At all time points, animals were recovered for 30 minutes after which the hearts were excised. Tissue extracts were prepared and measurements were made using the following groups; acute model with sham and 30 minute groups or an extended support/failure model with 60 minute and 120 minute groups. Cell extracts from left and right ventricles were used for immunoblot analysis to determine protein levels for the following heart failure markers; the extrinsic apoptotic pathway comprised of tumor necrosis factor receptor (TNFR) and caspase-8, the intrinsic apoptotic pathway comprised of caspase-9, cytochrome-C, or the stress related marker of heat shock protein-70 (Hsp70). Enzymatic activity was used to monitor the extrinsic pathway with caspase-8, or markers of stress with superoxide dismutase (SOD), and matrix metalloproteinase-9 (MMP-9) in all heart extracts. In the left ventricle acute model there was a rise in both 30 minute groups compared to the sham group for all the markers, except in the caspase-8 enzymatic assay where both groups showed less activity. The extended support/failure model showed that the use of DMVA during heart failure significantly attenuated the increase of protein content for TNFR, Hsp70, and caspase-8 at the 120 minute time point. The caspase-8 enzymatic assay showed a significant decrease in 120 minute DMVA assisted group compared to the heart failure group. For every marker in the extended support/failure model there was an increase in all the markers as the duration of the experiment increased. The right ventricle acute model exhibited an increase in both 30 minute groups for TNFR content. A significant rise in SOD levels for the 30 minute heart failure compared to both the sham and 30 DMVA groups. MMP-9 levels for both 30 minute groups rose significantly. In the extended support/failure model, the right ventricle showed a significant increase of both groups at 120 minutes when compared to the 60 minute heart failure group in TNFR and Hsp70 protein content. In the caspase-8 enzyme assay the 120 minute DMVA group had a significant rise when compared to the 60 minute heart failure group. DMVA provided evidence of the ability to attenuate the increase of pro-apoptotic signaling during heart failure. Evidence of this was mostly observed in the left ventricle samples, often significantly different. Both ventricles exhibited trends of attenuation by DMVA in many markers that did not differ significantly. It should also be noted that at no time point did DMVA appear to have a negative effect on the heart during failure. The validity of the heart failure model being used was also confirmed by the continuing increase in activation of known markers over time.
Advisors/Committee Members: Anstadt, Mark.
Subjects: Biology; Microbiology; Pharmacology; Toxicology
Keywords: apoptosis; cardiology; heart failure; DMVA
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20.
Lepera, Michael Anthony.
Response of Monovalent Cation Transporters to Pro-apoptotic Protein Kinase C Modulators in Human Lens Epithelial Cells.
Degree: MS, Pharmacology and Toxicology, 2011, Wright State University
► Protein kinase inhibition by staurosporine causes apoptotic volume decrease involving potassium (K)…
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▼ Protein kinase inhibition by staurosporine causes apoptotic volume decrease involving potassium (K) channels in immortalized human B3 lens epithelial cells (LECs). Here, the effect of two pro-apoptotic protein kinase C (PKC) inhibitors [12-O-tetradecanoyl-phorbol-13-acetate (TPA) and chelerythrine (CET)] were studied on membrane K transport in a fetal human LEC line (FHL124) by western blotting, immunofluorescence, ion flux, ATP, apoptosis, and biotinylation assays. Long term TPA exposure (0-6 h) inhibited 75% of Na-K-2Cl cotransport (NKCC). In contrast, short term (0-20 min) exposure to 50 µM CET reduced Na/K pump and NKCC by >90% and >70%, respectively, without retrieval from the membrane into the cytosol, loss of ATP and early signs of apoptosis. CET (10-30 µM) decreased cell K by 33%. Results suggest PKC modulation through the use of pro-apoptotic agents caused major early membrane changes independently affecting K channels, the Na/K pump and NKCC function, prior to rise of any significant apoptosis.
Advisors/Committee Members: Lauf, Peter.
Subjects: Pharmacology; Toxicology
Keywords: pharmacology; toxicology
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21.
Madhu, Malav Navinchandra.
Impact of Diabetes on ACE/ACE2 Balance and Angiotensin II Type 1 Receptor Expression in db/db Diabetic Mice.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Alterations in the renin-angiotensin system (RAS) are considered to be crucial for…
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▼ Alterations in the renin-angiotensin system (RAS) are considered to be crucial for the development of diabetic complications like hypertension and nephropathy. Our previous work demonstrated role of AT1 receptors (AT1R) in the development of hypertension in db/db diabetic mice. The aim of this study was to test the hypothesis that there is upregulation of renal AT1R and imbalance in renal ACE/ACE2 homeostasis in db/db mice. In addition, we hypothesize that treatment with an anti-hyperglycemic or an AT1R blocker will correct this imbalance. Five week old control and db/db mice were housed in metabolic cages for 24 hour collection of urine. At early age of 5 weeks, db/db mice were obese and hyperglycemic. Urinary albumin excretion was also significantly high in db/db mice. Changes in RAS were evaluated using enzyme activities, western blots and immunohistochemistry. There was a significant increase in urinary ACE2 activity and ACE2 content in db/db mice at 5 weeks. There was a significant increase in plasma ACE activity and Ang II content in db/db mice compared to controls at 8 weeks. Western blot analysis showed significant increase in AT1R protein expression in 8, 18 and 31 week db/db mice compared to controls. There was upregulation of ACE2 and down-regulation of ACE in kidney to compensate the effects of high plasma Ang II. To study the effect of reduction in blood glucose and AT1R blockade, mice were treated with metformin and losartan for 12 weeks. Chronic treatment with metformin (150 mg/kg/day) and losartan (10 mg/kg/day) significantly decreased urinary albumin and protein excretion. Metformin improved blood glucose and glucose tolerance db/db mice, but did not affect renal expression of ACE, ACE2 and AT1R. Although chronic losartan treatment did not alter blood glucose levels, it improved the morphology of pancreatic islets. There was a significant increase in renal AT1R protein expression and decrease in renal ACE2 protein expression following losartan treatment. Losartan treatment significantly increased urinary ACE2 activity. Western blot of concentrated urine from 8 week db/db mice revealed immunoreactive bands of ACE, ACE2 and AT1R protein. Conclusion: 1) There is upregulation in renal AT1R protein expression in db/db mice. 2) Chronic metformin treatment significantly reduces blood glucose and microalbuminuria in db/db mice without affecting ACE/ACE2 balance. 3) Chronic losartan treatment had no effect on blood glucose, but it up-regulates renal AT1R and down-regulates renal ACE2. 4) Enzyme activity and western blot shows increased excretion of ACE2 in the urine of db/db mice. These data show that urinary ACE and ACE2 provide good index of intra-renal RAS status and could be used in early diagnosis and prognosis of diabetic renal disease.
Advisors/Committee Members: Elased, Khalid.
Subjects: Pharmacology
Keywords: RAS, AT1 receptor, ACE, ACE2, diabetes, nephropathy
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22.
Nedderman, Drew Michael.
TISSUE SPECIFIC EFFECTS OF ADIPOSE STEM CELLS (ASC) IN A MELANOMA TUMOR ENVIRONMENT.
Degree: MS, Pharmacology and Toxicology, 2010, Wright State University
► This study determined the tissue-specific effect of adipose stem cells (ASC) within…
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▼ This study determined the tissue-specific effect of adipose stem cells (ASC) within a melanoma environment for development of a cell-based melanoma therapy. Analysis included a subcutaneous B16-F1 melanoma model using thirty-one C57BL/6 wild-type mice. Melanoma xenografts were treated with cell-based therapies of CFDA-SE-labeled human fibroblasts HF20x (control), non-differentiated ndASC or hematopoietic-differentiated HdASC. No tumor regression was observed in presence of cell-based therapies, thus, the HdASC group demonstrated an increase in tumor growth accompanied with an up-regulated macrophage response, and increased angiogenesis. In addition, this group demonstrated a decrease in Melan-A tumor marker and interferon-γ expression suggesting that ASC-supported tumor angiogenesis and macrophage immune response are accompanied with an inflammatory water influx and increased tumor porosity with no effect on tumor cell proliferation. In addition, histology demonstrated CFDA-SE-labeled HF20x, ndASC, or HdASC inside tumors with no signs of cell death or apoptosis suggesting an immuno-suppressive effect of human ASC on the mouse immune system.
Advisors/Committee Members: Hoffmann, Andrea.
Subjects: Pharmacology
Keywords: HdASC; TUMOR; ndASC; HF20x; MELANOMA; CELLS; Hematopoietic
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23.
Ochs, Sharon D.
Elucidating transcription factor regulation by TCDD within the hs1,2 enhancer.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► Immunoglobulin heavy chain (IgH) expression and Ig secretion is inhibited by the…
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▼ Immunoglobulin heavy chain (IgH) expression and Ig secretion is inhibited by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). Within the IgH gene, the 3' IgH regulatory region (3'IgH RR) has been identified as a transcriptional target of TCDD. The 3'IgH RR, which in part regulates transcription of the IgH gene, is composed of four enhancers in the mouse: hs3a; hs1,2; h3b; hs4 and three enhancers in the human: hs3a; hs1,2; hs4. In humans the hs1,2 enhancer has an invariant sequence (IS) containing a DRE, NF-κB, NF1 and AP-1 binding site. Also, the enhancer has an AP1.ETS and Oct site located 5' to the IS. The human hs1,2 enhancer is sensitive to TCDD-induced modulation but in contrast to the mouse hs1,2 and 3'IgH RR, TCDD activates the human hs1,2 enhancer. The current study demonstrates the complexity of how TCDD differentially induces modulation between mouse and human and what role these binding sites may have.
Advisors/Committee Members: Sulentic, Courtney.
Subjects: Immunology; Toxicology
Keywords: TCDD; hs1,2 enhancer; transcription factor binding sites
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24.
Oswal, Dhawal Pravin.
Effect of low-dose sarin exposure on the neurochemistry of different brain structures in mice.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Sarin (GB) is a toxic organophosphate (OP) nerve agent that was released…
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▼ Sarin (GB) is a toxic organophosphate (OP) nerve agent that was released in the Gulf War and was used in terrorist attacks in Japan. People who survive such attacks exhibit various long-term effects including alterations in neuropsychological performances. It has also been hypothesized that the Gulf War Illness could be a result of low level exposure to OP’s. In order to understand the effect of low dose exposure to GB on physiological and behavioral functions, we analyzed the levels of monoamines and their metabolites in different brain areas after exposure of mice to a sublethal dose of GB. Mice (male C57BL/6) were injected subcutaneously once a day for 2 days, with 0.05 LD50 or 0.4 LD50 of GB followed by behavioral testing in the open field environment, elevated plus maze and for fear potentiated startle. The mice did not show signs of cholinergic toxicity. They were sacrificed at 1, 4 and 8 weeks with collection of brains for neurochemical analysis. In both dose groups and time points, a significant decrease in the usage of dopamine (DA) was observed in the frontal cortex (FC) region of the brain which may account for a number of symptoms of the Gulf War veterans. There was an increase in the usage of DA in the amygdala at 4 weeks but not at 1, 8 weeks, indicating a reversible effect. No significant change observed in the DA activity of the caudate nucleus which was consistent with no change in the motor activity in the open field studies. In addition to this, the levels of serotonin (5HT) were transiently elevated in all the brain regions studied. The FC is innervated mainly by the A10 group of dopaminergic cell bodies located in the ventral tegmental area (VTA) of the brain. The amygdala, in addition to A10, also receives projections from the A8 group of dopaminergic cell bodies in the retrorubral nucleus (RRN) – accounting for the difference from the FC. The caudate nucleus is innervated mainly by the A9 group of dopaminergic cell bodies located in the substantia nigra (SN) region of the brain. Data strongly suggests that even low dose of sarin has potent long-term, region specific effects on other neurotransmitter systems. Further experiments are necessary to evaluate the relationship between these modifications and the neuropsychological disorders reported after asymptomatic exposure to OPs.
Advisors/Committee Members: Lucot, James.
Subjects: Armed forces; Pharmacology; Toxicology
Keywords: Sarin; low-dose; monoamines; dopamine activity; delayed-onset; Gulf War Illness
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25.
Paluri, Sesha Lakshmi Arathi.
Synthesis, Characterization and Manipulation of Creighton Silver Nanoparticles for Future Cytotoxicity Studies.
Degree: MS, Pharmacology and Toxicology, 2011, Wright State University
► Nowadays, 24% of the nanomaterial-based consumer products contain silver nanoparticles (AgNPs) and…
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▼ Nowadays, 24% of the nanomaterial-based consumer products contain silver nanoparticles (AgNPs) and exploit the well-known antimicrobial properties of silver. Although AgNPs have a wide range of biomedical research and industrial applications, very little is known about their toxicity. The main goal of this study is to synthesize, characterize and manipulate Creighton colloidal AgNPs for future cytotoxicity studies. These “naked” AgNPs were free from chemically aggressive capping/stabilizing agents, reaction byproducts or organic solvents. To achieve this goal, colloidal AgNPs were successfully: a) synthesized in large volumes (5L) using a slightly modified Creighton method by the reduction of silver nitrate with sodium borohydride, b) characterized using UV-Vis absorption spectrophotometry, flame atomic absorption spectroscopy, Raman spectroscopy and transmission electron microscopy (spherical AgNPs of 1-100 nm in diameter, moderately aggregated, free of impurities and having a surface plasmon resonance at 396 nm), and c) size-selected (mostly AgNPs of 1-20 nm in diameter) and highly concentrated (5 L of 14.98 ppm down to 4 mL of 7,461.65 ppm) in a small volume of water with minimal aggregation using tangential flow ultrafiltration. These homogenous and highly concentrated, “naked” AgNPs will be used in future cytotoxicity studies that will establish the median lethal concentration (LC50) of AgNPs and will identify AgNPs suitable for surface-enhanced Raman spectroscopy-based biosensing applications in our laboratories.
Advisors/Committee Members: Sizemore, Ioana.
Subjects: Chemistry; Nanoscience; Toxicology
Keywords: Silver nanoparticles, ultrafiltration, creighton synthesis, cytotoxicity
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26.
Penchikala, Madhuri.
ANGIOTENSIN AT1 RECEPTOR BLOCKADE PROTECTS THE BRAIN FROM ISCHEMIC DAMAGE.
Degree: MS, Pharmacology and Toxicology, 2007, Wright State University
► Angiotensin (Ang) AT1 receptors are considered to play an important role in…
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▼ Angiotensin (Ang) AT1 receptors are considered to play an important role in ischemic stroke via degenerative processes leading to cell death. Recent clinical and basic studies show that systemic blockade of Ang AT1 receptors reduces brain lesion in ischemic stroke. In this study we evaluated whether blockade of central Ang AT1 receptors protects the brain from ischemia and inflammation during ischemic stroke. Adult male C57BL/6 mice were divided into two groups for chronic intracerebroventricular (ICV) infusion of a selective Ang AT1 receptor antagonist, losartan (Los, n=18, 2 ug/hr) or isotonic saline (Con, n=20) using osmotic minipump. Twelve days post infusion, focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). The success of MCAO was verified by measurement of cerebral blood flow (CBF) using laser-Doppler flowmetry. Neurological deficits due to ischemic damage of neuronal cells were evaluated 24 hours after MCAO. Brains were removed 48 hrs after MCAO and the degree of damage due to ischemia was determined using triphenyltetrazolium chloride (TTC) staining. The expression of Ang AT1 receptors, matrix metalloproteinase-2 (MMP-2) and myeloperoxidase (MPO) was carried out using western blot analysis. Immunohistochemistry was performed to determine the inflammatory cell infiltration in control vs. losartan treated mice. Focal cerebral ischemia resulted in overexpression of Ang AT1 receptor ischemic hemisphere compared to non-ischemic hemisphere (27%, p<0.05) suggesting a role of Ang AT1 receptor in ischemia. We also found a significant increase in the expression of MPO and MMP-2 in ischemic vs. non-ischemic hemispheres. Pretreatment with losartan significantly improved neurological deficits and infarct volume compared to control mice (p < 0.05). Parelleling these effects on ischemia, losartan pretreatment also reduced (~50%) the reactive upregulation of MPO (P < 0.05) and inflammatory cells (neutrophils and macrophages, P < 0.01) in the ischemic area. These results support a role for Ang AT1 receptors in cerebral ischemia and inflammation produced by stroke.
Advisors/Committee Members: CHEN, YANFANG.
Subjects: Health Sciences, Pharmacology
Keywords: Stroke, angiotensin II, angiotensin type I (AT1) receptor, middle cerebral artery occlusion, ICV infusion, losartan
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27.
Plahovinsak, Jennifer Lee.
KINETICS AND PASSIVE PROTECTION EFFICACY INDUCED BY PURIFIED AVA HUMAN IMMUNOGLOBULIN G IN RABBITS AGAINST A Bacillus anthracis AEROSOL CHALLENGE.
Degree: MS, Pharmacology and Toxicology, 2006, Wright State University
► The present study was conducted to determine the half-life, assess the toxicity,…
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▼ The present study was conducted to determine the half-life, assess the toxicity, and passive protection efficacy of purified immunoglobulin G (IgG) from Anthrax Vaccine Adsorbed (AVA) vaccinated human donors. Half-life determinations were calculated from the reportable values obtained using the anti-PA ELISA assay and the Centers for Disease Control’s (CDC) “ELISA for Windows” software. For toxicity evaluations animals were observed clinical for one hour post administration and for 14-days post-treatment. The protection efficacy was determined based upon the mortality results from a lethal Bacillus anthracis aerosol challenge. While no protection was achieved in this delayed exposure scenario, the study yielded valuable kinetics data for use in future research.
Advisors/Committee Members: Casillas, Robert P.
Subjects: Biology, General
Keywords: ELISA; AIG; anti-PA; Naïve; IgG; LOQ; Diluent
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28.
Pyles, John Allen.
CHARACTERIZATION OF INFECTIVITY AND PATHOGENESIS OF PARTIALLY RECONSTRUCTED 1918 AND HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES IN THE BALB/c MOUSE MODEL.
Degree: MS, Pharmacology and Toxicology, 2009, Wright State University
► Influenza viruses are consistently responsible for an average of 20,000 deaths and…
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▼ Influenza viruses are consistently responsible for an average of 20,000 deaths and 114,000 hospitalizations per year. To a great extent, these viruses always stay one step ahead of the available vaccines and people’s immunity year after year because they have the ability to either mutate part of their genetic material, or to be transmitted from one species to another. That same genetic variability explains why highly pathogenic influenza viruses emerge that cause great mortality over several countries resulting in pandemics. Highly pathogenic strains of influenza A virus have emerged occasionally in recent history, producing pandemics such as the one in 1918. The Spanish influenza pandemic of 1918–1919 was uniquely severe, causing an estimated 50 million deaths worldwide. Also unique was the age distribution of its victims: the death rate for young, previously healthy adults, who rarely suffer fatal complications from influenza, was exceptionally high. More recently we have seen the emergence of influenza cases and fatalities involving the H5N1 avian influenza strains. Until an outbreak in Hong Kong claimed six human lives in 1997 (A/Hong Kong/156/97 [H5N1]), avian influenza viruses were thought to be incapable of infecting humans directly. However, the initial H5N1 outbreak has revealed that avian influenza viruses could infect humans without prior adaptation and even cause significant morbidity and mortality in the human population. It has been shown that the 1918 viral hemagglutinin sequence is more closely related to avian strains although it is a human HA. This indicates that the 1918 pandemic strain may have also jumped from avian to human with no prior adaptation or reassortment with a human virus. For these reasons, scientists have been interested in finding out what makes the 1918 virus different from all others, why avian influenza can be so pathogenic, and how to prevent and better treat infections with this virus. For this study the 1918 pandemic influenza was partially reconstructed by placing the 1918 influenza viral hemagglutinin and neuraminidase genes or the 1918 influenza viral hemagglutinin, neuraminidase and non-structural protein genes in the background of the low-pathogenic A/Texas/36/91 virus. The infectivity and pathogenesis of these two partially reconstructed 1918 influenza viruses were compared to each other as well as to the highly-pathogenic avian influenza A/Vietnam/1203/04 H5N1 virus in the BALB/c mouse model. The A/Vietnam/1203/04 influenza virus acted as a “positive” control for high infectivity and pathogenesis.
Advisors/Committee Members: Sabourin, Carol.
Subjects: Biology; Microbiology; Molecular biology; Virology
Keywords: INFLUENZA; TX91; virus; NS1; influenza A virus; HA; INFLUENZA VIRUSES
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29.
Rodwan, Naima Salem.
Light-Limited Access to Fructose Alters Metabolic Function and Adipose Tissue Catecholaminergic Activity in Mice.
Degree: MS, Pharmacology and Toxicology, 2012, Wright State University
► Consumption of high levels of fructose produces glucose intolerance, sympathetic nervous system…
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▼ Consumption of high levels of fructose produces glucose intolerance, sympathetic nervous system activation, and renal dysfunction. A diet high in fructose may play a role in the worldwide epidemic of obesity and diabetes. The aim of this study was to test the hypothesis that time (light/dark) limited access to fructose influences body fat, metabolic function, and adipose tissue catecholaminergic activity in mice. Male C57BL/6 mice were given standard chow and assigned to one of three groups: Control (n=10, water 24h); FL (n=11, 10% fructose solution during 12h light period); and FD (n=10, 10% fructose solution during 12h dark period). Metabolic parameters measured were body fat (BF, Echo-MRI), adipocyte cell size, and glucose tolerance as well as plasma glucose, adiponectin, insulin, leptin, triglycerides, and cholesterol. Catecholamine levels were measured directly in white adipose tissue (WAT) using high performance liquid chromatography (HPLC) with electrochemical detection. Immunochemistry was used for examining tyrosine hydroxylase (TH) staining in WAT and brainstem. Mice given fructose during the light phase (inactive period for mice) showed evidence for a diabetic like symptoms with prominent changes in adipose tissue fat. Results showed: 1) enhanced increase in BF and increased size of adipocytes in FL; 2) increased plasma insulin and leptin in FL without changes in glucose or glucose tolerance; 3) no change in cholesterol or triglycerides; 4) measurable amounts of norepinephrine (NE), epinephrine (EPI) in WAT without difference among groups; 5) enhanced staining for TH in WAT and brainstem locus coeruleus. The changes in adiposity occurred even though caloric intake was not different among groups. In conclusion, results document that restriction of fructose access to the light period produced pathological effects on metabolic function along with catecholaminergic activation in WAT and brainstem. These data may have clinical implications since the timing of intake may be important in the control of adiposity and the development of metabolic syndrome.
Advisors/Committee Members: Morris, Mariana.
Subjects: Pharmacology
Keywords: Light-Limited Access, fat tissue ,caticholamine
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30.
Ruark, Christopher Daniel.
Quantitative Structure-Activity Relationships for Organophosphates Binding to Trypsin and Chymotrypsin.
Degree: MS, Pharmacology and Toxicology, 2010, Wright State University
► Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino)…
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▼ Organophosphate (OP) nerve agents such as sarin, soman, tabun, and O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX) do not react solely with acetylcholinesterase (AChE). Evidence suggests that a wide range of cholinergic-independent pathways are also targeted, including serine proteases. These proteases comprise nearly one-third of all known proteases and play major roles in synaptic plasticity, learning, memory, neuroprotection, wound healing, cell signaling, inflammation, blood coagulation and protein processing. Inhibition of these proteases by OPs was found to exert a wide range of noncholinergic effects depending on the type of OP, the dose, and the duration of exposure. Consequently, in order to understand these differences, in silico biologically-based dose-response and quantitative structure-activity relationship (QSAR) methodologies need to be integrated. Here, QSARs were used to predict OP bimolecular rate constants for trypsin and α-chymotrypsin. A heuristic regression of over 500 topological/constitutional, geometric, thermodynamic, electrostatic, and quantum mechanical descriptors, using the software Ampac 8.0 and Codessa 2.51 (SemiChem, Inc., Shawnee, KS), was developed to obtain statistically verified equations for the models. General models, using all data subsets, resulted in R2 values of 0.94 and 0.92 and leave-one-out Q2 values of 0.9 and 0.87 for trypsin and α-chymotrypsin. To validate the general model, training sets were split into independent subsets for test set evaluation. A y-randomization procedure, used to estimate chance correlation, was performed 10,000 times resulting in mean R2 values of 0.24 and 0.3 for trypsin and α-chymotrypsin. The results show that these models are highly predictive and capable of delineating the complex mechanism of action between OPs and serine proteases, and ultimately, by applying this approach to other OP enzyme reactions such as AChE, facilitate the development of biologically based dose response models.
Advisors/Committee Members: Gearhart, Jeffery.
Subjects: Toxicology
Keywords: Quantitative structure activity relationship; QSAR; Trypsin; Chymotrypsin; Physiologically based pharmacokinetic; Biologically based dose response; Organophosphates
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