Master of Science / Department of Clinical Sciences / Elizabeth Davis / The first aim of the current study was to investigate the pharmacokinetics of oral meloxicam tablets and the gastrointestinal and renal effects after a 14-day treatment period. Meloxicam was orally administered to six adult horses once daily at a dosage of 0.6 mg/kg for 14 consecutive days. Blood was collected prior to each administration and at 20 and 40 min, and 1, 2, 4, 8, 12, and 24 hours after administration on days 1, 7, and 14 for the determination of meloxicam plasma concentrations by mass spectrometry. In addition, trough samples were taken on days 3 and 10. Complete blood count, serum biochemical analysis, urinalysis, and gastroscopy were performed at baseline and conclusion of the investigation. Complete blood count, serum chemistry, and urinalysis results were unchanged through the study period. Gastroscopy scores were not significantly increased. The Cmax was 1.82 ± 0.80 µg/mL at Tmax 3.48 ± 3.30 hr on day 1, 2.07 ± 0.94 µg/mL at Tmax 1.24 ± 1.24 hr on day 7, and 1.81 ± 0.76 µg/mL at 1.93 ± 1.30 h on day 14 (p = 0.30). The mean half-life was 4.99 ± 1.11 h. The second aim of the study was to compare the analgesic effects and gastrointestinal and renal adverse effects of oral meloxicam tablets (0.6 mg/kg) to oral phenylbutazone tablets (4.4 mg/kg) orally once daily for 4 days in induced and naturally occurring lameness in adult horses. The study was performed on 4 healthy but lame adult horses. Complete blood count, serum biochemistry, urinalysis, and gastroscopy were performed prior to entrance to the study. Lameness was exacerbated in two horses using lipopolysaccharide (LPS; E. coli O55:B5) injected into the right metacarpophalangeal joint. The remaining two horses had Grade 3 or Grade 4 lameness due to naturally occurring laminitis. Meloxicam or phenylbutazone was administered to two horses each in a blinded, randomized manner once daily for four days. Lameness was evaluated using a pressure mat system and contact pressure, force, and stride length were evaluated at baseline and twice daily. Complete blood count, serum chemistry, and urinalysis were unremarkable for all four horses except one horse with an increased GGT. This horse experienced hepatic rupture secondary to amyloidosis the final day of the study. Gastric ulcer scores did not change during the study period. Phenylbutazone administration resulted in a greater response (force and contact area) in the right front and left hind limbs compared to meloxicam administration. There were not enough data points to evaluate the other two limbs. A third aim of the study was two-fold and first evaluated the effects of ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with LPS on cyclooxygenase (COX) messenger RNA (mRNA) expression. The second portion documented the effects of LPS-induced joint inflammation and treatment with non-steroidal anti-inflammatory drugs on the mRNA and protein expression of COX-2 in PBMCs. The results indicate that LPS upregulates COX-2 gene expression in PBMCs. Additionally, injection of LPS into the metacarpophalangeal joint increases both COX-2 mRNA and protein expression in PBMCs at 24 hours after injection. The relative expression of COX-2 after treatment with meloxicam or phenylbutazone indicates a stronger inhibition with phenylbutazone; however, further study with additional horses is needed. Pharmacokinetic analysis of the oral tablet formulation of meloxicam indicates the pharmacokinetics are similar to the oral suspension formulation. Meloxicam appears to be inferior to phenylbutazone in its analgesic properties for induced lameness and naturally occurring laminitis, however the small sample size used in the study makes interpretation difficult.

The rate of injury and death inadvertently caused by medical treatment is too high and exacts enormous human and financial costs. Each year in Britain and the United States alone, hundreds of thousands of patients are injured, ten of thousands are killed and billions of dollars are spent on additional health care due to iatrogenic harm. Health care organisations remain predominately human-centred in their approach to safety-that is, methods of avoiding error rely primarily on the resolve and vigilance of individual clinicians to avoid bad outcomes. However, this approach is becoming increasingly inadequate in the face of the steadily rising complexity of modern health care and the increasing number of procedures carried out each year. In other high-reliability organisations such as aviation and nuclear power generation, safety results not from the sheer effort of “operators”, but from in-depth analysis of problems and the removal and redesign of dangerous aspects of systems-the so-called systems approach. Here I present an evaluation of the integrated drug administration system (IDAS) as an example of the systems approach, intended to reorganise the way in which anaesthetists give drugs to improve performance and facilitate safe practice. The problem of drug error in anaesthesia is an important subset of iatrogenic harm in medicine. From the prospective study of 10806 conventional anaesthetics I define the rate of drug error in anaesthesia as one error in every 133 anaesthetics conducted-a rate five times higher than anything previously reported. In addition, anaesthetists rated the risk of harming a patient through drug error in the course of their career as high. I discuss the principles of safe system design, the psychology of error, and advanced systems safety concepts with respect to the design of the IDAS and the future of safety in medicine. In clinical use, the IDAS saved time before and during anaesthesia, and was rated by anaesthetists as significantly safer and more useable than conventional methods of drug administration. This work supports the hypothesis that error in anaesthesia can be reduced through the systematic analysis of its causes and the implementation of appropriate countermeasure strategies. / Subscription resource available via Digital Dissertations only.

The rate of injury and death inadvertently caused by medical treatment is too high and exacts enormous human and financial costs. Each year in Britain and the United States alone, hundreds of thousands of patients are injured, ten of thousands are killed and billions of dollars are spent on additional health care due to iatrogenic harm. Health care organisations remain predominately human-centred in their approach to safety-that is, methods of avoiding error rely primarily on the resolve and vigilance of individual clinicians to avoid bad outcomes. However, this approach is becoming increasingly inadequate in the face of the steadily rising complexity of modern health care and the increasing number of procedures carried out each year. In other high-reliability organisations such as aviation and nuclear power generation, safety results not from the sheer effort of “operators”, but from in-depth analysis of problems and the removal and redesign of dangerous aspects of systems-the so-called systems approach. Here I present an evaluation of the integrated drug administration system (IDAS) as an example of the systems approach, intended to reorganise the way in which anaesthetists give drugs to improve performance and facilitate safe practice. The problem of drug error in anaesthesia is an important subset of iatrogenic harm in medicine. From the prospective study of 10806 conventional anaesthetics I define the rate of drug error in anaesthesia as one error in every 133 anaesthetics conducted-a rate five times higher than anything previously reported. In addition, anaesthetists rated the risk of harming a patient through drug error in the course of their career as high. I discuss the principles of safe system design, the psychology of error, and advanced systems safety concepts with respect to the design of the IDAS and the future of safety in medicine. In clinical use, the IDAS saved time before and during anaesthesia, and was rated by anaesthetists as significantly safer and more useable than conventional methods of drug administration. This work supports the hypothesis that error in anaesthesia can be reduced through the systematic analysis of its causes and the implementation of appropriate countermeasure strategies. / Subscription resource available via Digital Dissertations only.

The rate of injury and death inadvertently caused by medical treatment is too high and exacts enormous human and financial costs. Each year in Britain and the United States alone, hundreds of thousands of patients are injured, ten of thousands are killed and billions of dollars are spent on additional health care due to iatrogenic harm. Health care organisations remain predominately human-centred in their approach to safety-that is, methods of avoiding error rely primarily on the resolve and vigilance of individual clinicians to avoid bad outcomes. However, this approach is becoming increasingly inadequate in the face of the steadily rising complexity of modern health care and the increasing number of procedures carried out each year. In other high-reliability organisations such as aviation and nuclear power generation, safety results not from the sheer effort of “operators”, but from in-depth analysis of problems and the removal and redesign of dangerous aspects of systems-the so-called systems approach. Here I present an evaluation of the integrated drug administration system (IDAS) as an example of the systems approach, intended to reorganise the way in which anaesthetists give drugs to improve performance and facilitate safe practice. The problem of drug error in anaesthesia is an important subset of iatrogenic harm in medicine. From the prospective study of 10806 conventional anaesthetics I define the rate of drug error in anaesthesia as one error in every 133 anaesthetics conducted-a rate five times higher than anything previously reported. In addition, anaesthetists rated the risk of harming a patient through drug error in the course of their career as high. I discuss the principles of safe system design, the psychology of error, and advanced systems safety concepts with respect to the design of the IDAS and the future of safety in medicine. In clinical use, the IDAS saved time before and during anaesthesia, and was rated by anaesthetists as significantly safer and more useable than conventional methods of drug administration. This work supports the hypothesis that error in anaesthesia can be reduced through the systematic analysis of its causes and the implementation of appropriate countermeasure strategies. / Subscription resource available via Digital Dissertations only.

The rate of injury and death inadvertently caused by medical treatment is too high and exacts enormous human and financial costs. Each year in Britain and the United States alone, hundreds of thousands of patients are injured, ten of thousands are killed and billions of dollars are spent on additional health care due to iatrogenic harm. Health care organisations remain predominately human-centred in their approach to safety-that is, methods of avoiding error rely primarily on the resolve and vigilance of individual clinicians to avoid bad outcomes. However, this approach is becoming increasingly inadequate in the face of the steadily rising complexity of modern health care and the increasing number of procedures carried out each year. In other high-reliability organisations such as aviation and nuclear power generation, safety results not from the sheer effort of “operators”, but from in-depth analysis of problems and the removal and redesign of dangerous aspects of systems-the so-called systems approach. Here I present an evaluation of the integrated drug administration system (IDAS) as an example of the systems approach, intended to reorganise the way in which anaesthetists give drugs to improve performance and facilitate safe practice. The problem of drug error in anaesthesia is an important subset of iatrogenic harm in medicine. From the prospective study of 10806 conventional anaesthetics I define the rate of drug error in anaesthesia as one error in every 133 anaesthetics conducted-a rate five times higher than anything previously reported. In addition, anaesthetists rated the risk of harming a patient through drug error in the course of their career as high. I discuss the principles of safe system design, the psychology of error, and advanced systems safety concepts with respect to the design of the IDAS and the future of safety in medicine. In clinical use, the IDAS saved time before and during anaesthesia, and was rated by anaesthetists as significantly safer and more useable than conventional methods of drug administration. This work supports the hypothesis that error in anaesthesia can be reduced through the systematic analysis of its causes and the implementation of appropriate countermeasure strategies. / Subscription resource available via Digital Dissertations only.

The rate of injury and death inadvertently caused by medical treatment is too high and exacts enormous human and financial costs. Each year in Britain and the United States alone, hundreds of thousands of patients are injured, ten of thousands are killed and billions of dollars are spent on additional health care due to iatrogenic harm. Health care organisations remain predominately human-centred in their approach to safety-that is, methods of avoiding error rely primarily on the resolve and vigilance of individual clinicians to avoid bad outcomes. However, this approach is becoming increasingly inadequate in the face of the steadily rising complexity of modern health care and the increasing number of procedures carried out each year. In other high-reliability organisations such as aviation and nuclear power generation, safety results not from the sheer effort of “operators”, but from in-depth analysis of problems and the removal and redesign of dangerous aspects of systems-the so-called systems approach. Here I present an evaluation of the integrated drug administration system (IDAS) as an example of the systems approach, intended to reorganise the way in which anaesthetists give drugs to improve performance and facilitate safe practice. The problem of drug error in anaesthesia is an important subset of iatrogenic harm in medicine. From the prospective study of 10806 conventional anaesthetics I define the rate of drug error in anaesthesia as one error in every 133 anaesthetics conducted-a rate five times higher than anything previously reported. In addition, anaesthetists rated the risk of harming a patient through drug error in the course of their career as high. I discuss the principles of safe system design, the psychology of error, and advanced systems safety concepts with respect to the design of the IDAS and the future of safety in medicine. In clinical use, the IDAS saved time before and during anaesthesia, and was rated by anaesthetists as significantly safer and more useable than conventional methods of drug administration. This work supports the hypothesis that error in anaesthesia can be reduced through the systematic analysis of its causes and the implementation of appropriate countermeasure strategies. / Subscription resource available via Digital Dissertations only.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Michael D. Apley / The discovery of modern antimicrobials in the early 20th century revolutionized treatment of infectious diseases. Less than 100 years later, antimicrobial resistance has become a global threat to public health. With the rise of antimicrobial resistance, the question that remains to be answered is: Can dosing regimens provide maximal clinical efficacy, yet minimize the development of antimicrobial resistance? A pharmacokinetic / pharmacodynamic approach was utilized to investigate oxytetracycline regimens that would impart efficacy while minimizing the potential for resistance development due to plasmid transfer. An in vitro pharmacodynamic model was used to quantify the response of a Pasteurella multocida isolate to two oxytetracycline dosing regimens. The PK/PD index most closely related to efficacy was the Cmax:MIC. The in vitro pharmacodynamic model was then used to investigate the effects of antimicrobial exposure on plasmid transfer. A mixed population of oxytetracycline-susceptible and resistant bacteria was exposed to two dosing regimens and plasmid transfer was quantified. When oxytetracycline concentrations exceeded the MIC of the recipient, development of resistance was suppressed. The same donor and recipient bacteria were used in an in situ swine model to validate the in vitro findings. Following surgical implantation of porous membrane straws containing the mixed bacterial population, animal subjects in the treatment groups received one of two oxytetracycline treatments. Oxytetracycline concentrations in the plasma and interstitial fluid were quantified. Plasmid transfer within the implant membranes was quantified and correlated to pharmacokinetic measures in the animal. Plasmid transfer rates in the implant membranes did not correlate to the investigated pharmacokinetic parameters. The study methodologies in this dissertation should serve as a foundation for future studies in antimicrobial pharmacokinetic/pharmacodynamic research. The results presented here show that the bacterial response to oxytetracycline can be optimized in a concentration dependent manner and that antimicrobial resistance development through plasmid transfer can be suppressed in vitro when oxytetracycline concentrations exceed the MIC of the recipient bacteria. These results suggest that a proper balance between clinical efficacy and minimizing antimicrobial resistance can be achieved for oxytetracycline through appropriate dosing regimens and drug formulations.

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

Doctor of Philosophy / Department of Anatomy and Physiology / Timothy I. Musch / Matching local muscle O[subscript]2-supply to O[subscript]2-demand during the prodigious exercise-induced metabolic challenge is achieved through coordinated mechanisms of vascular control. The unique sensitivity of ATP-sensitive potassium (K[subscript]ATP) channels to cell metabolism indicates the potential to match energetic demand to peripheral O[subscript]2 transport. The aim of this dissertation was to determine the magnitude and kinetics of the K[subscript]ATP channel contribution to vascular control during exercise in health and heart failure. It was hypothesized that K[subscript]ATP channel inhibition via glibenclamide would, in healthy rats, 1) reduce exercising skeletal muscle blood flow and vascular conductance 2) speed the fall of microvascular O[subscript]2 driving pressure (PO[subscript]2mv; set by the O[subscript]2 delivery-O[subscript]2 utilization ratio) during muscle contractions and 3) in heart failure rats, augment the PO[subscript]2mv undershoot and delay the time to reach the contracting steady-state. A total of 55 male Sprague-Dawley rats were used under control and glibenclamide conditions (5 mg kg[superscript]-1). Hindlimb muscle blood flow (radiolabelled microspheres) was determined at rest (n = 6) or during treadmill exercise (n = 6-8; 20, 40 and 60 m min[superscript]-1, 5% incline). Spinotrapezius muscle PO[subscript]2mv (phosphorescence quenching) was measured in 16 heart failure (coronary artery ligation) and 12 healthy rats and during 180 s of 1-Hz twitch contractions (~6 V). The major effects of glibenclamide were, in healthy rats, 1) a reduction in exercising hindlimb skeletal muscle blood flow with the greatest effect in predominantly oxidative muscle fiber types and at higher running speeds 2) an increased prevalence of the undershoot of PO[subscript]2mv steady-state and doubled time to reach the steady-state and 3) in heart failure rats, a reduced baseline PO[subscript]2mv, an augmented undershoot of the steady-state and time to reach steady-state and a reduction in the mean PO[subscript]2mv during contractions. These data suggest that the K[subscript]ATP channel contributes substantially to exercise-induced hyperemia and may contribute to the slowing of VO[subscript]2 kinetics given the spatial and temporal effects of glibenclamide. The K[subscript]ATP channel-mediated protection against a severe O[subscript]2-delivery to O[subscript]2-utilization mismatch at the onset of contractions raises serious concerns for sulphonylurea treatment in diabetes which is likely to cause perturbations of [metabolite] and compromise exercise tolerance.