BACKGROUND: Hispanics make up one of the largest ethnic groups in the United States, and this traditionally underrepresented group faces medical and health disparities that make their disease burden disproportionally high. For stroke, this burden presents as increased incidence and increase negative outcomes. This burden is partially due to Hispanics taking longer to present to the hospital with their stroke symptoms, resulting in no rt-PA administration. LITERATURE REVIEW: The research shows that this increased time to presentation is due to poor understanding of the United States healthcare system, language barriers, and decreased access to healthcare. Poor understanding of the medical system stems from a higher level of fear and mistrust. The language barrier for Spanish-speaking Hispanics results from a lack of interpreter access as well as poor quality interpreters. Lastly, the majority of healthcare access issues result from higher rates of uninsured. However, there is limited data looking at what barriers Hispanic stroke patients cite as causing delays in stroke care. PROJECT AND METHODS: This study proposes to identify and analyze the barriers reported by Hispanics with a past medical history of stroke. In order to do so, patients will engage in open-ended interviews that will aim to cover barriers discussed in the literature. After the interviews, the qualitative data will undergo theme analysis, and then the themes will be organized via frequency. CONCLUSION: The advantage of this study design is the ability to more accurately identify the barriers in placed. The study design is not without limitations, which include selection bias, interviewer bias, and recall bias. SIGNIFICANCE: Once the study allows for accurate identification of the barriers, more effective Public Health campaigns can be created to target the Hispanic population. These more precise campaigns will work to lower time delay in acute stroke care thus increasing positive outcomes.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, which is characterized by impaired differentiation and uncontrollable proliferation of myeloid progenitor cells in the bone marrow. One-third of patients with AML have a mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. The FLT3 mutation most commonly occurs as an internal tandem duplication (ITD) and is associated with a poor prognosis. Patients with FLT3-ITD mutated AML are able to achieve complete remission at the same rate as patients with wild-type FLT3 but face a higher relapse rate and reduced overall survival. The standard treatment for FLT3-ITD mutated AML has been intensive chemotherapy with or without hematopoietic stem cell transplantation (HSCT). In recent years, there has been an emergence of various targeted first and second generation FLT3 inhibitors. The first-generation inhibitors lack specificity for FLT3 and include sunitinib, sorafenib, lestaurtinib and midostaurin. The second-generation inhibitors potently target FLT3 and include quizartinib, crenolanib and gilteritinib. Currently, midostaurin is the only FLT3 inhibitor that has been approved by the FDA to treat patients with AML. A barrier in AML treatment is drug resistance, which can lead to either a lack of efficacy or loss of prior efficacy. There are a variety of intrinsic and extrinsic mechanisms that underlie the acquisition of resistance. This literature review will explore the clinical implications and limitations of the treatment options for patients with FLT3- ITD mutated AML and discuss the negative impact that resistance has on efficacy.

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease with no known origin. Scar tissue continues to build up in the lungs diminishing pulmonary function. Without successful treatment, patients with IPF have a mean survival of 5 years after diagnosis. Though the exact etiology of IPF is unknown, studies have established a role of telomeres in 25% or more of familial or sporadic cases. Mutations in the genes that encode for the components of telomerase have been implicated as the cause of the telomere dysfunction seen in these cases. The mutations examined below include TERT, TERC, RTEL1, TINF2, and PARN. With this link in mind, the available and the researched treatment plans are discussed. Of the discussed therapies, the researched treatment options that target the telomere-specific cases of IPF are a synthetic sex hormone (danazol), stem cell therapy, and a small molecule activator (GRN510). The remaining treatment plans discussed target either the direct cause of symptoms or the symptoms themselves. These studied options include N-acetylcysteine, prednisone, azathioprine, pirfenidone, nintedanib (with and without statins), and low-dose inhaled carbon monoxide. The treatment options with the favored positive results are danazol and nintedanib.

Existing data have demonstrated that adequate levels of vitamin D are not only important for bone health but also support the development and maintenance of the immune system. As such, vitamin D sufficiency may be important in controlling the mucosal inflammation observed in patients with inflammatory bowel disease (IBD). Dietary intake of sufficient vitamin D can be daunting for many patients, and compliance with daily vitamin D supplements a challenge. As such, the goal of this project is to assess the safety and efficacy of high-dose oral interval vitamin D therapy in children and young adults with IBD. We chose to study this hypothesis in patients treated with Remicade (infliximab), which necessitates regular (monthly or bimonthly) office visits. Patients are eligible if they are being treated with Remicade, have a documented 25-hydroxyvitamin D less than 30.0ng/mL within 8 weeks of enrollment, and do not have a history of an underlying renal or liver disease that would complicate the assessment of potential adverse effects of study therapy.

The United States spends more than any other nation on healthcare by an exceedingly significant amount. The United States also ranks very poorly on health outcomes when compared to other economically developed nations. The reason why the United States spends so much more than other nations is because they spend more in all categories of healthcare. Hospital admissions and overnight stays are more expensive, medical staff salaries are higher than in other nations, procedures and treatments are ordered more frequently and cost more than in other nations, prescription drugs prices are multiple times greater in the U.S., the multi-payer private and public insurance payment schemes are time consuming and complex, and administrative costs are significantly higher in the U.S. than in other countries. These complexities and high costs create inefficiencies in America's healthcare system that can interfere with the quality of care provided. The major reasons why the United States is lacking in health outcome rankings can be attributed to an underdeveloped primary care sector and an underfunded social services sector, which make it difficult to coordinate care and practice preventative medicine. America's history of uninsured citizens lacked access to care so the U.S. saw repeats of emergency admissions and expensive hospitalizations for chronic conditions. These problems are preventable with a more robust primary care sector, increased access to care, and advocating public health awareness. Current reforms such as the Affordable Care Act are making strides in the positive direction by creating Accountable Care Organizations, increasing access to care, and regulating an online insurance marketplace. However, continued research and assessments of these new methods of delivering care are necessary in the near future to see if pilot studies can be scaled up on a national level.

BACKGROUND: Emerging evidence suggests that the population of children assisted with respiratory medical equipment and supplies (RMES) is increasing in size and is having a substantial impact on families, providers, and the health system. Little is known on a population level about children who use RMES to compensate for a deficit in the ability to breathe. This study addressed these gaps by assessing (1) the characteristics of children in Medicaid using RMES and (2) how the use of RMES influences healthcare utilization and spending across the care continuum. METHODS: A retrospective cohort analysis of 11,306 children using and 21,192 children not using RMES [propensity matched by age and complex chronic conditions (CCC)] who were age 0-to-21 years and continuously enrolled in Medicaid in 2013 from 10 states in the Truven Health Medicaid MarketScan Database. RMES use at home (not counting acute use in a clinic, emergency department, or hospital) was identified with Healthcare Common Procedure Coding System (HCPCS), billed by medical supply companies, and International Classification of Diseases (ICD9) codes, billed by clinicians and hospitals. RMES included oxygen, suctioning, apnea monitor, CPAP/BiPAP, tracheostomy, ventilator, cough assist, and vest. We regressed RMES use on total annual per member per year (PMPY) Medicaid payments, adjusting for enrollment reason, gender, age, race/ethnicity, and number of chronic conditions. RESULTS: Of children using RMES at home, 5% were identified with ICD9 only, 80% with HCPCS only, and 15% with ICD9 and HCPCS. Most (87%) children using RMES had a chronic condition (of any complexity); 71% had a complex chronic condition. Neuromuscular (32%) was the most common CCC. RMES usage among children included oxygen (47%), suctioning (28%), apnea monitor (23%), CPAP/BiPAP (22%), tracheostomy (17%), ventilator (8%), cough assist (5%), and vest (4%). PMPY payments in propensity-matched children using vs. not using RMES were $45,892 vs. $15,036, p<0.001. In adjusted analysis, payment increased significantly (p<.001) with use of CPAP/BiPAP (+$1,117), oxygen (+$3,525), cough assist (+$6,342), suctioning (+$8,569), tracheostomy (+$11,977), vest (+$11,999), apnea monitor (+$13,747), and ventilator (+$32,323). Of children using RMES, most payments were for hospitalization (57%), specialty care (24%), and medications (6%); <3% was for RMES or home nursing. CONCLUSION: RMES use can identify additional projected healthcare costs in children beyond consideration of chronic diagnoses. Because most of the cost of using RMES is due to inpatient and specialty care rather than the equipment itself, RMES may indicate – broadly - medical fragility and increased healthcare needs. Population health initiatives of children with medical complexity may benefit from consideration of RMES use in risk assessment for healthcare cost.

Osteoarthritis is a leading cause of disability for which there is no cure. We have discovered that the multidomain signalling protein Agrin, most commonly known for its requirement at the neuromuscular junction, strongly promotes chondrocyte differentiation and cartilage formation in vivo. Agrin is expressed in normal cartilage but absent in osteoarthritis. In vitro, Agrin knockdown resulted in the downregulation of the cartilage transcription factor SOX9 and other cartilage-specific extracellular matrix molecules. Conversely, the addition of exogenous Agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In contrast to other biological contexts where Agrin signalling requires the interaction with either LRP4 or α-dystroglycan, chondrocytes require the presence of both receptors. Our results identify Agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

ORAI and STIM genes are recently identified store-operated calcium channel molecules that play important roles in human physiology. In this thesis, the effects of oxidative stress conditions including high glucose, homocysteine and H₂O₂ on the expression of ORAI and STIM, Ca²⁺ influx, ORAI channel activity and potential underlying mechanisms were investigated using cell models and in vivo tissue samples from diabetic patients and mice. ORAI1-3 and STIM1-2 were detected in vascular endothelial cells and smooth muscle cells using RT-PCR, western blotting and immunostaining. Their expression was upregulated by chronic treatment with high glucose in cell models. The upregulation was also observed in human aorta from Type 2 diabetic patients and kidney tissues from streptozotocin-induced and Akita Type 1 diabetic mouse models. The high glucose-induced gene upregulation was prevented by the calcineurin inhibitor cyclosporin A and store-operated channel blocker diethylstilbestrol. H₂O₂ also upregulated ORAI1-3 and STIM1-2, however, homocysteine increased STIM1-2 expression, but downregulated ORAI1-3. Ca²⁺ influx and ORAI channel activity were investigated using Ca²⁺ imaging and whole-cell patch clamp. Chronic treatment with high glucose enhanced storeoperated Ca²⁺ influx in endothelial cells, but there was no effect if treated acutely. In HEK-293 cells overexpressing STIM1/ORAI1-3, high glucose had no acute effect on ORAI1-3 currents, but homocysteine decreased the currents. The cytosolic STIM1 movement was monitored by live-cell fluorescence imaging. Oxidative stress did not change STIM1-EYFP translocation and clustering after Ca²⁺ store-depletion. The effect of hyperosmolarity on STIM and ORAI expression and channel activity was also investigated. Hyperosmolarity inhibited ORAI1-3 currents and downregulated ORAI1-3 and STIM1-2 gene expression, but did not alter cytosolic STIM1-EYFP translocation. It is concluded that store-operated channel molecules, STIMs and ORAIs, are new proteins regulated by oxidative stress, especially in diabetes, which may provide a novel concept for the abnormality of Ca²⁺ homeostasis in blood vessels from patients with diabetes.

Electron transportation (ET) coupled with oxidative phosphorylation (OXPHOS) in the mitochondria produces limited, physiologic levels of reactive oxygen species (ROS). While this process is adaptive under normal conditions, excessive mitochondrial oxidative stress (mitoOS) has been correlated with a number of diseases, including atherosclerotic vascular disease in humans. However, definitive evidence of causation and cell-specific pro-atherogenic mechanisms of mitoOS require further investigation. The high level of interest in this topic, the human relevance, and the potential therapeutic implications prompted us to explore causation and mechanism with a focus on the key inflammatory cell type in atherosclerosis, the macrophage (Chapter 2). For this purpose, we used a recently described model, the mitochondrial catalase (mCAT) transgenic mouse, that decreases mitoOS in vivo. Normally, glutathione perioxidase is the endogenous mitochondrial enzyme that catalyzes the reduction of H2O2 and prevents its conversion into the most detrimental ROS hydroxyl nitrites. Catalase can carry out this role in peroxisomes, where it is exclusively located. The mCAT transgenic mouse expresses human catalase with a mitochondrial matrix-targeting motif, which quenches mitoOS and protects against mitoOS-induced damage. To focus on myeloid-derived cells in atherosclerosis, we used two strategies: transplantation of mCAT transgenic bone marrow cells into atheroprone Ldlr-/- mice and crossing Ldlr-/- mice with an mCATfl/-LysMCre model that expresses mCAT only in lysozyme M-expressing cells, notably differentiated macrophages. After 8 wk western type diet (WD) feeding, both models demonstrated evidence of decreased mitoOS in lesional macrophages, decreased atherosclerosis, suppression of Ly6chi monocyte infiltration, and lower levels of the monocyte chemotactic protein-1 (MCP-1). The decrease in lesional MCP-1 was associated with suppression of other markers of inflammation (iNOS and TNF-α) and with decreased phosphorylation of the critical transcription factor RelA (NF-κB p65), indicating decreased activation of the pro-inflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed MCP-1 expression by decreasing activation of the Iκ-kinase (IKK) - NF-κB (RelA) pathway. Taken together, we conclude that MitoOS in lesional macrophages amplifies early atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the early progression of atherosclerosis. The mitochondrial dynamic processes of fission and fusion influence and integrate with multiple physiologic and pathophysiologic processes. Mitochondrial fusion/fission dysregulation has been implicated in atherosclerosis, but little is known about the role of myeloid cell specific mitochondrial dynamics in the progression of atherosclerosis. Dynamin related protein 1(DRP1), a cytosolic GTPase family member, is one of the molecules that mediate mitochondrial fission. In the second part of this thesis (Chapter 3), we used western diet-fed Drp1fl/fl LysmCre+/-Ldlr-/-mice to determine the role of Mφ mitochondrial fission in both early atherogenesis and advanced atherosclerosis. Our data thus far show that: (1) Mitochondria in lesional Mφs are elongated in Drp1fl/fl LysmCre+/-Ldlr-/ mice by transmission electron microscopy (TEM) analysis; (2) Suppression of Mφ mitochondrial fission does not affect early atherogenesis; (3) Inhibition of Mφ mitochondrial fission leads to a striking increase of necrotic core area and the accumulation of apoptotic cells, which are likely due to the defective phagocytic clearance of apoptotic cells (efferocytosis) in the advanced stage of atherosclerosis in vivo; (4) DRP1-deficient Mφs are defective in efferocytosis in vitro and in vivo. (5) The phagocytic deficiency in DRP1-deficient Mφs is associated with a reduced level of uncoupling protein 2 (UCP2), a mitochondria protein required for continuous uptake and clearance of dead cells in phagocytes. We conclude that DRP1-mediated mitochondrial fission in Mφs promotes the clearance of apoptotic cells and thereby blocks necrotic core formation in advanced atherosclerosis. This study indicates that mitochondrial fusion/fission could be a new therapeutic target to stabilize the advanced plaques and prevent acute atherothrombosis in humans. In terms of mechanism, we hypothesize that mitochondrial fission stabilizes UCP2 in the inner membrane of mitochondria. Further studies are required to elucidate how DRP1-UCP2 pathway maintains the efferocytosis capability in phagocytosis. In summary, my thesis studies have revealed the pathological significance of macrophage mitoOS in early atherogenesis and a novel link of mitochondria dynamics to macrophage phagocytosis in the setting of advanced atherosclerosis.

BACKGROUND: Endothelial cells line the inner vessels of all organ systems. Endothelial cells share many similarities with cancer cells from their preference for glucose consumption even in the presence of oxygen availability (Warburg effect) to their incredibly fast proliferation through the mTOR pathway. Both cell types also rely heavily on glutamine, in addition to glucose, to maintain their metabolic activities. Glutamine is the most abundant free amino acid in the body, making it readily available to both endothelial and tumor cells as a source of carbons for the synthesis of biomass. Due to these overlapping similarities, there has become an interest in understanding how mutations of cells can allow them to survive in the absence of glutamine. A greater understanding of the mechanism of action from down regulation of specific genes can lead to many clinical applications. OBJECTIVE: To understand if down regulation of specific mTOR genes can affect the viability and proliferation of endothelial cells in glutamine deficient and glutamine supplemented media, respectively. METHODS: siRNA transfection was used as a secondary method to knock down specific genes in endothelial cell lines (ECFC and HUVEC). QPCR was used to validate knockdown efficiency. Cell progression was documented both visually and through cell counting over a long period of time. Cells were placed in both glutamine deficient and glutamine supplemented media to monitor mTOR activity and viability. RESULTS: Of all the genes tested, endothelial cells with CS, SLC7A11, WDR59, and WDR24 knockdowns were shown to have greater viability in glutamine deficient media than the controls. They were also shown to have suppressed proliferative behavior in complete media. CONCLUSIONS: Despite initial CRISPR screening results, not all of the knockdown genes selected from the list were able to survive in glutamine deficient conditions. However, we were able to show that knockdown of CS and SLC7A11 demonstrate similar viability in endothelial cells as previously published studies in cancer cells in glutamine deficient media. Interestingly, both GATOR2 complex genes (WDR59 and WDR24), which act as positive and negative regulators of the mTORC1 pathway via amino acid sensing, were able to survive without glutamine. Future studies could be done to understand the mechanisms behind their survival. / 2020-07-03T00:00:00Z