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, 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 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

Doctor of Philosophy, Miami University, 2013, Zoology

Drosophila Melanogaster is a well-established model for investigating neuro-developmental remodeling, as its nervous system undergoes extensive re-organization during the 4-day metamorphosis period as it shifts from a crawling larva to an adult capable of flight and walking. Studies described in this thesis examine the developmental fate and functional significance of a subset of embryonic/larval abdominal motor neurons (MNs) that expresses the dHb9 transcription factor. The present studies demonstrate that a subset of dHb9-positive larval MNs persists during metamorphosis to incorporate into the adult specific neural circuitry. Manipulation of this subset of neurons during metamorphosis results in eclosion defects such as a decrease in overall eclosion rate and an increase in the time required to eclose. Another subset of dHb9-positive larval MNs dies during early metamorphosis, and deletion of cell death gene reaper can prevent the programmed cell death (PCD) of these MNs until the newly eclosed adult stage, suggesting that the elimination of dHb9-positive larval MNs during metamorphosis is reaper dependent. Furthermore, this study shows that supernumerary dHb9 expressing MNs in the adult maintain their transcriptional identity, project axons to the periphery and are capable of making synaptic contacts with peripheral muscles. This thesis also demonstrates how the formation of the adult specific abdominal segmental nerve projection pattern is affected when the cell death machinery is inhibited in reaper null mutants.

Committee: Joyce Fernandes (Advisor); Kathleen Killian (Committee Member); Katia Del Rio-Tsonis (Committee Member); David Pennock (Committee Member); Richard Moore (Committee Member) Subjects: Academic Guidance Counseling

Doctor of Philosophy, The Ohio State University, 2008, Plant Pathology

The rice E3 ubiquitin ligase SPL11 was previously characterized as a negative regulator of both programmed cell death (PCD) and broad-spectrum disease resistance, suggesting its putative role in the ubiquitination/protein degradation machinery, or other ubiquitin-mediated pathway. A novel role for SPL11 in flowering time control was identified via its interaction with SPIN1 (SPL11-interacting protein 1), a nuclear, RNA/DNA binding protein and STAR (signal transduction and activation of RNA) family member. In addition to ubiquitinating SPIN1 in vitro, SPL11 downregulated the expression of Spin1 during the light phase to promote flowering. Overexpression of Spin1 inhibited flowering in both short and long days and affected Spl11 expression as well. These results link ubiquitination and RNA metabolism in flowering time control. Isolation of spl11-mediated cell death suppressor mutants (sds) provided additional evidence that PCD is under genetic control in the spl11 mutant. The sds mutants showed various levels of cell death suppression, had wild-type levels of resistance to bacterial blight, and repressed the elevated defense gene expression of spl11. The delayed flowering phenotype of spl11 in long days was partially repressed in sds plants independently of cell death suppression levels, suggesting that the PCD and flowering pathways may partially overlap. Another RNA binding protein was identified in a yeast two-hybrid screen designed to isolate SPIN1-interacting partners. Rbs1 (RNA binding and SPIN1-interacting 1) was shown to promote cell death when overexpressed in both Nicotiana benthamiana and rice. Rbs1 was upregulated in lesion mimic-expressing leaves of spl11, suggesting that it may contribute to the cell death phenotype. RBS1 may constitute a link between the flowering and PCD pathways controlled by SPL11. The data presented here provides the first evidence of a link between cell death control and the switch to reproductive development in plants.

Committee: Guo-Liang Wang PhD (Advisor); David Mackey PhD (Committee Member); Biao Ding PhD (Committee Member); Terrence Graham PhD (Committee Member) Subjects: Botany; Molecular Biology; Plant Pathology

Doctor of Philosophy, The Ohio State University, 2008, Molecular, Cellular, and Developmental Biology

During animal development, some key transcription factors, such as paired-box proteins (PAX proteins), regulate target gene expression in multiple tissues, and thereby control multiple biological processes. Given the high conservation of PAX proteins among animal species, studies using the simple organism Caenorhabditis elegans (C. elegans)can provide insight into the role of Paxgenes in development of all animals. In C. elegans, a Pax2/5/8related gene egl-38functions in multiple tissues. For example, egl-38activity is necessary for proper development of the hermaphrodite reproductive system and mating structure in the male tail. egl-38is also critical during embryogenesis. In the germline, EGL-38 and another Pax protein PAX-2 can influence germline cell death. Although multiple functions of EGL-38 have been identified, there is less known about the specific molecular details of how EGL-38 regulates target genes and how it is controlled by upstream regulators. To understand how a Pax protein mediates different outcomes in different cell types, it is necessary to elucidate the underlying logic behind EGL-38 tissue and target gene specificity. In addition, identification of new egl-38target genes may uncover novel roles for Pax2/5/8genes in C. elegansdevelopment. In Chapter 2 of this thesis, I show that EGL-38 coordinates with a protein complex formed between CES-2 and ATF-2 to modify the temporal transcription of a gene, lin-48. This combination influences the function of lin-48in determining excretory system development and anatomy, but not in its post-embryonic role in influencing excretory system physiology and salt tolerance. In appendix 2 and other work, we showed that EGL-38 and PAX-2 directly regulate ced-9, a bcl-2like anti-apoptotic gene, to influence germline cell death. In Chapter 3, the impact of the tyrosine receptor kinase (RTK) VAB-1 and its ephrin ligands on germline cell death is characterized. It is possible that VAB-1, acting through MAPK, regulate (open full item for complete abstract)

Committee: M. Chamberlin (Advisor) Subjects: Biology, Molecular

Master of Science, Miami University, 2011, Botany

Flooding of crop lands results in the displacement of soil oxygen, which causes physiological stress in the plants. Like some other legumes, flooded Glycine max cv. 'Yukihomare' seedlings develop a longitudinal cavity in the vascular cylinder beginning 1.0 cm behind the root tip and progress for varying distances toward the root base. We have investigated the transcriptome during vascular cavity formation in order to identify the involved genes. This study was focused around a 24 hour time course DNA microarray analysis of total RNA during vascular cavity formation. Results indicate 8529 genes are temporally up- or down-regulated two-fold or more during flooding. We found rapid changes in gene expression for ESTs involved in transcription and signal transduction and metabolism. There was two apparent waves of ethylene signaling in response to flooding, suggesting a multistage response. Hallmarks of programmed cell death were observed, and attributed to the formation of vascular cavities.

Committee: Daniel Gladish PhD (Advisor); Qingshun Li PhD (Committee Member); Chun Liang PhD (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Botany; Molecular Biology; Plant Biology; Plant Sciences

Doctor of Philosophy, Miami University, 2008, Botany

Parenchymatous cells in the center of the vascular cylinder of Pisum sativum (garden pea) primary roots degenerate in response to flooding at warm temperatures and form a long continuous cavity. Vascular cavities seem to provide a conduit for longitudinal oxygen transport in the roots. Many organisms are known to sacrifice specific cells by programmed cell death (PCD) to survive stresses. Presence of full-length cavities allows continued growth of the pea roots during flooding stress, and characteristic cytoplasmic degradation has been previously detected in the degenerating cells of cavity-forming roots. Hence, the hypothesis of this dissertation was that vascular cavity formation involves PCD. The present study shows that this cellular degradation initiates at around 3 h after flooding and continuous cavities usually develop within 6 h after flooding. The degenerating cells had thinner primary cell walls, less electron-dense middle lamellae, and less abundant cell wall homogalacturonans (HGs) in altered patterns, compared to healthy cells of roots without cavities. Changes in cell wall HGs have been associated with PCD-induced cortical aerenchyma formation in maize. Systematic DNA fragmentation, a hallmark character of PCD, was detected in vascular cavity-forming pea roots in the current study. DNA fragmentation occurred rapidly, within 3 h after flooding. The DNA fragments produced were about 20-30 kb, possibly corresponding to the size of chromatin loops in pea chromosomes. No low-molecular weight DNA fragments (inter-nucleosomal fragments) were detected. Release of cytochrome c from mitochondria into cytosol, a characteristic feature of a common PCD pathway, was detected in the cavity-forming roots 2 h after flooding. Hence, release of cytochrome c seems to be upstream to DNA fragmentation and cellular breakdown. All the cytological changes however, remained confined to the parenchymatous cells in center of the vascular cylinders, even after 24 h of f (open full item for complete abstract)

Committee: Daniel Gladish PhD (Committee Chair); John Kiss PhD (Committee Member); Qingshun Li PhD (Committee Member); Nancy Smith-Huerta PhD (Committee Member); Brian Keane PhD (Committee Member) Subjects: Biology; Botany; Cellular Biology; Molecular Biology

Master of Science, Miami University, 2008, Botany

This study reports the identification Glycine max varieties that form vascular cavities in their primary roots under warm and flooded conditions. Vascular cavity formation is a PCD-mediated process and this study explores the induction of caspase-like proteases, a hallmark of PCD. Injection of caspase inhibitors have resulted in suppression of vascular cavity formation under hypoxia. Substrate cleavage assays using synthetic caspase-specific substrates also show a marked increase in caspase activity after flooding. Further, the caspase like proteases are inhibited only by specific inhibitors and not by general protease inhibitors. Caspase-like proteases are pH sensitive and are effective under acidic pH conditions. Study of in vivo caspase-like protease activity employing peptide-based fluorogenic substrates show their activity in the cytosol of cells in the vascular cylinder of soybean roots. This study is expected to prompt a deeper understanding of hypoxia-induced PCD and add valuable data to existing knowledge of plant PCD

Committee: Daniel Gladish (Committee Chair); Qingshun Li (Committee Member); Alfredo Huerta (Committee Member) Subjects: Botany

Doctor of Philosophy, Case Western Reserve University, 2004, Pharmacology

The Bcl-2 oncogene encodes a protein that inhibits apoptosis or programmed cell death. Apoptosis is a process critical for development and tissue homeostasis and is of great importance to cancer chemotherapy. The molecular mechanisms of apoptosis are still poorly understood, but it is known that a number of events occur on mitochondria such as membrane permeabilization and release of intermembrane space proteins into the cytoplasm. These mitochondrial events are regulated by the Bcl-2 family of proteins. This family is made up of an anti-apoptotic subfamily (Bcl-2, Bcl-xL, Mcl-1) and two pro-apoptotic subfamilies, the multidomain pro-apoptotic proteins (Bax, Bak, Bok) and the BH3-only subfamily (Bid, Bim, Bik, Bad). These proteins were initially thought to function on the mitochondria to regulate mitochondrial events of cell death, but it has subsequently been shown that Bcl- 2 family members can regulate cell death when localized to the endoplasmic reticulum (ER). The work presented here shows that Bcl-2 localized to the ER can inhibit the activation of mitochondria-localized Bax, indicating that an intermediate must exist between the ER and mitochondria. This work also shows that Bcl-2 localized to the ER can bind to BH3-only proteins preventing them from activating Bax on the mitochondria. In addition to anti-apoptotic proteins, it was determined that the pro-apoptotic cleavage fragment of Bcl-2, deltaBH4 could induce apoptosis when localized to the ER, but with less potency than deltaBH4 localized to both the mitochondria and the ER. This work also demonstrates that deltaBH4 and deltaBH4 on the ER can induce the depletion of ER calcium, suggesting a probable mechanism for their toxicity. These data, taken together suggest a model whereby the Bcl-2 family works to regulate ER to mitochondrial communication during apoptosis.

Committee: Clark Distelhorst (Advisor) Subjects: