Transforming growth factor-beta (TGF-β) signaling in smooth muscle cells (SMC) contributes to normal vascular development but may also play a significant role in the progression of aortic disease. Accordingly, TGF-β signaling is emerging as a therapeutic target, with strategies to block TGF-β signaling proposed as a means of preventing human aortic disease, including aortic aneurysms and dissections. However, because TGF-β signaling contributes to normal vascular development, blockade of TGF-β signaling could be harmful. We hypothesized that elimination of TGF-β signaling in aortic SMC of adult mice would cause aortic disease. We used inducible Cre-lox recombination technology to knock out Tgfbr2 in adult murine SMC. Tgfbr2 encodes TGF-β receptor II, believed to be essential for all TGF-β signaling. Mice were euthanized 4-14 weeks after SMC Tgfbr2 knockout, and effects of Tgfbr2 knockout (compared to control mice) were assessed by gross observation of aortas as well as sectioning and staining of aortic tissue. We found intramural hematomas, penetrating aortic ulcers, aortic dissection, and aortic dilation as well as significant thickening of the medial wall and adventitia in the knockout mice. Our data strongly suggest that loss of TGF-β signaling in SMC has a negative effect on aortic health and support a cautious approach in pursuing blockade of TGF-β signaling as a therapeutic strategy.

Chronic allograft injury has replaced acute rejection as the leading cause of renal graft failure following transplantation. Graft failure is of special importance to pediatric patients since they typically require multiple grafts in their lifetime and therefore will benefit from graft life extension (Smith et al, 2010). A relationship has been demonstrated between subclinical Cytomegalovirus (CMV) and subclinical Epstein Barr Virus (EBV) and chronic allograft injury previously. This relationship could lead to insight into the mechanism of chronic allograft injury. To characterize the relationship between subclinical infection and chronic allograft injury we measured T-cell specific response to subclinical CMV and EBV infections. A panel measuring three aspects of T-cell response: activation, memory, and cytokine response, was proposed and validated. T-cell response was quantified using flow cytometry on stimulated whole blood samples from study participants in an intracellular cytokine staining assay. Analysis of T-cell activation, memory, and cytokine production was done using a Boolean gating strategy on FlowJo software. Twenty-six patients are currently enrolled, with whole blood samples being collected every six months over the course of twenty-four months. We are in the process of banking, processing, and analyzing the samples. After all of the patient samples have been analyzed for T-cell memory, activation, and cytokine production, we will quantify the relationship between T-cell response and subclinical viremia. This data will be compiled in conjunction with the GFR, kidney biopsies, and CMV/EBV PCR data to create a longitudinal history, allowing us to understand how CMV and EBV viremia contributes to chronic allograft injury.

Macrophage have roles in promoting and resolving inflammatory responses in the lung, and they do this in part by their ability to polarize into M1 or M2 cells depending on environmental signals. Lipopolysaccharide (LPS), a microbial initiator of inflammation, activates macrophages to the M1 polarized state, which drives the inflammatory response. M2 cells, which can be induced by cytokines IL4 and IL13, are involved in promoting wound repair responses and are considered reparative cells. Macrophages maintain their plasticity, and in vivo, pulmonary macrophages switch from M1 to M2 states during pneumonia resolution. In this project, we sought to determine the functional consequences of this macrophage repolarization using an in vitro model. We hypothesized that repolarization of LPS-treated macrophages (M1 cells) to M2 cells would alter their responsiveness to further LPS challenge. We cultured bone-marrow derived macrophages from mice. We exposed the macrophages sequentially to LPS, IL4 and IL13 or media alone, and then rechallenged with LPS. We harvested cells for RNA for analysis of gene expression of M1 and M2 markers using qRT-PCR. We found that repolarization of macrophages toward M2 states potentiated the immunoparalysis seen in LPS-treated macrophages. These results suggest that M2 cells may dampen macrophage inflammatory gene expression to promote resolution of inflammation but may also contribute to increased susceptibility to recurrent infection.

Barth Syndrome is a rare X-linked genetic disorder that can cause cardiac myopathy, muscle weakness, fatigue, immune dysfunction, and childhood mortality. Like many such diseases, Barth Syndrome is the product of a mutation encoding for a single protein, Tafazzin, a phospholipid-lysophospholipidtransacylase found primarily in cardiac and skeletal muscle, where it plays an integral role in modifying cardiolipin, a component of the mitochondria, or energy production organelles of the body. When Tafazzin, and consequently the modified form of cardiolipin are absent, the function of the mitochondrialelectron transport chain, the primary pathway by which the body converts food into useable energy, is reduced. We hope to rescue defective cardiolipin modification through Tafazzin protein therapy and learn more about tafazzin structure and function. To this end we used X-Ray Crystallography to obtain a three-dimensional electron density map corresponding to the diffraction pattern of the protein, based on the distance and angle between each atom. The determination of structural properties illuminated binding sites and chemical interactions in both functional and mutant Tafazzin. This was supplemented by the synthesis of enzymatically active and stable human and mouse Tafazzin using E. coli (BL21(DE3)pLysS). Overexpression of Tafazzin results in relatively pure inclusion bodies which we we isolated through protein metal affinity chromatography. We produced several protein coding variants of Tafazzin in order to study the enzymatic activity of different mutants. Understanding of structure-activity relationships in Tafazzin will direct future work in Barth Syndrome treatment by facilitating  in vivo studies of Tafazzin protein delivery.

Acute myeloid leukemia (AML) is a type of blood cancer. This disease is treated by chemotherapy and bone marrow transplantation, but mortality rates remain high and new therapies are needed. Because gene expression in AML differs from person to person, it is possible that personalized therapies can be designed by understanding how gene expression predicts drug sensitivity. Previous studies tested the sensitivity of 14 human AML cell lines and 30 primary patient samples to 160 chemotherapy drugs, and utilized computational approaches to identify genes that are correlated with the sensitivity to specific drugs. In my project, I am evaluating several of these predictions by silencing gene expression in AML cell lines with a tool called short hairpin RNA (shRNA), which is used to bind and degrade the expression products of genes called messenger RNA (mRNA). I will then test the ability of drugs to inhibit growth in cells that have undergone gene silencing versus control cells that have not undergone gene silencing. If we successfully validate these gene expression-drug sensitivity relationships, they could lead to personalized clinical treatment approaches in AML.

Tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), kills nearly 2 million people a year and is a major cause of morbidity and mortality worldwide. The increasing prevalence of multi-drug resistant (MDR) strains that fail to respond to all currently available drugs underscores the critical need for new therapeutic strategies. DNA topoisomerases (topos) are a class of enzymes responsible for maintaining the correct topological state of DNA molecules by modulating the degree of supercoiling through the breakage and reforming of covalent phosphodiester bonds in the DNA backbone. With the precedent-setting success of several anticancer and antibacterial drugs that target topos, focus in TB research has increasingly turned to these enzymes as new potential targets for chemotherapy. In this study, we isolated Mtb topoisomerase type IA (MtbTOPA), a subclass that only acts on single-stranded DNA (ssDNA), as our target of interest. At least one type IA topo activity must be present in every organism for resolving topological obstacles that require ssDNA cleavage, and fortuitously, Mtb happens to only encode one type IA, further validating MtbTOPA as a high-value drug target. We have cloned, expressed, and isolated highly purified recombinant MtbTOPA protein for enzymatic studies and work is currently underway to solve its crystal structure to aid in target-based approaches to drug development. In efforts to develop a high-throughput screen (HTS) for identification of small molecule inhibitors of MtbTOPA, we have collaborated with the Global Alliance for TB Drug Development (GATB) and leading structural genomics centers to gain access to large compound libraries as well as to accelerate the drug discovery process. We adopted a HTS method based on fluorescence resonance energy-transfer (FRET) and requisite MtbTOPA mutants were designed. It is our hope that the HTS will identify several hits that can be developed into novel TB drugs.

Supplementation with “good bacteria” (probiotics) has been promoted for various health conditions. Marketing and over-the-counter (OTC) access has also led to increased availability of these agents. Safety of probiotic supplementation for individuals with weakened immune systems, such as bone marrow transplant (BMT) recipients, is unknown. Since these patients are at increased risk for developing bloodstream infections (bacteremia), we sought to evaluate the incidence and outcome of bacteremia due to organisms common to OTC probiotic supplements in a cohort of BMT recipients. We reviewed all blood culture results from BMT recipients transplanted at the Fred Hutchinson Cancer Research Center in Seattle, WA between 2002-2011. Patients with at least one positive blood culture for common probiotic organisms (Lactobacillus species, Bifidobacterium species, or Streptococcus thermophilus) within one-year post-BMT were considered cases for this study. Patients with evidence of pre-transplant bacteremia from these agents were excluded. Data were collected from center databases, which contain archived laboratory data, patient demographics, and clinical summaries. We found that a total of 18/3799 (0.47%) BMT recipients developed bacteremia with Lactobacillus species within one-year post-HCT; no events with Bifidobacterium or S. thermophilus were identified. Positive blood cultures for Lactobacillus species occurred at a median of 84 days post-transplant (IQR: 34, 127), but the majority (n=12 [67%]) occurred within the first 100-days. The overall incidence rate of Lactobacillus bacteremia was 1.54 cases per 100,000 patient-days, but the highest incidence occurred during the first 100-days post-transplant (3.31 cases per 100,000 patient-days). Incidence did not deviate significantly by year of transplant. Most patients had a singular positive blood culture (n=17 [94%]); one patient developed prolonged bacteremia (30 days). No Lactobacillus-attributed mortality was observed during follow-up. We concluded that organisms frequently incorporated in available OTC probiotic supplements are infrequent causes of bacteremia after BMT. Studies evaluating the use of probiotics among these high-risk patients are needed.

Traumatic injuries can often lead to massive blood loss. When a patient’s mean blood pressure (MAP) drops due to bleeding, the standard protocol is to administer intravenous fluids with the goal of restoring normal blood pressure. However, increasing blood pressure may also increase the rate of bleeding, causing even more overall blood loss. There is no current evidence to show that MAP correlates directly to the velocity of blood exiting a wound. We hypothesize that a direct relationship exists between blood pressure and velocity of bleeding. We test this hypothesis using direct ultrasound imaging of a bleeding artery in a swine model of trauma. N=8 anesthetized pigs underwent bleeding from a femoral artery punch wound and were simultaneously given intravenous fluids to increase blood pressure and cardiac output. Ultrasound measurements of blood flow velocity from a femoral artery punch wound were taken at regular intervals. We found that MAP did not correlate with the bleeding velocity at the wound (R=0.27. p=0.068). However, cardiac output was significantly correlated with bleeding velocity (R=0.37, p=0.012). MAP and cardiac output were then stratified into normal or low categories based on their median values for the entire group. Average bleeding velocity was not different when MAP was low compared to when it was normal (normal=255.7 cm/s vs. 221.8 cm/s, p=0.18). However, bleeding velocity was significantly decreased when cardiac output was low vs. normal (normal=253.6 cm/s vs. 196.1 cm/s, p=0.01).This data does not support our hypothesis of a direct relationship between MAP and wound bleeding velocity. Alternatively, cardiac output as a measurement of blood flow, rather than pressure, may better reflect bleeding after injury and may be a more-useful guide for fluid resuscitation during active bleeding.

Mycobacterium tuberculosis (MTB) infection remains a major global health problem but the human immune response to this bacteria has yet to be fully understood. A challenge for vaccine development is the lack of an immunological correlate of protection from MTB. We hypothesize that studying adults that were highly exposed to MTB but failed to convert their tuberculin skin tests might help us understand protective immunity to MTB. We infected monocytes in-vitro with MTB and looked for unique gene expression signatures. We discovered that Ras-responsive binding protein 1 (RREB1) was suppressed in persistently skin test negative subjects when compared to skin test positive controls. Using the human monocytic cell line, THP-1, we are studying how in-vitro stimulation with IFN-γ, Toll-like receptor ligands, and bacteria change expression of RREB1. These studies will contribute to our understanding of the role of RREB1 in MTB pathogenesis.

Pericytes are mesenchymal cells that are attached to peritubular capillaries. The activation and differentiation of progenitor pericytes to myofibroblasts leads to matrix deposition, renal scarring, and is a major contributing factor leading to chronic kidney disease after injury. Connective tissue growth factor (CTGF) plays an important role in fibrogenesis by promoting the production and secretion of extracellular matrix components. The goal of this project is to dissect the contribution of CTGF/Wnt signaling pathways in pericytes during the development of kidney fibrosis and to examine the effect of using the canonical Wnt extracellular regulator Dickkopf related protein-1 (DKK-1) on CTGF/Wnt signaling. In-vitro signaling studies on purified kidney pericytes, migration assays, and immunological staining were used to characterize the roles of DKK-1 and CTGF in the regulation of renal fibrosis. Results show that CTGF induces Wnt/β-catenin signaling in pericytes. DKK-1 inhibits CTGF induced fibrotic gene activation, changes the morphology, and the capability of migration of kidney pericytes through JNK MAP kinase, and canonical WNT independent pathway.

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder of hematopoietic progenitor cells, also known as “blasts,” that fail to both differentiate normally and properly respond to normal proliferation regulators. The consequences include possible organ infiltration as well as impairment of normal blood cell production that can result in fatal infection or bleeding. Retention of leukemia cells within the bone marrow microenvironment enables survival from chemotherapy treatment. This retention is largely mediated by surface membrane receptors, such as the chemokine receptor CXCR4. CXCR4 facilitates cellular migration towards high concentrations of the stromal derived cell factor CXCL12 (SDF-1), a chemokine that is released from bone marrow stromal cells. A CXCR4 small molecule inhibitor, plerixafor, has been shown to improve response to chemotherapy treatment in murine leukemia models, by disrupting interactions with the microenvironment, releasing leukemia cells into the bloodstream, and conferring sensitivity to chemotherapy. Our objective was to assess the functionality of an anti-CXCR4 antibody, BMS-936564, to similarly enhance chemotherapy cytotoxicity. We assayed CXCR4 expression by the leukemia blasts and stem cells, observed the time course of mobilization of leukemic blasts and stem cells, and examined the induction of programmed cell death (apoptosis) in patient samples through analysis for Annexin V expression. Mobilization of leukemia blasts and stem cells was monitored by flow cytometry. We found a significant correlation between CXCR4 expression by leukemic blasts and patient response to therapy, with high CXCR4 expression (≥20%) linked to complete remission (p<0.05). It is intuitive that the antibody to CXCR4 would require CXCR4 to be expressed by blasts. These data provide a method to predict outcomes for patients undergoing combination treatment with anti-CXCR4 antibody and chemotherapy. Our ultimate objective is to overcome adhesion mediated chemotherapy resistance in order to improve outcomes for patients with AML.

Teledermatology is a clinical service program hosted by the VA Puget Sound to deliver expert dermatology care to underserved and rural veterans in the Pacific Northwest Region. During the four years since program inception, 25,830 consultations were completed on 13,249 unique Veterans resulting in the diagnosis and treatment of 23,332 conditions. The aim is to evaluate and improve Teledermatology processes and outcomes, rendering them non-inferior to face-to-face encounters with the few board-certified dermatologists in the VA healthcare system. Melanoma care is complex, requiring secure handoffs from primary care to dermatologists, general surgeons, surgical oncologists and other specialists, often in different locations. Dropped or missed handoffs risk allowing tumors to invade and spread; a system that safely focuses care in the primary care team might achieve better outcomes. The VA Teledermatology program educates and trains primary care providers to perform surgical procedures. The purpose of this study was to measure the uptake of the surgical education and assess its safety and benefit in reducing the amount of travel miles and time to receive care. 185 veterans identified as having malignant melanomas or severely dysplastic nevi between July 1, 2009 and June 30, 2013, met criteria for analysis. Of 361 surgical procedures performed for diagnosis and treatment 191 (53%) were performed by primary care clinicians with 1.95% biopsy complication rate compared to 3.33% for the equivalent procedure performed by dermatologists, general surgeons or specialty surgeons and a 5.41% wide-local excision complication rate compared to 12.12% and 9.38% for dermatologists and general surgeons respectively. These results show that a significant fraction of surgical procedures for diagnosis and treatment of malignant melanoma and severely dysplastic nevi can be safely performed in rural clinics by trained primary care providers. Future studies set will focus on the timelines for care and longitudinal follow up for outcomes.

The pancreatic islet β-cell secretes insulin in response to nutrients such as glucose. In type 2 diabetes, insulin secretion by β-cells is impaired, causing hyperglycemia. Our group investigates this impairment, with the goal of identifying and understanding the underlying mechanisms. Pancreatic islets contain an extensive capillary network, including endothelial cells, which produce soluble factors that may modulate β-cell function. Specifically, our group has shown that media from islet endothelial cells cultured under control conditions (5.5 mM glucose) and subsequently incubated with islets enhances insulin secretion from those islets. In contrast, media from islet endothelial cells cultured in diabetic conditions (high glucose; 25 mM) shows no effect on insulin secretion. Thus, we hypothesize that islet endothelial cells normally secrete paracrine factor(s) – signaling molecules produced by one cell that cause particular reactions in nearby cells – that enhance glucose-stimulated insulin release but that in diabetes, production of these paracrine factor(s) is reduced. The goal of this project is to identify one or more of these paracrine factors. We cultured islet endothelial cells under control and diabetic conditions. mRNA was extracted and sent for whole transcriptome analysis (RNA-Seq), creating an expression profile showing the expression amplitude for each gene. Analysis of RNA-Seq data revealed patterns of differential gene expression between control and diabetic samples. Changes in expression for genes of interest identified by RNA-Seq are being verified by quantitative real time PCR (qPCR). Subsequent protein analysis via ELISA or western blot will confirm the RNA-Seq and qPCR data. Viral vectors or siRNA will be used to either overexpress or silence respectively the gene(s) of interest in islet endothelial cells. Media will be collected from these cells and used on islets to determine its effect on glucose-stimulated insulin release. This project should reveal novel mechanisms by which islet endothelial cells influence insulin release.

The use of online news reports and social media as a means of public health surveillance such as the HealthMap web and smart phone applications have successfully provided real-time information to both the public and health officials. Providing up-to-date disease outbreak alerts relies heavily on compiling and processing large volumes of data from various official and unofficial news sources, and different settings require different techniques. For some regions of the world, global surveillance networks like HealthMap have had difficulty collecting data for use in their systems. We describe the process by which real simple syndication (RSS) feeds are integrated into the existing HealthMap infrastructure to increase incoming data from an area of comparatively limited coverage in the HealthMap system, Francophone Africa. Using integration of RSS feeds allowed for the collection of 143% more data than the traditional method of scraping Google News. While much of this data was irrelevant, as it was not related to disease events, this approach detected several disease-related events which were missed by the established approach. With additional refinement to reduce the volume of irrelevant alerts, the newly developed system will supplement the existing approach by capturing events that would otherwise be missed, thereby improving the overall sensitivity of the system.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to help lead to improved drugs. Most eukaryotic organisms synthesize sterols such as cholesterol and ergosterol for essential biological functions that can lead to cell death if these sterols were made incorrectly. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediate of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T.cruzi in vitro and are now the center of research regarding anti T.cruzi drug discovery. Further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of the research project I am presenting is to analyze the ERG3 homologs of T.cruzi in the context of the ergosterol synthesis. The putative homologs have been cloned and sequenced. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

Rotavirus is the main cause of viral diarrhea among young children. There exist licensed vaccines, but rotavirus still kills nearly half a million children yearly. Live, attenuated vaccines quickly lose potency at room temperature. My host lab is assisting a Seattle NGO callednPATH and the Bill and Melinda Gates Foundation with developing temperature-stabilized, inexpensive vaccines. The current industry standard assay for measuring vaccine potency is time-consuming, subjective, and low throughput. My goal is to develop an objective, inexpensive, automatable, and high-throughput assay of the potency of candidate, next-generation, temperature-stable rotavirus vaccines. To develop this assay, susceptible cells are infected with candidate vaccine-strain viruses manufactured in India and undergoing development at PATH. After cell membrane modification to allow antibody access to cytoplasmic viral proteins, an anti-rotavirus antibody and a fluorescent secondary antibody are used to detect viral protein within infected cells. Experimental samples undergo single-cell flow cytometry analysis of individual cells to differentiate infected and uninfected cells. The vaccine industry-standard potency assay is performed in parallel to directly compare assays. Comparison and titration of polyclonal and monoclonal rotavirus-specific antibodies identified the optimal antibody and concentration for specific intracellular recognition of rotavirus protein. Testing of secondary antibodies and fluorochromes resulted in cell fluorescence characteristics yielding a favorable signal-to-noise ratio without causing false-positive results. A direct relationship between the amount of live rotavirus input and the percentage of infected cells with good linearity and acceptable coefficients of variation was observed. Future work is focused on reducing material costs, comparing the flow cytometry assay to the industry standard, and adapting the assay to additional rotavirus vaccine strains. Based on the above results, flow cytometry is a promising candidate assay for quantifying rotavirus vaccine potency to assist the development of novel live rotavirus vaccine formulations and storage formats.

Malaria is an infectious disease caused by various species of the Plasmodium parasite which affects an estimated 207 million individuals, resulting in approximately 627,000 deaths per year. While this disease is primarily prevalent in developing nations, over half of the world’s population is at risk for malaria. Among existing malaria drugs, primaquine is uniquely effective against late-stage Plasmodium gametocytes and liver hypnozoites; however, it is dangerous to patients who are G6PD deficient. G6PD deficiency is caused primarily by point mutations in the Glucose-6-Phosphate Dehydrogenase (G6PD) protein—an enzyme that catalyzes the formation of NAPDH (a reducing agent) in the Pentose Phosphate Pathway. This condition affects around 400,000 individuals worldwide, most of whom live in areas at risk for malaria. Screening for G6PD deficiency is not readily available in many of these at risk areas and thus complicates treatment of malaria with primaquine. Therefore, screening for compounds that stabilize mutant forms of G6PD may yield potential candidates that can be used in combination with primaquine for a new therapeutic regimen. In a screen of ~100,000 compounds, 71 compounds were found to have stabilizing effects on mutant G6PD R454H (Andalus). Screening was done with a standard biochemical assay that measured enzyme activity through the detection of reaction product, NADPH (detected by fluorescence). Additional biochemical assays and studies on these 71 hits will follow to assess the mechanism and extent to which these compounds increase mutant G6PD Andalus activity. Candidate compounds that make it through these follow up experiments will then be tested with other G6PD mutants in search of a stabilizer of multiple G6PD mutants.

Obstructive sleep apnea (OSA) is a common clinical disorder and is linked to health-related morbidity. Positive airway pressure (PAP) reverses OSA-related hypoxia, yet less than half of patients are adherent to PAP treatment. Insulin-like growth factor 1 (IGF-1) is a neuropeptide that promotes neuronal growth and is essential for normal brain development. IGF-1 is reduced in OSA and may relate to improvement in symptoms following PAP treatment. This relationship has not yet been determined in a young cohort of patients with OSA. This was a prospective, observational study of 58 males, diagnosed with OSA. Auto-titrating PAP (APAP) was used as the treatment intervention. Adherence with APAP was objectively measured, as well as changes in the apnea-hypopnea index (AHI). I collected blood samples to determine changes in concentrations of IGF-1 and compiled clinical data regarding APAP usage and treatment responses. I then assessed these changes through one-way analysis of variance (ANOVA) analysis. There were 58 participants, all males, mean age 36.2±7.7, BMI 31.4±3.7, apnea-hypopnea index 19.1±19.2. There were no clinical differences between the APAP adherent and non-adherent groups. APAP adherence was achieved in 23 (39.7%); their usage of 5.7±0.9 hours was elevated compared to non-adherent patients at 1.1±1.8 hours (p< 0.001). IGF-1 concentrations at baseline were similar between APAP adherent and APAP non-adherent participants; however, at follow-up, adherent participants had concentrations of IGF-1 that were significantly higher than non-adherent participants (p <0.01). Young adult males with OSA who are adherent with APAP have increases in IGF-1 which could lead to improvements in brain functioning and overall quality of life. IGF-1 is a potential biomarker of adequate treatment of OSA and could be used to help in the assessment of proper treatment.

Pancreatic islets are comprised of ß cells that secrete insulin in response to glucose to maintain glucose homeostasis. The renin-angiotensin system is a regulatory hormone system active in many organs. Angiotensin (1-7) (Ang(1-7)), a product of the renin-angiotensin system, enhances glucose-stimulated insulin secretion (GSIS) in a rat model of type 2 diabetes. Since Ang(1-7) is cleaved into angiotensin (1-4) (Ang(1-4)) by neprilysin (NEP), an enzyme present in islets, it remains unknown whether Ang(1-7) or Ang(1-4) is responsible for enhancing GSIS. We sought to determine whether under normal conditions, absence of NEP attenuates the ability of Ang(1-7) to promote GSIS, and whether Ang(1-4) per se enhances GSIS. We cultured Wild-type or NEP deficient mouse islets for 48 hours in the absence (vehicle) or presence of 1 nM Ang(1-7) or 1 nM Ang(1-4) (4-7 experiments). We then measured insulin secretion in response to 2.8 mM (basal) and 20 mM (GSIS) glucose. Ang(1-7) enhanced GSIS in wild-type islets [Ang(1-7) 93.4±10.0 vs. Vehicle 59.0±6.8 pM/5 islets/h; p<0.05] but failed to do so in Nep deficient islets [Ang(1-7) 135.4±17.4 vs. Vehicle 145.1±23.1 pM/5 islets/h; p>0.05]. Ang(1-4) also enhanced GSIS in wild-type islets [Ang(1-7) 105.3±17.5 vs. Vehicle 44.9±9.5 pM/5 islets/h; p<0.05], while neither Ang(1-7) nor Ang(1-4) significantly altered basal insulin secretion from islets of either genotype. In conclusion, NEP is required for the increase in GSIS mediated by Ang(1-7), suggesting that a NEP-derived cleavage product of Ang(1-7) is responsible for its beneficial effects on ß-cell function. The enhancement of GSIS by Ang(1-4) supports this notion, suggesting that Ang(1-4) may be a novel therapeutic approach to improve GSIS in type 2 diabetes.

Affecting millions of people across the globe, the WHO deems myocardial infarctions (heart attacks) as the largest cause of death worldwide. The heart’s weak regenerative capacity leads to scarring of the dead tissue and a reduction in cardiac output that presents numerous health consequences. Tissue engineering is a promising outlook in restoring the structural integrity and contractile force of the damaged area. Recent findings have demonstrated that engineered heart tissues grown from stem cells can display significantly improved characteristics when subjected to mechanical stimuli. Inspired by these results, this project seeks to improve tissue performance even further by investigating a novel concept where stem cell-derived cardiac tissues are treated with the same mechanical profile experienced by native heart tissue. In order to simulate and implement these forces, tissues are grown and then anchored onto two posts that can vary in distance and stiffness. Upon contracting, post stiffness is increased providing a resistance to each beat. This resistance simulates the afterload stress that the ventricular wall contracts against to eject blood into the aorta. Next, the tissues are stretched by increasing the distance between each post. The stress produced by elongating the tissues simulates the preload stress exhibited by the ventricular wall as blood is pumped into the chamber prior to the next contraction. Finally, combining the stiffness (afterload) and stretching (preload) schemes allows the tissues to experience the entire force profile exhibited by the native heart. Simulating the cardiac mechanical environment gives the engineered tissues the stimulus to yield contractile forces and a cellular structure at par with their native counterparts. This investigation thus aims to advance regenerative medicine by bringing stem-cell derived cardiac tissues one step closer towards curing myocardial infarctions.

Currently, kinases are thought to function primarily in signal transduction cascades by phosphorylating target proteins. Based on emerging evidence, a novel role for kinases is promising: direct kinase binding of DNA and chromatin. To analyze the role of kinases in the nucleus and investigate whether direct kinase binding of DNA or chromatin takes place, I will generate genome-wide histone phosphorylation and kinase localization maps for multiple kinases in human embryonic stem cells. We first verified the presence of specific kinases in the nucleus of H1 human embryonic stem cells using mass spectroscopy. To visualize protein presence in the nucleus, I stained whole cells and whole nuclei using immunofluorescence. I then studied specific kinase-chromatin interactions and histone phosphorylation sites using ChIP-seq (chromatin immunoprecipitation coupled with high-throughput sequencing). Sequencing data are expected to show genomic locations where direct kinase binding of DNA and chromatin occurs. We predict we will see kinases present at promoter and enhancer elements, as phosphorylation of proteins in the complexes that bind these elements or the surrounding nucleosomes may be important for gene regulation.

 Acute myeloid leukemia (AML) is a blood cancer characterized by the failure of differentiation of white blood cells to their progeny (myeloid differentiation). MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs) for degradation or translational repression. In cancer, miRNA expression is altered and these changes contribute to impaired differentiation. In previous work we examined over 600 miRNAs to establish which miRNAs could promote differentiation or limit growth of AML cells. This work identified that miR-150 expression induces myeloid differentiation and that miR-150 expression is decreased in many AML patients. Because transcription factors are important regulators of myeloid differentiation, we examined the transcriptional regulation of miR-150 in hematopoietic cells to determine how miR-150 expression is decreased. We identified the minimal promoter region of miR-150 using luciferase reporter assays in THP-1 (AML) and K562 (chronic myeloid leukemia) cell lines. We identified DNA binding sites for the Krüppel-like factor (KLF) family of transcription factors as necessary for miR-150 promoter activity using site-directed DNA mutagenesis of the luciferase reporters. The current study aims to identify which KLF isoforms regulate miR-150 expression. We cloned KLFs 2, 3, 4, 5, 6, 7, 9, and 10 into lentiviral expression vectors. KLF2 and KLF4 overexpression increases miR-150 expression as measured by luciferase assays and quantitative PCR in THP-1 and K562 cells. Furthermore, we confirmed published results that KLF2 and KLF4 promote myeloid differentiation of THP-1 cells using flow cytometry and gene expression studies. Previous studies have determined that KLF2 and KLF4 expression are decreased in AML cells. This loss contributes to pathogenesis; however how miRNAs contribute remains unknown. Consequently, determining how miR-150, KLF2, and KLF4 promote differentiation will provide insight as to how loss of KLFs contributes to leukemia and how we may use miRNAs to overcome block in differentiation therapeutically.

MicroRNAs are a group of highly conserved non-coding RNAs that play critical roles in regulating networks of genes. MicroRNAs bind to target mRNAs and can inhibit translation and/or facilitate degradation. One of these miRNAs, microRNA-21, plays an important role in kidney disease as it has been shown to increase in animal models of disease. Recent studies have shown that microRNA-21 promotes interstitial kidney disease with fibrosis by silencing metabolic pathways and by promoting reactive oxygen species formation. Mice with microRNA-21 deletion show a decrease in kidney fibrosis.

Our hypothesis is that cells isolated from microRNA-21 knock out mice will have decreased expression of cytokines associated with fibrosis. I will look at the expression of reactive oxygen species by live imaging of cells where microRNA-21 has been blocked. Then I will use quantitative polymerase chain reaction and Western blots to analyze protein levels for known molecules associated with fibrosis and inflammation. We expect to see that cells treated with anti-microRNA-21 have a reduction in reactive oxygen species, leading to less fibrosis and inflammation. Blocking microRNA-21 is a potential new therapy to combat kidney disease.

Merkel Cell Carcinoma (MCC) is a rare, yet aggressive skin cancer with a disease-associated mortality of approximately 40%. Although surgery is typically a component of standard care, it is unclear how often patients should also receive radiation to control disease at the primary site. One retrospective study of 1254 patients found that of the patients who received surgery alone, 39% recurred locally compared to only 12% of the patients who received surgery in combination with radiation therapy. In contrast, in a cohort of 243 patients from New York treated with surgery alone, only 3.8% recurred locally. In order to establish a better standard of care for low risk MCC patients and attempt to resolve this discrepancy in the literature, we identified low risk patients within our cohort based on the following inclusion criteria: 1) primary tumor ≤1cm in diameter, 2) microscopically negative margins on the surgical excision, 3) absence of lymphovascular invasion in the primary tumor, 4) absence of profound, chronic immunosuppression, and 5) no microscopic evidence of nodal metastasis in the sentinel lymph node biopsy. Of the 29 patients who met these criteria, 17 were treated with surgery alone, while 12 received adjuvant radiation to the primary site after surgery. We found that 4 patients recurred locally (24%) among the patients treated with surgery monotherapy, while there were no recurrences among patients treated with surgery and adjuvant radiation. Even patients with low-risk MCC still have an appreciable chance of local recurrence if treated with surgery alone. While this risk may be acceptable to some patients, adding radiation therapy can lower the chance of local recurrence.

 Embryonic stem cells can be used to study development and diseases. Mouse embryonic stem cells (mESC) are used as a model in developmental research and display several pluripotent properties not seen in the current human embryonic stem cell (hESC) lines. A newly derived line of naïve hESCs is thought to recapitulate an earlier stage of development, and resemble mESCs. To understand how the naïve hESCs differ from typical hESCs we are investigating DNA methylation, a form of epigenetic regulation, on a global scale at nucleotide resolution. The underlying genomes of cells are highly similar, yet their epigenomes change with cellular differentiation and development. Epigenetic modifications are important factors controlling gene regulatory elements. To analyze whole genome DNA methylation of the naïve hESCs, I am using MethPipe, a computational tool, to determine how the naïve hESC line differs from a later stage hESC line, and if the naïve line is more similar to mESCs at the epigenetic level. I will compare global methylation levels, hypo/hyper-methylated regions, and partially methylated domains. I will determine which regulatory elements, such as promoters, enhancers and CpG islands, show similarities and differences. We also expect to determine differential non-CG methylation (a characteristic of ESC). This characterization of naïve hESCs will provide a foundation for studying the earliest stages of human development.

 In neurodegenerative diseases, such as Alzheimer’s disease, tau protein aggregation is observed. In Alzheimer’s disease and related conditions, accumulation of insoluble tau impairs physiological function that ultimately leads to neuron loss. Using a transgenic Caenorhabditis elegans model that expresses human tau protein in neurons, we are able to see accumulated, insoluble, phosphorylated tau, and uncoordinated movement (Unc) phenotypes in these organisms. To identify genes participating in aggregated tau and Unc phenotypes, we conducted a mutagenesis; an estimated 20,000 worm genomes were screened and we isolated seven promising strains of C. elegans that carried loss of function genes suppressing the tau-induced phenotypes. These strains are currently being sequenced to determine the identity of the loss of function genes, which may represent novel suppressors of tau pathology. Currently, the Kraemer lab has identified two genes suppressing tau-induced Unc phenotype and called them suppressor of tau 1 (sut-1) and suppressor of tau 2 (sut-2). Animals with sut-1 and/or sut-2 mutations are resistant to the negative effects of tau, meaning that the proteins made by these genes are necessary for tau phenotypes. With this knowledge and the additional information to be gained from the sequencing of seven novel sut alleles, we hope that potential neuroprotective approaches for treatment can be developed.

According to the World Health Organization, cardiovascular diseases are the leading cause of mortality worldwide. The low regenerative capacity of the heart renders it ineffective in healing itself after heart failure. Our lab has developed efficient methods to derive cardiomyocytes (cardiac muscle cells) from human embryonic stem cells (hESCs). However, it has become apparent that the cardiomyocytes derived using current differentiation methods exhibit immature morphological and functional properties. An important characteristic of mature cardiomyocytes is the presence of T-tubules, which are invaginations of the plasma membrane into the cell interior. These subcellular structures are critical for synchronized calcium release and contraction of mature cardiomyocytes. The protein caveolin-3 is a critical component of T-tubules, and thus its visualization can be used to assess the development of these important subcellular structures. I am currently evaluating the expression of caveolin-3 in hESC-derived cardiomyocytes using immunostaining techniques. For these studies, I expose the sample to anti-caveolin-3 as the primary antibody followed by a complementary secondary antibody conjugated to a fluorescent marker for visualizing the protein. Titration of several parameters, such as time of incubation and dilution of the antibodies allows for staining optimization. I have obtained detailed images of T-tubules aligning with striations in control heart tissue, but the staining is diffuse for the hESC-derived cardiomyocytes grown in standard 2D cultures. I am currently exploring whether this is a technical issue or the result of developmental immaturity. To assess the latter, I will stain hESC-derived cardiomyocytes that are more mature due to mechanical conditioning in 3D constructs. Optimization of this technique will facilitate our efforts to find novel agents that can accelerate the maturation of hESC cardiomyocytes. Finding ways to mature these cells will then provide a better platform for therapeutic applications such as drug screening, disease modeling and cardiac repair.

Myocardial infarction and resulting heart failure continues to be the leading cause of death worldwide. The heart does not respond effectively to injury through tissue regeneration because cardiomyocyte proliferation after differentiation occurs at a greatly diminished rate. The goal of this research is to study stem cell-derived cardiomyocytes to better understand the mechanisms and possible controls of cardiomyocyte proliferation. Achieving this goal would allow for the intitiation of cardiomyocyte proliferation, which would better direct new technologies aimed at regenerating cardiac tissue after injury. RUES2 human embryonic stem cells are differentiated into cardiomyocytes using a directed differentiation protocol that manipulates the TGFβ and Wnt signaling pathways, by the controlled addition of Activin A, BMP4, chiron, and Xav signaling factors. After twelve to fifteen days, cells are harvested and stained for cardiac troponin T (cTnT), which is a protein present in the sarcomere of cardiomyocytes. Flow cytometery is used to identify cardiomyocytes expressing cTnT and indicate the purity of the differentiated culture. Cardiomyocyte proliferation is measured by exposing cells to Bromodeoxyuridine (BrdU), which is a thymidine analog that is incorporated into synthesized DNA. A double immunohistochemistry stain for Beta Myosin Heavy Chain (β-MHC) and BrdU is used to indicate proliferating cardiomyocytes. RNA is isolated from cardiomyocyte cultures and analyzed using quantitative real time PCR. Specific sequences of RNA present in cells can indicate relative gene expression, and allow for genes of interest to be monitored through stages of differentiation to determine peak expression. Ongoing experiments are investigating the activity of the Notch signaling pathway during directed differentiation of cardiomyocytes and its relation to cardiomyocyte proliferation. Results from these experiments will better indicate time points for enhancing cardiomyocyte proliferation using Notch signaling and help further characterize methods for induced proliferation as a potential therapeutic after cardiac injury.