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

Glioblastoma (GBM) remains one of the most lethal human cancers, with conventional treatment offering only palliation. Like all cancers, GBMs display the Warburg effect, a preferential utilization of aerobic glycolysis for energy supplies. This metabolic shift reduces the cells oxygen dependence and provides a steady supply of anabolic material yet requires a steady supply of glucose. In this dissertation, I show that nutrient restriction contributes to tumor progression by enriching for glioblastoma stem cells (GSCs), the most treatment-resistant and regenerative of GBM cells. This enrichment is due to both preferential GSC survival and adaptation of non-GSCs through acquisition of stem-like features. GSCs outcompete for glucose uptake by co-opting the high-affinity neuronal glucose transporter isoform, type 3 (GLUT3). In the normal brain, glucose is an essential fuel for neuronal metabolism; yet vascular glucose delivery is physiologically stymied by the blood brain barrier. In response to this, neurons express GLUT3, allowing steady glucose uptake from a glucose-poor microenvironment. GSCs preferentially express GLUT3 and targeting GLUT3 inhibits GSC growth and tumorigenic potential, suggesting GLUT3 is a marker of CSCs. GLUT3, but not GLUT1, correlates with poor survival in brain tumors and other solid cancers in clinical patient datasets; thus, CSCs in many cancer types may extract nutrients with high affinity. Given the restricted expression profile of GLUT3 in non-malignant tissue and the critical role of GLUT3 in CSC biology, therapeutic targeting of GLUT3 may prove to be a viable treatment across multiple cancer types.

Committee: Jeremy Rich MD (Advisor); Xiongwei Zhu PhD (Committee Chair); Jan Jensen PhD (Committee Member); Robert Silverman PhD (Committee Member); Robert Weil MD (Committee Member); Anita Hjelmeland PhD (Committee Chair) Subjects: Biology; Biomedical Research; Cellular Biology; Molecular Biology; Neurobiology

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

Principal component analysis (PCA) is a data analysis technique used to reduce the dimension of a data set while retaining key patterns of variation by transforming the data to a lower-dimension space defined by orthonormal basis vectors that capture the directions of maximal variation. A novel technique named logistic PCA (LPCA) was developed that allows researchers to make use of benefits of PCA analysis in the study of data sets containing binary variables, allowing for more widespread use of these methods in areas of study frequently examining binary data, such as biomedical science and healthcare. We apply logistic PCA method to two data sets, the first comprised of data from tissue samples obtained from patients diagnosed with breast cancer and the second comprised of data from select genetic profiles of individuals diagnosed with brain tumor. An initial simulation study was performed to examine randomly-generated binary data from settings with a known clustering structure to evaluate retention of clustering in low-dimension plots created using PCA, LPCA, and t-distributed stochastic neighbor embedding (t-SNE), another frequently-utilized data analysis technique. Results revealed that LPCA consistently outperforms PCA in terms of reconstruction error in settings where probability parameters for clusters are close to 0.5 and that LPCA and PCA perform comparably in settings with more extreme probability parameters. LPCA and t-SNE also show comparable clustering in the two-dimensional plots. In analysis of the cancer-related data, two-dimensional plots for data embedding were generated, and principal component loadings were obtained from each of the data sets using LPCA and PCA, and used to provide interpretations of data patterns in the context of cancer-related biomedical science and healthcare. Analysis revealed that interpretations of LPCA loadings provide information consistent with established biomedical research findings as well as new information and that (open full item for complete abstract)

Committee: Yoonkyung Lee (Advisor); Asuman Turkmen (Committee Member) Subjects: Applied Mathematics; Biology; Genetics; Medicine; Oncology; Statistics

Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

Cancer is the second leading cause of global mortality and requires continuous research into novel treatment methods. Chemotherapy, radiation treatments, and surgery are often not adequate in part because of cancer stem cells (CSCs) that persist and cause tumor recurrence. Hence, researchers are examining natural remedies as complementary treatments. The turmeric ingredient curcumin is used in traditional medicine and exhibits potent anti-CSC properties but also low bioavailability that limits use in patients. Consequently, curcumin analogs with enhanced bioavailability and pharmacokinetics have been synthesized to suppress cancers. This project examined efficacy of curcumin analog HO-3867 in preparation for testing whether it also targets CSCs. Cells in gliomas, a type of brain cancer, contain circadian clocks that offer potential for suppressing tumor growth by manipulating the molecular mechanism that generates circadian rhythms. Here, we examined effects of HO-3867 on rat C6 glioma cells that are a model for human glioblastoma, the deadliest type of glioma. We tested whether the circadian clock regulates the anticancer effects of HO-3867. Our initial dose-response curves indicated the EC50 for HO-3867 in C6 cells is 26.3 μM. To test whether the circadian clock modulates HO-3867 anticancer effects, the circadian clocks within the cells were synchronized and one-day treatments of 20 μM HO-3867 were delivered at 6-hour intervals for two days. Efficacy was evaluated using an assay of cell viability and morphological measures of cell death. No circadian rhythm effect on HO-3867 efficacy was detected, indicating its delivery as a drug at any time of day is not likely to be restricted by a circadian rhythm in pharmacodynamics, although circadian rhythms in the body might modulate pharmacokinetics. The circadian rhythm in cancer cells might help them survive by evading immune surveillance that fluctuates in a circadian rhythm. Immunotherapy shows promise in cancer tre (open full item for complete abstract)

Committee: Michael Geusz Ph.D. (Committee Chair); Daniel Pavuk Ph.D. (Committee Member); Julia Halo-Knerr Ph.D. (Committee Member) Subjects: Biology; Cellular Biology; Neurosciences; Pharmacology

Master of Science (M.S.), University of Dayton, 2024, Biology

Objective: Glioblastoma (GBM) is a highly aggressive and malignant brain tumor that has limited treatment options and has an extremely poor prognosis (Waghmare et al. 2014). The amplification of Epidermal Growth Factor Receptor- VIII (EGFR-VIII) and activation of the Phosphatidyl Inositol 3-Kinase (PI3K) pathway are common genetic alterations observed in GBM patients (An et al. 2018). Our objective is to model GBM in Drosophila melanogaster and study the signaling pathways that promote GBM growth and inhibit cell death. Specifically, we aim to investigate the roles of MAPK, Hippo, and WNT signaling pathways in regulating GBM growth and Cactus expression, which regulates the JNK pathway. Methods: Our project involves genetic crosses that produce larvae with GBM, followed by brain dissections and immunohistochemistry to study changes in signaling pathways that promote GBM growth. Specifically, we are studying the early time points to understand the roles of signaling pathways like MAPK, Hippo, and WNT in promoting GBM growth and/or inhibiting cell death. By comparing our GBM models to experimental controls, we aim to generate initial data for designing further genetic experiments to identify specific signaling interactions that affect cell death and proliferation. We will use two lines, (1) y w UAS PI3K92E; +; Repo-Gal4 and (2) UAS GFP/TM3B,Sb, and (2) y w; UAS EGFRλtop/TM6C, to generate glioma in Drosophila, and investigate whether the Hippo pathway regulates Cactus, which also regulates the JNK pathway. Significance: The proposed research has significant implications for understanding the molecular mechanisms underlying GBM growth and identifying key molecules and pathways that drive this deadly disease. Using Drosophila as a model system allows for efficient genetic manipulation and provides a cost-effective way to study complex biological processes. Additionally, the results of this study will contribute to our understanding of GBM.

Committee: Dr. Madhuri Kango-Singh (Advisor); Dr. Amit Singh (Committee Member); Dr. Thomas Williams (Committee Member) Subjects: Biology; Cellular Biology; Genetics

MS, University of Cincinnati, 2024, Medicine: Cancer and Cell Biology

Glioblastoma multiforme poses significant treatment challenges, attributed to its distinctive characteristics and immune evasion mechanisms, and carries a grim prognosis despite advancements in therapy. Therefore, rigorous efforts are necessary to uncover additional therapies or other unconventional strategies. Recently, immunotherapies have gained significant traction due to their success in certain malignancies. Immunotherapies use the host's immune system to enhance the ability to combat cancer. These therapies have been in development for over 100 years, and many of them are currently under investigation for treating Glioblastoma multiforme in preclinical and clinical studies. Immunotherapies include antibody therapies, including immune checkpoint blockade, adoptive T cell therapies, cytokine therapies, cancer vaccines, and oncolytic viruses. A deeper understanding of the origins of immunotherapy, immune system dynamics in response to cancer, immune evasion mechanisms of glioblastoma, and the various immunotherapeutic strategies may facilitate the development of novel and effective treatment strategies for glioblastoma. Here, we review the different immunotherapeutic approaches and propose possible combinatory strategies.

Committee: Qing Lu Ph.D. (Committee Chair); David Plas Ph.D. (Committee Member); Biplab DasGupta Ph.D. (Committee Member); David Haslam M.D. (Committee Member) Subjects: Oncology

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

Neurological pathologies provide a distinctively challenging region for new therapeutic targets due to extra physical protection, resident brain cell responses, and the fragility of the irreplaceable brain itself. Novel ways to approach neurological diseases include considering smaller molecules such as metabolites and their effects and origins, and exploring broader, more complex connections such as the gut-brain-microbiome axis. In Chapter 1, I discuss gut-brain-microbial pathways and how these unique connections may provide us with myriad therapeutic targets in disease, including glioblastoma and Alzheimer's disease (AD). Chapter 2 explores the enrichment of trimethylamine-N oxide (TMAO), a metaorganismal metabolite, in the olfactory bulb, indicating an impactful role in olfactory perception. The evidence supporting this observation comes from orthogonal studies using both pharmacological inhibition and genetic loss of function (LOF) models. These findings carry implications for potential dietary and sensory interventions associated with neural changes in AD. Chapter 3 includes investigation into a new mechanism of immune suppression in the tumor microenvironment (TME) via the small molecule metabolite spermidine (SPD). Metabolites such as SPD that are produced and secreted by tumor cells have an indirect effect on tumor growth by targeting immune cells in the TME. This novel connection provides a window into yet another manner glioblastoma cells are able to manipulate their surroundings, building hope for new therapeutic targets. Although not fully comprehensive, these studies highlight some unique mechanisms regarding how some metabolites function in brain cancer and how other metabolites are working through the microbe-host connection to influence neurological symptoms.

Committee: Justin Lathia (Advisor); J. Mark Brown (Committee Chair); Jeannette Messer (Committee Member); Gabrielle Yeaney (Committee Member); Ofer Reizes (Committee Member) Subjects: Medicine; Microbiology; Molecular Biology; Neurology; Oncology

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Although there are many therapies against cancer, they involve devastating side effects. Hence, we need to understand the nature of cancer cells and the unique markers that exist within these cells that allow them to evade the immune system. These cell properties could be exploited to our advantage. CD47 is a cell membrane receptor protein widely expressed in most cells and is a versatile and crucial target in the tumor microenvironment for creating novel therapeutic approaches for cancer treatment. However, few studies have examined CD47 in glioma. CD47 is found on the surface of multiple cell types, and it usually protects the cells from being removed by phagocytes. It has been found that most cancer cells have high CD47 expression that prevents them from being engulfed by macrophages or activated microglial cells, essentially acting as a “don't eat me signal.” A second protein expressed on cancer cells, calreticulin (CALR), facilitates cell removal by phagocytes, serving as an “eat me signal.” In this project, we compared CD47 expression in glioma cancer stem cells (CSCs), which are negative for Hoechst 33342 nuclear staining (H-), and non-stem glioma cells (Hoechst-positive, H+) of the C6 cell line derived from a rat astrocytoma. We examined the colocalization of CALR with CD47 in both C6 cell types using immunocytochemistry and compared CALR and CD47 gene expression reported in the NCI-60 database of multiple human cancers. We found a significant difference in CD47 expression, with more CD47 in the H+ cells than the H- cells, which could imply that GSCs are more susceptible to CD47 immunotherapy. The highest expression of CD47 ( 50% above signal range) appeared to be in exosomes related to both cell types. We found a positive correlation between CD47 and CALR distribution in the H+ cells (p = 0.0204) and in both H+ and H- cells combined (p = 0.0121), suggesting that the cells might protect themselves from CALR-induced phagocytosis by increasing CD47. We al (open full item for complete abstract)

Committee: Michael Geusz PhD (Committee Chair); Julia Halo PhD (Committee Member); Paul Morris PhD (Committee Member) Subjects: Biology; Medicine

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Glioblastoma (GBM) is a malignant brain tumor with no current cure and a 5 year survival rate of 5%. It is a fast growing and invasive tumor that results in universal recurrence, even after treatment. The standard of care is surgery followed by radiotherapy and chemotherapy, mainly temozolomide. While GBM affects mainly adults, it can occur at any age and there is a need for new treatments to be developed. ZFTA fusion positive ependymoma (EPN-ZFTA) is an aggressive, pediatric brain tumor also with no curative measures. EPN-ZFTA is characterized by resistance to current chemotherapies, leaving surgery and radiotherapy as the only treatment options. These tumors have a 40% recurrence rate, and a poor prognosis correlating to the younger the patient is at diagnosis. Overexpressed cancer stem cell genes and the presence of cancer stem cells have been linked causatively to higher rates of recurrence. There is a need for a therapeutic drug that both targets EPN-ZFTA tumors and can be well tolerated in young patients. CBL0137 is a small molecule drug that inhibits NF-kB signaling, decreases expression of cancer stem cell genes, and activates p53. It has been shown in other tumor types to target cancer cells while being non-toxic to normal tissue. Here we investigate the potential for CBL0137 to be used as a new therapeutic for both GBM and EPN-ZFTA. In Chapter 2, we explore how CBL0137 increases the level of DNA damage and leads to tumor cell death. Further, we show that CBL0137 increases the efficacy of radiotherapy and survival in mouse models of GBM. In Chapter 3, we investigate how CBL0137 dynamically alters the tumor microenvironment in GBM. We see an increase in DNA damage and cell death, and a decrease in stem cell proteins after CBL0137 treatment in in vivo mouse models. Lastly, in Chapter 4 we explore if EPN-ZFTA is sensitive to CBL0137. We show CBL0137 treatment decreases NF-kB target genes, decreases stem cell genes and activates p53 in EPN-ZFTA. We investiga (open full item for complete abstract)

Committee: Monica Venere (Advisor); Daniel Stover (Committee Member); Christin Burd (Committee Member); Amanda Toland (Committee Chair) Subjects: Biomedical Research; Oncology; Radiation

Doctor of Philosophy, The Ohio State University, 2023, Biomedical Sciences

Glioblastoma (GBM), a grade IV treatment refractory aggressive brain tumor, is one of the deadliest forms of cancer with an average patient survival of 15 months and 5-year survival rate of less than 5%. This poor prognosis is despite an aggressive standard-of-care consisting of maximally safe surgical resection followed by radiation and temozolomide treatment. Standard-of-care has not changed over the past two decades. There is a clear unmet need for better therapeutic strategies to combat GBM. Genomic profiling of GBMs identified genetic alterations within three pathways: receptor tyrosine kinase (RTK)/Ras/PI3K; p53/ARF/MDM2/MDM4; and RB/CDK4/INK4A. Hyperactivation of the RTK/Ras/PI3K pathway leads to the persistent activation of diverse serine/threonine signaling networks that promote proliferation, metabolic reprogramming, and resistance to standard-of-care treatment. In addition, GBMs possess an enhanced signaling heterogeneity and plasticity which facilitates the activation of downstream STK signaling pathways. The underlying mechanisms that enable this signaling adaptability in GBMs remain poorly understood. A significant knowledge gap in our understanding of aberrant oncogene signaling is the role of serine/threonine phosphatases, in particular protein phosphatase 2A (PP2A). PP2A is a bona fide tumor suppressor, physiologically functioning as the counterbalance to the serine/threonine signaling networks activated downstream of RTK/Ras/PI3K activation. Unlike other tumor suppressors, such as p53 (~35% genetic alteration) and PTEN (~36% genetic alteration), PP2A (<1% genetic alteration) is genetically intact in GBM. However, it remains unclear how GBMs evade the tumor suppressor function of PP2A to maintain persistent activation of serine/threonine signaling networks. In this work, our goal was to elucidate regulatory mechanism(s) exploited by GBM to overcome the tumor suppressor activity of PP2A, with the aim of identifying actionable therapeutic targets. (open full item for complete abstract)

Committee: Arnab Chakravarti (Advisor); Deliang Guo (Committee Member); Steven Clinton (Committee Member); Nicholas Denko (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Radiation

Doctor of Philosophy, The Ohio State University, 0, Neuroscience Graduate Studies Program

Glioblastomas are highly fatal primary brain cancer lesions requiring careful clinical management. Despite standard primary intervention being maximal safe surgical resection followed by radiation with concomitant temozolomide therapy, later management is often ambiguous and ever-changing. In consequence, identification of markers and specific guidelines for post-chemoradiation management is critical to improve patient outcomes. Our overarching goal seeks to determine whether novel disease markers can be identified to better understand survival and novel radiologic imaging changes in patients with glioblastoma to improve current clinical management approaches. In the first component of our work, we collected retrospective clinical data from patients seen across several healthcare infrastructures to determine whether routinely collected information from the electronic health record is informative of long-term patient survival outcomes. Using an unsupervised machine learning approach, we discovered that white blood cell count during baseline treatment planning was associated with overall patient survival with an over 6-month median survival difference between the upper and lower quartiles of white blood cell count. Furthermore, using objective image analysis workflows, we merged quantification of PDL-1 immunohistochemistry to uncover associations of white blood cell load to tumor immune activity. Our findings support that incorporating white blood cell count and PDL-1 expression in brain tumor biopsies as simple biomarkers predictive of glioblastoma patient survival. In the second component of our work, we more closely examined the nature of novel radiologic imaging changes in patients by characterizing molecular features of truly progressive glioblastoma lesions against pseudoprogressive lesions that can be conservatively managed. Lack of objective clinical standards creates needs to validate the accuracy of current approaches in stratifying disease. We posited (open full item for complete abstract)

Committee: Jose Otero (Advisor); Leah Pyter (Committee Member); Raghu Machiraju (Committee Member); Monica Venere (Committee Member); Mireia Guerau (Committee Member) Subjects: Information Science; Neurosciences; Oncology

PhD, University of Cincinnati, 2022, Pharmacy: Pharmaceutical Sciences

Glioblastoma (GBM) is the most aggressive primary brain tumor with a dismal prognosis and a median survival of ~ 8 months. The efforts to develop novel therapeutic agents are impeded by (i) lack of novel validated GBM-specific targets and, (ii) failure of the anti-glioma agents to cross the blood-brain barrier (BBB). Our current research addresses these issues. 5' AMP-activated protein kinase (AMPK) is a major metabolic switch that controls a broad array of cellular biosynthetic and catabolic pathways. Recently, our group has provided evidence suggesting that AMPK is a “druggable” target for GBM therapy. The observations included: i) AMPK subunits were highly expressed in GBM cell lines, ii) AMPK depletion in vitro and orthotopic GBM xenografts significantly reduced cell viability and suppressed tumor growth, and iii) systemic AMPK knockout was well tolerated in adult mice with no discernible phenotype. Inhibition of AMPK by small molecules is mostly limited to Compound C (dorsomorphin), which has a poor target-selectivity profile, but a structurally related compound, SBI-0206965, an aminopyrimidine derivative shows greater specificity toward AMPK. As such, SBI-0206965 is being investigated for the treatment of GBM. We first characterized the systemic and brain pharmacokinetics (PK) in rats and mice and the metabolism of SBI-0206965 using liver microsomes. We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in rats. SBI-0206965 is quickly absorbed, and plasma and brain extracellular fluid (ECF) peak levels were attained within 0.25 – 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma, brain partitioning is ~ 0.6 - 0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in the mice, rats, and humans was 325, 76, and 68 mL/min/kg, respectively. SBI-0206965 metab (open full item for complete abstract)

Committee: Pankaj Desai Ph.D. (Committee Member); Biplab DasGupta Ph.D. (Committee Member); William Seibel Ph.D. (Committee Member); Nalinikanth Kotagiri Ph.D. (Committee Member); Gary Gudelsky Ph.D. (Committee Member) Subjects: Pharmaceuticals

PhD, University of Cincinnati, 2022, Pharmacy: Pharmaceutical Sciences

Glioblastoma (GBM) is one of the most aggressive malignant brain tumors with a dismal prognosis. Current treatment modalities for GBM include maximally safe surgical resection, followed by radiation therapy and concomitant administration of temozolomide (TMZ), the only chemotherapeutic agent approved for this purpose. However, this multimodal approach of treatment fails to eradicate the disease and tumors develop resistance to TMZ. Thus, there is an urgent unmet need to develop novel therapeutics for GBM to act as monotherapy and/or as a combination agent with TMZ. In this study we carried out translational studies on lead compounds that target 2 novel receptors abundantly expressed in GBM; ?-aminobutyric acid receptor-a (a5-GABAA) and aromatase, an estrogen synthase enzyme. The first compound, AML-101 is a newly discovered highly specific and selective agonist of a3- and-a5-GABAA agonist that has shown considerable activity against medulloblastoma. Here we show for the first time that AML-101 markedly potentiates TMZ activity against patient-derived GBM cells. Furthermore, employing microdialysis studies we simultaneously assessed the pharmacokinetics (PK) of AML-101 in the brain extracellular fluid and plasma in rats. Results suggested facile passage of the compound across the blood-brain barrier (BBB) and the oral bioavailability of AML-101 was 0.7. Thus, our PK studies strongly support further development of this compound based on the observation that it has excellent pharmacodynamic and PK properties to be an anti-GBM therapy. In parallel, my work focused on advancing efforts for repurposing letrozole (LTZ), a widely used drug in the treatment breast cancer, for the treatment of GBM. LTZ exhibits effective brain penetration, tumor localization and efficacy against C6 glioma cells in rat models. Employing patient-derived GBM lines, we assessed the influence of LTZ and TMZ on cell viability and neurosphere growth. Combination index analysis was performed (open full item for complete abstract)

Committee: Pankaj Desai Ph.D. (Committee Member); Gary Gudelsky Ph.D. (Committee Member); Soma Sengupta M.D. Ph.D. (Committee Member); Timothy Phoenix Ph.D. (Committee Member); Kevin Li Ph.D. (Committee Member) Subjects: Pharmaceuticals

Doctor of Philosophy, Case Western Reserve University, 2022, Molecular Medicine

Glioblastoma (GBM) is the most common primary brain tumor in adults and exhibits therapeutic resistance, recurrence, and poor prognosis. Hence, novel therapeutic strategies for outcomes in these patients are urgently needed. GBM tumor cells modulate expression of iron-associated genes to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The translocator protein 18kDa (TSPO) plays crucial roles in essential mitochondria-based physiological processes and is a validated biomarker of neuroinflammation, which is implicated in GBM progression. The TSPO gene has a germline single nucleotide polymorphism, rs6971, which is the most common SNP in the Caucasian population. We retrospectively analyzed the correlation of the TSPO polymorphic variant rs6971 with overall and progression-free survival in GBM patients using three independent cohorts. The rs6971 polymorphism was significantly associated with shorter overall survival and progression-free survival in male GBM patients but not in females in one large cohort and similar trends were observed in two other independent cohorts. GBM growth and progression can also be driven by enhanced iron uptake from the surrounding microenvironment by tumor cells. The HFE gene, encoding the iron sensing HFE protein, is upregulated in GBM and correlates with poor survival outcomes. However, the molecular mechanisms underlying these observations remain unclear. We interrogated the impact of cell-intrinsic Hfe expression on proliferation and tumor growth through genetic loss and gain of function approaches in syngeneic mouse glioma models. Loss of Hfe induced apoptotic cell death in vitro and inhibited tumor growth in vivo while overexpression of Hfe accelerated both proliferation and tumor growth. Analysis of iron gene signatures in Hfe knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Analyz (open full item for complete abstract)

Committee: Justin Lathia PhD (Advisor); Jennifer Yu MD, PhD (Committee Chair); Trine Jorgensen PhD (Committee Member); Erin Murphy MD (Committee Member); Jessica Williams PhD (Committee Member) Subjects: Biology; Molecular Biology; Oncology

Doctor of Philosophy, Case Western Reserve University, 2022, Pathology

Mitochondrial dynamics, the fission and fusion of mitochondria, are vital to cellular health and homeostasis. Altered dynamics are associated with a number of diseases, one of which is the most aggressive and prevalent brain cancer, glioblastoma (GBM). Once diagnosed, patients live a median of less than 15 months with nearly all experiencing tumor recurrence, highlighting the need for therapeutic advancement. Also, increased mitochondrial fragmentation is often observed within the GBM stem cells (GSCs), known to be resistant to conventional therapies and believed to contribute to tumor recurrence. The mitochondrial fission machinery, dynamin-related protein-1 (Drp1) and mitochondrial fission factor (Mff), have both been implicated in contributing to the GBM disease state. To understand the altered regulation of these proteins in GBM, we investigate the post-translation modifications, O-GlcNAcylation and phosphorylation on them, respectively. We found that Drp1 O-GlcNAcylation and Mff phosphorylation were both increased in patient GSCs. Specifically, elevated O-GlcNAcylation of Drp1 correlated with increased mitochondrial fragmentation and mass, as well as altered mitochondrial ETC complex function. We also found that adenosine monophosphate (AMP)-activated protein kinase (AMPK) was the primary kinase phosphorylating Mff in one, but not all, of the GSC patient samples examined, demonstrating the importance of individual patient tumor profiling. Altogether, these studies demonstrate the importance of proper PTM regulation of Drp1and Mff in GBM, which may serve as future therapeutic points of intervention.

Committee: Jason Mears (Advisor); Brian Cobb (Committee Chair); Clive Hamlin (Committee Member); Justin Lathia (Committee Member); Ruth Keri (Committee Member) Subjects: Biochemistry; Biology; Biomedical Research; Cellular Biology; Molecular Biology; Morphology; Science Education

Doctor of Philosophy, Case Western Reserve University, 0, Biomedical Engineering

Glioblastoma multiforme (GBM) is an aggressive, grade IV brain cancer. The current standard-of-care treatment for GBM patients is multimodal that includes surgical resection followed by radiotherapy and concomitant chemotherapy with temozolomide i.e. chemoradiation-therapy (CRT). However, in spite of such an aggressive treatment, GBM patients have a dismal median survival of 12-15 months and only <10% patients survive for over 5 years. Unfortunately, over 40% of GBM patients undergo disease progression within few weeks of CRT treatment. This poor prognosis can be attributed to genetic instability and intra- and inter-tumor heterogeneity of GBM that leads to treatment resistance, progression, and tumor recurrence. Although, isocitrate dehydrogenase-1 (IDH1) mutations, O6-methylguanine-DNA methyltransferase (MGMT) promotor methylation, and extent of resection have shown promise as prognostic biomarkers, to our knowledge, currently there are no validated image-based biomarkers that could apriori determine the risk of poor survival in a GBM patient. Each patient is unique with distinct morphological as well as phenotypical profiles. There is thus an unmet need to identify non-responder patients prior to CRT treatment and to predict progression-free survival, for personalizing treatment decisions in GBM patients. Radiographic imaging such as magnetic resonance imaging (MRI) is routinely used for clinical diagnosis and response assessment of GBM by manual visual inspection. Similarly, surgically resected tissue slides contain rich phenotypic information that could reveal the inherent intra-tumoral heterogeneity and thus has prognostic implications. Recent advances in computational techniques such as radiomics and pathomics have shown improved efficacy (over manual inspection) for prognosis and response assessment of GBM tumors from MRI scans and histopathology slides, respectively. However, there still remain a few open questions that need to be addressed in order to b (open full item for complete abstract)

Committee: Pallavi Tiwari (Advisor); Anant Madabhushi (Committee Chair); Manmeet Ahluwalia (Committee Member); Efstathios Karathanasis (Committee Member); Jennifer Yu (Committee Member) Subjects: Artificial Intelligence; Biomedical Engineering; Computer Engineering; Computer Science; Health Care; Medical Imaging

Doctor of Philosophy, The Ohio State University, 2021, Neuroscience Graduate Studies Program

KIF11, also known as Eg5 and kinesin-5, is a homotetrameric kinesin that is best-described for its role in mitosis. KIF11 binds anti-parallel microtubules during mitosis to form and maintain the bipolar spindle. The role of KIF11 in mitosis has been heavily studied and characterized, but there is mounting evidence that KIF11 is not simply a mitotic protein; rather, it has diverse functions in the cell and has the ability to impact widespread cellular functions. Here, we investigate the role of KIF11 at primary cilia, and how this role has potential implications for proper neurodevelopment. There are individuals with KIF11 mutations who present with neuropathological phenotypes that largely match patients with ciliopathies, though KIF11 has never been implicated as a regulator of primary cilia. In Chapter 2, we demonstrate, for the first time, that KIF11 is localized to the basal body of primary cilia, is involved in proper primary cilia expression, and regulates primary cilia length. Further, we provide evidence that this previously unknown role for KIF11 is independent of its role in mitosis. In Chapter 3, we begin to investigate how KIF11 impacts primary cilia expression, and demonstrate that there is a delay in primary cilia disassembly when KIF11 is depleted. In addition to studying a non-mitotic role for KIF11 at primary cilia and its potential consequences for proper neurodevelopment, we also study KIF11 as a target in the neoplastic brain. Due to its role in mitosis, KIF11 has been studied as a target in cancer before, and we have previously shown that it is a viable target in the most lethal primary brain tumor, glioblastoma (GBM). However, in our previous work treating GBM with a KIF11 inhibitor, tumors recurred after treatment was stopped, indicating that this may have had a cytostatic effect, rather than cytotoxic. Importantly, it has been reported that cells are most vulnerable to irradiation when they are in mitosis. Because inhibiting KIF11 arr (open full item for complete abstract)

Committee: Monica Venere (Advisor) Subjects: Cellular Biology; Developmental Biology; Molecular Biology; Neurosciences; Oncology

Doctor of Philosophy (PhD), Wright State University, 2021, Biomedical Sciences PhD

Carbon Nanotubes (CNTs) have beneficial properties for cell scaffolding, which has translated into effective growth of bone, muscle, and cardiac cells. However, loose carbon nanotubes can cause in vivo toxicity. To reduce this risk, our team has developed biomimetic scaffolds with multiscale hierarchy where carpet-like CNT arrays are covalently bonded to larger biocompatible substrates. In this study, we have tested such scaffolds in two distinct types of biomedical applications involving glioblastoma and keratinocyte cells. The growth of glioblastoma (GBM) cells on our CNT-coated biomimetic scaffolds was evaluated to check their suitability as a potential chemotherapy-loaded implant for GBM patient treatment. We utilized CNT carpets on flat carbon fiber cloths and porous carbon foams and identified differing effects on cell growth by altering the surface features, such as hydrophilicity. Synthesized CNT-coating is naturally superhydrophobic and prevents GBM cell growth initially, but over time cell proliferation increases to normal levels. When the CNT surface was modified to be hydrophilic, GBM cells followed a normal growth curve. These findings support the feasibility of developing a CNT-coated chemotherapy-loaded implant for post-surgical resection in GBM patients. Keratinocyte cell growth on CNT-coated carbon fiber cloth was investigated to assess its compatibility as a skin graft material for wound healing applications. Due to its covalently linked structure, biocompatibility, functionalizable topological features, and extensive surface area, CNTs could provide a suitable surface for skin cell proliferation. CNTs can also provide directionality, which can be important for supporting scaffolds used in wound healing applications. This project aimed to determine whether the use of CNTs attached to carbon scaffolds are capable of sustaining keratinocyte growth for future development of novel skin graft development. Studies demonstrated biocompatibility for (open full item for complete abstract)

Committee: Sharmila M. Mukhopadhyay Ph.D. (Advisor); Courtney E.W. Sulentic Ph.D. (Committee Member); David R. Cool Ph.D. (Committee Member); Saber Hussain Ph.D. (Committee Member); David R. Ladle Ph.D. (Committee Member); Tyler Nelson Ph.D. (Other) Subjects: Biomedical Engineering; Biomedical Research; Nanotechnology

PhD, University of Cincinnati, 2020, Medicine: Molecular Genetics, Biochemistry, and Microbiology

The goal of this dissertation is to improve our understanding of two important processes in carcinogenesis: First, the resistance mechanisms to tyrosine kinase inhibitors in glioblastoma (GBM). Second, the mechanisms involved in the metastatic spread of non-small cell lung cancer (NSCLC) to the brain. Malignant gliomas have a poor prognosis, as recurrence remains nearly inevitable despite aggressive treatment with a median survival time of 12-15 months. Primary GBM usually displays amplification or mutation of at least one receptor tyrosine kinase, most often the epidermal growth factor receptor (EGFR). Targeting EGFR for inhibition often provides an initial tumor response in patients, but recurrence is a main limitation of these therapies because secondary mutations emerge that lead to drug resistance. In this work, we identified a receptor tyrosine kinase, proto-oncogene ROS1 fusion, that drives the resistance to therapy and leads to tumorigenesis in GBM. In parallel, the aggressive metastatic phenotype is one of the hallmarks of recurrent tumors, and its estimated that 90% of all cancer deaths arise from the metastatic spread of primary tumors. Of all the processes involved in carcinogenesis, local invasion and the formation of metastases are clinically the most relevant, but the least well understood at the molecular level. This work also provides evidence for the use of circulating tumor cells as a biomarker for the metastatic spread of cancer, specifically in lung cancer. To uncover the molecular changes that govern the transition from a primary lung tumor to a secondary metastasis and specifically the mechanisms by which circulating tumor cells (CTCs) survive in circulation, we carried out whole genome sequencing of normal lung tissue, primary tumors, and the corresponding brain metastases from five patients with progressive metastatic NSCLC. We also isolated CTCs from patients with metastatic cancer and subjected them to whole genome amplification and Sanger (open full item for complete abstract)

Committee: Peter Stambrook Ph.D. (Committee Chair); El Mustapha Bahassi Ph.D. (Committee Member); Pankaj Desai Ph.D. (Committee Member); Rhett Kovall Ph.D. (Committee Member); William Miller Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Case Western Reserve University, 2020, Biomedical Engineering

Glioblastoma multiforme (GBM) is resilient to the current standard of care treatment of surgical resection followed by concurrent radiotherapy and temozolomide (TMZ) chemotherapy. GBM patient responses are poor and variable, resulting in more than 90% tumor recurrence and grim survival. The high mortality of GBM is attributed to its invasive peripheral growth, partially intact blood-brain barrier (BBB), regions of hypoxia, and high cellular heterogeneity that includes brain tumor initiating cells (BTICs) and immunosuppressive cells. These features of GBM work together to restrict the delivery of drugs throughout the tumor, suppress immune recognition of tumor cells, and facilitate tumor progression. Nanoparticles are well-suited to address limitations associated with the treatment of GBM by enhancing drug delivery to the tumor and reducing side effects. The overall objective of the work in this dissertation is to develop systemically administered nanoparticles that overcome barriers to drug distribution and cellular heterogeneity in GBM to improve therapeutic responses. In murine GBM models, the effective delivery of Doxorubicin (DOX) chemotherapy, BTIC inhibitor, and immune-stimulating agonists were evaluated using two distinct mesoporous silica nanoparticles (MSNs): 1) the Fe@MSN particle and 2) the immuno-MSN particle. First, drug release from the Fe@MSN particle was triggered using an external radiofrequency (RF) field to enhance the distribution of DOX and/or BTIC inhibitor across the partially intact BBB and into the tumor interstitium. The effective delivery of drugs facilitated by Fe@MSN particles translated into suppressed GBM growth, depleted stem-like cell phenotypes in hypoxic regions, and prolonged or cancer-free survival. Second, towards further improving GBM treatment strategies, the immuno-MSN particle delivered immune-stimulating agonists to dysfunctional immune cells in GBM to reverse the effects of immunosuppression. Immuno-MSN particles facilitat (open full item for complete abstract)

Committee: Efstathios Karathanasis Ph.D. (Advisor); Agata Exner Ph.D. (Advisor); Dominique Durand Ph.D. (Committee Chair); Jennifer Yu M.D., Ph.D. (Committee Member) Subjects: Biomedical Engineering; Immunology

Doctor of Philosophy, The Ohio State University, 2020, Biomedical Engineering

Tumor microenvironment (TME) is the local environment surrounding the tumor region and can interact with tumor cells to regulate a variety of tumor behaviors. In glioblastoma (GB), emerging evidences have shown that different components in the TME can interact with tumor cells and affect tumor cell proliferation, migration, and resistance to therapy. Therapies have been developed to target GB-TME interactions but received dismal improvements on patient outcome. Currently, animal models remain to be the gold standard in preclinical drug discovery and testing. However, animal models are expensive, time consuming, and feature an overcomplex TME, making it difficult to identify specific interplays between TME and tumor cells. In vitro models incorporating well-defined TME components have been introduced as a cost-effective tool to examine tumor-TME interactions. In this dissertation, we describe four engineered models designed to recapitulate critical players in the GB TME, including the extracellular matrix, stromal cells, compressive solid stress, and interstitial fluid pressure. Using these models, we successfully captured GB cell responses to the changes in the TME and associated such responses to molecular signaling pathways. We further present a novel cell labeling method which enables multiplex antigen labeling and offers a high throughput platform for cancer diagnosis.

Committee: Jessica Winter (Advisor); Otero Jose (Committee Member); Ghadiali Samir (Committee Member); Leight Jennifer (Committee Member) Subjects: Biomedical Engineering