Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

My research focuses on the diverse roles of programmed cell death (PCD) in host innate immune responses. My dissertation explores how PCD assists host antimicrobial defense, as well as contributes to the progression of sepsis-induced immunosuppression. The dissertation could be divided into two main sections and one derivative section. Firstly, I have identified a novel interaction between Pseudomonas aeruginosa and host necroptosis during in vitro and in vivo infection. Secondly, I have characterized the impact of a novel PCD regulator, NINJ1, in improving sepsis-induced immunosuppression by partially restoring the host defense to secondary infections. Quorum sensing (QS), a communication system evolved by Pseudomonas aeruginosa to monitor its density, is well-acknowledged to be involved in multiple activities during bacterial infection. Recent studies have revealed clues about link between Pseudomonas aeruginosa QS and host programed cell death. However, it remains limited understanding whether QS plays a role in host PCD process during the infection. In this study, I used rhl mutants of Pseudomonas aeruginosa to in vitro challenge multiple genetic knockout macrophages to explore the connection between QS and programmed cell death. According to the data from cell death assays and immunoblotting, I discovered these rhl mutants significantly promoted necroptosis which was unknown in this field. Additionally, I found that the increased necroptosis activation was caused by the upregulation of another QS subsystem, pqs, because the deletion of pqs in rhl-deficient Pseudomonas aeruginosa abolished macrophage necroptosis in vitro and in vivo. Therefore, this study revealed a novel rhl-pqs-necroptosis pathway. Sepsis is characterized by two dynamic stages occur during the initiation and progression, which are system inflammatory response syndrome (SIRS) in the acute phase and compensatory anti-inflammatory response syndrome (CARS) in the later phase. Recent study revea (open full item for complete abstract)

Committee: Haitao Wen (Advisor); Patrick Collins (Committee Member); Amal Amer (Committee Member); Daniel Wozniak (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2024, Molecular, Cellular and Developmental Biology

Pancreas is exposed to chronic stress for the duration of human life that can lead to extended inflammatory damage and risk towards development of different pancreatic diseases. Novel therapeutic interventions that could lessen inflammation and stress could significantly improve quality of life in patients. In this dissertation, the role of dietary metabolites in abrogating the progression of pancreatic diseases is explored. In the first part of the dissertation, chronic pancreatitis (CP), a fibro-inflammatory syndrome that develops upon persistent pathological responses to parenchymal stress, is studied. Complex dietary foods like soy and tomatoes have anti-inflammatory effects. Thus, we hypothesized that administration of a soy-tomato enriched diet can reduce inflammation and severity of CP. C57BL/6 mice were injected intraperitoneally with caeurlein to induce CP and administered a control or a soy-tomato enriched diet for 2 weeks. Mice fed a soy-tomato enriched diet had a significantly reduced level of inflammation and severity of CP compared to mice administered a control diet with reduced inflammatory factors and suppressive immune populations, improved total activity and health, and restored serum lipase and amylase levels. These pre-clinical results indicate that a soy-tomato enriched diet may be a novel treatment approach to reduce inflammation and pain in patients with CP. Next, the role of tomatidine, a small molecule tomato metabolite, in limiting pancreatic ductal adenocarcinoma (PDAC) was investigated. PDAC is an aggressive cancer with a five-year survival rate of 13%. The pancreas has a highly functioning endoplasmic reticulum (ER) and is subjected to ER stress. Activating Transcription Factor 4 (ATF4), a master regulator of cellular stress, is exploited by cancer cells to survive. We found evidence that high ATF4 expression correlates with worse overall survival in PDAC, and previous research indicated tomatidine was associated with inhibit (open full item for complete abstract)

Committee: Thomas Mace (Advisor); Christopher Coss (Committee Member); Monica Venere (Committee Member); Michael Kearse (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Oncology; Physiology

Master of Science, The Ohio State University, 2024, Anatomy

A traumatic brain injury (TBI) is caused by a forceful blow to the head or body and can lead to temporary or permanent brain damage, or even death. There are many complications that may arise from sustaining a TBI. Depending on the severity of the injury, TBI patients may experience a level of immune suppression, and this may result in an increased risk for developing nosocomial infections. Because the brain plays a role in the regulation of the autonomic nervous system (ANS), a TBI may also cause ANS dysfunction downstream and an upregulation of the sympathetic nervous system. The body's inability to maintain physiological balance after injury may create worse outcomes for patients long term. Currently, there are a limited number of studies that show reliable methods for predicting future outcomes for patients with TBIs. In recent years, heart rate variability (HRV) has been shown to be a non-invasive indicator of predicting patient outcomes after sustaining such injuries in the adult population. Heart rate variability is the variability in the time elapsed between each heartbeat and measures the body's ability to return to a baseline heart rate after experiencing a stressful event. This study sought to analyze the relationship between heart rate variability and the level of immunosuppression in pediatric TBI patients to see if there is a negative correlation between the two measures. Immune suppression was determined by a lipopolysaccharide (LPS) stimulated TNF – α production value of <520 pg/mL. HRV data was analyzed using a frequency and time domain analysis and the two measures were correlated. This study found a significant positive correlation between time domain measures and the LPS induced TNF – α response. Power in the low frequency band of heart rate (0.04-0.15 Hz) is used as a marker of sympathetic and parasympathetic activity. Power in the high frequency band of heart rate (0.15-0.40 Hz) is used as a marker of parasympathetic activity. Significant c (open full item for complete abstract)

Committee: Eric Sribnick (Advisor); James Cray Jr. (Committee Member); Jessica Blackburn (Committee Member) Subjects: Anatomy and Physiology

Doctor of Philosophy, Case Western Reserve University, 2023, Pathology

Advancements in therapy have improved remission and prolonged survival for patients with B cell malignancies. Unfortunately, for many challenging cancer subtypes such as mantle cell lymphoma, most patients will still relapse and die from the cancer. Chimeric antigen receptor (CAR)-T cell therapy uses genetically modified T cells expressing CAR protein to recognize and kill cancers expressing a specific antigen, such as CD19. Although CD19 CAR-T therapy has been very effective against relapsed/refractory B cell cancers, antigen escape and relapse still occur in up to 40% of patients treated with CD19 CAR-T. This work describes the creation and validation of a novel, BAFF ligand-based CAR that aims to prevent antigen escape by being able to bind multiple BAFF receptors expressed by the cancer cell, rather than one antigen alone. Using cytotoxicity assays, we demonstrate the functionality and specificity of BAFF CAR-T cells in killing several types of B cell malignancies, and we further confirm their efficacy in several mouse models of cancer. These results have helped bring BAFF CAR-T therapy to Phase I clinical trials, and we hope they prove efficacious for patients with relapsed/refractory B cell malignancies. Meanwhile, interest has rapidly increased in natural killer (NK) cell-based immunotherapies. NK cells have important roles in cancer immunosurveillance and offer advantages over T cells in clinical safety and time-to-treatment. However, NK therapies face challenges in efficacy and persistence, often due to the presence of TGF-β, a powerful immunosuppressive cytokine that is frequently elevated in cancer patients and a primary cause of NK cell dysfunction. In this work, we characterize an undiscovered role of cyclin dependent kinase 5 (Cdk5) and its coactivator p35 in NK cell cytotoxicity. Using genetic tools to modulate Cdk5/p35 kinase activity in human NK cells, as well as using NK cells from p35 knockout mice, we show that Cdk5/p35 negatively regulates NK (open full item for complete abstract)

Committee: Reshmi Parameswaran (Advisor); Alex Huang (Committee Chair); John Letterio (Committee Member); Clive Hamlin (Committee Member) Subjects: Biology; Biomedical Research; Immunology; Medicine; Molecular Biology; Oncology

Master of Science (MS), University of Toledo, 2022, Biomedical Sciences (Bioinformatics and Proteomics/Genomics)

As multi-omics studies gain popularity, more insight is gained on the biological mechanisms that cause and influence disease. This study combined a transcriptomic approach and functional proteomic approach to gain a holistic understanding of End-Stage Renal Disease (ESRD), renal allograft status, and immunosuppression status on a molecular level. Functional proteomics approaches like kinomics consider post-translational modifications such as phosphorylation that alter protein function. The kinome is the comprehensive list of all protein kinases within an organism and the study of these protein kinases is critical in the understanding of cellular communication. Kinase activity quantified by phosphorylation can reveal key biological pathways. This study aims to assess the active kinome profiles from peripheral blood mononuclear cells (PBMCs) from renal transplant patients. Active kinome profiles of PBMCs will provide information about the cellular communication that facilitates immune response. The information gathered from the PBMC active kinome profile may also be used to predict response to immunosuppressant drugs based on the observed activity of protein kinase signaling networks. The effects of immunosuppressive drugs are well understood on a transcriptomic level but have yet to be explored on a kinomic level. Immunosuppressive drugs perturb kinase signaling networks, therefore the baseline active kinome profile might act as a biomarker for trait-based immune system function. This can then allow for the prediction of a patient's potential allograft acceptance or rejection based on their active kinome profile and how it relates to immunosuppressant drug signatures. Additionally, PBMC samples can be treated with kinase inhibitors that model the effect of an immunosuppressant medication on the protein kinase signaling networks in vitro. The impact an inhibitor has on the protein kinase activity can be used to determine optimal treatment. Furthermore, active kinome p (open full item for complete abstract)

Committee: Robert Smith (Advisor); Stanislaw Stepkowski (Committee Member); Puneet Sindhwani (Committee Member); Kunal Yadav (Committee Member) Subjects: Bioinformatics; Biomedical Research; Immunology; Medicine; Molecular Biology; Statistics

MS, University of Cincinnati, 2017, Medicine: Clinical and Translational Research

Introduction: Long term outcomes of proteasome inhibitor (PI) based treatment for antibody-mediated rejection (AMR) and mixed-acute rejection (MAR) remain to be defined. Additionally, prognostic factors that determine allograft survival post-rejection have not been clearly defined in the literature with a homogenous treatment population. Therefore, the primary outcome of this study was to evaluate clinical responses to PI-based therapy for four rejection phenotypes (i.e. early AMR, early MAR, late AMR, late MAR) and to determine factors that predict allograft survival. Methods: Renal transplant recipients with first-time AMR treated with PI-based therapy between January 2005 to June 2015 at the University of Cincinnati and The Christ Hospital were evaluated. Patient demographic, immunosuppression, and laboratory information was obtained from an IRB-approved prospective database and electronic medical records. Categorical variables were compared with either the Kruskal Wallis test or ?2 test. Continuous variables were compared with the Wilcoxon signed-rank test, Mann-Whitney U test, or the Kruskal Wallis test. Univariate and multivariate logistic regression models were generated to determine risk factors for death-censored graft failure 3 years post-rejection. Results: 108 patients were analyzed with early AMR (n = 40), early MAR (n = 9), late AMR (n = 20), or late MAR (n = 39). Histopathologic improvement assessed using Banff component scoring was similar across the groups (early AMR 50.0% vs. early MAR 66.7% vs. late AMR 64.2% vs. late MAR 76.3%, p=0.25). Median percent reduction in immunodominant DSA (iDSA) was significantly different across the rejection phenotypes (early AMR 79.6% vs. early MAR 54.7% vs. late AMR 23.4% vs. late AMR 21.1%, p < 0.001). Death-censored graft survival (DCGS) was significantly different at 3-years post-rejection (early AMR 88.3% vs. early MAR 77.8% vs. late AMR 56.7% vs. late MAR 54.9%, p=0.02). Patients who achieved a > 50% r (open full item for complete abstract)

Committee: Erin Haynes Dr.P.H. (Committee Chair); Rita Alloway (Committee Member); Ervin Woodle M.D. (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 1980, Graduate School

Committee: Not Provided (Other) Subjects: Health Sciences

Doctor of Philosophy, The Ohio State University, 1977, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Chronic Lymphocytic Leukemia (CLL) is the most prevalent adult leukemia with estimations in the US of over 18, 900 newly diagnosed cases and 4,600 deaths for 2016. Patients suffer from profound defects in immune function, with secondary infection a leading cause of morbidity and mortality that is further exacerbated by frontline therapy. A deeper understanding of CLL-associated immunosuppression is needed in order to overcome immune compromise. Enhancing Natural Killer (NK) effector functions has emerged as a promising immunotherapy. NK cells are innate immune effectors that survey and kill pathogen-infected or malignant cells. These cells are a major component of anti-tumor response especially with monoclonal antibody therapeutics (mAbs) that engage NKs and mediate tumor killing. Activating NK anti-tumor functions depends on a tightly regulated network of inhibitory and activating receptors that detect “self” antigens, which are severely deregulated in CLL leading to tumor escape. MAbs that activate NK anti-tumor immunity represent a promising chemotherapy-free therapeutic option that would not only spare patients' immune systems but even enhance the anti-tumor response. Antibodies generated by glyco-engineering have improved capacity to recruit and activate NK anti-tumor response. Defucosylation is a form of glyco-engineering that removes a fucose moiety at the CH2 locus within the antibody constant region (Fc) and raises the affinity between antibody and NK resulting in more effective NK-mediated killing of targeted cancer cells. B-cell activation factor (BAFF) ligating to BAFF receptor (BAFF-R) triggers critical pro-survival signals in B cells and blocking this interaction represents a novel target for immunotherapy. Glyco-engineered anti-BAFF-R activated superior NK antibody-dependent cellular cytotoxicity (ADCC) over CD20 antibodies including glyco-engineered obinutuzumab, and activated additional innate immune response as demonstrated by TNFa release by mo (open full item for complete abstract)

Committee: John C. Byrd (Advisor); Natarajan Muthusamy (Advisor); Virginia Sanders (Committee Member); Susheela Tridandapani (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), Wright State University, 2014, Environmental Sciences PhD

Transcriptional regulation of the murine immunoglobulin heavy chain gene involves several regulatory elements including the 3'Igh regulatory region (3'IghRR) composed of at least four enhancers (hs3A; hs1,2; hs3B; hs4). Enhancers hs1,2 and hs4 contain binding sites for several transcription factors including NF-κB/Rel proteins and the AhR. Interestingly, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) profoundly inhibits 3'IghRR and hs1,2 activation induced by the B-cell activator lipopolysaccharide (LPS), but enhances the activation of the hs4. Within the hs4, the AhR binding site overlaps an NF-κB/Rel binding site suggesting that both the AhR and the NF-κB together may modulate of the 3'IghRR. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR following LPS stimulation and TCDD treatment on 3'IghRR, hs1,2, and hs4. In our studies we utilized the CH12.LX B cell line; the CH12.IκBαAA cell line, which expresses an inducible IκBα super repressor (IκBαAA); the CH12.γ2b-3'IghRR cell line that stably expresses a γ2b-3'IghRR-regulated γ2b transgene reporter; and splenocytes derived from B6C3F1 mice. The stimulation of the CH12.γ2b-3'IghRR cell line with Toll-like receptor (TLR) agonists LPS, Resiquimod (R848), or Cytosine-phosphate-Guanine (CpG)-oligodeoxynucleotides combined with a co-treatment of TCDD significantly inhibited the TLR-induced activation of the 3'IghRR. Utilizing transiently expressed luciferase reporters, we found induction of IκBαAA expression partially attenuated LPS-induced activation of the 3'IghRR and hs4, partially reversed the effects of a TCDD and LPS co-treatment on the activity of the 3'IghRR and hs4, and the addition of an AhR antagonist, CH223191, markedly reversed the LPS and TCDD induced inhibition of the 3'IghRR and inhibited the synergistic activation of the hs4. Chromatin immunoprecipitation analysis of CH12.LX and murine splenocytes demonstrated a LPS and a LPS co-treatment with TCDD-dependent increase in Rel (open full item for complete abstract)

Committee: Courtney Sulentic Ph.D. (Advisor); Michael Leffak Ph.D. (Committee Member); Mill Miller Ph.D. (Committee Member); David Cool Ph.D. (Committee Member); Rodney DeKoter Ph.D. (Committee Member); Don Cipollini Ph.D. (Other) Subjects: Environmental Science; Immunology; Molecular Biology; Toxicology

Doctor of Philosophy, The Ohio State University, 2013, Integrated Biomedical Science Graduate Program

The innate immune system is notoriously known for protecting the host against invading pathogens, especially those inhaled through the lung. The robust production of cytokines and other immune modulating proteins from innate immune cells contribute to bacterial clearance. Given the significance of this system in overall defense, we hypothesize that the most effective Gram-negative pathogens manipulate the immune response in favor of their own survival. This thesis focuses on the inability of host cells to respond effectively to Gram-negative bacteria. Two Gram-negative bacteria, Burkholderia cenocepacia and Francisella tularensis, are utilized in this study to explore this hypothesis. The first part of this thesis investigates the interactions between Burkholderia and airway epithelial cells, frequently overlooked participants in the immune system. Previously work from our lab detailed how monocytes were able to generate an efficient immune response that is driven by by NF-KB following following B. cenocepacia infections. Pro-inflammatory cytokine responses of airway epithelial cells after B. cenocepacia infection had not been previously explored. We demonstrated the ability of airway epithelial cells to produce pro-inflammatory cytokines after stimulation, yet IL-1b was not secreted despite increased transcript levels. Further examination showed that IL-1b failed to be secreted but instead was confined within the cytosol of epithelial cells. This occurred not only in cell lines but also in primary human bronchial epithelial cells. Caspase-1, the enzyme responsible for IL-1b processing, was extremely diminished in epithelial cells but restoration of this enzyme lead to proper IL-1b release. In the second portion of this thesis, we approached the host's inability to mount an effective response by investigating monocytes and Francisella interactions. Prior reports from our lab detailed that monocytes infected with virulent F. tularensis exhibit a mut (open full item for complete abstract)

Committee: Susheela Tridandapani (Advisor); Larry Schlesinger (Committee Chair); Mark Wewers (Committee Member); Prosper Boyaka (Committee Member) Subjects: Immunology

MS, University of Cincinnati, 2013, Medicine: Clinical and Translational Research

Previous studies associating non-adherence with donor specific antibodies (DSA) development in renal transplant recipients have used subjective assessments to classify non-adherence. No studies have examined the association between objective measures of non-adherence – primarily, standard deviation and/or coefficient of variation of immunosuppressive drug levels - and DSA development. We retrospectively analyzed the standard deviation and coefficient of variation in the five most recent tacrolimus or sirolimus trough drug levels at the time of DSA testing in 69 low-to-standard immunologic risk, first-time pediatric renal transplant recipients followed at a single US pediatric nephrology center. DSA prevalence in the population was 38% (n=26). DSA+ patients had significantly higher standard deviation (p=0.014) and coefficient of variation (p=0.024). There was a trend towards increased DSA prevalence in patients taking sirolimus vs. tacrolimus (p=0.14). Multivariate logistic regression demonstrated that coefficient of variation (p = 0.03) and black race (p=0.012) retained statistical significance in DSA development. For each 10% increase in coefficient of variation, the odds of developing a DSA increased by almost 40% (adj OR 1.38, 95% CI 1.09, 1.75). Future studies are needed to understand the extent and length of time variations in immunosuppressant medications that are tolerated prior to DSA formation.

Committee: Paul Succop Ph.D. (Committee Chair); Jens Goebel M.D. (Committee Member); Ahna Pai Ph.D. (Committee Member); Erin Nicole Haynes Dr.P.H. (Committee Member) Subjects: Surgery

MS, University of Cincinnati, 2011, Medicine: Clinical and Translational Research

Introduction: Current guidelines differ on screening for latent tuberculosis (LTB) infection prior to starting immunosuppressive therapy. We developed a Markov state-transition model to determine the most “cost-effective” screening strategy prior to initiating steroid treatment in a 5 year old with newly diagnosed idiopathic nephrotic syndrome. Methods: Using data from the published literature, universal purified protein derivative (PPD) testing was compared with targeted screening using a risk-factor questionnaire. A secondary model also included testing with the newer interferon gamma release assays (IGRAs), requiring only a single visit and having greater specificity than the PPD. Results: At an LTB prevalence of 1.1%, universal PPD testing was more costly and less effective than other strategies. Instead, we found that a targeted strategy using a simple risk assessment questionnaire to determine who should receive a PPD cost roughly $44,000 per quality-adjusted life year (QALY) gained compared to a no screening strategy. Conversely, at an LTB prevalence >12.8%, universal PPD became “cost-effective” compared to targeted screening. In the secondary model, targeted screening with a questionnaire followed by IGRA testing was the preferred strategy. Conclusion: Patients who live in areas with a higher prevalence of LTB will likely benefit from universal PPD testing. Once IGRA testing is approved in children, it may become a component of “cost-effective” screening options.

Committee: Paul Succop PhD (Committee Chair); Mark Eckman MD (Committee Member); Erin Nicole Haynes PhD (Committee Member) Subjects: Surgery

PhD, University of Cincinnati, 2007, Medicine : Toxicology (Environmental Health)

Polychlorinated biphenyls (PCBs) are persistent organic pollutants linked to numerous human health problems, including learning and memory deficits in children of exposed mothers. PCBs exist in the environment as complex mixtures. Both coplanar and non-coplanar PCBs are reported to have neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. We used a mixture of eight PCBs to model human exposures based on their reported concentrations in human tissue, breast milk, and the human food supply. Individual risk to PCBs varies by genetic makeup. We developed a mouse model to explore the role of two genes that may be responsible for some of the reported inter-individual differences: the aryl hydrocarbon receptor (AHR) and CYP1A2. There are >12-fold differences between humans with regard to AHR affinity and >60-fold differences in hepatic basal CYP1A2 levels. Our mouse model used Cyp1a2(+/+)wild-type and Cyp1a2(-/-)knockout mice with either a high-affinity or poor-affinity Ahrgene. We hypothesized that high-affinity Ahr bCyp1a2(-/-)would be most susceptible and poor-affinity dCyp1a2(+/+)most resistant to PCB-induced developmental neurotoxicity. Using GC-ECD, we determined that offspring of Cyp1a2(-/-)mothers were exposed to higher levels of coplanar PCBs during development and that Ahr bmice metabolized all PCB congeners more quickly. Differences in PCB exposure were closely correlated with changes in CYP1A1 and CYP1A2 mRNA and protein levels and with well-known endpoints of AHR-mediated toxicity: immunosuppression and hepatotoxicity. In addition, we conducted behavioral phenotyping of exposed offspring. We found significant deficits in learning and memory in Ahr bCyp1a2(-/-)mice, including impairments in novel object recognition and an increased failure rate in the Morris water maze. However, all PCB-treated groups and all genotypes showed significant differences in at least one measure of learning (open full item for complete abstract)

Committee: Dr. Daniel Nebert (Advisor) Subjects: