A main challenge of treating cancer is that everyone’s cancer is different and cells within tumors contain a unique set of genetic mutations. Because of this heterogeneity, the field of cancer research has seen a recent push toward personalized medicine, which depends on the ability to extract genetic information from a patient’s cancer. Tumors shed circulating tumor cells (CTCs) into the bloodstream, where they can migrate and create metastases. If physicians could harvest and analyze CTCs from patient blood, they would have a way to evaluate cancers without invasive biopsies. I work with Rarecyte, a biotechnology company specializing in finding and retrieving rare cells, in collaboration with Dr. Blau of UW’s Center for Cancer Innovation. We hope to develop techniques for those clinical applications. Our goal is to demonstrate that we can isolate a fraction of patient blood that contains the vast majority of the CTCs but minimal white blood cells (WBCs), from which single CTCs can be picked. We approach this work with a technique called sequential density fractionation, which exploits the different densities of blood components to isolate the component containing the majority of the CTCs. To develop this technique, I spiked model cancer cells into healthy blood to determine the optimal density of the desired fraction. Having found a density that works for the model cells, I repeated this method with samples from actual cancer patients. I measure the effectiveness of my experiments by comparing my CTC counts with blood from the same patient run with Rarecyte’s standard CTC-harvesting method. After demonstrating that we can collect a CTC-rich, WBC-sparse fraction across patients and cancer types, our next steps are to pick CTCs and carry out whole genome amplification and DNA sequencing to test if the isolated cells are viable for genetic analysis.

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 T.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 assaying genetic double knockouts of the ERG3 homologs against azole drugs to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs because the accumulation of sterol intermediates are not turned into toxic compounds.

Type 2 diabetes is characterized by impaired release of insulin and resultant malfunctions in glucose homeostasis. Insulin is produced in the pancreas by beta cells, which are a part of well-vascularized cell clusters called islets. Endothelial cells, a component of the islet vasculature, produce signaling factors that enhance insulin release. My mentor’s group has found that the immortalized islet endothelial cell line MS1 can be used as an in vitro model to study this paradigm. “Conditioned media” collected after exposure to MS1 cells has been shown to cause no change in insulin release under basal glucose conditions but resulted in a significant increase in glucose-stimulated insulin release from islets compared to non-conditioned media that had not been previously exposed to MS1 cells. This raises the question: Which factors present in conditioned media enhance insulin secretion from isolated pancreatic islets? We developed a new model to study this question which allowed rapid screening of conditioned media samples. Pancreatic islets were isolated from mice and dissociated to a single cell suspension that was plated in 6-well tissue culture plates containing conditioned media or non-conditioned media. After exposure to conditioned media, the insulin response of the dissociated islets was measured by determining cumulative release of insulin into the media during the culture period. An increase in cumulative insulin release by the islet cells in response to conditioned media was confirmed. As a next step, size fractionation using centrifugal filter units will help identify which molecular weight fractions are responsible for the effect. In-depth proteomics will then be performed on the size fraction which gives the strongest effect to enhance insulin release in an attempt to identify a candidate molecule. Identifying this molecule as an endothelial paracrine factor will further elucidate mechanisms by which endothelial cells support insulin release.

Over 29.1 million Americans have Type II Diabetes Mellitus (T2DM). Roux-en-Y Gastric Bypass (RYGB) achieves remission of T2DM in approximately 84 percent of cases, but the improvement in glucose homeostasis precedes the significant weight loss associated with the procedure. The mechanisms behind the ‘weight-independent’ correction of glucose homeostasis remain unclear, in part because large animal models of naturally occurring insulin resistance (IR) have been lacking. The purpose of the study was to examine the mechanisms behind improved glucose homeostasis and T2DM remission after bariatric surgery using a large animal model. Thirty-two Ossabaw swine received obesogenic diets and underwent RYGB (n=13), gastrojejunostomy (GJ) (n=10), gastrojejunostomy with duodenal exclusion (GJD) (n=7), or sham operations (n=2). Intravenous Glucose Tolerance Tests (IVGTT) and Meal Tolerance Tests (MTT) were performed pre- and post-operatively. Results were compared with 21 control Ossabaws that, received a regular diet, did not undergo an operation and received a one-time IVGTT and MTT. IR in this model was defined as the Homeostasis Model Assessment IR (HOMA-IR) >2 standard deviation above the regular diet group mean. Obesogenic-diet Ossabaws weighed more and 65.6% (n=21) had greater IR than controls. There was a positive correlation between weight and HOMA-IR (R²=0.08, p=0.05). RYGB was the only operation that induced weight loss, and resulted in increased in insulin/glucose area under the curve during MTT at 2 weeks (0.7±0.29) and 8 weeks (0.46±0.2) compared with baseline (0.28±0.07) (p=0.015, p=0.042 respectively). The largest reduction in IR was noted in the pigs that had the highest baseline IR. In Ossabaw swine, RYGB was the only procedure to induce both weight loss and IR improvement as measured by MTT. This animal models suggests a combination of upper and lower gut mechanisms improve glucose homeostasis postoperatively, and motivates further investigation into gastric and distal intestinal control of these effects.

Endurance exercise relies heavily on the utilization of fatty acids for energy production. In cells, the enzyme acetyl-CoA carboxylase (ACC2) produces malonyl-CoA, which inhibits the rate at which fatty acids are metabolized in the mitochondria for energy. Therefore, we hypothesized that deletion of the ACC2 protein would increase the oxidation of fatty acids and promote endurance exercise capacity. To test this, mice with the ACC2 deletion and controls were exercised until exhaustion on a motorized treadmill. Contrary to the hypothesis, mice with the ACC2 gene deletion had a significantly reduced endurance capacity compared to controls. The decreased exercise capacity was not associated with changes in blood levels of glucose, fatty acids, lactate, or triglycerides. Since ACC2 inhibitors are currently in drug development for the treatment of diabetes and obesity, these results suggest potential negative side effects. Current studies are testing whether chronic exercise training can reverse the impairment.

Ischemic heart disease, which results in irreplaceable loss of cardiac muscle and heart failure, is the leading cause of death in the world. Mammalian cardiac myocytes (CM) stop proliferating soon after birth, and the heart growth afterward predominately comes from hypertrophy, an increase in cell size, instead of hyperplasia, an increase in cell number. Because CM proliferation is required for the heart regeneration seen in lower vertebrates and neonatal mammalian injury models, we are interested in understanding the mechanism of CM cell cycle exiting and whether the exiting can be reversed. We hypothesized that Histone H3 Lysine 9 trimethylation (H3K9me3), a histone modification associated with heterochromatin and gene repression, is required for the silencing of cell cycle genes during CM cell cycle exiting. To test the hypothesis, we developed a mouse model where H3K9me3 is removed by Lysine-specific demethylase 4D (KDM4D) specifically in CMs. Using quantitative-reverse-transcription-PCR, we compared RNA expression of cell cycle genes in KDM4D-overexpressing and control adult cardiac myocytes (ACMs). In addition, we quantified ACM area and length using imaging to see whether the increased heart size in KDM4D-overexpressing mice is due to ACM hypertrophy or hyperplasia. The finding that cell cycle-related gene expression is increased in KDM4D-overexpressing ACMs indicates that H3K9me3 is required for CM cell cycle gene silencing. Image analysis and cell dimension quantification showed KDM4D overexpression did not alter ACM size, suggesting that the increase in heart size is due to ACM hyperplasia. Further studies need to be performed to better understand the mechanisms of cardiac growth and to determine if KDM4D can promote ACM regeneration in injury models; however, this finding helps advance the goal of developing regenerative therapies for myocardium after injury.

Idiopathic pulmonary fibrosis is a respiratory disease characterized by lung scarring, loss of tissue architecture, and impaired lung function. Attenuation of fibrosis has been an important research focus because no proven therapy exists. Integrins, a family of transmembrane proteins, may play a functional role in fibrosis. Certain integrins activate transforming growth factor beta (TGFb) signaling, an important profibrotic cytokine, by binding to the latent TGFb complex and releasing the active TGFb moiety. Integrin alpha 8 (itga8) is expressed in alveolar myofibroblasts, the cellular mediator of fibrosis, and is upregulated in lung fibrosis. Itga8 regulates fibroblast migration and adhesion, and it binds the latent TGFb complex. We hypothesize itga8 augments pro-fibrotic signaling in lung fibrosis through activation of TGFb. We examined the function of itga8 in transgenic mice where itga8 is deleted from a subset of fibroblasts. Platelet derived growth factor receptor-beta (PDGFRb) serves as a marker for a subset of fibroblasts that give rise to myofibroblasts in fibrosis. We generated transgenic mice where the itga8 gene is selectively deleted in PDGFRb+ cells (PDGFRb-Cre;a8 flox mice). PDGFRb-Cre;a8 flox mice (n=11) and littermate controls (n=10) underwent bleomycin lung injury and their lungs were harvested at day 21 after injury to evaluate various measures of fibrosis. In conclusion, we did not identify significant difference in scarring in transgenic mice where itga8 is deleted from PDGFRb+ cells, however a trend towards less fibrosis in the Cre+ group was observed in both histological evaluation and hydroxyproline content. Our results may be confounded by the heterogeneous injury pattern inherent to the bleomycin lung injury model. Furthermore, PDGFRb- fibroblasts also express itga8 and the biology of itga8 in fibrosis may be more relevant in this subset of fibroblasts. Future studies will focus on examination of itga8 function in PDGFRb- fibroblasts and conditional knockout of itga8 mouse models.