Doctor of Philosophy (Ph.D.), University of Dayton, 2023, Biology

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by cognitive decline, memory loss, and impaired daily functioning. It is the most common form of dementia, affecting millions worldwide. AD is characterized by the accumulation of amyloid-beta (Aβ42) plaques and neurofibrillary tangles in the brain. While the exact causes of AD are still unclear, a combination of genetic, environmental, and lifestyle factors is believed to contribute to its development. The Drosophila model has become a valuable tool for studying AD due to its genetic conservation with humans, short lifespan, simple nervous system, and available genetic tools. In this study, we utilized a Drosophila model expressing human Aβ42 in the developing retina to investigate the underlying mechanisms of Aβ42-induced neurodegeneration. We conducted a genetic screen and identified several modifiers that significantly affected Aβ42-induced neurodegeneration. One of the modifiers we identified is N-acetyltransferase 9 (Mnat9), known for its role in stabilizing microtubules and inhibiting the c-Jun-N-terminal kinase (JNK) signaling pathway. Overexpression of Mnat9 rescued the neurodegenerative phenotype caused by Aβ42 accumulation, while loss-of-function enhanced neurodegeneration. Importantly, we found that the neuroprotective function of Mnat9 was independent of its acetylation activity. The transgenic expression of human NAT9 (hNAT9) in Drosophila also suppressed Aβ42-mediated neurodegeneration, suggesting functional conservation between Mnat9 and hNAT9 in interacting with JNK-mediated neurodegeneration. These findings uncover a novel neuroprotective role of Mnat9 in downregulating the JNK pathway to ameliorate Aβ42-induced neurodegeneration. Another modifier we identified is miR-277 (hsa-miR-3660 in humans). Loss-of-function of miR-277 enhanced neurodegeneration, while its gain-of-function rescued the phenotype. Overexpression of miR-277 in the presence of Aβ42 reduced cell de (open full item for complete abstract)

Committee: Amit Singh Dr (Advisor); Madhuri Kango-Singh Dr (Committee Member); Potithos Pitychoutis Dr (Committee Member); Shirley Wright Dr (Committee Member); Shree Ram Singh Dr (Committee Member) Subjects: Biology; Genetics; Neurosciences

Master of Science (M.S.), University of Dayton, 2023, Interdisciplinary Studies

The SARS-CoV2 virus was responsible for the COVID-19 Pandemic, one of the most fatal international public health emergencies experienced in the past century. SARS-CoV2 induces symptoms like increased inflammatory response, severe acute respiratory syndrome (SARS), cognitive dysfunction like brain fog, and cardiovascular defects. Prolonged or long-term infection led to the emergence of Post-COVID-19 Syndrome, or PCS. PCS is characterized by chronic cardiovascular, autoimmune, and neurological manifestations and remains understudied. Individuals with pre-existing neurological insult like those with neuroinflammatory or neurodegenerative diseases are likely more vulnerable to such PCS effects. Furthermore, individuals with pre-existing neurological conditions often have comorbidities like obesity, hypertension, hyperlipidemia, and low activity levels. However, little is understood about the molecular effects of SARS-CoV2 on neuron in both healthy and neuro-compromised individuals. Currently, many individuals experiencing PCS-related neurological symptoms require management of their symptoms even though our knowledge in this area is still limited. Therefore, this study utilized an interdisciplinary approach to better understand how SARS-CoV2 impacts both neurons at a cellular level and clinically in neurologically compromised populations such as Multiple Sclerosis (MS). This interdisciplinary approach sheds light on how translational work is being done where basic science efforts complement efforts made clinically to make connections and identify relationships between observed effects and known science. To do so, SARS-CoV2 proteins were misexpressed in the Drosophila eye and through a forward genetic screen evaluated for changes to cellular structure or function. To corroborate these findings, SARS-CoV2 proteins were also transfected into Neuro-2a cells to assess how these proteins affected cellular functioning. Furthermore, SARS-CoV2 protein structure-function analys (open full item for complete abstract)

Committee: Kurt Jackson (Advisor); Mrigendra Rajput (Advisor) Subjects: Biomedical Research; Neurology; Neurosciences; Physical Therapy; Virology

Doctor of Philosophy (Ph.D.), University of Dayton, 2022, Biology

Hippo pathway is an organ size regulating pathway that has implications in organogenesis, cell competition, compensatory proliferation, and regeneration. Previous studies have identified the role of impaired Hippo pathway in cancer and Alzheimer's. Down-regulation of pathway causes over proliferation by upregulation of target gene expression that promotes proliferation and prevents apoptosis. On the contrary, pathway hyper-activation causes cell death by upregulating a pro-apoptotic protein Hid and without affecting the expression of Diap-1, a target protein that prevents apoptosis. However, down-regulation of Hid fails to significantly rescue Hippo pathway mediated cell death. Work from a previous graduate student in our lab has shown that Hippo signaling regulates a key cell death gene dronc which is a homolog of the initiator caspase-9 in mammals. When Hippo levels are high, Yki is sequestered in the cytoplasm and dronc levels are high and cell death is the result. In contrast to that when Hippo is downregulated, Yki is free to move to the nucleus and cause changes in gene expression and dronc is also downregulated and the result is proliferation of cells. But the phenomenon between Yki and dronc are poorly studied. The aim of my work was to thus identify how Yki that interacts with Hippo pathway to regulate cell death via dronc regulation. Dronc is also a common target to the Ecdysone signaling which is an important steroid hormone in insects that allows spatio-temporal of gene expression to regulate events of cell death and growth during molting and metamorphosis. But the relationship between Hippo Signaling and Ecdysone signaling to regulate a common target such as dronc is underexplored. To attain this goal, we tested for genetic interaction between known Ecr pathway components and the Hippo pathway components to determine if there is any genetic epistasis between the components. Our experiments led to the identification of a feedback loop in which the downst (open full item for complete abstract)

Committee: Madhuri Kango-Singh (Advisor); Pitychoutis Pothitos (Committee Member); Andreas Bergmann (Committee Member); Amit Singh (Committee Member); Mark Nielsen (Committee Member) Subjects: Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2019, College of Sciences and Health Professions

Differentiation and apoptosis are coordinately regulated biological endpoints in many cell types, including skeletal myoblasts. In adult skeletal muscle, the muscle transcription factor MyoD is necessary for the regeneration of damaged tissue. When myoblasts encounter differentiation cues, mimicked in vitro by removing serum from culture media (differentiation media; DM), ~70% of cells undergo differentiation, while ~30% undergo apoptosis. We have previously reported that the expression of apoptotic versus differentiation-associated genes is temporally distinct, with PUMA expression (apoptosis) and myogenin expression (differentiation) detected after 3 or 18 hours of culture in DM, respectively. The underlying goal of the current study was to identify distinctions influencing MyoD induced transcription of PUMA from MyoD induced transcription of myogenin. We hypothesized that epigenetic modifications of histones at MyoD-responsive promoter regions and post-translational modifications of MyoD may influence MyoD's ability to promote differentiation vs. apoptosis. We first discovered higher levels of acetylated histones surrounding the MyoD-responsive element in the PUMA promoter. Chromatin immunoprecipitation showed MyoD binding at the PUMA and myogenin promoter regions at 3 and 18 hours in DM, respectively. However, MyoD was not detected at the PUMA promoter after 18 hours in DM despite the fact that the PUMA promoter remained highly acetylated. We next assessed the effect of the cell cycle position. By synchronizing myoblasts, we determined that cells in early S-phase have a greater propensity to undergo apoptosis in response to DM, than cells in any other cell cycle phase, while cells in G1 or G2 undergo differentiation. Since MyoD becomes phosphorylated at serine200 near the G1/S border, we hypothesized that MyoDPser200 may be responsible for driving PUMA expression in response to differentiation cues. Herein, we report that a MyoD mutant (MyoDs200a) is unable to i (open full item for complete abstract)

Committee: Crystal Weyman Dr. (Advisor); Anton Komar Dr. (Committee Member); Aaron Severson Dr. (Committee Member); Roman Kondratov Dr. (Committee Member); Aimin Zhou Dr. (Committee Member); Michelle Longworth Dr. (Committee Member) Subjects: Biology; Cellular Biology; Molecular Biology

PhD, University of Cincinnati, 2014, Medicine: Pathobiology and Molecular Medicine

Cancer is a persistent global health concern despite advancements in prevention, detection, and treatment. Acquired chemotherapy resistance and dose-limiting toxicities continue to plague current cancer treatment modalities, indicating a desperate need for new and improved molecularly targeted anti-cancer therapies. Proliferating cell nuclear antigen (PCNA) is a non-oncogenic mediator of DNA replication. For this reason, PCNA function is essential to the uncontrolled proliferation of cancer cells. PCNA is highly expressed in tumors, determining it as a reliable marker of proliferation and a potentially desirable target for cancer therapy. Functional PCNA is a ring-shaped homotrimer which is loaded onto chromatin by replication factor C (RFC). PCNA plays a crucial role in DNA replication by providing replicative DNA polymerases the high processivity required to duplicate the entire genome. Furthermore, PCNA functions as a scaffold protein, binding a multitude of protein partners involved in many vital cellular processes such as DNA replication, DNA repair, and cell cycle control. Collectively, these many functions of PCNA and its localization at the replisome put PCNA in a central position for determining the fate of the replication fork. The homotrimeric structure of PCNA is crucial for its function. Therefore, we performed an in silico screen of a drug-like chemical library for small molecule compounds that would bind the monomer-monomer interface of PCNA in attempts to identify compounds that would alter homotrimer stability. The PCNA-inhibitors (PCNA-Is) identified were found to bind PCNA homotrimers with a high affinity and promote homotrimer stabilization by serving as a “linker” at the monomer-monomer interface. This increased stability resulted in a decrease in PCNA chromatin-association in various tumor cell lines. Treatment with PCNA-Is inhibited DNA replication and growth of tumor cells. Additional studies using PCNA-I1, the most pote (open full item for complete abstract)

Committee: Zhongyun Dong M.D. Ph.D. (Committee Chair); Susan Waltz Ph.D. (Committee Member); Shao-Chun Wang Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member); Matthew Wortman Ph.D. (Committee Member) Subjects: Oncology

PhD, University of Cincinnati, 2008, Medicine : Molecular and Developmental Biology

Primordial germ cells (PGCs) are the embryonic precursors of the gametes, and are essential for continuation of generations in all eukaryotes. Using the mouse as a model system, our lab has described key points in the mechanism and regulation of mammalian PGC migration. During gastrulation, newly formed PGCs migrate through the posterior primitive streak into the hindgut endoderm. Shortly thereafter, they exit the hindgut, into the midline region of the dorsal body wall. From there, they migrate bilaterally to the genital ridges, which will become the gonads. By making movies of migratory PGCs, we observed that some germ cells fail to reach the genital ridges, but remain in midline structures including the hindgut and dorsal body wall. These ectopic PGCs rapidly fragment and disappear, suggestive of apoptotic cell death. Programmed cell death, or apoptosis, is an important mechanism during development that allows ectopic cells to be efficiently removed. All extragonadal germ cell tumors (EGCTs) occur within midline structures in humans, and it is thought that these arise from ectopic migratory PGCs that fail to undergo apoptosis. This thesis addresses two general questions.1) What is the mechanism of removal of PGCs that fail to reach the genital ridges during migration? 2) What are the consequences of the failure of these mechanisms? I show that PGCs are dependent upon Steel/c-Kit signaling for survival as they migrate towards the genital ridges, and that the withdrawal of Steel expression in the midline is the cause of midline germ cell death. Further, this death is dependent upon the pro-apoptotic factor, Bax, and the loss of Bax is sufficient to rescue apoptosis caused by the withdrawal of Steel. I then show that in the absence of Bax, PGCs outside the genital ridges, and which would normally die by apoptosis, instead survive and persist in the same regions that infantile EGCTs occur. Lastly, I demonstrate that the behavior of these extragonadal PGCs differs dep (open full item for complete abstract)

Committee: Christopher Wylie PhD (Committee Chair); David Hildeman PhD (Committee Member); George Mutema MD/PhD (Committee Member); Leslie Myatt PhD (Committee Member); James Wells PhD (Committee Member); David Williams MD (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 2008, Chemical Engineering

Biotechnology-derived protein drugs, usually referred as biologics, represent a significant part of the whole pharmaceutical market. Typically, biologics are produced in genetically modified animal cells, which are regarded by many engineers and practitioners in the pharmaceutical industry as extremely sensitive to hydrodynamic forces. Since during research or normal operation in the biopharmaceutical industry cells are exposed to a range of hydrodynamic forces, this "shear sensitivity" idea often leads to very mild, sub-optimal designing and operating conditions.To determine the actual levels of hydrodynamic stress capable of affecting the metabolism or viability of a cell line in bioprocessing or analytical devices, a microfluidic contracting-expanding device was developed in our group that exposes cells to controlled, well-defined hydrodynamic forces by means of keeping the flow in laminar conditions. Using this device, changes in cell behavior can be determined as a function of the local energy dissipation rate (EDR). EDR is a scalar value that is intrinsic to any moving fluid, is independent of the flow regime (turbulent/laminar) and accounts for both shear and extensional components of three-dimensional flow. It represents the rate at which work is done on a fluid element or a cell. If laminar flow is maintained, EDR can be reliably calculated using well-established equations for simple geometries or computational fluid dynamics (CFD) software for more complex problems. The microfluidic device, consisting of a micro-channel bored in a stainless steel sheet in sandwich between two polycarbonate plates, was used in different setups to imitate the environment cells will experience in both bioprocessing and analytical equipment. As a model for analytical devices, it was selected a Fluorescent Activated Cell Sorter (FACS), where cells are forced through a nozzle and interrogated by a laser beam. This instrument was mimicked by passing the cells once through the mic (open full item for complete abstract)

Committee: Jeffrey Chalmers PhD (Advisor); Kurt Koelling PhD (Committee Member); Andre Palmer PhD (Committee Member) Subjects: Chemical Engineering; Pharmaceuticals

Doctor of Philosophy, Case Western Reserve University, 2008, Pharmacology

Bcl-2 is the founding member of a large family of apoptosis regulating proteins. The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1, 4, 5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide (peptide 2) based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Furthermore, peptide 2 enhances ABT-737-induced cell death in chronic lymphocytic leukemia cells. The interaction of Bcl-2 with IP3R's contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2. We also investigated the region of Bcl-2 responsible for interaction with the IP3R. Based on results of coimmunoprecipitation and GST pull-down experiments the BH4 domain of Bcl-2 is necessary for interaction with the IP3R. A synthetic peptide corresponding to the BH4 domain of Bcl-2 interacts with the same IP3R domain as full length Bcl-2. TAT-BH4, formed by fusing a peptide corresponding to the BH4 domain of Bcl-2 with the protein transduction domain of HIV TAT, enters cells and functions like full length Bcl-2, to inhibit cytoplasmic Ca2+ elevation and apoptosis induced by T cell receptor (TCR) activation. Two experimental findings establish that these actions of TAT-BH4 are mediated through interaction with the IP3R. First, TAT-BH4 inhibits Ca2+ elevation induced by a cell permeant IP3 ester. Second, peptide 2 that blocks Bcl-2-IP3R interaction reverses the inhibitory effect of TAT-BH4 on both Ca2+ elevation and apoptos (open full item for complete abstract)

Committee: Clark Distelhorst (Advisor); Anthony Berdis (Committee Chair); Shigemi Matsuyama (Committee Member); William Schilling (Committee Member); George Dubyak (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Oncology; Pharmacology

Doctor of Philosophy (Ph.D.), University of Dayton, 2024, Biology

The Drosophila model is an extremely useful tool to study development and disease. It offers several advantages with its short lifespan, high number of genetically identical offsprings, repertoire of genetic tools and conservation in developmental pathways and processes. With high genetic conservation and ease of screening phenotypes, the Drosophila eye offers an excellent model for studying development and disease. In this study, we have used the Drosophila eye to study the regulation achieved by a transcription factor, and transcriptional pausing factor in eye development with implications in human craniofacial development. In addition, we also assayed the impact of various proteins of SARs-CoV2 using the Drosophila eye model. In the first study, we focused on the role of a dorsal selector gene - defective proventriculus, that encodes a K-50 homeodomain containing transcription factor. Axial patterning is required to establish antero-posterior, dorso-ventral and proximo-distal axes and is crucial for the formation of a 3D organ. In the eye, dorso-ventral axis is the first to form. Previously, we characterized dve as a dorsal selector gene based on (1) its expression in the dorsal head vertex region of the eye disc, (2) its gain-of-function phenotype showing complete eye suppression and (3) its loss-of-function phenotype showing dorsal eye enlargements, as in other dorsal eye genes. We also reported that differences in Dve expression impact the placement of eyes on the head of an organism resulting in diversity across animal groups. The human ortholog of Dve is SATB1 and it is highly conserved. Misexpression of SATB1 in the developing eye results in eye suppression and ectopic Wg expression, as in dve gain-of-function. SATB1 is highly expressed in cancers and metastasis. As part of this thesis, I have performed structure-function analysis of Dve protein to dissect its diverse roles in development and growth. We identified N-terminal+ULD+Hox1 as the minimal domain (open full item for complete abstract)

Committee: Amit Singh PhD (Advisor); Madhuri Kango-Singh PhD (Committee Member); Mrigendra Rajput PhD (Committee Member); Shree Ram Singh PhD (Committee Member); Pothitos Pitychoutis PhD (Committee Member) Subjects: Biology; Developmental Biology; Genetics; Molecular Biology

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, Biomedical Sciences

The innate immune response to infection involves a complex series of signaling events, threat recognition molecules, and effector proteins that act together to eliminate pathogens. As part of the patrolling immune cells, monocytes are often the first responders to infection and are specifically equipped to recognize a diverse range of pathogens and quickly respond to threats. One of these mechanisms involves a series of proteins known as inflammasomes that are critical for producing an adequate immune response to fend off pathogen infection while simultaneously being regulated to prevent hyper inflammation and inflammatory diseases such as sepsis. Inflammasomes are multimeric complexes that become activated upon pathogen detection by an upstream sensor molecule that can be in response to both bacterial and viral infection. Once activated, a highly regulated signaling cascade occurs that results in the eventual enzymatic activation of the pore forming protein Gasdermin D (GSDMD). Once activated, GSDMD traffics to cellular membranes to form pores often leading to an inflammatory form of cell death known as pyroptosis. While cell death is necessary to maintain homeostasis and cell turnover, dysregulated cell death in the case of GSDMD induced pyroptosis can lead to severe inflammatory diseases such as sepsis and acute respiratory distress syndrome (ARDS). Despite significant advances in unraveling the mechanism of GSDMD activation and designing targeted therapeutics against inflammation, gaps in knowledge still persist as evident by the high mortality rate of sepsis and inflammatory disorders. As such, it is essential to improve our understanding about the signaling events leading to GSDMD activation, the impact of active GSDMD on inflammation and cell death, and the regulators involved with GSDMD activation. Our lab has recently discovered a novel avenue of GSDMD mediated cell injury through the release of monocyte derived exosomes (EVs) containing active GSDMD that a (open full item for complete abstract)

Committee: Anasuya Sarkar (Advisor); Yutong Zhao (Committee Member); Susheela Tridandapani (Committee Member); Jacob Yount (Committee Member) Subjects: Biomedical Research

Doctor of Philosophy (PhD), University of Toledo, 2023, Experimental Therapeutics

Triple-negative breast cancer (TNBC), the most lethal and aggressive subtype of breast cancer, lacks estrogen receptors, progesterone receptors, and human epidermal receptors, rendering it unsuitable with targeted-based treatment. TNBC has higher relapse rate, worst prognosis and higher metastasis rate compared to non-TNBC because of their tendency to resist to apoptosis, a form of programmed cell death, induced by chemotherapy. Hence, non-apoptotic cell death inducers could be a potential alternative to circumvent the apoptotic drug resistance. In this study, we discovered two novel compounds, TPH104c and TPH104m, which induce non-apoptotic cell death in TNBC cells. These lead compounds were 15 to 30-fold more selective in TNBC cell lines and significantly decreased the proliferation of TNBC cells compared to normal mammary epithelial cell lines. TPH104c and TPH104m induced a unique type of non-apoptotic cell death characterized by no cellular shrinkage, absence of nuclear fragmentation and f apoptotic blebs. Although TPH104c and TPH104m produced the loss of the mitochondrial membrane potential, TPH104c- and TPH104m-induced cell death did not increase total cytochrome c and intracellular ROS, lacked caspases activation, and was not rescued by pan-caspase inhibitor, zVAD-FMK. Moreover, TPH104c and TPH104m significantly downregulated mitochondrial fission protein, Drp1 and its levels determined their cytotoxic efficacy. Studies have shown that protein, Bcl-2 interacting protein 3 (BNIP3), mediates a non-apoptotic, necrosis-like cell death similar to that produced by TPH104c and TPH104m that lacked activation of caspases and reduced mitochondrial transmembrane potential. Therefore, we determined the effect of TPH104c and TPH104m on various mitochondrial functions, in triple negative breast cancer (TNBC) cells, BT-20 and MDA-MB-231. TPH104c and TPH104m (2 and 5 μM), compared to vehicle, significantly increased the levels of reactive oxygen species (ROS) BNIP3 and c-Jun (open full item for complete abstract)

Committee: Amit K. Tiwari (Committee Chair); Aniruddha Ray (Committee Member); Ana Maria Oyarce (Committee Member); Frederick E. Williams (Committee Member) Subjects: Pharmacology

Bachelor of Science, Marietta College, 2023, Chemistry

Parabens are commonly used as preservatives in regularly used topical products, but their safety is under discussion since small amounts of paraben have been found in tumor tissue. Mononitroparaben causes cell death in melanoma cells with an LC50 value of 7.02mM after twelve hours of treatment. This study focused on how mononitroparaben affects the cellular lipid content during induced cell death. The experiment was conducted by growing the M624 melanoma cells, dissolving the paraben in methanol, and then leaving 0 mM, 5 mM, and 10 mM concentrations of paraben on the melanoma cancer cells for twelve hours. The paraben was then removed from the cells, and the cells were lysed. Colorimetric cholesterol assays and ceramide assays were completed to determine the changes in cellular cholesterol and ceramide content and the role of cellular lipids in cell death signaling. The results showed that cholesterol concentrations did not significantly change among cells treated with mononitroparaben. However, ceramide significantly increased for cells treated with 10 mM mononitroparaben, which indicated that apoptosis occurred.

Committee: Suzanne Parsons Ph.D. (Advisor); Adam Jacoby Ph.D. (Committee Member); Rakibul Sarker Ph.D. (Committee Member) Subjects: Biochemistry

Doctor of Philosophy, Miami University, 2022, Chemistry and Biochemistry

This work investigated the impact of polymer conjugation on the activity and stability profile of a lipase enzyme. An industrially important enzyme, Candida antarctica lipase B (CalB), was conjugated with different functional polymers, including those with hydrophilic, hydrophobic, cationic, and anionic characters. The study demonstrated enhanced CalB activity when conjugated with hydrophilic and cationic polymers against a hydrophobic substrate, p-nitrophenol palmitate (p-NPP), due to the possible non-covalent interaction between the polymer and the substrate. On the contrary, conjugating with hydrophobic polymer showed significant activity inhibition, likely due to binding to the catalytic site. Hence, CalB conjugation with this diverse range of polymers demonstrated the significance of polymer composition, polymer-substrate interactions, and protein-polymer interactions in deciding protein catalytic performance. With this in mind, this dissertation also showed the manipulation of these protein-polymer interactions to tune a protein's catalytic performance in different pH environments. In addition, this thesis also explored work focusing on combating coronavirus. In one project, the receptor-binding domain of spike protein of SARS-CoV-2 was modified with different polymers to explore the sensitivity of interactions between the modified RBD and ACE2 protein, which may be beneficial in the downstream development of inhibitors targeting the RBD-ACE2 interaction. In another project, spike binding peptide (SBP1) was immobilized into a covalent crosslinked network system to develop a novel peptide-functionalized network to capture the spike protein of coronavirus. The development of such functionalized network could provide us materials with potential antiviral properties, which can provide us the opportunity to mitigate coronavirus spread. Hence, this thesis work highlighted different functional consequences of protein-polymer interaction ranging from tuning activity a (open full item for complete abstract)

Committee: Rick Page (Advisor); Dominik Konkolewicz (Advisor); C. Scott Hartley (Committee Chair); Gary Lorigan (Committee Member); Jason Berberich (Committee Member) Subjects: Analytical Chemistry; Biochemistry; Polymer Chemistry

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

Lytic cell death occurs in an accidental or regulated manner and causes plasma membrane rupture along with release of cellular danger signals and cytokines. A recently described family of proteins called gasdermins drives a majority of programmed, lytic cell death in a process termed pyroptosis. Pyroptosis occurs downstream of protease activation of gasdermins, whereby they form pores on the plasma membrane and initiate cell disassembly. Inflammatory caspases activate GSDMD downstream of PAMPs and DAMPs, with concomitant cytokine processing and release, and apoptotic caspases activate GSDME downstream of extrinsic signals or mitochondrial damage, such as from chemotherapy. Here, we describe a series of interconnected pathways between gasdermins. We find that GSDME can be activated at the inflammasome and recapitulate both the lytic effects and cytokine release of GSDMD. Notably, this process can occur independently of cell lysis in a sublytic phase with limited cell death and may overall contribute more cytokine-based effects over time. Conversely, we find that GSDMD can substitute for GSDME in response to chemotherapy. While most studies have focused on GSDME as the ultimate effector of chemotherapy-induced pyroptosis, we show that instead GSDMD is the terminal effector when GSDME or an apoptosis activated channel PANX1 do not reach a lytic threshold. When either of GSDME or PANX1 does, it supersedes GSDMD as the main cell death effector. These studies establish an interconnected lattice of cell death proteins that is striking in contrast with the typical one pathway one effector doctrine.

Committee: Stanley Adoro (Committee Chair); Tsan Xiao (Committee Member); Arne Rietsch (Committee Member); Clive Hamlin (Committee Member); XiaoXia Li (Committee Member); Derek Abbott (Advisor) Subjects: Cellular Biology; Immunology

Doctor of Philosophy, Case Western Reserve University, 2017, Pathology

Pyroptosis is a regulated mode of lytic inflammatory cell death that promotes anti-microbial host defense but may contribute to sepsis. Pyroptosis requires canonical inflammasome assembly to mediate caspase-1 activation. Active caspase-1 cleaves gasdermin D (Gsdmd) to relieve an autoinhibitory interaction between the N and C-termini enabling N-terminal Gsdmd (N-Gsdmd) to oligomerize, insert into the plasma membrane (PM) as lytic pores, and execute pyroptotic cell death. To further characterize N-Gsdmd-dependent changes in plasma membrane (PM) permeability, we assayed propidium2+ (Pro2+) influx kinetics during NLRP3/Pyrin inflammasome activation in murine bone marrow-derived macrophages (BMDM). BMDM were characterized by rapid Pro2+ influx after initiation of NLRP3/Pyrin inflammasomes by nigericin (NG) or C. difficile toxin B (TcdB), respectively. No Pro2+ uptake in response to NG or TcdB was observed in Caspase-1-/- or ASC-/- BMDM. The cytoprotectant glycine profoundly suppressed NG and TcdB-induced lysis but not Pro2+ influx. The absence of Gsdmd expression resulted in suppression of NG-stimulated Pro2+ influx and pyroptotic lysis. Extracellular La3+ and Gd3+ reversibly blocked the induced Pro2+ influx and markedly delayed pyroptotic lysis without limiting upstream inflammasome activation. These caspase-1-induced pre-lytic, N-Gsdmd PM pores also facilitated the efflux of cytosolic ATP and influx of extracellular Ca2+. Whether N-Gsdmd also partitions into membranes of intracellular organelles to facilitate pyroptotic cell death signaling remains undefined. We stimulated WT, Gsdmd-/-, and Nlrp3-/- immortalized macrophages (iBMDMs) with NG, and also used La3+ as an inhibitor of PM pyroptotic pore activity to investigate the contribution of intracellular PM pore-independent perturbations to active Gsdmd-mediated cell death. The absence of Gsdmd attenuated NG-induced decreases in redox homeostasis. Inhibition of N-Gsdmd PM pore activity with La3+ uncovered a ROS-dri (open full item for complete abstract)

Committee: George Dubyak (Advisor); Alan Levine (Committee Chair); Clifford Harding (Committee Member); Pamela Wearsch (Committee Member); Carlos Subauste (Committee Member); Clive Hamlin (Committee Member) Subjects: Cellular Biology; Immunology; Pathology

Doctor of Philosophy, Case Western Reserve University, 2016, Pharmacology

Accumulation of extracellular adenine nucleotides is a key regulator of the purinergic anti-tumor immune response that is triggered after treatment with certain chemotherapeutic agents. ATP released from dying tumor cells has been shown to engage P2 purinergic receptors on nearby leukocytes to activate this response. Conversely, accumulation of adenosine within the tumor microenvironment causes suppression of the immune response. The balance between ATP and adenosine in the tumor microenvironment can dictate the aggressiveness of the cancer and its response to chemotherapy treatment. To examine the regulated release of ATP and its metabolites, as well as their extracellular accumulation, we conducted three main studies. In the first study, we used human leukemic Jurkat T cells to characterize the role of Pannexin 1 (Panx1) channels in the release of ATP during treatment with three chemotherapeutic agents: doxorubicin (Dox), Etoposide (Etop), and staurosporine (STS). These diverse pro-apoptotic drugs were able to induce functional activation of the Panx1 channel by cleavage of the autoinhibitory C terminal domain. Activation of Panx1 led to the release of ATP, but the majority of nucleotide release was in the form of ADP and AMP. Our second study examined the how different rates of adenine nucleotide release and ectometabolism dictate the composition of pro- or anti- inflammatory nucleotides using Jurkat T cells that included clonal variants lacking either FADD or RIP1and treated with pro-apoptotic chemotherapeutic drugs. Despite similar apoptotic induction, the accumulation of ATP+ADP+AMP was decreased in the clonal variants and this decrease was linked to increased expression of the ectonucleotidase CD73. Finally, we explored the role of Panx1-mediated ATP release in the murine lymphoma cell line, EG7, that have been used for in vivo studies of the purinergic anti-tumor immune response. We discover that while the EG7 cells activate Panx1 and release adenine nucleot (open full item for complete abstract)

Committee: George Dubyak (Advisor); Ruth Keri (Committee Chair); Vera Moiseenkova-Bell (Committee Member); Clark Distelhorst (Committee Member); Thomas Kelley (Committee Member) Subjects: Pharmacology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Heart disease is the major cause of mortality in the United States and other parts of the world. Heart transplantation is the treatment of choice for patients with end stage heart failure. However, transplanted organs fail due to either acute or chronic rejection. This acute and chronic rejection impacts distinct compartments of cardiac allografts. Acute rejection is characterized by infiltration of mononuclear cells whereas chronic rejection is characterized by progressive narrowing of coronary arteries. In a minor histoincompatibility mismatch mouse model we found hearts transplanted from male to female C57BL/6 mice undergo an acute rejection with diffuse interstitial infiltrates at 2 weeks that resolve by 6 weeks when about half of the large arteries develop CAV. These processes are dependent on T cells because no infiltrate developed in T cell deficient mice. Markers of M1 macrophages were upregulated in the interstitium acutely and then decreased as markers of M2 macrophages increased chronically. Interstitial and arterial infiltrates were microdissected and expression of an array of 86 genes was screened by real time PCR. Programmed cell death protein 1 (PD1), a negative costimulator, and its ligand PDL1 were highly upregulated in the interstitium during the resolution of acute rejection. Flow cytometry analysis of graft infiltrating cells confirmed an enrichment of macrophages expressing PDL1. Treatment with a blocking antibody to PDL1 in the acute phase increased interstitial T cell infiltrates. In the arterial compartment, Toll Like Receptor 4 (TLR4) was upregulated at 6 weeks. Hyaluronan, an endogenous ligand of TLR4, was increased in arteries with neointimal expansion. Injection of hyaluronan fragments increased intragraft production of chemokines. Our data indicate that negative co-stimulatory pathways are critical for the resolution of acute interstitial infiltrates. In the arterial compartment recognition of endogenous ligands including hyaluronan by i (open full item for complete abstract)

Committee: William Baldwin, III PhD (Advisor); Christine Moravec PhD (Committee Member); Booki Min PhD (Committee Member); Barsanjit Mazumder PhD (Committee Member); Girish Shukla PhD (Committee Member); Carol De la Motte PhD (Committee Member) Subjects: Biology; Immunology; Molecular Biology

PhD, University of Cincinnati, 2014, Medicine: Neuroscience/Medical Science Scholars Interdisciplinary

An estimated 35 million diabetic patients worldwide take antipsychotic medications for a variety of co-morbid mental illnesses. Diabetic patients frequently experience hypoglycemic episodes ranging from mild to severe. Interestingly, the combination of antipsychotic medications and hypoglycemia has remained largely unstudied. Both antipsychotic medications and hypoglycemia modulate extracellular glutamate creating the potential for glutamate-mediated excitotoxicity. The typical antipsychotic medication, haloperidol, has been shown to elevate extracellular glutamate levels through its binding pharmacology at the D2 and 5HT1A receptors. Conversely, quetiapine, an atypical antipsychotic medication, is not predicted to elevate extracellular glutamate through its binding pharmacology at the D2, 5HT1A, and 5HT2A receptors and may be less likely to induce this toxicity when combined with hypoglycemia. We hypothesized that haloperidol and hypoglycemia, not quetiapine and hypoglycemia, would elevate neuronal death and microglial activation. We predicted that this would occur through glutamate-mediated excitotoxicity potentiated by activated microglia. We investigated this hypothesis by combining these medications with a range of different hypoglycemic episodes: acute, severe; single, moderate; and multiple, moderate. The combination of severe hypoglycemia with these antipsychotic medications showed that both medications elevated neuronal death as compared to hypoglycemia alone. This suggests that the predicted abilities of quetiapine to attenuate extracellular glutamate levels and prevent glutamate-mediated excitotoxicity of neurons are overshadowed in instances of severe hypoglycemia. However, when moderate hypoglycemic episodes were combined with antipsychotic medications, no changes in neuronal death or microglial activation were detected. Overall, these data suggest that severe hypoglycemia and antipsychotic medications induce neuronal death, but the effect (open full item for complete abstract)

Committee: Gary Gudelsky Ph.D. (Committee Chair); Steve Danzer Ph.D. (Committee Member); Andreas Loepke M.D.Ph.D. (Committee Member); Neil Richtand M.D. (Committee Member); Renu Sah Ph.D. (Committee Member) Subjects: Neurology

PHD, Kent State University, 2014, College of Arts and Sciences / School of Biomedical Sciences

Ischemic postconditioning has been shown to reduce injury in response to ischemia/reperfusion. Because of limitations on the clinical use of ischemic postconditioning protocols, pharmacological agents that elicit a postconditioning (PostC) effect are highly desired. Previous studies have shown that stimulation of alpha1-adrenergic receptors (α1-ARs) is cardioprotective, thus, the first aim of this dissertation was to examine the effects of post-ischemic stimulation of α1-ARs on cardiac myocyte cell death. Adult rat ventricular myocytes and HL-1 cardiac myocytes were subjected to simulated ischemia-reperfusion injury. Cell membrane permeability, evaluated by measuring released lactate dehydrogenase (LDH) or propidium iodide uptake (PI), was used as an estimate of cell death. Lower amounts of LDH and PI uptake were detected when α1-ARs were stimulated at the onset of reperfusion. Further, lower levels of apoptosis were measured using TUNEL and DNA laddering to evaluate DNA cleavage and Annexin V staining to evaluate outer membrane phosphatidylserine. Prior studies suggest that increased autophagy following ischemia is protective. The second aim of this dissertation was to determine whether post-ischemic α1-AR stimulation inhibits cardiac myocyte death through modulation of autophagy. Alpha1-AR-mediated reductions in cell death were reversed in the presence of ATG inhibitor, 3-Methyladenine. Western blot for autophagosomal marker, LC3-II indicated modulation of autophagy, and two methods were used to measure autophagic flux. LC3-II turnover examined with and without autophagosome-lysosome fusion inhibitor chloroquine revealed an increase in autophagic flux or induction. HL-1 cells transfected with plasmid to express a tandem fluorescent-tagged LC3 molecule also indicated an increase in autophagic flux or induction. Finally, the third aim of this dissertation was to examine the molecular pathways stimulated by α1-ARs that lead to decreased cell death. PI fluorescence as (open full item for complete abstract)

Committee: June Yun Ph.D. (Advisor); J. Gary Meszaros Ph.D. (Committee Member); Angelo DeLucia Ph.D. (Committee Member); Werner Geldenhuys Ph.D. (Committee Member); Joel Hughes Ph.D. (Committee Member) Subjects: Biology; Biomedical Research