Lipid peroxidation is a key contributor to the pathophysiology of traumatic brain injury (TBI). Traditional antioxidant therapies are intended to scavenge the free radicals responsible for either the initiation or propagation of lipid peroxidation (LP). However, targeting free radicals after TBI is difficult as they rapidly react with other cellular macromolecules, and thus has a limited post-injury time window in which they may be intercepted by a radical scavenging agent. In contrast, our laboratory has begun testing an antioxidant approach that scavenges the final stages of LP i.e. formation of carbonyl-containing breakdown products. By scavenging breakdown products such as the highly reactive and neurotoxic aldehydes (often referred to as “carbonyls”) 4-hydroxynonenal (4-HNE) and acrolein (ACR), we are able to prevent the covalent modification of cellular proteins that are largely responsible for posttraumatic neurodegeneration. Without intervention, carbonyl additions render cellular proteins non-functional which initiates the loss of ionic homeostasis, mitochondrial failure, and subsequent neuronal death. Phenelzine (PZ) is an FDA-approved monoamine oxidase (MAO) inhibitor traditionally used for the treatment of depression. Phenelzine also possesses a hydrazine functional group capable of covalently binding neurotoxic carbonyls. The hypothesis of this dissertation is that carbonyl scavenging with PZ will exert an antioxidant neuroprotective effect in the traumatically injured rat brain mechanistically related to PZ’s hydrazine moiety reacting with the lipid peroxidation (LP)-derived reactive aldehydes 4-hydroxynonenal (4-HNE) and acrolein (ACR). Data from our ex vivo experiments demonstrate that the exogenous application of 4-HNE or ACR significantly reduced respiratory function and increased markers of oxidative damage in isolated non-injured rat cortical mitochondria, whereas PZ pre-treatment significantly prevented mitochondrial dysfunction and oxidative modification of mitochondrial proteins in a concentration-related manner. Additionally, PZ’s neuroprotective scavenging mechanism was confirmed to require the presence of a hydrazine moiety based on experiments with a structurally similar MAO inhibitor, pargyline, which lacks the hydrazine group and did not protect the isolated mitochondria from 4-HNE and ACR. Our in vivo work demonstrates that subcutaneous injections of PZ following TBI in the rat are able to significantly protect brain mitochondrial respiratory function, decrease markers of oxidative damage, protect mitochondrial calcium buffering capacity, and increase cortical tissue sparing without decreasing neuronal cytoskeletal spectrin degradation. These results confirm that PZ is capable of protecting mitochondrial function and providing neuroprotection after experimental TBI related to scavenging of neurotoxic LP degradation products.

The most recognized risk associated with the HeartMate II is thrombosis. In the presence of developing clot, the HeartMate II Display Monitor and System Controller senses a decrease in pump flow and is accompanied by audible and visual alarms when flow rates drop below a certain threshold; however, when flow is completely inhibited, the Display Monitor and System Controller both fail to indicate that flow has reduced to zero and does not produce any corresponding alarms. To test the efficacy of the HeartMate II alarms, the Donavan Mock was used to simulate the hemodynamics of a typical heart failure patient. The hemodynamics were then improved by the addition of the HeartMate II LVAS. Partially occluding the inflow and outflow of the HeartMate II did display changes in flow and presented with alarms when appropriate; however, complete occlusions of the device failed to produce any alarms or accurate changes in flow.

Many common cancers have a predisposition for bone metastasis. Tumor occupation of bone is both destructive and a source of debilitating pain in cancer patients. As a result, cancer-induced bone pain (CIBP) is the single most common form of clinical cancer pain. Opioids remain the golden standard for the management of CIBP; however, >30% of cancer patients do not experience adequate pain relief with opioids. Furthermore, clinical reports have suggested that opioids can exacerbate bone loss and increase the likelihood of skeletal-related events. To date, there is no known direct mechanism for opioid-induced bone loss (OIBL). We hypothesized that opioid off-target activation of toll-like receptor 4 (TLR4), an innate immune receptor that is expressed in bone, mediates an increase bone loss and associated CIBP. In the 66.1-BALB/cfC3H murine model of breast cancer bone metastasis, TLR4 expression is upregulated in tumor-burdened bone. Chronic morphine treatment exacerbated spontaneous and evoked pain behaviors in a manner paralleled by bone loss: we identified an increase in spontaneous fracture and osteolysis markers including serum collagen-type I (CTX) and intramedullary receptor activator of nuclear κ-B ligand (RANKL). Administration of (+)naloxone, a non-opioid TLR4 antagonist, attenuated both exacerbation of CIBP and morphine-induced osteolytic changes in vivo. Morphine did not alter tumor burden in vivo or tumor cell growth in vitro. Importantly, morphine produced the in vitro differentiation and activation of osteoclasts in a dose-dependent manner that was reversible with (+)naloxone, suggesting that morphine may contribute directly to osteolytic activation. To improve opioid management of CIBP, we then posited and evaluated three novel adjunct therapeutic targets: cannabinoid receptor-2, adenosine 3 receptor and sphingosine-1-phosphate receptor 1. These pharmacological targets were identified as having a multiplicity of anti-cancer, osteoprotective and/or neuroprotective effects in addition to analgesic efficacy in chronic pain. Targets were tested in the 66.1-BALB/cfC3H model of CIBP and demonstrated to have stand-alone efficacy as antinociceptive agents. Taken together, this work provides a cautionary evaluation of opioid therapy in cancer-induced bone pain and seeks to mitigate opioid side effects through the identification of innovative adjunct therapies that can ultimately improve quality of life in patients suffering from cancer pain.

Many common cancers, including breast, prostate and lung cancers, have a propensity to metastasize to bone. Although these cancers go undetected in their native tissues, bone metastases often produce excruciating pain, the etiology of which is poorly understood. Cancer-induced bone pain (CIBP) is not well-controlled with existing medications, severely compromising patient quality of life. While CIBP is multifaceted, increased level of the excitatory neurotransmitter glutamate in the bone-tumor microenvironment may contribute to the pain state. Here, we demonstrate for the first time a relationship between reactive oxygen/nitrogen species, glutamate in the bone-tumor microenvironment and pain behaviors. The murine mammary adenocarcinoma cell line 66.1 is found to release glutamate via the cystine/glutamate antiporter system xc⁻. In a syngeneic model of breast CIBP in which 66.1 cells are inoculated into the femur intramedullary space, administration of sulfasalazine, an established system xc⁻ inhibitor and anti-inflammatory agent, reduces femur glutamate level and attenuates CIBP-related behaviors. Peroxynitrite, a reactive nitrogen species known to be generated in breast tumors, is shown to drive 66.1 system xc⁻ functional expression and tumor cell glutamate release. The elimination of peroxynitrite with the redox modulators FeTMPyP or SRI10 not only modulates tumor cell system xc⁻ functional expression in vitro and in vivo, significantly altering glutamate levels, but also assuages CIBP. In sum, we demonstrate that pharmacological inhibition of system xc⁻ transport attenuates CIBP-related behaviors. These data support a role for tumor-derived glutamate in CIBP and validate system xc⁻ an analgesic target in this pain state.

Foram estimados custos diretos de diferentes opções de tratamento para artrite reumatóide com base em dados de eficácia obtidos por revisão de literatura. O modelo analítico de tomada de decisão para o tratamento e desfechos durante um período de 48 meses baseado no modelo de Markov foi fundamentado em protocolos clínicos recomendados pela Sociedade Brasileira de Reumatologia, com esquemas alternativos para cinco ciclos de tratamento durante o período de quatro anos. O paciente pode permanecer em algumas das etapas ou migrar entre elas, de acordo com a resposta à terapia. Foram analisados custos diretos (medicamentos, materiais médico-hospitalares e exames laboratoriais). As doses dos medicamentos e o monitoramento foram baseados no Consenso Brasileiro para o Diagnóstico e Tratamento da Artrite Reumatóide, considerando-se o peso médio do paciente de 70 kg. Na comparação entre o custo das cinco etapas de tratamento os resultados mostram que a etapa que usa o medicamento infliximabe tem um custo superior às outras, este causado pelo preço de aquisição do medicamento. O custo do monitoramento tem impacto nas etapas que utilizam os medicamentos com preço de aquisição menor. O ciclo com a menor razão custo/efetividade foi o 1 (respondedor ao MTX). Aquele que usa o biológico desde o início do tratamento é responsável pela maior razão de custo/efetividade. A variação do custo entre os outros ciclos não foi muito grande, mas todas devem ser consideradas quando da escolha do prescritor. Estes resultados se mostraram robustos com a análise de sensibilidade. / The costs of the different rheumatoid arthritis therapy options were estimated and they were compared by cos/efficacy reason, through the development of an analytical model for a 48-month period. For the calculation of these costs, it was developed an analytical decision model based on the Markov Analysis, where five different therapy stages were elaborated based on clinical protocols recommended by the Brazilian Society of Rheumatology, and then five therapy cycles, where patients may continue in some of those stages or shift between them according to the therapy response, for a time horizon of 4 years. Only direct costs with drugs, medical-hospital materials for drug administration and laboratory examinations required for the patient monitoring because of the use of some drugs, were comprised in the analyzed data. The doses of drugs and the monitoring were based on the Brazilian Consensus for the Diagnosis and Treatment of Rheumatoid Arthritis, considering the average patient weight of 70 kg. When comparing the cost of the five treatment stages, the results show that the stage in which the infleximabe drug is used has a cost higher than that of the other stages and that the impact is caused by the drug acquisition price. The monitoring cost impacts the stages that employ drugs with smaller acquisition price. The cost-effectiveness cycle was the number 1 (good response with MTX). When we use de biologic in the beginning, it is responsible for the higher cost-effectiveness reason. The cost variation between cycles 2, 3 and 4 was smaller, but they have to be considered for the decision maker. The sensitivity analysis confirm this results.

Severe congenital heart disease (CHD) is diagnosed in the United States 147.4 times per 100,000 live births, excluding still births and abortions^1. With the advancement of diagnostic methods, prenatal care, and screening modalities, the total CHD birth prevalence has increased substantially^2. In turn, this increases the number of cardiac surgery cases. With the advancement of technology and cardiac surgery, smaller and younger patients are undergoing more complex cardiac procedures that involve cardiopulmonary bypass (CPB). Neonates and infants undergoing CPB are susceptible to adverse effects of CPB on the coagulation cascade due to their smaller weight and hematologic immaturity^3,4. In addition to these physiological issues in neonates and infants, CPB decreases circulating coagulation factors and anti- thrombin III levels to 50% and platelet counts to 70%^5, which can contribute to the post-operative bleeding.During CPB, neonates' and infants' coagulation factors become extremely diluted causing multiple coagulation defects^6. Optimizing the CPB circuit volume and the use of anti-fibrinolytic, packed red blood cells (pRBCs), platelets, cryoprecipitate, and ultrafiltration are among the most widely used methods in preserving and aiding coagulation factors^3,7-9. Another method of improving hemodilution-related coagulation dysfunction bleeding is by transfusing Fresh Frozen Plasma (FFP)^10. However, there are only a small number of articles focusing on the effect of FFP in post-operative bleeding in neonates and infants following complex cardiac surgery with CPB. I postulate that adding FFP during CPB will lower the possibility of patients to experience post-operational bleeding, thus, shortening their length of stay (LOS).

PART I Background: For over forty years, depolarizing, hyperkalemic cardioplegia solutions have served as the standard of care for cardiac surgery. While effective in inducing cardiac arrest, potassium-based solutions are associated with an array of negative consequences, such as coagulopathies, conduction dysfunction, inflammation, coronary vasoconstriction, myocardial edema, and ischemic injury. Adenosine-lidocaine-magnesium, a non-depolarizing, non-potassium-containing solution, has recently entered the clinical arena. Animal research suggests that this agent may provide a method of diastolic arrest that is as effective as potassium-based cardioplegia but with improved protective benefits.Purpose: The aim is to assess the safety and efficacy of adenosine-lidocaine-magnesium as a cardioplegia solution in terms of overall patient outcomes.Methodology: In June 2014, Banner University Medical Center Tucson became the first American institution to adopt the use of PolarShot (ALM)--adenosine-lidocaine-magnesium - as a cardioplegia solution. This one-year, retrospective study compares patients receiving adenosine-lidocaine-magnesium to those receiving high-potassium/low-potassium cardioplegia during adult cardiac surgery. Cases compared in this study include isolated coronary artery bypass, isolated aortic/mitral valve repair/replacement, and combination coronary artery bypass/valve replacement surgery only. A propensity-weighted regression model was used for analysis to determine whether or not cardioplegia treatment affected clinical outcome. To assess overall clinical outcome, major morbidity and mortality and post-procedural length of stay were chosen as primary endpoints. Results: In terms of treatment (adenosine-magnesium-lidocaine vs. high-potassium/low-potassium), no statistically significant difference was found between groups in regard to major morbidity and mortality event occurrences nor was a significant difference found between post-procedural length of stay. Discussion: After comparing postoperative outcomes between cardioplegia treatment groups, PolarShot (ALM) cardioplegia produced postoperative outcomes that were statistically similar to those of high-potassium/low-potassium cardioplegia. The confidence in these results is limited by low case volume, surgical case variability, and retrospective nature of this study. Conclusion: According to this propensity-weighted regression model, PolarShot (ALM) cardioplegia appears to be a safe and effective alternative to traditional potassium-based cardioplegia for the purpose of adult cardiac surgery. More research, including prospective randomized trials, is necessary to confirm or deny the findings of this study. PART II Background: Historically, surgical cardioplegia compounding was accomplished by filling patient-tailored prescriptions on-demand. Modern day compounding has become a manufacturing process to improve quality and accommodate physician demand. Additionally, sterile compounding standards have become more stringent, further necessitating a standardized compounding approach. In 2013, scrutiny of sterile drug compounding increased with passage of the Drug Quality and Security Act (DQSA) and subsequent Federal Drug Administration oversight. This federal mandate requires all compounded sterile preparations distributed by 503B Outsourcing Facilities be tested for potency, stability, and sterility. To accomplish this, compounders must significantly reduce batched formula variability. Purpose: A review of 2014 sales data from a large 503B outsourcing facility and cardioplegia compounder will be conducted. The study will identify solution differences and detail its findings. The aim of this study is to assess cardioplegia variability on a national level. Methodology: Results will be summarized by cardioplegia strategy (Buckberg, high-potassium/low-potassium, crystalloid, del Nido, Adenocaine, and microplegia), dilution strategy (4:1 blood-crystalloid, 8:1 blood-crystalloid, 1:4 crystalloid-blood, all-blood, and all-crystalloid), formula constituents (base solutions, additives, buffers), potassium concentrations. Any observed patterns in formula usage will also be reported, geographical or otherwise. Results / Discussion: Based on institutional use, high-potassium/low-potassium (two-solution) multidose strategy was the most common. Based on solutions ordered, the most common cardioplegia ingredient was potassium chloride, present in almost ninety percent (89.64%) of all units sold. After looking at potassium content, extensive variability was noted in terms of potassium added to the bag (undiluted) and potassium to-be delivered (post-dilutional). Additionally, unique solution formulations identified in multiple institutions were often found in neighboring states or within a single state. Conclusion: The results of this analysis illustrate the extent to cardioplegia formula variability nationwide. Variability exists in both methodology and formulation on a state-to-state, institution-to-institution, even across-single-institution basis. This formula customization appears to be institution- and surgeon-specific, suggesting empirical influence in formula adaptation. Formula standardization may be necessary to combat the compounded issue of formula customization moving forward.

Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a short, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of experiments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines. Stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with microglial activation. Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of sigma receptor activation and HUCBC therapy in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to oxygen glucose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain over time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brain in appreciable numbers at any timepoint. IL10 and IFN?; levels were found to significantly increase by 96hours post MCAO. This increase in IL10 and IFNγ expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the relative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatments to modulate the peripheral immune systems response to injury.

The prevalence of antibiotic resistant bacteria has made the choices for topical treatments for patients who experience burns wounds extremely limited. The Staphylococcus genus is naturally occurring in and on the human body but can become harmful once it enters the bloodstream. A novel antimicrobial gel has been shown by our laboratory to inhibit both the planktonic growth and biofilm formation of Staphylococcus aureus in previous studies. The antimicrobial gel is made of seven natural compounds including antioxidants (vitamin C and E). We wanted to examine the effects of the antimicrobial gel on numerous other Staphylococcal species because it is prevalent on the body and becomes harmful when the immune system is compromised. The species tested were Staphylococcus capitis, Staphylococcus epidermidis, and Staphylococcus saprophyticus. A planktonic broth challenge test, biofilm attachment test, and biofilm maturation test were all performed in order to test this hypothesis. These tests showed a significant inhibition of the Staphylococcus species as a result of the effects of the antimicrobial gel. The antimicrobial gel inhibited the attachment, maturation, and growth of Staphylococcus colonies in a 10% antimicrobial gel solution. The antimicrobial gel shows promise as an option in treating burn patients and should be considered in further testing for its uses in other areas of medicine.

<p>Allosteric modulators are a newly emerging concept in the field of drug discovery which have shown remarkable success in their ability to alter G-protein coupled receptor (GPCR) activity in a precise and subtle manner. A GPCR of particular interest for allosteric targeting is the dopamine D2 receptor. This receptor has repeatedly been implicated in the etiology of complex neurological and neuropsychiatric disorders including Parkinson’s disease and schizophrenia. Previous studies from our lab have effectively developed allosteric modulators targeting the D2 receptor based on the pharmacophore of the endogenous tripeptide L-prolyl-L-leucyl-glycinamide (PLG). PLG and its potent peptidomimetics, particularly 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) (PCT/CA2011/000968), have shown robust preclinical efficacy in treating models of Parkinson’s disease, depression, tardive dyskinesia and schizophrenia. These ligands modulate agonist binding to the D2 receptor in a biphasic manner, although further information on their mechanisms of action are currently unknown. Therefore, the overarching objective of this thesis was to enhance our knowledge on the mechanisms of action of the promising D2 allosteric ligands PLG and PAOPA. Results of the studies presented here show PAOPA to cause significant upregulation of D2 regulatory proteins and downstream signaling kinases, as well as cause an increase in D2 internalization. Additionally, the PLG allosteric binding site was narrowed down to be localized between transmembrane domains 5 and 6 on the D2 receptor. The collection of work presented here enhance our understanding of the mechanisms of action of the potentially therapeutic D2 allosteric ligands PLG and PAOPA, progressing them closer to helping clinically affected populations. The findings of these studies prove globally significant as they highlight the diverse cellular pathways which could be affected by allosteric modulators, and bring to light the importance of studying these candidate ligands for eventual improvements in the treatment of human health.</p> / Doctor of Philosophy (Medical Science)