Doctor of Philosophy, Case Western Reserve University, 2021, Molecular Medicine

Objective – The dietary contribution to human health and disease has been long appreciated. However, the metabolism of dietary substrates by commensal gut microbiota and ultimately the impact of microbe-derived metabolites on host physiology, remain poorly understood. Approach and Results – Here, we interrogated the role of a single microbial metabolite stemming from a flavonoid-rich diet, 4-hydroxyphenylacetic acid (4- HPAA), in a mouse model of high fat diet-induced obesity. Subcutaneous administration of 4-HPAA led to a marked reversal of hepatic steatosis when compared to scaffold control treated mice resulting from hepatic activation of AMPKa and its downstream effectors. Furthermore, we revealed the rarity of the bacterial genes required to catabolize dietary flavonoids into 4-HPAA in human metagenomic datasets. Next, we used a multi-omics approach to identify microbe-derived metabolites stemming postprandially from a single meal equivalent of a human-relevant fast food diet. Here, we provided mice with control drinking water or antibiotic drinking water to ablate the gut microbial community prior to a single oral gavage of either chow control diet or fast food. Using untargeted mass spectrometry, we identified several putative metabolites that were postprandially enriched in the portal blood of fast food-fed mice in a microbe-dependent manner. Furthermore, we utilized an unbiased RNA sequencing approach to survey diet and microbe-associated hepatic transcriptional changes, highlighting the influence of both diet and the gut microbial metabolome on the liver transcriptome. Finally, we developed a novel surgical technique to continuously administer gut microbial metabolites intraportally, thus recapitulating the natural delivery of gut microbes from the mesentery to the liver via the portal vein. Here, we show the suitability of this method for metabolites with a high degree of hepatic first rate metabolism. Conclusion – These (open full item for complete abstract)

Committee: Jan Claesen Ph.D. (Advisor); J. Mark Brown Ph.D. (Advisor); Christine McDonald Ph.D. (Committee Chair); Philip Ahern D.Phil. (Committee Member); Betul Hatipoglu M.D. (Committee Member) Subjects: Microbiology; Nutrition

Doctor of Philosophy (PhD), University of Toledo, 2018, Biomedical Sciences (Molecular Medicine)

Insulin resistance has long been considered to play a crucial role in the pathophysiology of metabolic syndrome that is the leading cause of mortality and morbidity worldwide. Metabolic diseases consist of a group of metabolic abnormalities that increase the risk of health problems, such as type 2 diabetes (T2D) and cardiovascular disease. Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and metabolic syndromes. It is the fastest growing cause of liver dysfunction. Its progressive form nonalcoholic steatohepatitis (NASH) is associated with hepatic fibrosis that can develop into cirrhosis. In addition, there is a growing body of evidence that among risk factors that promote atherosclerosis, metabolic syndrome is a potent predictor of cardiovascular events. Insulin resistance seems to play a major role in the pathophysiology of atherosclerosis in relation with metabolic syndrome. Given that patients with NAFLD/NASH are at a high risk to develop atherosclerosis; these two diseases may share some pathology. However, precise molecular mechanisms underlying the pathogenesis and progression of these diseases are not well understood. Thus studying the molecular link between them would pinpoint sites of more effective pharmacologic interventions. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a protein that is markedly reduced in the liver of patients with NASH, promotes insulin clearance to regulate insulin action. Whether or not CEACAM1 links hyperinsulinemia to NAFLD/NASH and atherosclerosis still needs to be determined. CEACAM1 enhances the rate of uptake of the insulin-receptor complex into the clathrin-coated vesicles of hepatocytes. It also plays a major role in mediating the negative acute effect on fatty acid synthase (Fasn) activity. As such, CEACAM1 regulates insulin and lipid metabolism. Mice with global null mutation of CEACAM1 (Cc1–/–) display hyperinsulinemia resulting from impaired insulin clearance, insulin resist (open full item for complete abstract)

Committee: Sonia Najjar PhD (Committee Chair); Guillermo Vazquez PhD (Committee Member); Jennifer Hill PhD (Committee Member); Rajesh Gupta MD (Committee Member); David Kennedy PhD (Committee Member); Steven Haller PhD (Committee Member) Subjects: Biomedical Research

PhD, University of Cincinnati, 2024, Engineering and Applied Science: Biomedical Engineering

Thermal ablation, including radiofrequency (RFA) and microwave ablation (MWA), is a potential first-line treatment for many patients with small liver tumors. Real-time monitoring and control methods can potentially reduce under- or over-treatment rates. Here, echo decorrelation imaging was expanded from two- to three-dimensional (3D) imaging, and its performance was assessed in ex vivo and in vivo. First, 3D echo decorrelation with three normalization approaches and 3D integrated backscatter (IBS) were used to monitor ex vivo bovine liver RFA. Receiver-operating characteristic (ROC) curves, comparing these imaging parameters to ablation zones, indicated that all four were potentially good predictors of local RFA. Tissue temperatures, recorded at four thermocouples locations, weakly correlated with co-located measured decorrelation. In tests predicting ablation zones using a weighted K-means clustering approach, echo decorrelation performed better than IBS, with smaller root mean square (RMS) volume errors and higher Dice coefficients between predicted and measured ablation zones. Further, RFA in samples from normal, steatotic, and cirrhotic human liver parenchyma was monitored and controlled using 3D echo decorrelation imaging feedback. For each liver type, N=14 samples underwent ablation per RFA generator's settings, monitored by echo decorrelation imaging (uncontrolled). In N>14 trials, RFA was controlled, automatically ceasing if the average cumulative decorrelation within the target region of interest surpassed the control threshold (successfully controlled), otherwise, ablation continued like the uncontrolled trials (unsuccessfully controlled). ROC curves showed good prediction performance for all liver and control conditions. Significantly higher control success rate and generally higher measured ablation volume were observed in controlling normal vs. diseased liver. The same monitoring and control algorithms were used in in vivo swine liver RFA, (open full item for complete abstract)

Committee: T. Douglas Mast Ph.D. (Committee Chair); Ali Kord M.D. M. (Committee Member); Stacey Schutte Ph.D. (Committee Member); Kevin Haworth Ph.D. (Committee Member) Subjects: Biomedical Engineering

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

The gut microbiome constitutes billions of microorganisms consisting of bacteria, viruses, fungi, and archea which colonize the gastrointestinal tract. These microbes have a profound impact on host physiology due to their ability to modulate immune responses, metabolize dietary compounds, and produce metabolites which can enter circulation. As a result, the microbiome can influence the pathogenesis of various diseases, including alcohol-associated liver disease (ALD). ALD remains one of the most prevalent liver diseases worldwide, contributing to increases in mortality through its ability to affect multiple organs in the host. Recently, much attention has been brought to the role of alcohol-induced disruptions to the microbiome, and the subsequent modulations which either prevent or promote the progression of this disease. However, the vast mechanisms by which the microbiome can influence disease means that much is still left unanswered in regard to this axis. In this dissertation, our objective was to further elucidate mechanisms by which ALD-associated disruptions to the microbiome influence the course of disease. To do so, we employ clinical data from patients with ALD along with novel methodologies of targeting microbial metabolites for the elucidation of microbiome-related mechanisms in mice exposed to ethanol. In Chapter 2, we use mass-spectrometry to profile changes in circulating microbial metabolites in patients with ALD. We find that meta-organismal metabolites are decreased in patients with severe ALD and the exploration of bulk RNA-seq datasets unveil that this is potentially a result of dysregulated hepatic phase II metabolism in patients with ALD. In Chapter 3, we use synthetic bacterial communities to test the effects of the deletion of a bacterial tryptophanase gene. Here, we identify bacterial species susceptible to ethanol as well as a mechanism by which bacterial tryptophanase expression protects against ethanol administration through the promotio (open full item for complete abstract)

Committee: Mark Brown (Advisor); Laura Nagy (Advisor); Daniela Allende (Committee Member); Philip Ahern (Committee Member); Christine McDonald (Committee Chair) Subjects: Biomedical Research; Immunology; Microbiology; Molecular Biology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2023, College of Sciences and Health Professions

Throughout my doctoral research, I have focused on three projects examining the role of RNase L, in liver and kidney diseases. In the first project, we explored the involvement of RNase L in the development of nonalcoholic fatty liver disease (NAFLD). We induced NAFLD in RNase L wild type (WT) and knockout (KO) mice using a high-fat, high-cholesterol diet (HFHCD) and assessed their progression to nonalcoholic steatohepatitis (NASH). Surprisingly, RNase L WT mice exhibited more severe NASH, characterized by macro-vesicular steatosis, hepatocyte ballooning degeneration, inflammation, and fibrosis. Further analyses revealed that RNase L contributes to the expression of some key genes associated with lipid metabolism, inflammation, and fibrosis signaling. The findings suggest that targeting RNase L expression and activity may represent a novel therapeutic approach for NAFLD intervention and treatment. In the second project, we investigated the role of RNase L in kidney function and acute kidney injury (AKI) recovery. AKI is a prevalent clinical disorder with substantial morbidity and mortality, posing a significant healthcare burden. In the experiments, AKI was induced in RNase L WT and KO mice with folic acid (FA), and the renal function, histological structure, and recovery were evaluated. Our findings demonstrated that RNase L deficiency attenuated kidney damage caused by FA and promoted renal recovery from AKI through reducing systemic inflammation. Mechanistically, the lack of RNase L increased the activation of the EGFR/AKT signaling and suppressed apoptosis in the kidney after FA exposure. In the third project, we studied the potential relationship between RNase L and kidney aging in mice. Aged RNase L WT and KO mice were evaluated for physical and renal changes, with the KO mice demonstrating signs that are associated with slower kidney aging. To determine how RNase L is involved in kidney function and renal diseases, we performed immunoprecipitat (open full item for complete abstract)

Committee: Aimin Zhou (Advisor); David Anderson (Committee Member); Michael Kalafatis (Committee Member); Bin Su (Committee Member); Aaron Severson (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Experiments; Medicine; Molecular Biology; Pathology; Physiology

PhD, University of Cincinnati, 2023, Medicine: Immunology

The unabated obesity pandemic drives increased incidence of non-alcoholic fatty liver disease (NAFLD). NAFLD has enacted an enormous health and financial burden worldwide. Yet, therapies for this disease are lacking in part because etiology and progression of this disease are underdefined. Inflammation is a critical link between obesity and NAFLD. Moreover, inflammatory mediators and immune cells are invoked in the transition from benign steatosis to inflammatory non-alcoholic steatohepatitis (NASH). Hence, understanding mechanisms that regulate inflammation during obesity driven NAFLD may allow for development of novel therapeutics. In this dissertation, we focus on two factors that regulate immune cell inflammation and NAFLD progression, cellular metabolism and thermoneutral housing. Among the myriad immune cells, CD4+ T cells are garnering attention as key contributors to NAFLD progression. We show that exploitation of CD4+ T cell glutamine metabolism via exogenous glutamine supplementation negatively regulates their inflammatory capacity in obesity driven NAFLD. Indeed, CD4+ inhibition of glutaminolysis uniquely increased their inflammatory vigor, activation of IL-17 signaling axis and exacerbated hepatocellular damage. Notably, we show that hepatocellular IL-17RA expression was central to amplified hepatocellular damage and NAFLD. Although inflammation is essential to NAFLD pathogenesis, pre-clinical research is plagued by the lack of experimental models that recapitulate human disease. Here, we expand upon previous findings in demonstrating that augmented immune cell inflammatory vigor and exacerbated NAFLD prompted by thermoneutral housing (Tn) are conserved in multiple experimental models of NAFLD. Hence, addressing both metabolism and inflammation should be a two-pronged approached when investigation mechanisms of NAFLD pathogenesis. Notably, Tn housing modifies both in mice and should be utilized as a tool for interrogating cellular metabolic mechanisms (open full item for complete abstract)

Committee: Senad Divanovic Ph.D. (Committee Chair); Ian Lewkowich Ph.D. (Committee Member); Nikolai Timchenko Ph.D. (Committee Member); Joseph Qualls Ph.D. (Committee Member); Rajat Madan Ph.D. M.B.B.S. (Committee Member) Subjects: Immunology

MS, University of Cincinnati, 2022, Medicine: Clinical and Translational Research

Background: Recent evidence shows that hepatic steatosis can occur as early as in utero. There is minimal data on maternal nonalcoholic fatty liver disease (NAFLD) during pregnancy, and its link to early-onset pediatric hepatic steatosis has not been investigated. Our primary objective was to evaluate maternal NAFLD as a risk factor for fetal hepatic steatosis. Methods: This was a prospective observational study of mother-fetus dyads in the 3rd trimester of pregnancy. Inclusion criteria were age ?18 years, healthy singleton pregnancy, and gestational age (GA) ?28 weeks. Women were considered high-risk for NAFLD if they had pre-pregnancy obesity (PPO), excessive weight gain during pregnancy, gestational diabetes (GDM), and/or NAFLD. If no risk factors were present, participants were designated as no-risk. Enrolled participants underwent magnetic resonance imaging (MRI) with proton density fat fraction (PDFF) to measure maternal and fetal hepatic PDFF, placenta PDFF and fetal abdominal wall subcutaneous fat thickness. Fasting labs and 3-day diet recalls were obtained. General linear regression models were used to compare fetal hepatic and placenta PDFF separately, based on maternal NAFLD status, controlling for GDM and PPO. Results: There were 42 participants included, 36 in the risk group and 6 in the no-risk group. The majority of participants were non-Hispanic with a median age of 31 years (IQR: 28, 34) and a median GA at time of MRI of 34 weeks (IQR: 33, 35). In high-risk women, NAFLD was seen in 17% (n=6/34) with a median maternal liver PDFF of 11% (IQR 7, 15). PPO and GDM prevalence were not different in high-risk women with or without NAFLD. There was no significant correlation between maternal and fetal steatosis (r=-0.13, p=0.44). Hepatic fat fractions of fetuses of high-risk women with NAFLD were 60% of those for fetuses of high-risk women without NAFLD. In high-risk women without NAFLD versus those with NAFLD, the placenta PDFF (1.6% vs 1.7%) (open full item for complete abstract)

Committee: Scott Langevin Ph.D. (Committee Member); Marialena Mouzaki Ph.D. (Committee Member); Lin Fei Ph.D. (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2021, Comparative and Veterinary Medicine

Therapeutic estrogens address multiple facets of non-alcoholic steatohepatitis (NASH) pathophysiology. Their potential, however, is limited by undesirable side effects caused by persistent estrogen receptor a (ERa) activation. Estrogen receptor b (ERb) is functionally unique, capable of inducing estrogen's benefits while limiting ERa-mediated side effects. We hypothesized that OSU-ERb-12, a novel carborane selective estrogen receptor modulator with affinity for ERb, would demonstrate therapeutic benefit in preclinical NASH models at doses absent of ERa-mediated effects (ERb-selective). Our objectives were first to describe OSU-ERb-12's drug-like properties by characterizing its ERb-selectivity, pursuing early but critical preclinical safety studies, describing its pharmacokinetic properties, and finally by identifying ERb-selective doses to apply to future studies. We then assessed OSU-ERb-12's therapeutic potential in two pre-clinical models of relevant NASH pathophysiology while simultaneously gaining mechanistic insight into drug efficacy. We applied several in vitro techniques to demonstrate OSU-ERb-12's drug-like properties. We established its ERb-selectivity first through a competitive radioligand-binding assay to demonstrate ligand affinity for ERb, then through a luciferase reporter assay to assess its preferential activation of ERb-mediated transcription. In both experiments, OSU-ERb-12 demonstrated selective properties comparable to LY500307, another ERb-selective agonist. We then put OSU-ERb-12 through nuclear hormone receptor and CYP-inhibition assays to identify potential off-target effects and drug-drug interactions, respectively; none were found suggesting compound safety. Multi-species microsomal stability assays and in vivo pharmacokinetic studies not only demonstrated remarkable OSU-ERb-12 stability in most species, but that the drug has superior pharmacokinetic properties to LY500307. Finally, to establish in vivo ERb-selective doses, we utiliz (open full item for complete abstract)

Committee: Christopher Coss (Advisor); Heather Shive (Committee Member); Moray Campbell (Committee Member); Steven Clinton (Advisor) Subjects: Endocrinology; Medicine; Oncology; Pharmaceuticals; Pharmacology

MS, University of Cincinnati, 2021, Medicine: Clinical and Translational Research

Background: Pediatric patients with end-stage liver disease (ESLD) are subjected to increased waitlist morbidity and mortality due to a lack of appropriately sized donor allografts. To combat waitlist mortality, increased utilization of Technical Variant Grafts (TVGs) has been proposed. However, recent literature suggests recipients of ex-vivo reduced allografts experience worse graft survival and postoperative complications than in-situ split allografts. The goal of this study was to determine if there are significant differences between pediatric patients who receive in-situ split and ex-vivo reduced allografts. Methods: The prospectively maintained pediatric liver transplant database was queried for all TVG recipients between 2015-2020. Baseline patient demographics, clinical characteristics, intra-operative benchmarks, post-operative complications, and survival curves were compared between in-situ and ex-vivo TVG recipients. Results: In 70 consecutive TVG LT's, 40 (57.1%) received ex-vivo reduced and 30 (42.9%) received in-situ split allografts. Recipients of in-situ split allografts were more likely to be younger (p<0.01), shorter (p=0.04), weigh less (p=0.02), receive a living donation (p<0.01), and a left lateral segment graft (p<0.01) than ex-vivo reduced recipients. In-situ recipients were exposed to less cold ischemia (p<0.01) and warm ischemia (p<0.01) time. Despite this, there was no difference in estimated blood loss (p=0.26), blood transfusions (p=0.32), or postoperative vascular and biliary complications (all p>0.05). Furthermore, with a median follow-up of 1010 days, there was no difference in patient or graft survival between cohorts on Kaplan-Meier analysis (p>0.05), and ex-vivo reduced allografts were not associated with an increased hazard of death or graft failure on multivariable cox-regression (p>0.05). Conclusion: Ex-vivo reduced allografts have similar intra-operative, postoperative, and long-term survival outcomes as in-situ spli (open full item for complete abstract)

Committee: Scott Langevin Ph.D. (Committee Chair); Shimul Shah M.D. (Committee Member) Subjects: Surgery

Master of Science, The Ohio State University, 2019, Allied Medicine

LEARNING OUTCOME: To identify the dietary patterns of varying stages of non-alcoholic fatty liver disease (NAFLD) that could inform behavior modifications utilized during medical nutrition therapies (MNT) implemented by a Registered Dietitian Nutritionist (RDN). BACKGROUND: NAFLD is a leading cause of liver disease worldwide and due to poor efficacy of pharmacotherapy, lifestyle modification and dietary change are the primary treatment options. Dietary education must be tailored to mitigate the progression of NAFLD; therefore, elucidating current dietary patterns associated with NAFLD and its progressive stages will identify areas for behavior modification. METHODS: A cross-sectional study including 28 clinic patients with a diagnosis of NAFLD, non-alcoholic steatohepatitis (NASH), or cirrhosis confirmed by imaging and/or biopsy was conducted. Nutrient intakes and dietary patterns were assessed via a validated graphical food frequency questionnaire (FFQ). The Healthy Eating Index 2015 (HEI-2015) and alternative Mediterranean diet (aMED) scores were compared across diagnoses using descriptive statistics and a Kruskal-Wallis H test for significance. RESULTS: Patients with NAFLD had higher fruit intake among HEI and aMED indices in relation to NASH and cirrhosis (p<0.05). Total HEI-2015 scores were significantly different between diagnoses (p<0.05) and were poorest in patients with cirrhosis (58.0 ± 7.6). Saturated fat and total energy intakes differed between diagnoses (p<0.05) and were highest in cirrhosis patients. Adults with varying stages of NAFLD had significantly different sodium intakes (p=0.005), with cirrhosis patients having a two-fold higher median intake. CONCLUSIONS: Patients with cirrhosis had poorer diet quality compared to NASH and NAFLD. This may be associated with lower fruit consumption, increased sodium, and higher saturated fat intakes noted in cirrhosis patients. RDNs should target behavioral strategies to improve and modify so (open full item for complete abstract)

Committee: Kristen Roberts (Advisor); Marcia Nahikian-Nelms (Committee Member); Chris Taylor (Committee Member) Subjects: Health; Nutrition

Doctor of Philosophy, The Ohio State University, 2019, Integrated Biomedical Science Graduate Program

The primary function of adipose tissue (AT) was previously thought to be a passive storage site for excess caloric intake. However, increasing evidence suggests that inflammatory processes taking place within AT play a fundamental role in the development of obesity-related complications. Within mouse models of obesity, there is an accumulation and shift in the inflammatory profile of the immune cells that traditionally constitute the lean AT from anti-inflammatory macrophages (M2-like), immunosuppressive regulatory T cells (Tregs), T helper type 2 cells (Th2) and innate lymphoid type 2 cells (ILC2) to proinflammatory M1-like macrophages, T helper type 1 cells (Th1), CD8+ cytotoxic T cells and proinflammatory neutrophils. This shift to a more pro-inflammatory milieu in states of chronic over-nutrition has been linked to the development of insulin resistance and fatty liver disease in mouse models. However, the inflammatory microenvironment in human AT remains largely unexplored. Thus, our laboratory has developed a more comprehensive profile of the human AT microenvironment and has identified multiple immune cells that contribute to an obesity-related pro-inflammatory state that fosters metabolic disease. Similar to mouse AT, T cells and macrophages comprise a significant proportion of the immune cells within human AT, and obesity leads to a pro-inflammatory shift within these immune cell populations. While the total number of T cells within the lean and obese state remains fairly constant, the number of the anti-inflammatory Tregs is reduced in obesity. They undergo rapid and acute depletion in lean patients who are placed on a hypercaloric diet with about a 50% reduction in subcutaneous AT T cells within 2 weeks. In addition to a change in specific T lymphocytes, AT macrophages are highly prevalent in obesity, contributing to ~10% of the AT stromal vascular fraction (SVF). We discovered that human AT macrophages are mainly comprised of two distinct populations: (open full item for complete abstract)

Committee: Willa Hsueh MD (Advisor); Martha Belury PhD (Committee Member); William Lafuse PhD (Committee Member); Peter Mohler PhD (Committee Member) Subjects: Biomedical Research

Doctor of Engineering, Cleveland State University, 2018, Washkewicz College of Engineering

Hepatocellular carcinoma (HCC) is an invasive and aggressive cancer of the liver that arises due to chronic cirrhosis. Research into understanding HCC has focused on two-dimensional (2D) and three-dimensional (3D) technologies to simulate the liver microenvironment and use animal models to model how HCC affects the rest of the body. 3D hydrogel models are desired because they can mimic the transport behavior observed in vivo by structurally mimicking the extracellular matrix (ECM) without the ethical concerns of animal models. However, hydrogels can be toxic to cells and require optimal procedures for appropriate handling. In this study, we created 3D models of liver diseases on high-throughput platforms. First, we optimized hydrogel attachment on micropillar chips by coating them with 0.01 w/v % PMA-OD in ethanol. Next, we optimized the protocol for encapsulation of viable Hep3B cells PuraMatrix peptide hydrogel, using a higher seeding density (6 * 106 cells/mL) and two post-print media washes. Then, we established the ability to transduce adenoviruses in situ in encapsulated cells and successfully demonstrated their dose-response behavior towards six compounds. In the second part, we scaled up to using the microwell chip platform and optimized the polymerization of oxidized methacrylated alginate (OMA) for Hep3B encapsulation. First, we plasma-treated microwell chips for 15 minutes at high RF to minimize bubbles. Then, we optimized micro-scale photopolymerization conditions at 45 % methacrylated OMA (OMA-45) and 2 w/v % OMA with 0.05 w/v % PI and reflective background under either low intensity, long duration (2.5 mW/cm2 for 2 minutes) or high intensity, short duration (4.0 mW/cm2, 30 seconds) light by testing cell viability at these conditions. Third, we used these OMA conditions to develop a high-throughput, real-time 3D cell migration assay on a newly engineered 384-pillar plate with sidewalls. We first developed a set of a protocols where out-of-focus cells ar (open full item for complete abstract)

Committee: Moo-Yeal Lee Ph.D. (Committee Chair); Joah Belovich Ph.D. (Committee Member); Nolan Holland Ph.D. (Committee Member); Chandra Kothapalli Ph.D. (Committee Member); Xue-Long Sun Ph.D. (Committee Chair) Subjects: Biomedical Engineering; Biomedical Research; Chemical Engineering; Materials Science; Nanoscience

MS, University of Cincinnati, 2018, Medicine: Immunology

Obesity and aging are associated with metabolic dysfunction including, type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Altered immune responsiveness in both obesity and aging promote the development of metabolic derangements. Additionally, gender impacts the development of obesity and pathogenesis of obesity-associated and aging-associated metabolic derangements. However, the mechanisms underlying aging and gender-dependent effects in obesity development and pathogenesis remain under-defined, in part due to the inability to induce severe obesity in female mice. Here, young and aged male and female mice, fed a regular or high fat diet (HFD) were examined for the development of obesity and pathogenesis of obesity-associated sequelae. Further, via utility of thermoneutral housing we examined the development and pathogenesis of obesity-associated sequelae in both young and aged female mice. We show that both aging and gender promote differential responses to HFD induced weight gain and metabolic function. Specifically, aged male mice, compared to young counterparts, had lower propensity for diet-induced weight gain and glucose dysmetabolism, but had a similar level of hepatocellular damage. Aged female mice, compared to young counterparts, had a higher propensity for diet-induced weight gain and hepatic steatosis but similar glucose dysmetabolism. Under thermoneutral housing conditions however, aged female mice, compared to young counterparts, had reduced NAFLD severity but similar glucose dysmetabolism. Mechanistically, aging and gender-specific effects in metabolic dysfunction correlated with tempered inflammatory responses including increased tissue inflammation, Treg numbers, and IL-10 levels. In sum, our data demonstrate that both aging and gender modulate development of diet-induced obesity and metabolic dysfunction. Notably, modulation of these pathways holds potential for development of novel therapeutic strategies.

Committee: Senad Divanovic Ph.D. (Committee Chair); David Hildeman Ph.D. (Committee Member); Jonathan Katz Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy (PhD), Ohio University, 2018, Molecular and Cellular Biology (Arts and Sciences)

Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity, both of which are exceedingly prevalent worldwide. Obesity facilitates a state of low-grade inflammation that is critical for the pathogenesis of NAFLD which constitutes a disease spectrum that consists of the very early stage, hepatic steatosis, involving the ectopic deposition of lipid in the liver to the advanced stage of nonalcoholic steatohepatitis (NASH) that is marked by immune cell infiltrate, hepatocyte damage, and/or fibrosis. While NAFLD is the most common liver disease in the Western hemisphere, only a small percentage (2-3%) of NAFLD patients progress to NASH, and thus the progression from hepatic steatosis to NASH is not well understood. Until now, hepatic steatosis was considered to be a benign stage of NAFLD, however, we now know that hepatic inflammation and insulin resistance is present during hepatic steatosis. It is the inflammation in early NAFLD that is crucial for the progression to NASH. A high fat (HF) diet-induced model of NAFLD promotes an increase in circulating free fatty acids (FFAs) that bind to and activate toll-like receptor 4 (TLR4) signaling leading to the production of pro-inflammatory cytokines which can exacerbate lipid accumulation and cause insulin resistance. FFA-induced TLR4 signaling in hepatocytes is the predominate facilitator for the development of hepatic steatosis, by targeting TLR4 signaling with novel small molecule inhibitors of inflammation we have prevented and reversed HF diet-induced NAFLD.

Committee: Kelly McCall (Advisor) Subjects: Biology; Molecular Biology; Molecules

Doctor of Philosophy, Case Western Reserve University, 2017, Molecular Medicine

Ethanol consumption disrupts the intestinal barrier and sensitizes hepatic macrophages to inflammatory stimuli. We have recently shown that hyaluronan (HA) with an average molecular weight of 35kDa (HA35) prevents sensitization of TLR4 signaling in lipopolysaccharide (LPS) challenged Kupffer cells isolated from ethanol-fed rats. To determine whether HA35 would prevent liver injury in response to ethanol, eight to ten week old female C57BL/6J mice were fed a control or ethanol (2d of 1% and 2d of 6% v/v) containing Lieber-DeCarli diet. Mice were treated with HA35 or sterile saline by daily gavage for the last three days. HA35 treatment prevented ethanol-induced increases in hepatocyte apoptosis and expression of proinflammatory cytokine and chemokine mRNA in liver. HA35 prevented ethanol-induced decreases in miR181b-3 a suppressor of NF-kB responses through targeted downregulation of the nuclear transporter importin a5 in non-parenchymal cells. Ethanol increased the nuclear translocation of phosphorylation of NF-kB subunit p65 in the livers of ethanol-fed mice, but HA35 prevented translocation comparable to control. HA35 also protected the intestine from ethanol-induced injury. Ethanol feeding increased intestinal permeability as assessed by increased the portal concentration of endotoxin but not in mice treated with HA35. Short-term ethanol feeding increased the thickness of the lamina propria in the distal colon and decreased the co-localization of ZO-1 and occludin at tight junctions in both the proximal and distal colon. HA35 prevented these effects of ethanol. In differentiated Caco-2 cells, ethanol decreased the localization of ZO-1 and occludin at tight junctions and increased FITC-inulin permeability. Pre-treatment with HA35 prevented these changes. When the hyaluronan receptor Layilin was knocked down in Caco-2 cells, HA35 was no longer able to protect from ethanol-induced loss of tight junctions. Taken together these data identify HA35 as a therapeutic for (open full item for complete abstract)

Committee: Laura Nagy (Advisor); Thomas McIntyre (Committee Chair); Thomas Hamilton (Committee Member); Alexandru Almasan (Committee Member); Arthur McCullough (Committee Member) Subjects: Biology; Molecular Biology

Doctor of Philosophy, Case Western Reserve University, 2014, Molecular Medicine

Macrophage migration inhibitory factor (MIF) exhibits both cytokine and chemotactic properties and is expressed by many cell types, including hepatocytes and nonparenchymal cells. Excessive alcohol abuse can lead to alcoholic liver disease (ALD), which has multiple stages of pathogenesis including hepatic steatosis and hepatic fibrosis. During steatohepatitis, several precipitating events, including triglyceride accumulation in the liver, hepatocyte damage and leukocyte infiltration, lead to further pathogenesis of ALD. Hepatic fibrosis is dependent on interactions between hepatocytes, hepatic stellate cells (HSC) and leukocytes. Recruitment of monocytes to the liver is a key contributor to hepatic fibrosis. Due to MIF's chemotactic properties, we hypothesized that MIF would be a key contributor to ALD pathogenesis. Female C57BL/6 or MIF-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet for 25 days. Expression of MIF messenger RNA was induced after 25 days of ethanol feeding. After chronic ethanol feeding, chemokine expression and monocyte recruitment was increased in wild-type mice, but not MIF-/- mice. In a model of 12 liver fibrosis, female C57BL/6 or MIF-/- mice were challenged with carbon tetrachloride (CCl4). Expression of mRNA for MIF, as well as plasma MIF content, were increased in female wild-type mice in response to an acute dose of CCl4. a Smooth muscle actin (aSMA) mRNA and protein, indicators of HSC activation, were increased in liver of wild-type after chronic CCl4 challenge. This response was dampened in MIF-/- mice. Despite lower activation of HSC, MIF deficient mice developed similar fibrosis after CCl4 exposure when compared to wild-type mice. Inflammatory scar-associated macrophages (SAMs) were recruited to the liver from the periphery in response to chronic CCl4 in wild-type and MIF-/- mice, but later converted to a restorative SAM phenotype during fibrosis resolution. Fewer inflammatory and restorative monocytes were (open full item for complete abstract)

Committee: Laura Nagy PhD (Advisor); Maria Hatzoglou PhD (Committee Chair); Pierre Gholam MD (Committee Member); Booki Min DVM, PhD (Committee Member); Xiaoxia Li PhD (Committee Member) Subjects: Biology; Biomedical Research; Health Sciences; Immunology

PHD, Kent State University, 2013, College of Arts and Sciences / Department of Chemistry

Preeti Pathak Department of Chemistry and Biochemistry RETINOIC ACID RELATED ORPHAN NUCLEAR RECEPTOR a (RORa) REGULATES DINURAL RHYTHM AND FASTING INDUCTION OF STEROL 12a- HYDROXYLASE (CYP8B1) IN BILE ACID SYNTHESIS. Directors of Dissertation: John Y. L. Chiang, Ph.D. and Dr. Diane Stroup, Ph. D. Abstract Sterol 12a-hydroxylase (CYP8B1) is involved in cholic acid synthesis and plays a role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone disease and dyslipidemia. In this study, we investigated the underlying mechanism of a fasting-induced and cholesterol activated nuclear receptor and core clock gene RORa in regulation of circadian rhythm and fasting induction of CYP8B1 expression. In free fed mice, CYP8B1 expression was reduced to the lowest level at the onset of the dark cycle when RORa expression was the lowest. However, fasting stimulated, while re-feeding reduced expression of CYP8B1 mRNA and protein expression. Interestingly, fasting and feeding had little effect on the diurnal rhythm of RORa mRNA expression, but fasting increased, whereas feeding decreased RORa protein levels in mouse liver. Adenovirus-mediated transduction of RORa to mice strongly induced CYP8B1 gene expression, increased 12a-hydroxylated bile acids in bile acid pool and serum and liver cholesterol. Reporter assay and mutagenesis analysis of the CYP8B1 promoter identified a functional RORa response element. Mammalian two-hybrid assay showed strong interaction of RORa with cAMP response element binding protein-binding protein (CBP). Chromatin immune-precipitation assay showed that RORa recruited CBP to the CYP8B1 promoter to stimulate histone acetylation. CAMP-activated protein kinase A phosphorylates RORa and increases its half-life. In conclusion, RORa is a key regulator of circadian expression and fasting induction of CYP8B1 to increase 12a-hydroxylated bile acids in the bile acid pool, and serum and liver cholestero (open full item for complete abstract)

Committee: Stroup Diane Ph.D. (Committee Chair); Chiang John Ph.D. (Committee Co-Chair) Subjects: Biochemistry

MS, University of Cincinnati, 2006, Medicine : Epidemiology (Environmental Health)

The prevalence of nonalcoholic fatty liver disease (NAFLD) in children and young adults is unknown, as population-based studies are often limited by use of serum aminotransferases as surrogate markers, lacking imaging or biopsy data to confirm NAFLD. This study determines the prevalence and determinants of NAFLD in young adult females using magnetic resonance imaging (MRI) to measure hepatic steatosis. Prevalence was hypothesized to be 15%. Methods: 278 female subjects, ages 25-27 years, were recruited from a population-based cohort. Exclusion criteria included weight >300 lbs, daily alcohol intake >20 gm/day, hepatotoxic medications, or serologic evidence of hepatitis B, C or autoimmune hepatitis. Anthropometric indices, serum lipids, insulin, glucose and aminotransferases (ALT, GGT) were measured. The hepatic fat index (HFI) was measured using a biopsy-validated MRI technique. Descriptive statistics and nonparametric tests were used, analyzing races separately to control for higher prevalence of obesity in the black females. Results: 38% of the cohort was obese (BMI > 30 kg/m2), but only 4% had abnormal ALT (>35 IU/L). Black females (N=157) had a higher prevalence of obesity (47% vs. 27%, p<0.001), abnormal waist circumference (40% vs. 25%, p=0.02), hyperinsulinemia (54% vs. 30%, p<0.001) and metabolic syndrome (10% vs. 5%, p =0.15) compared to white females. Mean HFI was 3.0 ± 2.6 (range 0 - 26) with no significant racial differences. Prevalence of abnormal HFI was 2.5%, with no significant racial differences (blacks 1.9%, whites 3.3%). BMI, waist-hip ratio, waist circumference, triglycerides, and GGT directly correlated with HFI. Of subjects with abnormal HFI, 100% were obese and hyperinsulinemic, 80% had abnormal waist circumference, but only 29% had abnormal ALT. Conclusions: Despite a high prevalence of obesity, significant hepatic steatosis is not very prevalent in young black and white adult women. Black women have a significantly higher prevalence of risk (open full item for complete abstract)

Committee: Dr. James Heubi (Advisor) Subjects:

MS, University of Cincinnati, 2004, Medicine : Epidemiology (Environmental Health)

Current practice is to monitor prothrombin time as an indicator of vitamin K sufficiency in cholestatic liver disease. Since prothrombin time is a surrogate marker, it may underestimate the actual prevalence of vitamin K deficiency in this population. This study investigates the frequency of vitamin K deficiency among a convenience sample of children and adults with cholestatic liver disease by determining plasma levels of protein induced in vitamin K absence II (PIVKA-II), and assesses the relationship between PIVKA-II levels and markers of cholestasis, measured prothrombin time, and vitamin A, E and 25-hydroxyvitamin D levels. Methods: Subjects with cholestatic liver disease were recruited from the Cincinnati referral area. Subjects with decompensated cirrhosis, malignancy, concurrent disease that results in fat malabsorption and AIDS were excluded. All subjects had blood collected for liver function tests, prothrombin time (PT), INR, bile acids, 25-hydroxyvitamin D, vitamin A, vitamin E and PIVKA-II levels. Plasma PIVKA-II concentrations were measured using a commercially available ELISA kit. Results: 31 subjects were enrolled (age range 0.5-54 years). Nine subjects (29%) had prolonged prothrombin times, while 21 (68%) had elevated PIVKA-II levels. All patients with prolonged PT had PIVKA-II elevations. Of the 21 patients with PIVKA-II elevation, 15 were on supplemental vitamin K therapy (range 7.8-700 μg/kg/day). PIVKA-II levels were positively correlated with serum conjugated bilirubin, bile acids, AST, ALT and measured PT and negatively correlated with serum 25-hydroxyvitamin D levels. Seven subjects (22.5%) had vitamin A deficiency, 5 subjects (16%) had vitamin D deficiency and 2 subjects (8%) had vitamin E deficiency. Conclusions: Despite vitamin K supplementation, vitamin K deficiency, as measured by PIVKA-II, is common in cholestatic liver disease. Better strategies for vitamin K supplementation and dosing guidelines are needed.

Committee: Dr. James Heubi (Advisor) Subjects:

Bachelor of Science (BS), Ohio University, 2010, Biological Sciences

Nonalcoholic fatty liver disease (NAFLD), defined as hepatic steatosis with less than 20 g of daily alcohol intake, is the most prevalent liver disease in the world because of its close association with obesity and metabolic disorders. Currently, the only available treatment for NAFLD is diet and exercise. Our lab has found that treatment with growth hormone (GH) or insulin-like growth factor-1 (IGF-1) significantly reduces liver triglyceride content in diet-induced obese and diabetic mice exhibiting NAFLD. The goal of this study was to characterize the molecular mechanisms at the mRNA level of these novel treatments using One-color Agilent 4x44K Whole Mouse Genome Arrays. Two major treatment regimens were used. In treatment regimen #1, mice were subjected to 16 weeks of low-fat (LF) or high-fat (LF) diet, which induces obesity, diabetes, and NAFLD, followed by three weeks (21 days) of twice daily injections. The LF and HF controls received phosphate-buffered saline (PBS). The 3 experimental treatments were GH, IGF-1, and a combination of GH/IGF-1 (Combo). The GH and Combo treatments lowered fat mass and liver triglyceride content, and increased lean mass, plasma IGF-1, and glucose tolerance. The IGF-1 treatment lowered liver triglyceride content and increased lean mass. A subset of four mice from the LF and HF controls and the GH and IGF-1 treated groups were selected for microarray experiment #1 (LF, HF, GH21 & IGF21, respectively). A total of 910, 372, and 256 genes were differentially expressed when comparing HF versus LF, GH21 versus HF, and IGF21 versus HF, respectively. In treatment regimen #2, diet-induced obese and diabetic mice were treated with twice daily injections of PBS, GH, IGF-1, or switched to a LF diet (diet reversal, DR) for three weeks (21 days). A subset of each experimental treatment groups was sacrificed at 2, 7, and 21 days of treatment. The DR treatment lowered fat mass and liver triglyceride content. Four mice from the HF control, GH treat (open full item for complete abstract)

Committee: Edward O. List PhD (Advisor); Darlene E. Berryman PhD (Advisor) Subjects: Biomedical Research